Antigen-antibody reactions and cation transport in biomembranes: Immunophysiological aspects

Biochhnica et Biophysica Acta, 415 (1975) 173-229 (') Elsevier Scientific Publishing C o m p a n y , Amsterdam - Printed in The Netherlands BBA ,~5147

ANTIGEN-ANTIBODY BIOMEMBRANES: PEIER

REACTIONS

AND

IMMUNOPHYSIOLOGICAL

CATION

TRANSPORT

IN

ASPECTS

K. L A U F

Department of Physiology & Pharmacology and Division ~f hmmmology, Duke University Schoo! of Medicine. Durh,.#n, N. C. 27710 (U.S.A.) (Received January 23rd, 1975)

CONTEN]'S I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. General propertics of reacting c o m p o n e n t s

. . . . . . . . . . . . . . . . . . . .

174 175

A. Antibody and lectins: site recognition and effector function . . . . . . . . . . .

175

B. Antigens, receptors related to m e m b r a n e permeability functions

. . . . . . . . .

180

C. N a t , K ÷ transport and ATPase prior to immunological alteration . . . . . . . . .

181

III. Modification of cation transport and (Na+,K-)-ATPase by antigen-antibody reactions and lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

185

A. Stimulation of cation transport . . . . . . . . . . . . . . . . . . . . . . . .

185

I. Association of Na +, K + transport polymorphism with the ML-isoantigens in erythrocytes of some ungulates . . . . . . . . . . . . . . . . . . . . . . . . . .

1~5

a. Genetics of cation transport polymorphism . . . . . . . . . . . . . . . .

186

b. Biophysical basis o f c a t i o n transport bimodality . . . . . . . . . . . . . .

186

c. Biochemical differences of m e m b r a n e (Na*, K*)-ATPase . . . . . . . . . .

190

d. Physiology of maturation of cation polymorphic ungulate red cel!s . . . . . .

191

e. Membrane structural aspects and other physiological parameters . . . . . . .

192

f. Genetics of the MI,-antigen system . . . . . . . . . . . . . . . . . . . .

194

g. I m m u n o c h e m i s t r y o f a n t i - M and the M-antigen . . . . . . . . . . . . . .

195

h. The effect o f a n t i - L on the N a * , K ' F'ump and ATPase in LK red cells of sheep, goats and cattle . . . . . . . . . . . . . . . . . . . . . . . . . . . .

197

i. Immunochemistry of anti-L and the L-antigen . . . . . . . . . . . . . . .

202

2. Alteration of cation transport by lectins in nucleated cells . . . . . . . . . . .

205

a. Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

205

b. T u m o r Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

210

B, I n h i b i t i n g or "'silent" antibodies against N a * , K " transport and ATPase

. . . . . .

211

IV. Cellular metabolism, membrane i m m u n e events and ion permeability . . . . . . . . .

213

V.

217

I m m u n o l o g i c a l alteration o f ion transport in lipid model membranes . . . . . . . . .

Vl. Conclusion and prospectus Acknowledgments References

. . . . . . . . . . . . . . . . . . . . . . . . . . .

219

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

220

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

220

174 I. INTRODUCTION Immunological tools are now being increasingly applied in investigations on ion permeability in membranes of living cells as well as in artificial lipid membrane systems. For various reasons the immunological approach has been aimed at studying particularly transport of cations rath6r than anions. Membranes are much less permeable to cations than to anions, and it is known that immunological reactions do alter the low cation permeability of membranes. Furthermore, the immunological modulation of the energy dependent active cation transport is presently being intensely investigated since considerable information has accumulated about the physiological and biochemical properties of the membrane components involved in binding and translocation of monovalent cations. The purpose of this article is to review the recent progress in this field. A prerequisite lbr such studies is the presence of surface antigens or receptors through which specific antibodies (via antigen-antibody reactions) or other immunological probes such as lectins interact in a manner that modifies membrane cation permeability. Antigens or receptors will be considered as being close to or part of the membrane constituents comprising cation transport systems. The intimacy of such structure-activity relationships has been also analyzed on isolated membrane components still capable of at least partial reactions of the in situ system. Cellular Metabolism

Ion Permeability "

•

5

Membrane Structure

x

Antigens, Receptors Antibodies, Lectins Diagram I In intacl cells, cation permeability is a function of membrane structure and is interdependent with cellular metabolism. Thus, as shown schematically in Diagram I, two principal approaches in this newly emerging field of membrane immuno-physiology can be envisaged and will be illuminated throughout this review. First, direct (Path 4) or indirect (Path 2 and 3) effects of antibodies or of lectins (Path I) on cation permeability may be studied. Membrane cation permeability may be modified by such molecules in several ways: activation or inactivation of transport sites, changes of kinetic parameters, effect on active and/or passive cation transpolt processes, to name a few. It is possible that antigen-antibody reactions affect cellular metabolism via ion permeability changes (Path 5) and that amplifier mechanisms cxist (Path 5-7-3).

175 Second, manipulation of cellular metabolism, which is known to influence cation permeability and membrane structure (Path 6 and 7), may be investigated in its effect on distribution and steric conformation of membrane surface antigens (Path 8) and their interactions with immunological probes. Amplification may also be at play here (Path 7-3-5-7-8). These approaches do not necessarily require that the antigen-antibody reaction must occur solely at the outer membrane surface, since, for example, it is now possible to incorporate large molecules such as antibodies into red cell ghosts by reversed hemolysis followed by a resealing process [1 ]. Inverted membranes (inside-out vesicles [2])should also prove useful in the study of immunological effects on cation permeability from the inner surface of the membrane. Finally, antigen-antibody reactions are, of course, instrumental in the isolation and identification of membrane constituents comprising the cation transport system and are powerful tools to study conformational transitions of the isolated cation transport components during partial reaction steps. In the following sections a discussion of the general properties of the reacting components i.e. antibodies, lectins, antigens and the Na+,K ÷ transport system will preceed the analysis of effects of the antigen-antibody reaction on cation permeability in cell membranes. In addition to reviewing extensively the work of this laboratory*, other systems will be considered such as the changes in cation transport of some nucleated cells by lectins, the problem of the anti-Na ÷ pump antibody, and immunological alteration of ion permeability in artificial lipid membranes. It is not only the mere "scio ut nescio" but also the magnitude of the tasl~ to gather for the first time immunochemical and physiological aspects of the effect of antibodies on cation transport which may result in omitting "sine malevolentia" some literature references.

II. GENERAL PROPERTIES OF REACTING COMPONENTS

HA. Antibody and lectins: site recognition and effector function Although entirely different in their origin and structure, antibodies and lectins will be shown in this review to be capable of modulating cation transport processes in biomembranes. Antibodies (immuno-globulins) raised against antigenic membrane constituents are structurally similar to thgse produced by injection of any soluble, nonmembraneous antigen, but they are very different from the lectins, which occur naturally in plants and seeds. The common structural basis for affecting membrane functions is, however, the bi- or multivalent "combining sites" of these molecules i.e. regions on an antibody or lectin which will specifically recognize and bind membrane ligands. In the case of the antibody, an antigenic or haptenic group is the ligand. while lectins generally bind to certain carbohydrate structures, which may also be antigenic and thus recognized by antibodies. Both antibodies and lectins may agglutinate cells, a macroscopic phenomenon not necessarily affecting cation trans* See Acknowledgement

176

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F(ab)

....... N H 2 ~ Variable

Hinge Regi0n

~IConstonl

?CA ~ - - - - ~ ' ~

Fc . . . .

"~

I

"

CHO

F

I

I

CO,C,,F

I

COOH

Si I

Heo'q Chain

'l

P C A ~ NH 2 ~

F(ab')~

Lighl Chore - - J

! I

I

S

Clio

I I

,CJ- E

I,

.' i

Pepsin l)iagram 2

port processes. Because of their cell-aggregating property, lectins are usually termed agglutinins, or hemagglutinins if red cells are involved. On the molecular level, however, it appears evident that some antibodies do tilter cation transport in nucleated and enucleate cells, just as some lectins modify cation permeability in lymphocytes and induce cellular transformation and mitosis. In regard to the latter events, the terms lectins and mitogcns are used synonymously. There is ample evidence that antibodies and lectins, when applied to cell suspensions, exert their effects after binding from the outside of the cell membrane, and for simplicity', any uptake into tile cell by phagocytic processes will be considered as secondary events unrelated to primary changes of membrane permeability processes. Since some structural properties of antibodies and lectins (mitogens), such as the valency of their combining sites, are instrumental in eliciting membrane permeability changes, a brief recapitulation of their general structure features is justified. For more detailed information the ~eader may consult reviews on the structure and functions of antibodies [3-7] and lectins [8-9]. The basic unit of immunoglobulins (Diagram 2) consists of four polypeptide chains: two heavy (H) molecular weight chains (M~ of each ~ 50-75000 depending on the isotype or class) and two light (L) molecular weight chains (M~ of each _~ 22500) structurally joined by at least three interchain disulfide bonds and non-covalent threes. Reduction by mercaptans and alkylation by iodoaeetamide in presence of dissociating agents cleaves the interchain disulfide bridges and generates the individual polypeptide chair.s [10], and is generally associated with a dramatic loss of antigen combining activity [11]. A typical representative of the four chain basic unit is imrnunoglobulin G or IgG (see Diagram 2). which has a molecular weight of ca. 160 000 containing two N-terminal sites each capable of combining specifically with an antigen. The division into live major immunoglobulin classes or isotypes, namely IgG, IgA, IgM, IgD and IgE, is based primarily on differences in the H-chains of these proteins, such as primary sequence, carbohydrate content, antigenic property and molecular weight. L-chains occur in two isotypes, z or 2, either of which may

177 be associated with any of the immunoglobulin classes. All five immunoglobulin istotypes exist as monomers (basic unit) or dimers except IgM which is a pentamer of five covalently linked basic units [12]. Proteolytic enzymes have been shown to cleave lgG at unique points. Papain, by attacking at the hinge region N-terminal to the inter H-chain disulfide bridges, generates two monovalent F(ab) fragments Mr ca. 50000), each composed of one L-chain and a portion of the H-chain, and the Fc fragment, containing the remaining C-terminal portion of the H-chains, which is incapable of antigen recognition [13]. Pepsin hydrolyses peptide bonds beyond the inter H-chain disulfide bridge, thus cleaving a bivalent F(ab')2 fragment (Mr -- 100000) from an Fc' fragment consisting of the C-terminal part of the H-chain [14]. Within the F(ab) portion, the N-terminal halves of the L-chain and the H-chain fragment are genetically variable and, in some segments, "hypervariable" in their amino acid sequence [15,16]. It is this particular alignment of the two variable polypeptide portions which is related to the unique antibody specificity and diversity [17]. Thus immunization with an antigen induces the selective formation of an antibody molecule which, in its combining site or F(ab) fragmenl, is specifically complementary to the immunogenic group on the antigen. This immunologic specificity is the basis of the classical immune-precipitation and immune-agglutination reactions [18]. In generating either monovalent or bivalent antibody fragments, these proteolytic techniques are important tools for determining whether membrane functional changes brought about by antigen-antibody reactions require the presence of one or mul'i point-interactions and thus relate to number and mobility of the antigenic ligands in the surface membrane. Diagram 3 demonstrates that these and other techniques were instrumental in delineating the various functions of the antibody molecule. It is also evident that the Fc portions of the molecule, being inert with respect to antigen binding, carry a variety of physiological properties. It has been customary to distinguish the F(ab)or F(ab')2 related "simple recognition functions" of the antibody molecule from its Fc associated "effector functions". During our discussion of antigen-antibody reactions and their effect on cation transport in biomembranes a third category of "complex recognition-associated effector functions" must be established. "Simple recognition functions", for example, involve "sensitization" of the cell surface with a specific antibody directed against a membrane component. Whether or not cell agglutination occurs is decided by the nature of the antigen, size and valency of the antibody molecule as well as the electric surface charge of the cell membrane. This complex interaction per se is not necessarily accompanied by changes in cation permeability. Among the effector functions of the antibody molecule, unrelated to the F(ab) portion but related to the Fc fragment, are the fixation of complement [19], the basophile and mast cell sensitizing properties of lgE [20], the capability of IgG to traverse the placental barriers and its capacity to bind to macrophage membranes [21,22] and liposomes [23]. These phenomena, although partially understood, have physiological aspects, particularly the resultant effects such as complement-mediated immune cytolysis, lgE mediated histamine release [24]

178 "Simple" Recognition Functions

Complex Recognition Associated Effector Functions

Soluble Antigen-Antibody Complexes

Alteration of Membrane Substrate and Ion Permeability and Transport Enzymes

Immune Precipitation Effects on: "Sensitization" of Membrane Surfaces

Erythrocytes Nucleated Cells

Cyto-Agglutination of Antigenic Cells

Membrane Na~K ÷ ATPase F(ab) or F(ab')2 Combining Sites

SOME FUNCTIONAL ASPECTS OF ANTIBODY S T R U C T U R E Fc-Fragment Effector Functions Complement Fixation

Cytolysis

Placental Interaction

Transport across Placenta

Macrophage Sensitization ~

Permeability Changes

Liposome Sensitization IgE Binding to Basophiles

~-~ Histamine Release

Diagram 3

or the change in solute permeability as Fc fragments or whole lgG protein are inserted into artificial liposomal membranes [23]. "Complex recognition associated effector functions" require the antigen-antibody reaction per se in the first step followed by an effect on membrane functions. Examples of such kind of sequential reactions are: (a) the L-antibody (anti-L)

179 induced stimulation of active Na+,K + transport in red cells of ungulates; (b) the anti-Na ÷ pumpantibodyand (c) the antibody against isolated peptides of the (Na ÷, K+). ATPase system. These effects will be discussed below (see Section III). In analogy to the experiments on liposomes [23] the Fc fragments may be instrumental in bringing about cation permeability changes in lymphocytes. This binding of specific antigens to Fc-mediated membrane-bound antibodyon primed lymphocytes cart be considered as another example ofeffector functions involving a specific recognition process. Certainly, there are a number of other effects of antigen-antibody reactions not affecting membrane cation transport processes which also have their place in the above category: the stimulation by an antibody of a defective fl-galactosidase found in Escherichia coil mutants [25-30], the activation of penicillinases from Bacillus licheniformis by anti-penicJllinase antibodies [31], the stabilizing effect ofanti-acetylcholinesterase antibodies on the activity of heated bovine red cell acetylcholinesterase [32-35] and the inhibiting action of antibodies against bovine pancreatic ribonuclease [36-40] or Vibrio cholerae neuraminidase [41]. It should be pointed out, however, that antibodies to any antigen tested are heterogenous and that in the course of immunization a selection of higher affinity antibody populations against membrane functions may contain a spectrum of activating, inhibiting or even only blocking antibodies (e.g. antibodies which prevent others from binding and acting). This introduces a considerable complexity into the analysis of the effect of antigen-antibody reactions on cation transport in biomembranes just as it has been shown in the ribonuclease anti-ribonuclease system [39,40]. (For in depth review consult [42]) The basic structural and functional properties are quite similar in all immunoglobulins, but have not yet been established for lectins. Of the hundreds of lectins known [43] only a few have been thoroughly studied with respect to their structure and biological effects. These are the lectins from the red kidney bean (phytohemagglutinin [44], concanavalin A from the jack bean [45,46], wheat germ agglutinin [47] and the lectin from the common lentil [48]. The structural common denominator for these molecules (molecular weights in the range from 104 to 105) are two or more combining sites which react preferentially with a variety of terminal sugars of membrane glycoproteins or glycolipids. Their combining sites have been estimated to be somewhat more shallow than those of immunogiobulins which may accommodate up to pentamers of amino acids, monosaccharides or nucleotides [17]. It is well known that blastogenic transformation oflymphocytes can be induced by phytohemagglutinin [44], concanavalin A [49], and the lentil lectin [50-52]. It will be the topic of Section III A.2. to showthat one of the earlyevents accompanying blast cell transformation, the dramatic change in membrane cation permeability and ATPase activity, occurs upon exposure of lymphocytes to phytohemagglutinin or concanavalin A. Thus lectins prcmise to be powerful tools in the study of early cation permeability changes during cell transformation, although the ubiquitous occurrence of their sugar ligands and their rather high equilibrium binding numbers (106 or more per lymphocyte [52] make it presently difficult to reconcile their action on cation transport with a direct binding to its structural constituents.

