Studies of the antigenic properties of the catalytic and glycoprotein subunits of Na+,K+-ATPase

ARCHIVES OF BIOCHEMISTRY Vol. 221, No. 2, March,

Studies

pp.

AND BIOPHYSICS 371380, 1983

of the Antigenic Properties of the Catalytic Glycoprotein Subunits of Na+,K+-ATPase WILLIAM J. BALL, JR.,~ JOHN LOIS K. LANE, AND ARNOLD

Lkpartment of CoUege

H. COLLINS, SCHWARTZ

Pharmacdogy and Cell Biophysics, University of Medicine, 221 Bethesda Avenue, Cincinnati, Received

August

and

of Cincinnati Ohio 45267

2, 1982

Antibodies were raised against isolated, delipidated catalytic [a] and glycoprotein [p] subunits of the Na+,K+-dependent ATPase purified from lamb kidney medulla. The specificity of each antiserum was confirmed by agar double-diffusion precipitation, immunoeleetrophoresis, and polyaerylamide gel electrophoresis. A solid phase adsorption assay was also employed to determine antibody binding titers and to further test the specificity of these antisera. Antibodies raised to the cy subunit had a strong reactivity and similar titer values for both the holoenzyme and the (Y subunit and a lowaffinity cross-reactivity with the /3 subunit. In contrast, b-subunit-directed antibodies had little reactivity or binding with the holoenzyme and a low-affinity cross-reactivity with the cx subunit. Competition binding studies revealed that about 80% of the LYsubunit-specific antibodies bound to the holoenzyme, indicating that similar sets of antigenic sites are exposed in the lipid-embedded holoenzyme complex and in the isolated (Y subunit. Competition binding studies also suggest that the subunit cross-reactivities of the antisera may not result from simple contamination of the respective antigens, but that there may be partial homologies of some antigenic sites. In addition, the b-directed antibodies had no effect on Na+,K+-ATPase activity, while the o-directed antibodies were effective inhibitors of activity. This indicates that at least some funetionally important antigenic sites of the a! subunit may be unaltered by its isolation and delipidation.

The membrane Na+,K+-activated ATPase is responsible for active transport of Na+ and K+ across the cell membrane. This enzyme also specifically binds and is inhibited by cardiac glycosides and is thought to be the pharmacological receptor for digitalis. The enzyme consists of two major subunits. The digitalis and ATP binding sites reside on the (Y subunit (i& - 100,000). Although the other major peptide, the p subunit (1M, - 50,000), is closely associated with the LY subunit, it has not been demonstrated as yet to have a specific function 1 Author dressed.

to

whom

correspondence

should

be

(see reviews (l-3)). In addition, a third y or proteolipid component (1M, - 12,000) has been identified and isolated from Na+,K+ATPase preparations (4,5). Several investigators (4, 6, 7) have found that labeling of the enzyme by photoactive derivatives of the glycoside ouabain covalently labels both the LYsubunit and the y component. It is possible, although controversial, that the y may be a part of the digitalis receptor site of the Na+,K+-ATPase. Enzyme-directed antibodies have the potential of being specific and sensitive probes of the structural and functional properties of Na+,K+-ATPase. Since the first report by Averduck et al. (8) of an-

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371

0003-9861/83/040371-10$03.00/O Copyright All rights

0 1983 by Academic Press, Inc. of reproduction in any form reserved.