180

liB. Antigens, receptors related to membrane permeabili O,./'unction~ In the light of tile effects discussed below of antibodies or lectins on membrane transport processes, one should expect that antigenic groups or receptors arc intimate structural parts cf the molecules involved in the translocation of cations, Recent reports on the production of antibodies against purified ( N a ' , K ' ) - A T P a s e preparations from various sources [53] suggest that some, if not all, postulated subunils of the enzyme contain antigenic regions through which antibodies bind and m~ly or may not alter the enzymatic process associated with the active cation transport. It is of interest that these antigenic specificities appear to be mainly located at the inner portion of presumably transmembraneous polypeptide chains [53,54]. Since these antisera contain antibodies which either inactivate the ( N a * . K ) - A T P a s e [53], detect conformational changes [55,56] or bind without any effect at all [54], a considerable heterogeneity of the antigenic groups has to be assumed provided they are indeed part of the transport polypeptide chtfins. The interspecies heterogeneity of these sites, however, appears to be less because anti-kidney enzyme antibodies, for example, crossreact with kidneyenzyme of other species [57]. Similarly, organ specificity is not pronounced since anti-kidney enzyme antibodies have been showntointeractwith brain-derived enzyme and, furthermore, altered cation processes when incorporated into erythrocyte ghosts [53]. Antibodies against antigenic groups at the outer aspect ot the Na+,K + transport system have not been demonstrated ~ls yet when purified (Na+,K ~)-ATPase preparations were used as antigens. Unequivocal presence of an antigen at the outer surface membrane has been demonstrated only on cation polymorphic sheep red cells, and its structure activity relationship with the Na+,K ~ pump system in these ceils will be discussed in Section III A. It is now fairly well established that a variety of mitogenic substances such as plant agglutinins, anti-lymphocyte sera as well as antigens to lymphocytes primed in vivo can activate membrane functions, in particular active Na~,K ~ (pump) transport as well as the (Na~,K *)-ATPase of isolated microsoma[ membrane fractions derived from these cells. The effect of anti-lymphocyte antisera ol antigens requires the presence of specific antigens or antibody combining sites on the lymphocyte membrane, respectively, and thus is based on classical ~mtigen-antibody reactions. In the case of the lectins carbohydrates constitute the membrane ligands. From monosaccharide inhibition studies it is known that various lectins differ in their sugar specilicity and thus must react with different carbohydrate moieties on the membrane. For example, terminal galactose residues as well as the penultimate sugar sequence (i.e. mannose) are crucial in binding phytohemagg[utinins from Phaseoh~s vulgaris or Robinia [58], while ~t-D-gluco or ~-D-mannopyranosyl residues are the principal structures involved in binding of concanavalin A [59]. These sugar residues ~Jre known to occur on membrane glycoproteins and to some extent on glycolipids as part of small oligosaccharide side chains. The number of these receptors (10s-10~'/ cell) equals or is somewhat greater than the number of glycoprotein copies and thus larger by an order of magnitude than the number of transport polypeptide chains. Similarly, the number of antibody receptors is probably much larger than the number

181 of cation pump sites per cell. This apparent numerical discrepancy as well as the fact that glycop~oteins or glycolipids are the target molecules for mitogenic substances poses the question as to how the alteration of active cation transport is brought about, for example, in lymphocyte membranes. Since the major polypeptide chains of the (Na+,K+)-ATPase enzyme complex (i.e. the 100000 dalton moieties) are not glycoproteins, they are obviously not candidates to carry binding sites for lectins. Thus more complex interactions between the carbohydrate bearing membrane constituents and the Na+,K + pump system have to be considered. To name a few possibilities: adjacent glycophorin molecules [60-64] may be crosslinked by multi-valent lectins, or the minor carbohydratebearing components associated with the larger transport polypeptide chains may bind lectins. These events may ultimately affect lipid-protein or protein-protein interactions and thus membrane fluidity [65,66]. That even sodium periodate oxidation of membrane carbohydrates in intact ceils seems to affect membrane permeability [67] and induce cellular transformation invalidates to some extent the restricted sugar specificity of the various lectins used and introduces a quite non-specific aspect of transport stimulation by these substances. Presently, it appears a "fait accompli" that any perturbation of the outer surface membrane of lymphocytes or their carbohydrate-water shell may affect the membrane structure in the vicinity of the cation transport loci. ilC. Na +, K + tran.sport and A TPase prior to #nmunological alteration

in iso-osmotic plasma the cation steady state of the cell and its volume are regulated by diffusion-controlled, or leak fluxes and energy dependent, active cation transport mechanisms, while exchange diffusion processes, known to occur in various magnitudes in different ceils, are functionally unimportant in this respect. The model schema of a cell at steady state shown in Diagram 4 shall merely illustrate the

KS NO+

,.-Z.

NOo'~f~ ~NoT Diffusion

Cl#

--

+

÷

-

'

'

'

'

A;K NoTCI;

t ~'~

I

ATP ~'---~

L

~2K

~g~t~,1~; ,fl

Ko] I .."' "~

ATP

/

3N°T

I I

k Pump~

.I

.___~"~S I ........ O uobo,n x,d. •4-

Diagram 4

4-

NO o ; no K o

+

182 general aspects of membrane permeability functions discussed below which may be the target for antigen-antibody reactions. The driving forces which move ions across the cell membranes are partially of chemical (osmotic) and partially of electrical nature. As, for example, in red ceils the permeability of the membrane to small anions (Ci-, HCO3- ) is about 106-fold higher than that to cations, the Donnan ratio of their concentrations in plasma versus cytoplasm is a function of the large non-penetrating intracellular anions (mainly hemoglobin, ATP and 2,3 diphosphoglycerate) and thus is the principal determinant of the slightly negative membrane potential. Hence in these cells Goldman's constant field equation for the passive distribution of ions across the membrane can be practically reduced to the Nernst equation in which the chloride concentratiou ratio sets the membrane potential to about --- 10 mV, a value quite in agreement with recent direct measurements by microelectrode techniques [68,69]. In some nucleated cells [70], however, the membrane is not as impermeable to cations as in the case of red cells and cation leak fluxes have been found to be larger than in erythrocytes. In this situation the negative membrane potential was computed to be more in the directicn of a K + equilibrium potential [70]. As the distribution of cations between cell and plasma strictly observes electroneutrality, their tendency to move downhill across the membrane is affected by the membrane potential which is controlled by the anion permeability. Principally, this concept has not been changed by more recent findings suggesting a carrier-mediated and electrically silent anion exchange pathway through which anions pass at least 104 times faster than through the electric or conductance pathway [71-73]. The cation distribution of a high K + cell in a high Na + plasma, however, is far from equilibrium. Active, energy dependent transport processes arc required to compensate for the continuous loss of K + from the cell and gain of Na + from the surrounding plasma. This functional impermeability of the cell membrane is gravely impaired when antigen antibody reactions lead to activation of membrane attacking complement factors[74,75]. In some of these reactions (own unpublished data) the (Na ~, K+)-ATPase seems to be unaffected while the membrane becomes several orders of magnitude more permeable to cations [76,77}. The ensuing hemolysis of the cells is ofcolloid osmotic nature, whereby the lesion produced causes more than a simple increase in non-selective cation permeability [78]. This effect, therefore, may be unlike that seen in red cells which have been made non-selectively permeable to Na + and K~ by exposure to the antibiotics gramicidine A [79,80] and nystatin [81]. The antigen-antibody reactions discussed in this review affect almost exclusively the active energy dependent movement of ions across the membrane, it is therefore relevant to outline briefly some major characteristics of the N a ' , K + pump and its apparent biochemical correlate, the (Na+,K+)-ATPase. Several recent reviews [82-89] have dealt extensively with the biophysical and biochemical properties of the two aspects of the cation transport system and should be further consulted by the reader. It is obvious that a de'ailed description of all complex facets of the cation transport system cannot be fully honored in this review.

183 The Na+,K + pump and the (Na+,K+)-ATPase have a number of properties in common which are the basis of our tacit assumption that both processes may be identical. In plasma of high sodium concentrations, (Na+)o, the red cell maintains its high K + gradient by an ATP-driven pump that exchanges three intracellular (Na+)l for two extracellular (K+)o as one ATP is bound and hydrolyzed to ADP and inorganic phosphate (P0 [90-92]. The standard free energy of hydrolysis of the y-phosphate group of A T P available in the cell was computed to be sufficient to fulfill the energy requirements of the Na+,K ÷ pump [93]. The coupling in the opposite direction of the Na + and K + movements is tight, and may be close to unity [94] or deviate from it somewhat as the intracellular ion composition is varied experimentally. Metabolically depleted red cells fail to maintain their high K + steady state concentrations. As the ATP concentration decreases within the cell, K + pump influx diminishes [95]. Sachs has shown that glucose deprivation of human red cells causes a fall in both ouabain sensitive K + influx and Na ÷ eft]ux [96]. Restoration of the original ATP level by incubation in glucose-containing media causes a reappearance of active transport activity [95]. The finding that ouabain inhibits the phosphoglycerate-kinase-mediated ATP synthesis established that the Na+,K + pump may act as a pacemaker of cellular metabolism [97,98]. In addition, a compartmentalization of some of the phosphoglycerate-kinase-derived ATP in the vicinity of the cation pump may occur [99,100]. Since the product of the vectorial phosphoglycerate action, 3-phosphoglycerate, is ultimately converted into lactate, part of the glucose metabolism can be related directly to the Na+,K ÷ pump rate [101]. Further evidence of the ATP dependence of the Na +, K + pump comes from experiments with resealed ghosts. When ATP is incorporated during the resealing process, the ghosts acquire the capability of actively extruding Nai + [102]. The general scheme for the (Na ÷, K÷)-ATPase activity was derived from investigations on brain and kidney membranes [103,104]. It consists of (a) ATP binding to the membrane ATPase [105]; (b) ATP hydrolysis and Na+-dependent formation of high energy Pj intermediates, and (c) K+-stimulated and ouabain-sensitive dephosphorylation and can also be applied to the membranes of red cells or other, nucleated cells. Partial step (a) and (b) when studied under low ATP concentrations may be referred to as the Na+-dependent activity [106]. The activity of the ouabain sensitive K+-dependent p-nitrophenyl phosphatase (K+-phosphatase) of isolated membrane fragments is believed to be related to partial reaction (c) [107,108]. In spite of some reports [109], there is still a lack of unequivocal evidence that the K ÷ phosphatase can be induced to pump K + into and Na ÷ out of the cell [108]. It has recently been shown that the nature of the chemical bond between the enzyme and the energized Pl group is a y-acylphosphate of L-aspartic acid [110]. The distinction into Na + and K+-dependent partial reactions, as well as the physiologic vectorial transport of K + into and Na + out of the cells, reflects the asymmetry of the Na+,K + pump system. Although the Na+,K ÷ coupling ratio may be different in other non-red-cell systems, all membrane pumps hydrolyze ATP at the inside (cis) and are inhibited by cardiac glycosides such as ouabain from the

184 outside (trans) of the membrane [1 I1-113]. Thus inside-out red cell ghost vesicles are not able to bind ouabain [I 14]. The activation kinetics by internal Na" and external K + exhibit complex similairites in both the Na ~, K ~ pump and the (Na ~, K+)-ATPase as studied mostly using red cells. In presence of high (Na ~ )o the K + pump influx is activated as (K+)o is increased and plateaus beyond 6-8 mM (K')o. The external activation curve is sigmoidal between 0 and 8 mM (K')o, This observation precludes Michaelis-Menten type kinetic analysis. Half maximum stimulation of K "~ pump influx (Kt,2) occurs between 1--2 mM ( K ' ) o in human red cells [115.116] and around 3 mM (K+)o in sheep red cells [117]. At very low (Na~)o, K1.,, is shifted to much lower (K+)o and the K" activation curve assumes the shape of a rectangular hyperbola [116] which may be described adequately by Michaelis-Menten kinetics [I 17]. The response of the N a ' , K + pump to (Na"),~ has been explained in terms ot" an interference by Na + with the binding of two K~o at the K ~ loading (trans) site [I 16,118]. Under certain experinaental conditions Na ~ and K ~ may move down their concentrations gradients via the membrane cation pump normally transporting these ions uphill against their electrochemical gradients. In media containing cnly Na+o and no K+o this reversal of the pump is tightly coupled and causes net synthesis of ATP as demonstrated in intact red cells [I 19.120] and rcsealed ghosts [121,122]; both processes are inhibited by ouabain. Antigen-antibody reactions have been shown to affect this reversed pump as well as ouabain sensitive K ~: K ~ exchange [123], while ouabain sensitive Na": K" exchange [116,124] or glycoside insensitive Na*: K ~ exchange [125], which may or may not be mediated by the pump, have not been studied in this respect. Half maximum saturation of the Na + loading site at the internal (cis) side of the pump has been found around 30 mM (Na~)~ using red cells whose (Na+)~: (K~)~ ratio was altered by cold storage [126] or the p-chloromercuribenzoate sulfonate method [127]. it follows that in a normal high K ~ cells the pump operates at or below half maximum activity and that an increase of (Na ~)~ activates the Na+,K pump system severalfold until it reaches a plateau. Apparently K+~ competes with Na+~ at the Na ~ loading site, ~,n effect that is much more pronounced in LK type red cells of sheep, goats and cattle (discussed in Section I11). This internal Na + activation of the cation pump has been shown to be independent of the type of external cation present [I 17}. The concentrations (Na~h and (K~)~ required to half maximal activate the (Na+,K+)-ATPase compare with those observed for the activation of the Na~,K * pump [I 28]. although kinetic analysis of the cation activation of this enzyme in broken membrane preparations is complicated by the loss of sidedness [129]. In resealed Na~-rich ghosts, suspended in Na~-o media, addition of K~o results in a sigmoidal activation of the (Na'.K")-ATPase, which becomes hyperbolic when N a ~ is omitted [129], a finding comparing favorably with experiments on intact red cells. Both the Na+,K ~ pump and the (Na+,K ')-ATPase are 50~, inhibited by about 0.5 • 10 SM ouabain. Thus ouabain, by virtue of its action at a still undefined outer

135 surface receptor, relates the two activities well. At low concentrations it can be shown that [3H]ouabain binds specifically and stoicheiometrically to the Na+,K + transport system, i.e. the inhibition of the Na+,K + pump or the (Na+,K+)-ATPase is proportionally related to the number of cardiac glycoside molecules bound. Ouabain inhibits not only the normal vectorial Na+,K + translocation but also the reversed Na + pump [120], further demonstrating the mutual interdependence of ATP utilization and Na+,K + transpolt. From [3H]ouabain binding studies about 200--300 Na+,K + pump sites have been computed for human erythrocytes [130,131] or their hemoglobin free ghosts [132] and at least an order of magnitude higher for lymphocytes [133] and other nucleated cells [134--136]. In sheep and goat red cells, where a cation pump polymorphism exists (Section [!!) the number of [3H]ouabain binding sites per cell are lower. However, as these latter cells aze smaller and nucleated cells are larger than human red cells, the average pump density in all these cells may not deviate too much from a few sites per/~2 surface area. Binding of ouabain is reversible and depends in a complex fashion on the ionic composition at the cis and trans side of the membrane. The binding is believed to obey the law of mass action whereby the association reaction of the drug with its receptor is considered to be a bimolecular process [134,137,138] with forward rate constants in the order of about 5" 104M-t "s -~ [138,139] and a dissociation rate constant which varies considerably between tissues [138,140]. Differences in the equilibrium dissociation constant Ko (about 0.5 • 10-'~M) are thus mainly explained by differences in the rate of dissociation of the complex which appears to be somewhat more thermally dependent than the forward reaction [138]. Monovalent cations affect the affinity of the membrane receptor for ouabain [141,142] and thus affect the number of molecules bound at a particular time. The dissociation rate of ouabain from its receptor may be enhanced when (K+)o is increased; (Na+)~ in the presence of ATP seems to affect this process inversely. The requirement at the inside of the membrane for, and cation dependence of ouabain binding to the membrane suggest that the Na +, K + pump must be in a specific conformation to accept the cardiac glycoside. In the presence of K+o required to run the pump, any change in the affinity of the pump for ouabain is difficult to analyze since, as stated, K+o, like Rb+0 or Cs+o, affect the rate of dissociation of the ouabain-pump complex [141]. It is interesting that half maximal inhibition of( I ) ouabain-binding (2) ox brain (Na*, K*)-ATPase is caused by 2 mM (K+)o, a concentration necessary for half maximum activation of the enzyme activity [143}. For further detailed information on the kinetics of [3H]ouabain binding and its dependence on structural features of the molecule, the reader is referred to recent reviews and detailed reports [87,144,145]. i11. MODIFICATION OF CATION TRANSPORT AND (Na*,K~)-ATPASE BY ANTIGENANTIBODY REACTIONS AND LECTINS

IIIA. Stimulation of cation tran,~port HIA-I. The association of the Na~,K + transport polymorphism ML-isoantigens in ervthrocytes of some ungulates

with the

186

a. The genetics of the cation transport polymorphism. Since the first investigations of Abderhalden [146] in 1898 it has been known that not all mammalian species possess red cells with a high (K+)L steady state composition. Abderhalden's quantitative analysis of "whole blood organics and inorganics" on a variety of species brought to light that as in humans [147], the blood of horse, pig and rabbit contains higher K + concentrations than that of cattle, sheep, goats, dogs and cats. Conversely, the blood Na + concentration was significantly higher in these animals. In 1937 Kerr [148] extended these studies and found that in sheep the variability in lhe blood cation composition can be explained by an intraspecies red cell cation polymorphism : group I sheep had low (K+)i, high (Na+)~ (LK) red cells, group II sheep high (K~)i, low (Na+)i (HK) cells, and group III sheep possessed red cells with about similar (K÷)i and (Na+)~ values. In 1954 Evans [149] showed that the distribution of Kerr type 1 and I1 varied among Scottish Blackface sheep, Cheviots and Merinos and, on the basis of progeny studies [150], concluded that the HK property is of recessive nature,while LK red cell-possessing animals "may be either homozygous or heterozygous for a gene affecting the electrolyte type of red blood cells". From the data of Abderhalden [146], Widdas [I 51] and Hallman [152] and his own studies, Evans [153,154] concluded that simple Mendelian inheritance determines the LK-HK bimodality in sheep red cells. Analysis of large numbers of sheep has revealed that the red cells of heterozygotes appear to have a (K÷)i slightly higher than that found in red cells of homozygotes [155,156], and it was suggested that the dominance of the gene controlling the LK property may be incomplete [155]. This finding should not be confused with the rare intermediate subtype, Kefl and KeA, corresponding to group III of Kerr's original study. The gene for LK property has been termed Ka as opposed to the recessive allele ka responsible for the H K character [156]. The L K - H K bimodality also applies to goat red cells [154] and has been now firmly established for red cells of cattle [157,158] and Indian buffalos [159]. While the L K - H K polymorphism has also been reported in red cells of the Australian phalanger [160], the dog and cat have only L K red cells [161,162]. Table 1 summarizes some of these genetic aspects of red cell cation polymorphism. b. Biophysical basis of cati,m transport bimodality. In order to learn more about the evolutionary cause of the intra-species red cell cation differences in sheep, goats and cattle and to understand the effect of antibodies on the Na÷,K ÷ transport system in their red cells, it is imperative to consider the physiology of the cation transport processes maintaining the low or high K + steady state concentrations in the mature enucleate red cells of these species. Furthermore, since peptide bond formation during protein synthesis appears to depend on (K÷)~ [163], the precursor of the mature I_K red cell in the bone marrow is expected to be of HK nature. The transition from H K erythroblasts and reticulocytes to the adult enucleate LK red cell must involve fundamental changes in the cation transport properties. In addition, the correlation between the active cation transport and the activity of the (Na",K+) ATPase in ghosts derived from HK or LK red cells must be considered, and it is relevant to ask whether the Ka-locus affects other structural and functional para-

-+*

-r -~--r

t--

-T --~--

Sheep

Low High Low High Low

High Low

Relative Na +, K + pump and ATPase activities M M L L M L **H' **S

Transport associated antigens Anti-M Anti-M Anti-L Anti-L Anti-M Anti-L Anti-M Anti-L

Sheep antibody tested None None Stimulation Stimulation None Stimulation None Stimulation with some sera

Na+,K + pump and ATPase

Effect of sheep antibodies

Hemolysis Hemolysis Hemolysis (dosage effect) Hemolysis (metabolically dependent) Hemolysis No hemolysis Hemolysis Hemolysis

In presence of complement

* Heterozygote ** In cattle there is no clear cut relationship between (K+), and the H' and S antigens. Sheep anti-M, however, lyses H' positive cattle cells while sheep anti-L never lyses S negative cattle cells. (Consult ref. 222).

Cattle

Goat

Red blood cell cation type HK LK

Ungulate

BASIC RELATIONSHIP BETWEEN CATION TRANSPORT POLYMORPHISM, M E M B R A N E S U R F A C E ANTIGENS AND EFFECT O F ANTIBODIES IN UNGULATE ERYTHROCYTES

TABLE 1

OO

188 meters of the LK erythrocyte membrane. These aspects have been studied most in sheep red cells, but more recently also in goat and cattle red cells. In their classic paper Tosteson and Hoffman [94] laid tile foundation for our understanding of the volume regulation in HK and LK sheep red cells. To maintain their high potassium steady state compositions HK red cclls have been found to pump cations four times faster than LK red cells. Since N, the Na+:K + coupling ratio, is approximately unity in both HK and LK red cells, it was concluded that the control of the dift'ercnt potassium steady state compositions occurs by the same mechanism. Kinetic analysis [117] of the external activation ol the ouabain sensitive Na ~,K ~ pump showed that in Na" medium the pump rate of both types of sheep red cells is sigmoidally related to (K"),, with a K a : of about 3mM. a value higher than that observed in human or goat cells (see Section IIC. and [I 18.164]). In Na ~ free media the (K*)o activation curves become hyperbolic and obe). the Michaelis-Menten equation w i t h a K ~ . 2 f o r HK cells of 0.6 and for LK cells of 0.2 mM (K')o [117]. Tiffs transition of the external activation curve to a rectangular hyperbolic function is compatible with the assumption [I 18] stated above (see Section !1 C.) that sodium competes for two potassium loading sites. It is of interest that in the absence of sodium the maximum K" pumping rate is reduced to about one half in sheep [I 17] and human red cells [165]. A more complex picture emerges from the analysis of the internal cation activation kinetics by Hoffman and Tosteson [117]. Using the para-chloromcrcurylbenzoyl sulfonate method of Garrahan and Rega [127], it became apparent that at their physiological intracellular cation composition, active cation transport in HK and LK red cells is considerably less than when (K+)~ was reduced to practically zero values by replacement with Na ~ ions. Although lo~'ering (K+)~ by replacing with Na + somewhat complicates a rigorous kinetic analysis, the data have revealed that the sodium loading site of LK cells appears to be very IK'), sensitive: K' pump influx approaches zero in these cells as (K')~ is raised to about 40". of the total inlracellular monovalent cation concentrations. The exponential rise in the K' pump activity in LK cells when (K')~ was reduced to values less than 1 raM is different from the internal activation curve tk)r H K cells and reaches a maximum which is about 1/6 of the maximum rateobserved in HK red ceils [I 17]. F'rot-n curve fitting analysis of Hoffman and Tosteson's data [I 17] Schmidt [166] concluded that the best fit of these data was obtained by assuming that the altinity of the sodium loading site in LK cells was twice for K" ions than tk)r Na ~ ions. This analysis is based on the assumption that two K" ions are required to inhibit each site. Another stud.',' of the internal activation kinetics of HK cells, although of some bimodal shape, concludes that tile Na* loading site favors Na ~ over K ' ions by a large margin [164]. One of tile main conclusions from these studies is that tile HK-LK cation transport polymc~rphism resides in cation aflhaity difl'erences of tile N a ' . K " pump. Thus LK sheep red cells ha,,e a low altinity for Na" at the internal loading site and are more responsive to extcrnal Nit' ions. since in the absence of (Na')o the K~.2 for

189 (K+)0 is about 1/3 of the value for HK cells. Only homozygous LK sheep red cells have been used in these kinetic studies. For the final analysis, it would certainly be of interest to know whether heterozygote LK sheep red cells exhibit internal activation curves of intermediate type between those of HK and homozygous LK cells (see also lllA. l.f). Finally, it is important to note that, although the actual pump rate is a function of internal and external cation composition and concentration, the activation at the internal or cis side was found to be independent of the ions at the external or trans side of the pump [117]. Based on this independence principle Hoffman and Tosteson concluded that the Na+,K ÷ pump in sheep red cells is not of sequential type [117]. Thus the number of cis sites occupied with Na ÷ ions is independent of the trans ion concentration. In addition to the qualitative differences of HK and LK pumps, the greater pump maxima in HK cells are also due to a greater number of pump sites [167]. Using [3H]ouabain the mean n u m b e r o f pumps per HK and LK sheep red cell were found to be 42.4 and 7.6, respectively, suggesting a density of about one pump per /~2 membrane surface area in HK red cells. These values were obtained by extrapolating the number of [H3]ouabain molecules bound at 100°~i, K ~ pump inhibition. The studies based on the assumption that (a) in sheep red cells only one ouabain niolecule binds per pump site, and that (b) only pump sites are being labelled at the low [3H]ouabain concentrations used (between 10 8 and 10-7 M). Labelling of "'nonspecific" sites at higher [3H]ouabain concentrations (10 -°) was believed to be reduced to negligible values by the use of cesium chloride. Turnover numbers of about 6 • 103 ions • site -~ • min ~ were computed for both HK and LK red cells [167]. Using a modification of the technique of Kepner and Tosteson [168] to separate [3H]ouabain labelled red cells from their incubation medium by centrifuging through a water-immiscible organic phase, we have recently investigated the binding kinetics of [3H]ouabain to the two types of red cells [169,170]. The studies were done in the absence of CsCI since cesium, like potassium ions clearly decrease the rate of specific [3H]ouabain binding to the Na +, K + pump. The effect of Cs" or K + is so pronounced that at the low [3H]ouabain concentrations used (5.10 8 M) saturation of all pump sites is only achieved after extended incubations of the cells in [3H]ouabain. [31-t]ouabain binding saturates at about 120, 50-70 and 30-40 molecules per HI<. and homozygous or heterozygous LK red cell, respectively. Furthermore. HK red cells have a higher affinity for ouabain than homozygous LK cells while heterozygous LK cells are of intermediate affinity [169,171]. The biophysical differences between HK and LK red cells are not limited to the active cation transport system. It was already pointed out by Tosteson and Hoffman [94] that the K + leak fluxes are dissimilar in the two types of red cells. The passive permeability ratio of Na+/K + (c~) is higher in HK than in LK red cells. Tiffs is apparent as a higher ouabain-insensi~ive K + leak flux in LK cells, a difference important in considering the different cation steady state composition in these cells [94]. Tosteson [172] has shown that (K')~ determines ~xas well as fl, the ratio of K + pump

190 to leak influx. Thus the a and fl parameters have been found to be 2-3 and more than l0 times higher in HK than in LK sheep red cells, respectively. The inverse proportional behavioul of K + pump and leak flux was the subject of genetic consideration [172], and it remains to be seen whether a and fl, i.e. in simpler terms the ratio of pumps to leaks, is genetically controlled as recently suggsted [167]. Inasmuch as pump and leak fluxes seem to be the determinants of the differences in the cation steady state composition in HK and LK sheep red cells, cation exchange diffusion processes have not been shown to be involved in the control of cell volume [94]. Whether the rather large and ouabain-insensitive sodium exchange diffusion in LK sheep red cells, first pointed out by Tosteson [94] and recently attributed by Motais [173] to a sulphydryl-group dependent carrier mediated process, may also be considered as controlled by the Ka/ka 1oci[156] has to await further studies. c. Biochemical differences ~" the red cell membrane (Na, K)-ATPase. The difference observed in the transport properties of HK and LK red cells was complemented by Tosteson's finding that the Na+,K+-stimulated ATPase activity was fourfold greater in hemoglobin-free membranes of HK than in those of LK red cells, a difference ascribed to the maximum velocities of the enzyme rather than to the Km values of the reaction [172]. From this important finding Tosteson concluded that the physiological transport difference between HK and LK sheep membranes may reflect a structural change in the ATPase molecule inasmuch as the HK enzyme may be more responsive to a cation or ATP-induced conformational change than the LK enzyme [172]. Although the effect of cations is somewhat difficult to study in broken membrane preparations, this hypothesis was consistent with the later detailed tracer flux studies on the asymmetric effects of Na + and K + on the cis and tra,s side of intact red cells [I 17]. Using low ATP concentrations (0.2 ,uM) Blostein confirmed this observation, finding an HK: LK activity ratio of 10 for the N a ' and 13 for tile N a * , K ' stimulated enzyme [I 74] and extended the studies to an examination of the K ~ activation kinetics and tile partial reactions involved in tile (Na',K+)-ATPase in both types of sheep red cell membranes. The response curves of the basic (Na~) ATPase activity to K ÷ in H K membranes consist of an activating( I m M) and inhibiti ng (beyond I mM K +) segment presumably reflecting the activation and inhibition by K ÷ at the trans and cis sides o1" the enzyme complex. In contrast, the (Na')-ATPase of LK membranes was only slightly stimulated, if at all. by K + (0.05-1 mM) and at higher K ' concentrations inhibition was much more pronounced than in HK membranes. However, in the presence of only I mM (Na*) the HK (Na-)-ATPase reacted to the varying of tile K + concentration as the LK enzyme did at 20 or 50 mM Na + [175]. These findings relate well to the observations of Hoffman and Tosteson on the ci.s effect of K ' in intact LK sheep red cells [117]. In addition, Tosteson's hypothesis that tile cations and ATP may determine in a complex manner the maximum velocity of tile enzyme in the two t~pes of red cell membranes [172] was supported by Blostein's observation of increased K" activation and/or decreased K' inhibition as the ATP concentration is raised in HK membranes [176].

191 Analysis of the partial reactions revealed a quantitative difference between H K and LK sheep red cell membranes: the Na+-dependent steady state levels of phosphorylated intermediate was sevenfold greater in H K than in LK membranes. An estimate of the number of phosphorylation sites per cell compared well with the earlier reported number of pump sites per cell [167], however, appears to be too low and the HK : LK ratio too high in light of our recent data on [3H]ouabain binding [169-171]. The partial reactions were not only quantitatively but also kinetically different since the ratio of the sodium stimulated ADP: ATP exchange reaction in HK versus LK red cell membranes was only 2.7 [174]. Blostein's studies thus clearly established that the larger (Na ÷, K÷)-ATPase activity in HK membranes is probably a complex function of a greater number of catalytic sites plus a kinetic distinction. The latter may be a consequence of different subst~ate and ion affinities and perhaps a cause for different responses of the two membranes to the inhibitory action of ouabain [176] and oligomycin [175]. Thus, ;n the two types of sheep red cells, there appear to be clearly quantitative as well as qualitative differences of the active Na ÷, K ÷ transport system. d. Physiology of maturation of cation polymorphic ungulate red cells. The complexities of the distinct biophysical and biochemical aspects of the cation transport system in the cation polymorphic sheep red cells become even more evident if one considers its changes during cellular maturation. Red cells obtained from peripheral blood of post-natal lambs exhibit an H K steady state composition regardless of the cation phenotype predicted by the progeny [177,178]. Similarly, stimulating erythropoiesis in LK sheep by massive hemorrhage causes the release of young cells into the circulation which have a (K+)~ close to that found in mature red cells from HK sheep [179,180]. It has been reported that phenotypically LK goats or cattle also contain H K red cells postnatally [181,158]. Working either on red cells from newborn, or from adult animals massively bled by phlebetomy, a number of workers have looked at the transport and cell physiological aspects of the H K - L K red cell transition in sheep [182-185] or in cattle [158.186]. In newborn genotypically I_K lambs, the fall in (K+)~ and the rise in (Na+)~ occurs somewhat laler (tl/2 ---- 20 days) than the decrease in the parameter fl and the (Na +, K+)-ATPase activity [182,183] (tj/2 = 10 days). These findings are consistent with the assumption that fetal HK cells are gradually replaced by mature LK red cells [183] associated with a reduction of the number of cation pumps per cell as measured by [3H]ouabain binding [184]. There is no hard evidence for the claim [184] of an interconversion c f pump sites into leak sites during the lifespan in peripheral circulation of an HK lamb cell. In contrast, the persistence of HK type kinetics in LK lamb crythrocytes during a4-week observation period, i.e. when the (Na+)-ATPase activity approaches that of mature LK red cells, supports the replacement hypothes;s [183]. In further agreement with this view is the fact that the time course of the disappearance of fetal hemoglobin in post-natal lamb red cells follows very closely the inactivation of the (Na÷,K÷)-ATPase activity, i.e. ahead of the changes in cation composition [188]. However, this correlation apparently does not necessitate that all young cells contain hemoglobin F.

192 since in studies of LK calf red cells Israel and co-workers [158] showed that young cells maycontain hemoglobin A and still be of HK type. It seemsthen that the hemoglobin F ,Atransition superimposed on the H K - L K transition m;~y be only ofcoincidental nature reflectingsimilar time dependencies of two different genetic processes. Thc quantitative and qualitative changes in the properties of thc Na+,K + pump system observed during the H K - L K transition in cattle p~rallels with that in sheep which makes it likely that this process is quite similal between the two species. The mechanism of the involution of the active transport and (Na+,K')-ATPase in LK lamb and calf red cells awaits further studics, particularly using long term incubation techniques [168] to decide in vitro whether the hypothesis of cellular replacement is valid or. alternatively, crythroblast maluration has to occur. Massive bleeding by phlebetomy [158.182,183] provokes an L K - H K - L K transition in the peripheral red cells of these animals that seems to mimic in part the H K - L K transition in red cells of phcnotypic LK lamb and calf. The HK reticuIocytes produced during the anemia of these animals have different cell volumes with a greater membrane surface area and apparemly a different surface charge density [186]. Because of the relatively fast appearance of HK red cells in bled LK shecp, Tosteson [183] suggested that the LK membrane properties develop relatively late in the erythrocyte differentiation, perhaps at the ultimate or penultimate cytodivisions. Thus the HK cells entering the circulation may not have undergone the last differentiation step during which the LK properties of the membrane is developed. A recent observation of Blostein and co-workers [175], howcver, in which the INa*)ATPase in membranes from reticulocytes of anemic LK or HK sheep shows LK type K" inhibition characteristics, raises some questions as to the comparability of the H K cell character induced by bleeding with that naturally occurring in the postmttal period. It is possible that HK red cells in genotypic LK lambs are arrested at a differentiation step prior to that observed in reticulocytes of anemic adult LK sheep, i.e. at a last step in the cellular maturation where the LK kinetic characteristics of the pump (i.e. the high affinity of the Na" loading site for K'~) are already fully expressed. However, this hypothesis does not agree with the findings on the K + sensitivity of the (Na +)-ATPase in reticulocytes of anemic H K sheep requiring further experiments on the nature of the switch mechanism. Analyses on single cells certainly would also clarify whether all HK reticulocytes in anemic LK and HK sheep contain hemoglobin C justifying the proposal that during ancmic stress the LK-H K transition obeys similar mechanistic principles as the hemoglobin A-C switch [175]. e. Membrctne structural a.spects aml other ph)'.~'iological parameters. The preceding discussion naturally poses the question whether membrane structural differences can be associated with the differences in cation transport properties in the rcd cells of sheep, goats and cattle. Moreover, can one relate other cell physiological parameters to the action of the Ka/ka genes'? Under the light microscope HK and LK sheep red cells are indistinguishable. Unlike human red cells, they are spherocytic rather than discoid indicating that normally thesc cells are closer to their critical hemolytic volume. Accordingly, the

193 mean osmotic fragility lies at higher salt concentration than in human cells. Among some 8 Dorset sheep tested, fresh LK red cells seem to have a somewhat higher mean osmotic fragility (around 190 mOsm) than fresh HK cells (around 180 mOsm) when measured in Na + media, an observation corroborating Evans [187] data but not those of others [I 88]. It is of interest that membranes from osmotically hemolysed HK or LK red cells are more fragile than human red cell membranes. Fragments prepared from H K and LK membranes aggregate to a similar extent in solutions containing bi- or trivalent ions [189], a finding not unexpected in light of our observation that membranes from adult HK and LK sheep red cells have identical amounts of sialic acid at their cell surface [190]. Biochemical differences in the overall lipid composition have not been detected in the two types of sheep red cells [191,192] and the polyacrylamide electrophoretic separation profiles of mature LK and H K membranes and their n-butanol-extracted proteins were similar [I 93,194] Membranes of HK and LK cattle red cells also seem to be similar in lipid composition and protein pattern [158]. in freeze-fractured sheep red cells, Kirk and Tosteson [195] found questionably small differences of A-surface particles between the two types of sheep red cells and the finding of a slight but significant reduction of these particles by ouabain awaits further experimental interpretation. in general, attempts have failed to establish a functionally meaningful association between a variety of other cell physiological parameters and the cation transport processes. The values of (K+)~ are higher in hemoglobin A-type HK and LK redcellsthan in AB or B-type cells [196]. However, there is a positive correlation between HK red cell property and hemoglobin B in Indian sheep [197]. No significant correlation between carbonic anhydrase activity, hemoglobin type and (K+)~ steady state level was found [198] indicating that large differences in the (K+)i values do not have the effect on the carbonic anhydrase activity observed in human red cells [199]. In sheep the red cell level of reduced glutathione (GSH) are controlled by a single pair of autosomal alleles, of which the gene for high GSH levels ( > 90 mg 70) is dominant to that for low GSH concentration ( < 50 mg ~o) [200]. it is of interest lhat the glutathione reductase is considerably lower in high GSH sheep hemolysates [201] as well as in cattle hemolysates but red cells of rats, which do not exhibit cation polymorphism, contain also little GSH reductase as compared to man, guinea pigs or rabbits [201]. In spite of this seemingly negative association the findings of a low GSH reductase activity in sheep red cells [201] may be relevant to the findings of Dick et al. [202] that, in broken membranes, the ouabain-sensitive (Na+,K+)-ATPase activity can be quite effectively inhibited by oxidized GSH. How much the observed positive correlation between high GSH levels on the one hand, and higher (K+)l [200,203] and Mg:+-dependent ouabain-insensitive ATPase in HK and LK sheep red cells [204] on the other, will contribute to our understanding of the cation transport polymorphism in these cells awaits further investigation. Finally, metabolic studies on HK and LK sheep red cells failed to show significant differences which could be related to the cation polymorphism. Lactate pro-

194 duction is identical in both cell types (T. J. McManus, personal communication.) and the recently discovered inability of some sheep red cells to ulilize inosine could not be related to the actions of the Ka locus [205]. The levels of 2,3-Pz-glycerate are equally low in HK and LK sheep red cells; temporarily high levels are found in the post-natal period [206]. The preceding discussion attempted to provide a picture of the complex biophysical and physiological-chemical aspects of the cation-polymorphism in red cells of sheep, goats and cattle. It became evident that membrane functional properties are principal results of the genetic difference as the cells originate in the bone marrow, mature and enter the peripheral circulation. Some of the physiological parameters discussed may be relevant to the cation bimodality. However. in light of the small numbers of cation transport sites involved, minute molecular changes such as amino acid point mutations, small differences in lipid-p~otein interactions, or reductions in functional groups, (e.g. sulfhydryls) leading to inactivation of transport sites or asymmetric ion affinity changes, may escape our present analytical means. The recently-reported differential agglutinability of HK and LK sheep red cells by con-A [207] is interesting but requires further interpretation on the membrane molecular level. In the following, newly developed immunological techniques permitting us to probe deeper into the molecular mystery of the H K - L K dimorphism will be discussed. f Genetics of the ML-antigen system. Rasmusen and Hall's [156] discovery that red cells of sheep homo-or heterozygous for the ka gene are blood group M-antigen positive and red cells from sheep homozygous for Ka are M-antigen negative, proved to be of extraordinary importance to further investigations on the molecular nature of the cation transport differences in red cells of sheep, goats and cattle. By immunization of LK sheep with HK red cells an isoantibody, anti-M, was obtained which in the presence ef guinea pig serum complement hemolyzed all red cells of 45 HK (ka/ka) animals tested and red cells of 48 (Ka/ka, heterozygotes) out of 70 LK sheep. The distribution of the M antigen was consistent with the Hardy-Weinberg law for random mating populations in genetic equilibrium. The M system in sheep occurs in multiple alleles [208,209] and appea~s to be homologous to the S-system in cattle [210]. Cattle red cells of serological type $2 and U_, are hemolyzed by anti-M. Using ovine anti-M and complement, the M-antigen was also found in HK and LK goat red cells; however, M negative LK goat red cells may contain considerably higher (K+)~ levels than sheep LK red cells of genotype ka/ka [210]. Tucker [211,212] confirmed the association of the M property with the ka gene and showed that at birth MM and Mm cells react only weakly with the M-antibody. As adult HK or (heterozygous) LK cells, tYee of fetal hemoglobin, enter the circulation "full" M antigen activity was detected [212]. Because of the negative correlation between the appearance of the M-antigen and the fall in (K+)i levels in Mm-cells after birth, Tucker concluded that it must be the m-gene determining the final cation steady state composition in the red cells of all genotypically LK lambs [212].

195 By iso-immunization of H K (MM) type sheep with LK (ram) type sheep red cells Ellory and Tucker [213] obtained anti-m, an isoantibody which in the presence of complement hemolyzes all LK (Mm and ram) red cells. Independently, Rasmusen described an antibody produced with LK (Mm) Hampshire sheep red cells in HK (MM) Suffolk sheep, which exclusively reacted with LK sheep red cells and was designated anti-L [214], a notation now adopted by other workers in this field. (There has been a recent change in the notation for gene M and m (L) to M a and M b, respectively, which clarifies the terminology for the blood group geneticist but will not preclude the use of M and L by the physiologist, see ref. 215). The M/L antigen system appears to be genetically closed like the unrelated M/N system in human red cells [216,217]. A dosage effect, i.e. less hemolysis by anti-L in heterozygous ML positive LK red cells, has been pointed out by Rasmusen [214] as well as Tucker [218], a finding indicative of differences in the amount of L-antigen on these cells (see below). As discussed later in this review, it is the L-substance which directly appears to be involved in the modification of active Na÷,K ÷ transport in LK sheep red cells. Like the M-substance the L-substance appears to be not fully developed on lamb red cells at birth [219,220]. Maximum hemolytic scores were obtained about 40-50 days after birth when all fetal cells were replaced b y mature adult LK red cells. It should be pointed out that the development of the M/L antigens on post-natal red cells was measured by direct hemolytic tests. Since differences in the response of newborn and adult red cells to complement are not considered in these reports [220,221], only further quantitative data on the binding of labeled anti-L or anti-M will provide a conclusive test as to whether or not the L and M activities are truly weak or absent on the eldest fetal-type red cells. Like anti-M the L reagent has also been found to cross-react with LK goat and cattle red cells [221], an interaction involving the Na+,K ÷ transport system further discussed below. Cattle red cells of serological type S are lyzed by sheep anti-L, while S-negative cells were never positive for sheep anti-L [222]. g. lmmunochemistry of anti-M and the M-antigen. The M-antibody used in early studies was mainly o f l g M nature [193] and in the presence of guinea pig serum complement of high lytic potency, reacted equally well towards both MM and ML positive sheep red cells. Adsorption studies of anti-M to MM (HK) membranes indicated a rather high affinity interaction with the M-antigen in hemoglobin-free membranes derived from HK cells by osmotic hemolysis [223]. Using a second M-antiserum the M-antibody activity was only present in the IgG~ fraction of the immunoglobulins, i.e. among the lgG antibodies, due to a higher net negative surface charge, of faster electrophoretic mobility. The immunological distinction into rather acidic and electrophoretically fast moving lgGl proteins and more basic and electrophoretically slower migrating IgG2 proteins is particularly well known in sheep [224-227], but also in other species, a phenomenon which generally may be related to the overall net charge of antigens [228-230]. It is conceivable that within the mostly acidic immunoglobulin fraction the occurrence of M-antibody activity (and

196 as shown later also of all the I_-antibody activity) reflects some basicity of the overall structure of the ML-antigens. Although kinetic analysis of immune hemolysis of anti-M and complement did net reveal ally significant differences between M M and M L red cells, antibody absorption experiments using the hemolytic assay for testing of residual M-antibody activity clearly showed that MM (HK) cells appear to have some 2-3 times more M antigenic substance than M k (k K) cells [231 ]. Experiments with ~3~l-labelled anti-M going on in this laboratory seem to confirm these original observations: at equilibrium about 3 ]03 molecules anti-M were bound per MM (HK) cell and only 607,; pcr ML (LK) cells [232]. The equilibrium association constant for binding of the M-antibody is high and the maximum number of M-antigenic sites in M positive HK and LK sheep red cells appears to bc about an order of magnitude greater than that found for the number of N a ' , K " pump sites by the 3[H]ouabain method. This finding is not surprising in light of our earlier observations that absorption of M-antibody molecules seems to be about equal to membranes incubated in the presence or absence of 10 4M ouabain [193] and that anti-M affected neither active and passive cation transport nor the ( N a ' . K')-ATPase in HK sheep red cells [233--235]. Togethcr these findings are consistent with Tucker's earlier suggestion [212] that the M-substance in sheep red cells of animals with the Ka gene apparently is not dircctly involved in the control of the cation steady state composition <1t"these cells. Although it cannot yet be ruled out that in some way the M-antigen may be part of the structural subunits comprising the cation transport system, our tindings pose the question as to how tightly M and H K properties are coupled. The M-antigen was found to be an integral protein constituent of MM (HK) membranes. Treatment of intact MM (HK) red cells with ncuraminidase [235], controlled trypsin digestion (unpublished data) or phospholipase A and B prepared from small intestine of mice and provided by Dr A. Otto!enghi [236] did not inactivate the M-antigenic activity as measured by absorption studies, nor was the M-antigen lost when hemoglobin-free membranes were exposed to trypsin. In hemoglobin-free ghosts of HK red cells the M-antigen resisted solubilization by alteration of ionic strength [194] indicating that neither ionic bonds nor weak hydrophobic forces are anchoring the antigen within the insoluble membrane matrix. Likewise the use of chaotropic anions (I , SCN ) promoting exposure o f a p o l a r groups to the aqueous environment by rupturing hydrophobic bonds at the surface membrane [237] did not result in solubilization of significant amounts of M-antigen [194]. That relatively strong hydrophobic bonds seem to be involved in the maintainance of the M-antigen was also indicated by the finding that 6 M urea or guanidine • HCI [238,239] did not result in solubilization of tile antigenic membrane components [194]. However, solubilization and gel filtration on Sephadex G 100 in 150 mM desoxycholate resulted in a clear separation of membrane proteins from phospholipids and cholesterol. About one-fourth of the M-antigen activity was detected in the protein fraction while the lipid peak was inactive. Upon recombination of the protein with lipid from either H K or L K membranes full restoration of M-antigenic activity was achieved [194],

197 suggesting that the M-antigen is a lipid-dependent membrane protein or glycoprotein. Both phospholipids as well as cholesterol were required for full reactivation and there was no preference for a particular phospholipid. The lipid removal by desoxycholate seems to be a milder procedure than lipid-extraction with organic solvents such as n-butanol [240,241] or it is possible that the structural similarities to cholesterol protect some labile regions of the M-protein during solubilization. Exposure of HK membranes in guanidine • HCI to mercaptoethanol followed by alkylation with iodoacetamide [238] completely inactivated the M-antigen, an effect not observed in the absence of dissociating agents [194]. In summary these studies have shown that the M-antigen in H K sheep red cells most likely is a protein. Presence of membrane lipid and integrity of deeply located sulphydryl groups or disulphide bonds are the structural requirements to maintain the M-antigenic determinant at the membrane surface. These characteristics of the M-antigen resemble some of the structural features known for (Na +, K+)-ATPase preparations of various tissues. The importance of sulphydryl groups for the activity of the (Na+,K÷)-ATPase in H K sheep membranes was pointed out earlier and an absolute lipid requirement for the partial reaction of the Na+,K+)-ATPase activity is known [242-248]. In our studies phosphatidylserine was as effective in the activation of the M-antigen as any other phospholipid. Whether this phospholipid plays such a particular role in the activation of (Na +, K+)-ATPase preparation is still the subject of intensive investigations [249,250]. h. The effect o f anti-L on the Na +, K + pump and ATPase in L K red cells o f sheep, goats and cattle Sheep redcells. Tucker's [212] hypothesis that it is not the M but rather the m (subsequently called L) gene which is involved in the modification of the Na+,K + transport system in LK sheep red cells was fully supported by the subsequent finding that anti-L reacted with all LK sheep red cells by stimulating the ouabain-sensitive K + influx and the (Na+,K+)-activated ATPase activity [213,251]. Anti-L was without effect in H K red cells. This observation raises the crucial question as to whether the L-antibody confers on LK cells the properties of H K cells as originally suggested [213]. Thus the effect of anti-L on the kinetic characteristics of the red cell membrane transport system have to be considered, i.e. Na+,K + transport as a function of cations, substrates and inhibitors at the cis and trans side of the LK red cell membrane. Of particular importance are the effects of anti-L on (1) the number of membrane pumps, (2) the cation-affinities of the pump, (3) the partial reactions of the (Na+,K+)-ATPase and (4) whether there is a quantitative relationship between the number of antigenic sites and membrane pumps. Since the L-antibody appears to detect L-antigenic groups on LK red cells from goats and cattle, and much of the recent work to elucidate the L-antibody effect has been done also on goat and cattle red cells, the following discussion will contain a comparative element by considering the action of anti-L on the red cells in all three ungulate species. The remarkable stimulation of cation transport in sheep and goat LK red cells by anti-L is mainly due to the action of the antibody on the Na+,K + p u m p [213,251,252].

198

STM I ULATJONOFACTIVEK*-TRANSPORTINLK(LL} -~ SHEEPREDCELLSBYANTJ-LORNON-M I MUNESERUM "~ 151 LK(LL)'BSSRBC

o ,L N

0

15

/

/ 30

60 90 ['Mi,utes 0t 37°C]

120

Fig. 1. (Published with permission of journal, ref. 253, G. Thieme, Publishers, Stuttgart).

Fig. 1 illustrates the K + pump-stimulating effect of anti-L on LK sheep red cells [253]. The effect depends on rather high L-antibody concentrations [252,253]. In practically undiluted L-antiserum the ouabain-sensitive K "-pump influx in LK goat red cells was stimulated 3-5 fold [254]. In LK sheep red cells incubated for long periods of time at 37 °C in the presence of anti-L a net accumulation of K+~ and loss of Na+~ was observed excluding the possibility that the antibody exerts a significant influence on cation exchange diffusion processes in these cells [252]. The response of LL and LM type LK sheep to the K + pump stimulatory action of anti-L is different. At highest antiserum concentrations LL cells show a K+-pump activation by the antibody which is significantly higher than in LM cells, a finding which could be at least partizdly explained by absorption experiments: about 3 times more LM cells were required to absorb out 50 ~o K+ pump stimulating L-antibody activity than LL cells. This would indicate that LM sheep red cells have fewer L-antigenic sites [253,255]. A similar conclusion was reached using immunological tcchniques: anti-L hemolyzes to a greater extent LL than LM sheep red cells [253,255] which can be related to a smaller number of L-antigenic sites on LM cells as demonstrated by absorption cxperiments. Although one has to await further studies using radiolabelled anti-L, the dat~l thus far obtained seem to indicate that in sheep LM cells have somewhat less L-substance than LL cells [231,255]. Since mature LK sheep red cells have a lower number of membrane pumps than H K red cells [167,169], and anti-L was first reported to increase the number of [3H]ouabain molecules bound per cell [213,252], it was suggested that the L-antigenantibody interaction causes unmasking of latent pump sites. The variation in the numbers of pump sites in the presence and absence of anti-L as reported from [3H]ouabain bindingexperiments by the groups at Cambridge and Duke was unsatisfying, particularly in light of the possibility that the L-antibody may change the rate of

199 ouabain binding rather than the actual number of sites. Whether the number of sites or the kinetics, or both, are affected by anti-L were then considered. Our recent studies on [3H]ouabain binding showed that anti-L did not change the maximum number of [3H]ouabain molecules bound to LL or LM red cells but indeed altered the rate of ouabain uptake [169-171]. If one takes the rate of association of the ligand with its receptor as an indicator of the affinity of the p u m p for ouabain, then anti-L confers an HK-iike affinity upon LL and LM (LK) red cells. A close correlation existed between the rate of [3H]ouabain binding and the rate of K ÷ p u m p inhibition [169,170]. These findings do not support the new pump-site hypothesis but rather point to an affinity change of each pump site for ouabain. At least two interpretations of our findings are possible. First, anti-L increases the cation turnover per pre-existing and ouabain-labetled p u m p site. Second, there are active and inactive p u m p sites in LK sheep red cells, both are being labelled by [3H]ouabain. Anti-L could turn on the inactive p u m p sites and change the affinity of all pumps for ouabain. In this case the cation turnover number would remain the same. It is interesting that LM cells have a smaller number of [3H]ouabain binding sites per cell and that the affinity for the glycoside is between that of H K and LL (LK) sheep red cells [170,171 ]. Since in the presence of anti-L the rate of ouabain binding to LM sheep red cells also becomes similar to that in H K cells, the antibody appears to have a smaller effect on this process in the heterozygous cells. It has been pointed out above that anti-L stimulates K ÷ pump influx in LM cells less than in LL red cells. This observation could be reconciled with the possibility that LM cells are kinetically (i.e. qualitatively) closer to H K cells (i.e. as shown by a higher late of [3H]ouabain binding and, experimentally yet unproven, by a lower affinity for K ÷ at the Na ÷ loading site) although the total number of pump sites seems to be even lower than in LL cells. Further evidence for anti-L induced change in kinetics was obtained from studies on the inhibition of the internal Na ÷ loading site by (K+)i : at around 35 mM (K+)l K ÷ pump influx ceases in LL type (LK) red cells, but not in the presence of anti-L [252,256]. The shape of the internal Kj ÷ activation curve appears to be somewhat different in L-antibody treated LK cells as opposed to control cells, and on the basis of the earlier ouabain-binding data these kinetic findings were related to an activation of new but H K pump sites [252,256]. Two hypotheses have been put forward to explain the L-antibody action on the cis side of the membrane. Assuming that anti-L generates new membrane pumps, Schmidt [166] suggested that the antibody increases the affinity of the LK pump for Nai ÷ to about one-third of that seen in H K cells. This model requires inhibition of the LK p u m p by two Ki ÷ ions. Glynn and Ellory [164], discussing our data [252], attributed the anti-L effect on the internal activation kinetics to a competition by K ÷ for Na ÷ at a single internal site. Although it has been reported that anti-L does not affect the affinity of the K ÷ loading (trans) site for external K ÷ when assayed in the presence of (Na+)o [251] or in the absence of Na ÷ (Mg 2÷ replacement) [252], recent experiments in which the antigen-antibody reaction equilibrium was not perturbed also indicate an increase in the affinity of the L K p u m p system for K+0 in the presence of anti-L: the K1/2 values fell from about

200 2.6 to 1.5 mM (K+)0 [231]. Thus it is possible that anti-L may affect both ion loading sites of the LK pump in sheep red cells. The effect of anti-L on the Na",K + transport system in LK sheep red cell membranes is further complicated by the observation that the Na+-dependent ATP hydrolysis by tile (Na~,K')-ATPase is markedly increased and the response of the antibody stimulated enzyme to K + resembles that of L K rathel than of H K membranes [257]. These results imply that anti-L increases the number of enzyme units without altering tile response of the presumable Na + loading site to the inhibitory action of K-, a conclusion not necessarily in conflict with above discussed hypothesis of an L-antibody induced qualitative changes of the LK pump if the L K / H K transition is incomplete [258]. In comparing the reported eflects of anti-L on the (Na+,K~)ATPase in sheep [257] and goat LK red cell membranes [254] one should bear in mind that the former studies were done at very low ATP (0.2/~M) concentrations, the latter not. Anti-L also stimulates the (Na+,K*)-ATPase at "physiological" concentrations of ATP (1 m M), but variations were considerable between experiments on the same membrane preparation [234]. This is partly due to the fact that the LK (Na~,K~)-ATPase constitutes less than 10-20!',~i of the total membrane ATPase activity in sheep red cell membranes. In LK goat red cell membranes ouabain inhibits 70~, of the total ATPase activity [164]. Varying the Na + concentration at constant (K +) in tile medium Glynn and EIIory [164] showed that anti-L increased the sensitivity of the ATPase to Na +, thus enhancing only the apparent affinity of the Na + loading site for Na + since 10 mM (K ÷) presumably saturated the K + loading site. Unless the stimulation of the (Na+)-ATPase by anti-L is related to an apparent affinity change rather than to an increase in the number of ATP hydrolysing enzymes, it remains somewhat paradoxical that the effect of anti-L on the inner aspect of the Na+,K ~ pump in LK sheep red cells can be best correlated only with the antibody's action on the Na + loading site of the (Na +, K~)-ATPase in goat LK red cell membranes. As the experiments with low ATP concentrations [257] were aimed at the Na'-activated portion of the (Na+,K+)-ATPase system, measurements of the ouabain sensitive and K+-dependent p-nitrophenyl phosphatase activity should provide information concerning the effect of anti-L on the K ' affected partial reaction steps of the Na+,K f translocation system. Thus EIIory and Lew [259] showed that anti-L stimulated the K'-sensitive p-nitrophenyl phosphatase activity in LK goat red cell membranes without affecting the K + affinity of the enzyme. As expected Na + plus ATP increase significantly the affinity of the acylphosphatase for K + at low (K ~) and, due to competition with Na + at tile "inner surface" site, inhibition by higher (K +) was found. When anti-L was present the maximum K + phosphatase activity was increased by a factor of almost three, while the sensitivity to K - was unchanged. This inability of anti-L to reduce the affinity of the cis site for K + was attributed to a second inhibitory site at which K- must act on the enzyme [259]. Goat and cattle red cells. Analogous to sheep red cells, it has been shown that anti-L also alters in LK goat red cells the relative affinity of the Na+,K + pump

201 to (K÷)l [254,260]. As in the sheep system the antibody seems to increase the apparent affinity of the pump in LK goat red ceils for Na ÷. Whether this is due to an absolute decrease in the sensitivity to Ki ÷ or to an increase in the Na ÷ affinity cannot yet be decided. In their studies on the internal activation kinetics, Sachs and co-workers [260] replaced (Na÷)~ or (K+)~ with choline using the para.chloromercurylbenzoyl sulfonate method [127]. At very low (K+)l anti-L only slightly increased the apparent Na ÷ affinity of the pump making the shape of the activation curve superimposable on that of an H K Na + activation curve at comparatively low (K*)l. At high (K+)~ anti-L exhibited a much more powerful effect on the apparent Na ÷ affinity of the pump but the Na + activation curve obtained is not that of an HK cell with similar cation composition. Likewise, internal K ÷ activation curves in the presence of anti-L did not reveal the biphasic effect of K~÷ on the pump found in H K goat red cells. This was interpreted in terms of the potent inhibitory effect of K÷l at very low (K÷)i. The variation found in the maximum K ÷ pump influxes of LK goat ceils at very low (K+)~ could not be attributed to an effect of low (K÷)~ on anti-L binding [260]. However, it could reflect differences in the genotype of the LK goat cells used, i.e. LL cells vs. LM cells as discussed previously for the sheep system. Provided the maximum number of ouabain binding sites and therefore pumps per cell does not change, the turnover rate of cations must be the same in very low (K+)~ LK goat red cells and its increase at higher (K+)~ reflects only the change in the relative affinities of the pump for (Na+)~ and (K+)~. As in sheep red cells [169-171] the rate of both ouabain binding and K ÷ pump inhibition is higher in H K than in LK goat red ceils [261] and treatment of LK goat red cells with sheep anti-L increases the rate at which inhibition occurs to that observed in HK cells [261]. Since, however,.anti-L does not affect the rate of K ~ p u m p inhibition by ouabain at very low (K÷)~, Sachs et al. [261] conclude that anti-L apparently modifies the rate of ouabain binding by altering the affinity of LK goat cells for K~÷. The number of ouabain binding sites and therefore pumps per cell is somewhat greater in goat cells than in sheep ceils because goat cells have a mean cell volume of 16.7 ~ 3 which is about one half of that in sheep. In contrast to sheep red cells [169-171] the calculated number of ouabain binding sites was similar in H K and LK goat red cells (55 and 57 per cell, respectively [261]). Anti-L increased slightly the calculated number of ouabain molecules bound in control LK goat cells but did not in para-chloromercurylbenzoyl sulfonate-treated [127] cells with very low (K*)~ [261]. Although these data were obtained by extrapolation to 100 K inhibition and the differences seen are small, the authors postulate that in goat red cells LK pumps are heterogeneous in their ouabain binding rate and pump rate, both being very sensitive to the inhibitory action of K~+ [261] As to the stoicheiometry between the number of L-antigenic sites and Na+,K + pumps in LK sheep or goat cells, recent advance by Kropp and Sachs [262] suggest the requirement of about 60 anti-L molecules to maximally stimulate Na+,K ÷ transport in LK goat red cells. Comparing this number with that of pump sites estimated from [3H}ouabain binding (i.e. 55/ce11) the author's conclusion [262] seems

202 to be justified that anti-L combines with the pump or with an antigen present in the same amount as the pump. In sheep, the situation is somewhat more complex since more L-antigens appear to present (P. K. Lauf and W. W. Sun. experiments in progress) of which only a fraction may be involved in the L-antibody mediated K + pump alteration. Heterogeneity of sheep L-antigenic sites is evident from a range of association equilibrium constants obtained thus far in this laboratory. Anti-L changes the (Na +, K+)-ATPase activity in membranes of LK cattle red cells [263], but as compared to the sheep and goat system, the relief of the K + inhibition of the enzyme by the antigen-antibody reaction was only hardly apparent. Evidence now exists that some, but not all, anti-L sera may stimulate also active K * transport in LK cattle red cells (B. A. Rasmusen and J. C. Ellory, personal Communication). in conclusion, the observation made particularly in sheep and goat LK red cells is consistent with the tentative hypothesis that the L-antibody combines with L-antigenic sites whicl~ are either part of or numerically and structurally closely related to the Na*,K ÷ pump. This specific antigen-antibody reaction then causes a conformational alteration of the Na+, K * pump as evident from the kinetic changes discussed in this section. i. hmnunochemistry of anti-L and the L-antigen. From the foregoing discussion it is clear that aside from its cation composition, the LK sheep red cell character can be defined by immunological reactivity. In the absence of complement, sheep anti-L stimulates active Na÷,K + transport in these cells; in the presence of complement. however, the hemolytic reaction by far outweighs the effect on the N a ' . K * pump. This dichotomy raises the question as to how many and different L-antigenic receptors are on an LK red cell and as to how many L-antibody specificities do exist. On the basis of limited proteolytic digestion of intact LK sheep erythrocytes two L-antigen and/or antibody specificities were suspected: trypsin treatment of LK red cells prevented the effect of anti-L on the LK membrane pump but did not abolish the hemolytic response to anti-L and complement [190]. The L-antigenic site binding the hemolytically reactive L-antibody was called Lev and that involving the modulation of the pump, L o [190]. Independently. EIIory and Tucker [221] were able to adsorb to and elute from LK goat red cells mainly K + pump-stimulating L-antibody, reacting of course in the sheep system where it had been produced, and not the L-antibody hemolysing LK sheep red cells. Even up to 8 absorptions ~ith LK goat red cells did not remove the anti-sheep L-antibody [221]. The latter of the two antibodies, therefore, must be quite sheep-L-antigen specific, while the former must uncover an L-antigenic group common to I.K goat and sheep red cells. The observation that booster injections of [.K red cells into H K sheep produce an L-antiserum with an increased lytic but declining K ~ pump stimulatory activity was taken in support of the assumption of at least two antibody spccificities, anti Lp and hemolytic anti-L [2211. However, these interpretations are not fully sufficient to explain the disparity in the reaction of LK sheep and goat red cells with ovine anti-L. For example, it

203 must be demonstrated that the L-antibody involved in lysis of L K sheep red cells has no effect on the membrane pump in the absence of complement. One should explore whether the decline of K + pump stimulatory action of anti-L obtained from prolonged immunized sheep is due to the appearance of an L-antibody population that only hemolyses LK sheep red cells but does not affect K ÷ transport in LK goat red cells. If so, then the L-antibody raised early during immunization reacts with the L-substance associated with the Na÷,K + pump in all ungulates studied, i.e. sheep, goat, cattle, leading to Na ÷, K ÷ pump and ATPase activation on the basis of a high and cross-reacting specificity for these species. As immunization continues, the specificity of ovine anti-L may be confined to the sheep L-antigen resulting in a decrease of cross-reactivity. This interpretation would be compatible with general immunological experience that prolonged immunization leads to a selection of cell clones which produce antibodies of higher affinity and/or of differing specificity. However, recent experiments from this laboratory [231,255] make it somewhat doubtful that the L-antibody involved in hemolysis is different from that activating the LK pump: LL and LM sheep red ceils differed to about the same degree in their capacity to absorb hemolytic and K ÷ pump stimulating L-antibody. Explanations other than one implying a tight coupling between LLy and Lp sites have to be sought. For instance, it is possible that the sheep anti-sheep L-antibody is a mixed population of molecules with similar affinities for the same L-antigen receptor site but different heavy chains, i.e. a fraction of the total population has H chains which will not fix complement as it is known from studies on human lgG subclasses. The L-antibody reacting with LK goat cells also may not fix complement indicating a triple heterogeneity of antibodies based on the assumption that the L-antibody specificities for sheep and goat red ceils are different. Lastly, goat red cells may not be hemolyzed by complement even if the proper L-antibody binds to the K + pump-associa'ed L-antigen, indicating that the target sites for attack of the late complement components are either too remote from the L-antigenic site or somehow influenced and reduced in their reactivity by the L-antigen-antibody reaction in these cells. Whatever the explanations are, future experiments, including, in particular, goat L-antisera should bring interesting insight into the correlation between the immunologically developing specificities and the physiological effects of these L-antibodies. Such studies are also important to elucidate whether or not the capacity of an HK sheep to produce an antibody against the LK pump is related to the genetic Na ÷, K + transport and antigen polymorphism. The L and M isoantibodies do not agglutinate sheep or goat cells and by this rather historic criteria they are incomplete antibodies. Does this mean that both F(ab) combining sites of the lgG ~-type L-antibody molecule are required to react with two L-antigenic ligands in order to modulate active Na+,K ÷ transport? A number of studies have been directed to answer this question, and although there are some differences in the observations [264--267], evidence for anti-L binding to more than one antigenic ligand has been obtained, as described in the following: Using monovalent Fab-fragments derived from an anti-L IgG preparation by the controlled

204 papain hydrolysis method of Porter [13], we found an almost complete lack of response of the Na+,K ~ pump in LK sheep red cells [264,265]. Even at highest concentrations of monovalent F(ab) fragments used, only a minimal K ' pump stimulation was observed. That the K" pump activation by intact anti-L IgG, was not blocked by F(ab) fragments could be interpreted in terms of a lowered binding affinity, or no binding of the F(ab) fragments at all. The F(ab')z fragments obtained by pepsin hydrolysis [14], however, showed full K + pump stimulatory effect. If indeed F(ab) fragments bind but do not affect the membrane pump while F(ab')2 fragments, i.e. bivalent L-antibody fragments, stimulate Na+K ~ transport, it must be concluded that two closely adjacent L-antigenic ligands react with one L-antibody molecule. This may change the thcrmodynamic equilibrium of the L-substance controlling the activity of the pump or directly the Na+,K - transport system, if it carries the L-antigen. It is conceivable that binding of the bivalent L-antibody alters proteinprotein or protein lipid interaction in the vicinity of the pump i.e. affects membrane fluidity [65], since maximum change of the K ~ transport rate is reached only after 15-20 rain. although binding occurs faster at the conccntrations of anti-L used [266]. That the bridging property of two antibody combining sites on one molccule may be involved in this effect was supported by one experiment in which an anti-F(ab) antibody was added to anti-L F(ab) presensitized LK sheep red cells resulting in a significant although not complete recovery of the K ~ pump stimulation [231]. This finding also indicates that F(ab) fragments of anti-L lgG~ molecules indeed bind to the L-substance but are unable to affect the pump. Recently it has been reported that F(ab) fragments as well as F(ab') preparations obtained by reductive cleavage of Ftab')2 derived from anti-L IgG induced slightly less than half of the K + pump stimulation observed with native anti-L or its F(ab')2 fragment [267]. Since maximum stimulation of the K + pump flux by native anti-L and F(ab~)2 (8.5-fold) and by F(ab) (4-fold) was obtained at about the same molar concentrations of univalent binding sites, it was concluded that the lower degree of K ~ pump stimulation by monowdent F(ab) fragments cannot be attributed to lower binding affinity. The differences in the findings of the two groups [264,265,267] could be due to various reasons such as ~ntibody variability, incomplete IgG~ digestion or milder handling of proteolytic and reductive cleavage processes, and the like. However, it remains common to the two reports that the monovalent Lantibody fragment, whether F(ab) or F(~b ~) have a considerably reduced capacity to modulate active K + transport. It is interesting that in contrast to the native L-antibody, F(ab) fragments do not induce a different response of the K ' pump in LL versus LM (LK) cells [267]. Furthermore in LM cells F(ab) fragments are still less effective than native anti-L suggesting that also in LM cells anti-L exerts its full effect by doubly attaching to the L-substance [267]. The L antigen can be considered as a lipid dependent integral component of the LK sheep red cell membrane and thus behaves analogously to the M-antigen [194]. As with the M-antigen, solubilization in high or low ionic strength or in urea was unsuccessful. The L-antibody always adsorbs to the insoluble membrane residues.

205 Inactivation or loss of L-antigenic activity occurs at pH values below 4 and above I I indicating a rather broad pH stability [231]. Removal of lipid by n-butanol [240,241] completely inactivates the L-antigen [231]. Attempts to fractionale Lactive material from the butanol soluble lipid by ethanol or to recombine the total lipid with the membrane proteins have failed thus far. Desoxycholate inactivates the L-antigen at concentrations lower than those used for solubilization of M-antigen [ 231]. Similarly, other extlaction procedures such as phenol/water [268] or pyridine [269,270] produced negative results. These attempts are handicapped by the probability of a very low number of L-antigenic copies per cell (see Section Ill. 1. h.). Complete loss of L-antigenic activity after reduction and alkylation in 6-8 M urea indicates the importance of sulphydryl groups or disulphide bonds in more hydrophobic "buried" membrane areas for maintenance of the L-antigenic groups at the membrane surface [231 ]. A more promising approach to obtain information concerning the surface portion of the L-stubstance is the analysis of L-antibody binding and effects following enzyme treatment of intact LK red cells. Neuraminic acid appears not to participate in tormation of the L-antigenic groups since neuraminidase did not affect the physiological action of anti-L [190] nor interfere with binding of the L-antibody. When, however, LK sheep red cells were exposed to trypsin, complete loss of the K + pump stimulatory effect of anti-L occurred at the time when most of the trypsin labile components were released from the intact cell [190,235]. Since, however, L-antibody could still be adsorbed to trypsinized cells to almost the same extent as to untreated cells and since no L-antigen active material was released during the enzyme treatment, it may be concluded that trypsin cleaves an important peptide bond through which anti-L modulates the membrane pump, or the membrane environment around the L-antigenic determinant and thus prevents the action of the L-antibody on the Na +, K ÷ transport system. Trypsin, however, does not decrease the anti-L mediated complement hemolysis of LK sheep red cells [190,266]. In the light of these findings it is surprising that other proteolytic enzymes such as papain, chymotrypsin, nagarse [220] or pronase [266] did not influence the K ÷ pump stimulating effect of anti-L. Certainly it will be of interest to extend these studies to goat and cattle LK red cells. IIIA-2. Alteration of cation tran,sport by lectins in nucleated cells a. Lymphocytes. Since the early discovery of Nowell [271] and Hungerford and co-workers [44], a group of immunological[y reactive molecules have been identified which upon binding tomammalian lymphocytes produce "lymphocyte activation', "blast cell transformation", "lymphocyte induction" etc. i.e. morphologically defined cellular events which are accompanied by a variety of biochemical changes. These in vitro phenomena require the presence of distinct receptor molecules at the lymphocyte surface membrane able to interact with lectins, antibodies and antigens. The differential in vitro reactivity of lymphocytes with lectins (such as phytohemagglutin or concanava[in A) and antigens or anti-immuno-globulin sera was instrumental lbr (a) the understanding of the in vivo induction of

206 cellular and humoral immune response to antigenic challenge and (b) in the classification of thymus derived (T) and thymus independent (B) lymphocytcs. While lectins bind to carbohydrate-containing structures (see Section II A.) at tile lymphocyte surface, antigens and anti-immunoglobulin sera require the presence of immunoglobulin-type receptors [272-282]. Tile latter contain the important features described for humoral antibodies (Section ilA) such as bivalent combining sites and tile Fc portions possessing antigenic regions somewhere along the H-chains and the ability to attach the whole molecule to the lymphocyte plasma membrane. Multivalent interactions between lectins, antigens or antibodies and membrane bound receptors appear to be the basis of such morpholog;cal events as receptor site clustering [283] and "cap" formation. For example, in the "capping" process fluorescing bivalent anti-immunoglobulins have been found to move laterally to one pole of the cell, while monovalent antibody fragments failed to do so [284,285]. As in a number of systems mentioned above (i.e. IgE mediated histamine release, antibody binding and complement tixation, K* transport stimulation by bivalent L-antibodies, or surface charge redistribution induced by anti-spectrin antibodies [286])a "bridging" immune reaction appears to play tile important role in bringing about these morphological events leading to early increases in RNA and protein symhesis [287], changes in carbohydrate metabolism [288,289] and phospholipid turnover [290-294]. While it is beyond the scope of this contribution to review these morphological and biochemical phenomena (see reviews 295-297), the early effects of these mitogenic agents on the cation permeability will now be considered in more detail. Apparently. induction of lymphocyte transformation requires contact and attachment of mitogens only to the plasma membrane of [ymphocytes, not necessarily intrace[[u[ar uptake [298]. In light of the yet scarce information that K~~ seems to be required for intracellular protein synthesis [299-301], the question is more than academic whether or not mitogens may induce early permeability changes in lymphocytes preceding all other biochemical events. Thus Quastel and Kaplan [302,303] were first to show that 4 . 10 6 M ouabain prevented transformation and biochemical changes in lymphocytes preincubated for 1 h in phytohemagglutinin. The/5o of ouabain to block [3H]uridine, ['~H]thymidine. [~'~C]leucine and methionine incorporation into RNA, DNA and protein, respectively, was found to be around 5" 10 8 M. The phytohemagglutinin-stimulated activity was strongly inhibited by ouabain, much less the incorporation in the absence of phytohemagglutinin. Similar concentrations of ouabain affected the mixed leucocyte reaction between leucocytes from unrelated donors [303]. Raising (K~)o from 5 to about 26 mM decreased the /so values to about 5 • 10 ~ M. and washing of the cultured cells previously exposed to about 10 -v M ouabain to block biochemical changes, did reverse the inhibition: the cells resumed their characteristic chemico-morphological transilion in response to playtohemagglutinin stimulation [303]. These data were compatible v,ith later observations that phytohemagglutinin considerably stimulates an ouabain sensitive K'influx while the ouabain insensitive K' leak tlux appears to be unaltered [304]. Although these data do n o t permit the exact calculation of exacl cation fluxes, a

207 rough estimate indicates that in the absence of phytohemagglutinin the K* pump influx per lymphocyte is abaut 15-fold higher than in a human red cell and is increased further by a factor of 2-3 in the presence of phytohemagglutinin. Assuming a mature lymphocyte is a sphere with a maximum diameter of 18/z [305], its surface area would be about 103,u 2 which is about six times that of the human erythrocyte membrane [306]. If, due to folding, the lymphocyte surface membrane area is actually larger by a factor of two, the fifteen-fold higher ouaba;n sensitive K ÷ influx in lymphocytes could be accounted for without invoking a higher membrane pump density per unit surface area than known for human red cells (Section 11 C.), provided the cation turnover numbers are comparable. Since phytohemagglutinin did not change K~,,2 of the K + p u m p influx for K0 +, the mitogen-increased V of active K + transpor~ was initially explained in terms of (1) an activation of previously formed but inactive membrane pumps, or (2) by some "conformational modifications of the Na+,K p u m p " [307]. As far as the possibility of increased synthesis of pumps is concerned, the effect of phytohemagglutinin on V of K + transport was evident within less than an hour after its addition to cultured lymphocytes and a de novo biosynthesis of transport sites was excluded because inhibitors of RNA and protein synthesis such as actinomycin D and cycloheximide, respectively, were without influence on the phytohemagglutinin-induced permeability changes [307]. Thus it remains possible that the increase in V is due to a conformational change at the cell membrane level resulting in exposure of previously "cryptic" K + binding sites [308]. The observation that increasing (K+)o dramatically reduced the inhibitory effect of ouabain is compatible with the findings of others [136-138] that the ouabain binding rate is inversely dependent on (K+)0. The maximum amount of ouabain bound near equilibrium, however, appears not to be affected even if it takes many hours to achieve near-equilibrium binding at low ouabain concentrations [309]. Quastel and Kaplan's washout experiments [303] to remove ouabain and restore the phytohemagglutinin susceptibility (i.e. stimulation of protein and RNA synthesis) of the lymphocytes indicate a rather high dissociation rate constant for ouabain, and it would be of interest to know whether washing out of ouabain may also result in a full recovery of the phytohemagglutinin dependent K + transport activity. In view of the complex action of Na + and K + at the inner and outer aspect ot" the Na+,K + translocation system, the authors claim [308,309] that "cryptic" membrane K + binding sites are being unmasked by phytohemagglutinin must be treated with caution and await further experiments on tile effects of (K+)~ and (K*)0 o11 tile Na+,K + pump activity and [3H]ouabain binding. If the phytohemagglutinininduced increase in V indeed reflects a higher cation turnover per pump site, then analogous to the situation in the ML antigen-antibody system in HK and LK sheep red cells, one would expect a change of cation or substrate affinities at the inncr surface of the Na +, K + transport system subsequent, to binding of phytohemagglutinin. Otherwise, an increase of the (K+)~ steady state concentration by phytohemagglutinin in lymphocytes would cause a decline in flux rates provided the K ' leak transport remains unchanged. However, ill discord with this hypothcsis of a

208 conformational change in the lymphocyte Na+,K ~ pump system by phytohemagglutinin are recent findings by the same group that phytohemagglutinin does not affect the rate of [3H]ouabain binding but rather increases about twofold the total amount bound [309], an increment in the order of that found in the K + influx experiments indicating an increased turnover per cell but not per pump site. This, however, is in contrast to recent studies in our laboratory (see below and ref. 133). It appears to be important to define in one way or the other the mode of molecular action of these mitogens on the membrane transport level prior to interpreting cation permeability changes either as triggering events for, or as parallel phenomena of complex cell physiological changes, The observation that the phytohemagglutinin-stimulated DNA synthesis seems to be also a function of (K~)o with a similar K1/2 for K0 + [304,308,309] emphasizes the important role of monovalent cation and their transport in the transformation of lymphocytes. Recently we have confirmed the K + pump stimulatory action of phytohemagglutinin on human lymphocytes and extended our studies on the effect of this and of other mitogens on K ~ transport in intact cells and the membrane ATPase present in microsomal membrane preparations [131,310-313]. Among the other mitogens tested wcre concanavalin-A, anti-lymphocyte sera, sheep antihuman IgG sera and human IgG as specific antigen used to prime in vivo sheep lymphocytes, in human lymphocytes the order of potency of these mitogens to alter K- transport was concanavalin A > phytohemagglutinin - anti-lymphocytic sera :::- anti-lgG and may be related to the type of lymphocytes (T or B) present in the cell preparation obtained from peripheral blood. The ouabain-sensitive K ~ pump influx, measured under conditions of initial velocity increased from about 22 to 61 mM K + per liter cells per hour in lymphocytes exposed to concanavalin A. about 2-fold with phytohemagglutinin (Fig. 2), antilymphocyte sera and somewhat less with anti-lgG, a finding well compati-

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Fig. 2. (Published with permission of journal, ref. 133). <, in presence of PHA; O, control; ordinate, relative K+ uptake; abscissa, ruin)

209 ble with the magnitudes of fluxes observedlby Quastel and Kaplan [302]. The ouabaininsensitive K ÷ influx was only sligh'ly increased. The observed stimulation of K ÷ pump influx is accompanied by an enhanced active extrusion of Na~ ÷ with a coupling ratio (N) of about 1. [aH]ouabain binding studies at low temperature or in the presence of sodiumazide at 37 °C reveal a complex picture but appear to be consistent with the hypothesis that mitogens change the affinity of the Na +, K ÷ pump for ouabain [133]. However, these studies do not yet rule out that the total number of Na+,K ÷ pumps may also be increased, a possibility difficult to verify experimentally when the cell is metabolically fully active [133]. Incubation of microsomal membrane preparations from human lymphocytes with concanavalin A, phytohemagglutinin, anti-lymphocyte sera or anti-lgG caused a marked 3-4 fold increase of the ouabain-sensitive (Na ÷, K+)-ATPase while the ouabain-insensitive Mg2~-dependent ATPase appeared to be increased twofold. The latter finding may be attributed to the possibility that (a) ouabain did not have complete access to all binding sites in the membrane preparations which are probably vesicularized or (b) that indeed the Mg z÷ enzyme is also stimulated by these mitogens. Our observations further support Quastel and Kaplan's hypothesis that one of the initial event in phytohemagglutinin-stimulation may be the activation of the energy dependent Na ÷, K ÷ transport system. They are, however, in contrast to results of others [314315]. Novogrodsky [315] found that concanavalin A affected only the Na+K+-independent ATPase of rat lymphocytes which could be prevented by methyl-a-D-mannopyranoside, a sugar specifically bound by concanavalin A. In lymphocytes of species other than human, such as rat, ouabain is a much less effective inhibitor of phytohemagglutinin-stimulated cell transformation [316]. It has been reported that 45Ca 2÷ accumulates in lymphocytes as early as 38 min after addition of phytohemagglutinio [317], indicating additional changes in membrane permeability to bivalent cations. From Ca 2÷ transport studies in red cells and other tissues it is known that Ca 2÷ diffuses along its electrochemical gradient into the cell, and that the low cellular Ca 2÷ steady state concentration (10-6M) is maintained by the (Ca2+)-ATPase pumping Ca 2÷ out of the cell [318,319]. Calcium influx into lymphocytes was found to be saturable with a Km value of 0.74 mM and explained in terms of a facilitated diffusion process [320]. Subsequent to phytohemagglutinin binding to lymphocytes, which per se was found to be Ca 2÷independent [321], the rate constan~ of aSCa2+ entry increased from 0.029 to 0.042 " min -1. This increased 45Ca 2+ entry was explained by a decrease of the Km value for Ca 2+ to 0.30 mM [320]. While Mn 2+ was a competitive inhibitor, Mg 2+ had no effect on Ca 2+ uptake. Phytohemagglutinin obviously changed the turnover number of the carrier rather than increasing the number of carriers since no change in V was observed. In the preceding discussion some evidence was provided that alteration of membrane cation permeability may be one of the earliest if not first physiological events induced by mitogens in lymphocytes followed by further cellular differentiation, mitosis and blastogenesis. It is not the aim of this review to interrelate the observed

210 cation permeability changes with a variety of other early phenomena such as activation of adenylcyclase, raising of cyclic AMP levels [322] or P-incorporation into membrane phospholipids, particularly phosphatidyl inositol [323] etc., events which experimentally may be measured earlier than cation tracer movements. One may ask why such different substances as concanavalin A, phytohemagglutinin, anti-lymphocyte sera or anti-lgG sera produce a common change in membrane cation permeability once they are bound to their respective receptors. Although the immunochemical specificity of these mitogens is apparently different, any of these reactions will induce membrane permeability changes. This may imply that none of the mitogenic substances is required to bind directly to polypeptides of the cation transport apparatus. Treatment of lymphocytes with carbohydrate attacking enzymes [324,325] and chemicals such as sodium periodate [325,327] similarly produces blast cell transformation. These treatments apparently affect membrane cation permeability since we could confirm Brossmer and co-workers' finding [67,328] that periodate, a carbohydrate oxidizing reagent, drastically changes K- permeability in human red cells, whereby the K + leak flux is more affected than the K + pump flux (unpublished experiments), it is conceivable that binding of mitogens to their carbohydrate receptors affects aggregation of intra- or transmembraneous constituents resulting, for example, in altered lipid-protein interactions which in turn lead to increases in ion transport activities. Compatible with this view are recent findings of fluidization of membrane lipids in lymphocytes exposed to concanavalin A [66] only for a few minutes. b. Tumor Cells. Because of their short generation cycle [329] mouse Ehrlich ascites tumors have long been recogmzed as suitable cell source for transport physiological [330] and immunological [331] studies. In their classic papers Goldberg and Green [332-335] showed that. only in the presence of antitumor cell antibody, complement induced rapid K ÷ leakage followcd by loss of macromolecules from thc damaged cells. Since albumin did not prevent cation equilibration but inhibited cytolysis, a colloid osmotic swelling was suggested. Herc again, as in the case of complement dependent hemolysis, we have little understanding as to thc underlying molecular mechanism leading to the dramatic change in cation permeability. The irreversibility of the membrane damage produced appears to create still formidable experimental problems. With the lectins, however, the membrane permeability change caused in Ehrlich ascites tumor cells is transient and does not seem to affect the viability of the cell. Aull and Nachbar [336] have recently shown that within minutes after addition concanawdin A caused a net gain of Na~ ÷ and loss of K," followed by ouabain sensitive net loss of Na~ ÷. The authors suggest that concanavalin A first increases passive cation permeability followed by a stimulation of Na~ ÷ extrusion due to higher (Na+)~. It has been pointed out above (see Section I1 C.) that increases in (Na+)~ in red cells causes stimulation of active cation transport. It certainly would be of interest to know how Ehrlich ascites tumor cells with different (Na÷)~ respond to the effect of concanavalin A. Although experimentally not proven it is possible that such an

211 mitiai effect of concanavalin A on passive cation permeability occurs also in lymphocytes. However, activation of the (Na+,K+)-ATPase in isolated microsomal membranes from these ceils would argue against this possibility. IIIB. Inhibiting or "'silent" antibodies against Na +, K + transport and ATPase Antibodies are now being recognized as useful tools to ascertain further the asymmetric organization of the membrane Na+,K ÷ transport system and its conformational state. The ideal situation, namely to obtain antibodies against inner and/or outer aspect of the Na÷,K ÷ pump, the Na÷,K ÷ dependent partial reaction steps of the (Na+,K+)-ATPase and/or glycoside receptor function, requires the presence of antigenic groups on the transport polypeptide chains themselves. Such a direct structure-antigen relationship may also facilitate immunological studies of conformational equilibria since the various ligands affecting the Na+,K + pump and ATPase system may also influence its immunological interaction with an anti-transport enzyme antibody. Thus a change in the antigen-antibody reaction would monitor conformational changes in the transport system. The latter approach has been successfully used by Anfinsen and co-workers who studied the interaction Staphylococcal aureus nuclease with its respective antibody [337-339]. Specific antibodies against (Na+,K¢)-ATPase can be divided into two groups: inhibiting antibodies and "silent" antibodies which apparently bind to the enzyme but failed to interfere with its activity. The first report of cation transport and ATPase inhibiting antibodies came from a German group in 1969; Averdunk and co-workers [340] raised antihuman red cell stroma antisera in rabbits which inhibited almost entirely both the (Na +, K ÷) and the Mg 2+ dependent ATPase in broken human red cell ghosts. Intact human red cells cold stored for 4 days failed to exchange intracellular sodium for external potassium when incubated in the presence of the rabbit antiserum for 4 h at 37 °C with glucose, while control nonimmune serum did not affect the reattainment of the original cation steady state. The effects were dependent on antiserum concentration and may be interpreted in terms of a dual specificity of the antiserum toward the inner and outer aspect of the Na ÷, K ÷ translocation system. However, no further efforts were undertaken by this group to analyse in detail the binding and effect of these anti-ATPase antibodies with respect to the sidedness of the membrane. The rabbit anti-rat brain (Na+,K +) ATPase antibody of Askari and Rao [57] completely inactivated the (Na+,K +) enzyme activity while the K ÷ dependent paranitrophenylphosphatase was unaffected. Furthermore, the Na ÷ dependent phosphorylation of the enzyme was inhibited but apparently not the K + sens;tive Na + dependent activity. The effect of the antibody was therefore different from that of oligomycin which does not inhibit Na ÷ dependent phosphorylation but inhibits the (Na ÷, K+)-ATPase. The data were interpreted in terms of an immunological distinction between the component involved in Na ÷ dependent phosphorylation of the complex by ATP on the one hand, and the K ÷ para-nitrophenylphosphatase and the K ÷ dependent dephosphorylation on the other [57].

212 All anti-Na + pump antibody prepared by immunization of rabbits with highly active membrane (Na+,K~)-ATPase preparations from the outer medulla of pig kidney was reported by Jorgensen and co-workers [53]. The antiserum has been well defined with respect to its action on the asymmetric aspects of the Na¢,K + pump. While this "anti-Na + pump" antibody inhibited dose dependently the (Na+,K+) ATPase in brain preparations from pig, rabbit and ox, it inactivated Jn human red cell ghosts ouabain-sensitive Na p efl]ux only after inclusion into the ghosts prior to resealing. This observation indicates that the antibody (a) detects a specificity common to (Na+,K~)-ATPase of a variety of tissues, {b) acts only at the inner aspect of the cation transport system, and (c) does not affect the ouabain-insensitive diffusion controlled Na ¢ efflux. The ineffectiveness of the antiserum at the outward facing aspect of the Na¢,K ' pump was explained by a possible in vivo adsorption of such an antibody specificity to the rabbit's own red cells (i.e. in terms of a crossreactivity). Attempts, however, have not been undertaken to recover such an antibody, for example, byelution experiments. The anti-Na + pump antibody also inhibited ouabaJn-sensitive Na-:Na" (reverse pump) and K ~ :K + exchange as well as Na + efl]ux into Na- and K + free solution [341] suggesting the limitation of the immunological approach in further separating these ouabain-sensitive ion exchange processes, if one does not assume they are expressions of one and the same ion Transport system. In contrast to the work with rabbit antibody against red cell ghosts [340] no effect by this antibody on the (Mg2+)-ATPase was observed [341] although in broken membranes the (Na+,K+)-ATPase was inhibited. The (Ca'+)-ATPas¢ was unaffected [341]. When the anti-Na' pump antibody was used in connection with anti-L on LK goat red cell ghosts, the mode of inactivation of the L-antibody stimulated Na ~ pump indicated no increase in the number of pump sites in these cells by anti-L [341], a finding supporting the concept of L-antibody-induced affinity changes (see Section III A. 1.). Rabbit anti-canine kidney N a ' , K + enzyme antibodies apparently sensitive to the conformational state of the Na +, K ~ enzyme complex were reported by McCans and co-workers [55,56]. The antibodies were absorbed to native purified Na~,K ' enzyme and purified by acid elution techniques. The eluted antibody preparation contained two specificities: one that inhibited catalytic activity when the enzyme was in "turnover condition", and another that decreased [3H]ouabain binding. Since the first antibody was bound when the dephosphorylalion step of the enzyme was blocked by ouabain, but the latter failed to bind, it was concluded that the anti-catalytic site antibody may be specific for the sodium dependent partial reaction of the enzyme, while the other antibody (anti-digitoxin receptor antibody) monitors the ouabain sensitive dephosphorylation of the enzyme [55]. It is not yet known against which of the two groups of polypeptides comprising the (Na",K')-ATPase [342] the two antibodies are directed. It is evident from these studies that antibodies can be effective tools for delineating the conformational states associated with the sequence of partial reactions of the N a ' , K + transport enzyme. The (Na +,K+)-ATPase conformer specificity of these antib(~ties will be of invaluable help in further understanding

213 cation and substrate dependent conformational changes of the Na ÷, K ÷ translocation system as they affect turnover of the enzyme as well as its inhibition by cardiac glycosides. "Silent" antibodies which bind to one of the two larger polypeptides of the (Na ÷, K÷)-ATPase but do not inhibit the enzyme were recently reported by Kyte [54]. As in McCans and co-workers' [55,56] studies the antibody was raised in rabbits against native canine renal (Na÷,K÷)-ATPase or its large polypeptide chains. As measured by complement fixation techniques bolh antibodies detected an antigen on one of the two large polypeptide chains of the enzyme complex. Using ferritinlabelled antibodies Kyte established that the antigenic determinant must be located exclusively at the inside surface of the plasma membrane. At antibody excess no inactivation of the (Na÷,K~)-ATPase was observed. Since the large chains are supposed to span the membrane, and antibody binding from the inside does not inhibit the enzyme, Kyte's conclusion is not unexpected in that active Na+,K + transport does not require a gross reorientation of the (Na÷,K÷)-ATPase and certainly no diffusion or any translocation of the carrier across the membrane during cation transport [54]. At present, it appears that immunization with native (Na÷,K÷)-ATPase produces a variety of antibodies indicating an equal variety of antigenic groups on the (Na÷,K÷)-ATPase complex. When large polypeptide chains are used as antigen, only "silent" antibodies are obtained, and in the case of native enzyme additional inhibiting antibodies are formed. It remains to be seen whether the inhibiting antibodies exert their effect only on the "native" complex or also on the isolated polypeptide chains. From an immunological point of view, it would be interesting to know whether in general Burnet's law of forbidden clones [343] does not prohibit formation of inhib:ting or activating antibodies against outward facing "self" antigenic receptors of such physiologically vital systems as the Na+,K ÷ pump and (Na÷,K÷)-ATPase. It is not unlikely that the biochemical isolation and purification of a highly active (Na÷,K+)-ATPase exposes "hidden" antigenic sites on the cis aspects of the membrane constituents comprising the Na÷,K + pump, which under normal in vivo conditions are not available and therefore immunologically not recognized.

IV. CELLULAR METABOLISM, MEMBRANE IMMUNE EVENTS AND ION PERMEABILITY In the preceding chapter it was shown that (1) some antigen-antibody reactions alter cation transport in certain red cells and in lymphocytes and (2) that chemical or enzymatic modification of membrane surfaces may cause loss of antigenic receptors or membrane perturbations interfering in some way with the effect of immunological reactions on cation transport. The structure of membranes in intact ceils, however, depends also on the cellular metabolism. Its alteration per se may effect reversible or irreversible changes in the dynamics of interaction of the various membrane

214 constituents. Thus it is conceivable that metabolic disorders or manipulation of the metabolic state of a cell may severely affect the surface equilibrium of antigens through which antibodies modulate membrane functions. This correlation is frequently overlooked in studies of immunological reactions on cell surfaces causing dramatic passive permeability changes and cytolysis. In fact, most of our present understandi0g of the molecular mechanisms of immune hemolysis is derived from experiments which for "'reproducibility" reasons used erythrocytes of considerable age, thus cells which are depleted of high energy phosphates essential for glycolysis, active cation transport and overall membrane conformation and cellular shape. Experiments on metabolic depletion and energy restoration (repletion) have been instrumental in defining "membrane-integrity" a term reflecting our lack of knowledge about the membrane regulatory role of metabolism. It is. however, fairly well established that Ca 2- is crucially at play in energy dependent membrane phenomena. Depletion of ATP by starvation for 20 h, storage for several weeks or short time exposure to NaF, NaAsO,,, 2-deoxy-o-glucose or iodoacetamide results in a gradual disc-sphere transition of the cell [344-352]. Until a critical residual ATP level is reached, from which the full ATP level can be regenerated, this morphological transition is reversible by repleting the cells in media containing nucleosides :~ glucose, except for iodoacetamide treatment which irreversibly alkylates the glyceraldehyde-phosphate-dehydrogenase. Metabolically depleted cells have lost their deformability [348,349] and filterability [350,351] and due to an estimated 60-70~,', decrease in surface area [352] the area/volume ratio becomes smaller. Concomitantly, the mean osmotic fragility increases indicating that the cell is closer to the critical hemolytic volume, which is a function of the surface/volume (S/V) ratio [353]. The presence of Ca 2+ in the medium during the depletion phase promotes these effects [353-355]. As intracellular ATP falls, the Ca 2+ pump and ATPase cease to function in maintaining the low Ca 2- steady state [318.319,356], and the ATP concentration becomes insufficient to chelate Ca 2+ [357]. The increased (Ca2+h is believed to affect via inner membrane constituents such as spectrin [358,359], other membrane protein or lipid-lipid interactions. Differences in surface tensions between inner and outer membrane layers may effect "contraction" or folding of the membrane over the same volume. Loss oI lipid, particularly cholesterol [360], as well as of protein [361] or reorganization of membrane protein [362,363] accompany metabolic depletion of the cell. Failure to acylate rising lysophosphatidyl choline levels [364] may enhance lipid phase changes responsible for a smaller S/V ratio. When ATP is depleted below the minimum concentration necessary to again stimulate glycolysis by adding adenine - inosine [365,366] the phenomena mentioned become irreversible and the cell is doomed to hcmolyse. That membrane changes observed during ATP depletion of red cells are accompanied by alterations in immunological reactivity and ion permeability has been recognized for a while. However, not many experiments have been conducted as to how metabolism, immunological reactivity and ion permeability interrelate on the molecular level. From the few reports on erythrocytes, it appears that the metab-

215 olic state of these cells may influence antigen-antibody reactions occurring at the cell surface and affect cation permeability either in a separate or interdependent manner. For example, a correlation was found between the rate of agglutination by basic polyaminoacids and the red cells surface charge. Aged human red cells have a lower electrophoretic mobility and a higher rate of agglutination than younger red cells [367]. Furthermore, old human red blood ceils are agglutinated by anti-A, anti-B or anti-Rh antibodies at a higher rate than young red cells [368] and this difference is not due to an increase in the number of antigenic sites [368]. In contrast are other findings that stored human red cells have a decreased specific hemagglutmability by anti-A which also was not due to a loss of antigenic sites [369]. Obviously a metabolically dependent redistribution of membrane surface charge may alter locally ionic repulsion. Bessis has shown that agglutinability by anti-A of sickled human type A cells is reversible. Only the oxygenated cell can be agglutinated [370]. It would be interesting to see whether metabolic depletion of sickle cells leading to irreversibly sickled cell shape [371] would also ~bolish the A-antibody agglutination. It should be pointed out that an interdependence between metabolic state and agglutinability has also been reported in transformed tibroblasts: concanavalin A preferentially agglutinates cells of low ATP content growing in high culture density [372]. When red cell antigen-antibody reactions activate serum complement, immune hemolysis ensues. While the process of activation of complement is fairly well understood, no thorough analysis of the membrane molecular basis of the lyric process has enlightened us since the first discovery of this phenomenon. As to the activation of complement as the hemolytic principle, the reader is referred to reviews [74-76]. As immunhemolysis is preceded by changes in the cation steady state of the cell, membrane permeability changes and immune reactions are closely interrelated and thus both may be dependent on the metabolic state of the cell. Thus old red cells hemolyse in the presence of Forssman-antibody and complement earlier than young red cells. Human red cells depleted of ATP by incubation for 24 h at 37 °C in glucose free media are more susceptible to complement lysis than controls, an observation which has been related to a diminished ATP dependent acyltransferase activity involved in lysolecithin-lecithin interconversion [373]. It is possible that red cells used in these experiments were metabolically depleted beyond the reversible steps where greater hemolytic susceptibility should be attributed to increased cell volume, i.e. a shorter time interval required for cell swelling prior to hemolysis. We have recently reported that the interaclion of LK sheep red cells with anti-L, the antibody shown above to affect active cation transport, and complement is clearly dependent on the metabolic state of the cell [374,375]. Depletion of ATP by starvation (16 h at 37 °C), 2-deoxy-D-glucose or iodoacetamide led to a several-fold increase in the hemolytic susceptibility of these cells to the combined actions of anti-L and complement which could not be attributed to a change in the relative amount of L-substance but rather to an increased efficiency of complement. This

216

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phenomenon was partially reversible upon repletion of ATP by incubating depleted cells in adenosine plus glucose [375]. Fig. 3 shows that LK (LL) sheep red cells are significantly more susceptible to hemolysis by anti-L complement when preincubated for 16 h at 37 ' C in media containing glucose lower than 10-¢ M. [mmunologically this observation may be explained in terms of a change in the proximity of L-antigenic sites thus facilitating doublet formation of anti-L lgG antibody molecules required to fix the activated complement component [376]. This is plausible because of the probable low density of L-antigenic sites per cell. It is also possible that a different subclass of L-antigen lgG molecules binds to the L-antigens capable of a better utilization of complement components. Physiologically the ATP depleted LK sheep red cells could have gained water, facilitating lysis by changing tile threshold volume I¥om which tile immune lytic process takes off. However, this was not found to be the case: metabolically depleted LK cells had about the same or less cell water. This finding and the increased osmotic fragility indicates that the S / V ratio must have decreased at constant volume, an event which may decrease the actual critical hemolytic volume in these cells and thus makes them more prone to hemolysis. It should, however, be recalled that Ca z+ required for activation of complement was present in tile incubation medium (10 -'~ M). in ATP-depleted human red cells (Ca2')i increases several-fold the rate constant of K ~ efflux [377-379] a phenomenon known as the Gardos-effect [377] and recently shown by Blum and Hoffman to be ouabain inhibitable and thus possibly mediated by the carrier normally involved in ATP dependent Na*: K + exchange [380, 381]. Thus Ca 2+ ions could play a dual role in activating the complement sequence in the final phase and also by entering the cell altering membrane cation permeability promoting subsequent cation exchange and wz,tcr entry and ultimately immune hemolysis. The Ca 2+ uptake mechanism was reported to be similar in red cells of human, sheep, goat, cow, guinea pig, coypus, rat and dog, and independent of the cation polymorphism in red cells of the ungulates tested [382]. However, the effect of Ca 2~ on K + efl]ux in ATP depleted red cells of sheep, cow and goat was only small indicating the absence of

217 Ca 2+ sensitive K- channels in these species [382]. This finding would make it unlikely that the higher susceptibility of LK sheep red cells to immune-lysis by anti-L and complement involves this process, a hypothesis supported by our observation that enhancement of lysis also occurred in Na+-free, K ÷ containing media. It is of interest that ATP depletion of H K sheep red cells did not significantly change their response to anti-M, indicating that the metabolic dependence of immune lysis in lhis system may be more relevant to the membrane properties of LK sheep red cells and the L-antigen/L-antibody interactions.

v. IMMUNOLOGICAL ALTERATION OF ION TRANSPORT 1N LIPID MODEL MEMBRANES Although progress has been made in incorporating various enzymatically active or inactive proteins into artificial membrane systems such as lipid mono- and bilayers and black lipid membranes, only a few experiments were performed in which the effect of antigen-antibody reactions has been studied. This is partly due to the intrinsic difficulties of introducing functionally or antigenically active molecules into phospholipid membranes and partly reflects a lack of "cross-fertilization" of the fields of lipid membrane biophysics and membrane immunology. The inseltion of proteins into the monolayer generally leads to surface pressure changes which will depend on the penetrability of the molecule. In black lipid membranes, interaction with proteins may cause an increase in membrane conductance, and in liposomes permeability changes are monitored by measuring the eMux of trapped markers. From the discussions of the effect of antigen-antibody reactions in lymphocytes, it is evident that two approaches can be taken in studying immunological reactions in artificial lipid membrane systems. One is to incorporate the antigen or hapten into the lipid membrane, followed by subsequent addition of specific antibody. The other possibility is to insert the antibody molecule first, and then let it react via its F(ab) combining sites with antigens applied to the bathing solution. The tacit assumption in both approaches is that the molecules combining with the lipid membrane first have particular structural properties to bind and penetrate such membranes, keeping the ligand in the proper conformation at either surface to react with the fitting probe added in the second step. For example, various membrane lipids known to be potent antigens, such as glycolipids containing the major human blood group antigens A and B [383], and the pentasaccharide carrying sphingolipids with Forssman-antigen activity [384], or acidic phospholipids such as cardiolipin can be considered as ideal candidates to be inserted into phospholipid membranes. It has also been known for a long time that one can prepare antibodies against cholesterol [18] or its derivatives [385], and antibodies against phosphorylcholine are available from monoclonai mouse myeIomas [386]. Recently anti-sphix*gomyelin-antibodies have been reported [387]. Using liposomes, Kinsky and co-workers [388-395] (see also the review by S. C. Kinsky in this journal [396]) have studied the immunological release of trapped

218 glucose from liposomes containing Forssman antigen or lipopolysaccharides; when complement was added to antibody exposed and antigen containing liposomes, their lysis commenced. Although this phenomenon remarkably resembles the immune lysis process brought about by the same antigen-antibody reaction in sheep red cells, our advancement in understanding the membrane molecular and ionic processes during immune lysis is still very slow. In particular, there are no vigorous experimental data on incorporation of complement components into artificial membranes and their effect on membrane ion conductance to justify the assertion that these proteins are being inserted into the lipid bilayer prior to changing membrane permeability and onset of hemolysis [75]. Thus these techniques may be useful to understand events at the molecular level of the membrane. It now appears possible to incorporate ),-globulin molecules into artificial lipid membranes. Colaccio and co-workers [397,398] studied the interaction of various proteins with lipid monolayers and showed theft y-globulin incorporation from the subphase increased the surface pressure of the monolayers. Weissmann and coworkers [23] recently demonstrated the association of mildly heat-treated human y-globulins with liposomes. Of particular interest is that Fc fragments prepared by papain hydrolysis from native IgG associated with the liposomes as well as the native protein indicating that it is this portion of the antibody molecule which associates with the liposomal membrane and is operative in bringing about permeability changes in the liposomal membranes [23]. There was a clear preference of these liposomes for certain y-globulin subclasses: igG~ protein was most reactive, while IgA, and lgM proteins were inert. This work opens the way to use specific antibodies and study their interaction with their respective antigens added in a second stage. There are only three reports which have dealt with the effect of antigen-antibody reactions on black lipid films. Del Castillo and co-workers [399] showed a reduction in the transmembrane electric resistance when albumin and anti-albumin immuneserum were added to the inner compartment of their system. The effect seemed to be antibody dose dependent but independent of the order of addition of the two reaction partners. Barford and co-workers [400] used insulin, ribonuclease and lysozyme as antigens, but antisera against these proteins plus or minus complement had to be on the opposite side of the bimolecular leaflet to change membrane conductance. They concluded that either part of the antigens or of the antibody molecule protrudes through the membrane in order to facilitate the antigen-antibody reaction responsible for increased ionic flux. Tosteson and co-workers [401] demonstrated that addition of glycoprotein prepared from blood group 0 human red cell membranes to the solution bathing the outside of the lipid bilayer membrane led to a steady increase of membrane conductance up to 25-fold above that of the untreated control. When concanavalin A ~,as added in the presence of Ca -,+ ions (1 raM) a further 7-8-fold increment in membrane conductance was observed. Concanavalin A alone was ineffective. Though these three reports are promising beginnings, further work is required on (a) the mode of incorporation of these antigenic polypeptides into lipid bilayer

219 membranes and (b) whether it is the immunologically specific interaction of the antibodies or lectins with their ligands which causes the observed change, it would be of particular interest to see whether anti-ATPase antibodies will alter conductance changes observed with this enzyme [402,403] and whether such studies are also feasible with purified (Na +, K+)-ATPase preparations and their antibodies discussed above.

VI. CONCLUSION AND PROSPECTUS By experimental device the in vitro modification of cation transport by antigen/ antibody reactions or binding of lectins certainly constitutes an "artificial provocation" since the cells studied never come into in vivo contact with the antibodies produced by immunization or the lectins extracted from often exotic plant seeds. Yet the immunophysiological approach appears now to have considerable potential for enhancing our understanding of cation transport processes in biomembranes. Antibodies proved to be important tools in the elucidation of the cation transport polymorphism in ungulate red cells and appear to be equally useful in further defining the active Na+,K + pump, in general. Fortunately, the erythrocyte may serve again as a model system for investigations of permeability changes in nucleated ceils brought about by antigen/antibody reactions or membrane/lectin interactions. In red ceils the effect of antigen/antibody reactions on the Na+,K + pump supports at least in part and extends our existing models of active cation transport. In nucleated cells one has to ask whether cation permeability changes are at the beginning of and thus trigger cellular transformation, or constitute merely one of multiple simultaneous changes accompanying cellular metamorphosis. Obviously, a model awaits its conceptualization which establishes a causal relationship between permeability changes and subsequent biochemical events. There are a number of other ill-defined immunological membrane events accompanied by initial cation permeability changes (for example, immune cytolysis) stressing the relevance to study in depth the effect of immune reactions on cation transport. Furthermore, among a host of erythrocyte abnormalities those distinguished by tendency to auto-hemolysis or short in vivo survival (altered membrane permeability?) combined with genetically documented antigen deficiencies are prime candidates for transport physiological and immunological investigations. A typical example is the Rhnu. red cell, supposedly lacking the Rh antigenic determinants [404-406], which has been shown to be part of a membrane integral lipoprotein complex [407,410]. In its physicochemical properties [407-409] (sulfhydryl group and phospholipid requirements) the Rh antigen seems to resemble the Na+,K + pump-associated M and L antigens [231]. Recently Smith et al. [411] showed that, following phospholipase A 2 modification, Rh,,~, red'cells, in contrast to the Rh cell. failed to exhibit increased l-anilino-naphthalene-8-sulfonate fluorescence and N-[~4C]ethylmaleimide incorporation into two major proteins. A conformational

220 change of the constituents carrying the R h - H r antigenic d e t e r m i n a n t s related to the decreased in vivo red cell survival is an alternative hypothesis [411] to that proposed by Levine et al. [412] that the biosynthesis of the Rh-antigen is suppressed in the R h , , u erythrocyte. There is a long but promising path leading from the investigations on i m m u n o l ogically affected cation transport systems reviewed above to their application to clinical red cell disorders. Clearly, m e m b r a n e physiology and i m m u n o l o g y will pave the way to u n d e r s t a n d i n g and even therapeutic solution of pathophysiological phenomena. However, to achieve such a goal. further detailed studies are necessary on the exact molecular interrelationship between the cation transport system and antigenic determinants on both sides of asymmetric biomembranes.

ACKNOWLEDGMENTS The main thrust into this new field of m e m b r a n e i m m u n o - p h y s i o l o g y would have been impossible without the moral and experimental support of Professors D. C. Tosteson, B. A. Rasmusen, R. Blostein and R. Averdunk, as well as my coworkers P. Shrager and J. J. Snyder, my students C. H. Joiner and W. A n d e r s o n and my research assistants M. P. Dessent and W. W. Sun. The final critical reading by my colleagues B. A. Rasmusen, R. Blostein, T. J. M c M a n u s , S. Simon and C. H. Joiner is greatly appreciated as well as the excellent typing of the m a n u script by Mrs. M. Kaplan. Supported by a United States Public Health Career Development Award K 4 - G M 50, 194 and PHS 2 POI-HL-12, 157.

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