372

BALL

tiserum directed against the Na+,K+ATPase, several investigators have raised antibodies against various holoenzyme preparations and also against the isolated, denatured (Yand /3 subunits of the enzyme. These antibodies have been used to study the organization of the enzyme complex in the membrane, to study subunit functions, and to detect ligand-induced conformational changes. However, these antibodies have had varying and sometimes even apparently contradictory effects upon measurable functions of the enzyme (9-15). The wide variability in the results of these studies is generally attributed to the variability between animals in their immunological response to this complex antigen, to the difficulties caused by using heterogeneous populations of antibodies, and probably to the incomplete characterization of the antibodies utilized. The effects of antibodies on enzyme function may be caused by a single antibody binding to a specific site on the enzyme or by the cumulative effects of many different antibody species binding to many sites. Because such large amounts of antibodies appear to be needed to cause these functional changes, it is difficult to determine which of the above explanations is correct. Clearly, a better understanding of the basic antigenic nature of the holoenzyme and its subunits is needed before more meaningful information concerning the enzyme can be obtained with an immunological approach. In this study antibodies were raised to the SDS’-solubilized (Y and /3 subunits of the lamb kidney Na+,K+-ATPase. The antigenic properties of these proteins were analyzed by using a solid-surface assay system that detects both “active” and “silent” antibody binding. This technique can be used to determine the relative levels and affinities of the antibodies raised and the extent to which the antigenically active sites of the subunits resemble those exposed in the native enzyme. The binding specificity of the antibodies was tested us’ Abbreviations used: SDS, sodium dodecyl sulfate; EGTA, ethylene glycol bis(@aminoethyl ether) NJ’tetraacetic acid; PAS, periodic acid/Schiff.

ET

AL.

ing agarose gel immunoelectrophoresis, double-diffusion precipitation techniques, and polyacrylamide gel electrophoresis. The ability of the antibodies to inhibit the Na+,K+-ATPase activity was also determined. MATERIALS

AND

METHODS

Preparation of Na+,K+-ATPase and s&units. The membrane-bound enzyme was purified from the outer medulla of frozen lamb kidney according to the procedure of Lane et al. (16). The a and fi subunits of the enzyme were then separated by gel filtration in a 5 mg sodium phosphate, 1 mM 2-mercaptoethanol, 0.1% SDS solvent (pH 8.0) following solubilization of the holoenzyme with SDS at 10 mg/mg protein by procedures described by Lane et al. (16) and Reeves et al. (5). The SDS was removed from the proteins by chromatography on AG-l-X2 resin in a buffer containing 6 M urea, 0.05 M sodium phosphate, and 0.01% NaNa, pH 8.0. The protein sample was then dialyzed extensively against a 10 mM Tris-buffer containing 0.5 mM EGTA, 1 mM fl-mercaptoethanol, and 0.01% NaN3 solution, pH 7.3. Immunization of animals and preparation of antise-rum. Antibodies were raised in rabbits by injecting an emulsified suspension of complete Freunds adjuvant and the antigen (1 mg/ml) (17). The suspension (1 ml) was initially injected subcutaneously (multiple sites) in the back of the rabbits weekly for 4 weeks, followed by 0.2 ml injected intramuscularly into each haunch for 2 weeks. Animals were then bled bimonthly from the ear marginal vein, with booster injections being administered on the weeks between bleedings. The antisera complement was heat inactivated for 30 min at 5’7°C. The antisera were then centrifuged and filtered through 0.45- and 0.22-pm Millipore filters before use. The immunoglobulin fraction was precipitated from the immune serum four times with 40% (NH,)zSO, and the globulin fraction was resuspended in and dialyzed against a 150 mM Tris-buffer, pH 7.4, containing 0.5 mrd EGTA and 0.01% NaNz. Determination of antibody binding. The procedure described below is similar to that developed by Engvall (18). Microtiter plate wells (Cooke flexible 96-well plates) were treated for 30 min with 100 rl/well of test protein at 0.10 to 0.5 mg/ml concentrations. The protein solutions were then removed and the wells were washed three times with a 10 mM Tris-Cl-saline solution (pH 7.4), containing 5 mg/ml bovine serum albumin (Buffer A) and then incubated for 10 min with an excess of this buffer in each well. The buffer was removed, and lOO-~1 aliquots of antiserum were added per well, and the wells were incubated at room temperature for 90 min. The wells were then emptied, rinsed with Buffer A, and 100 pl of ‘%I-labeled protein

IMMUNOCHEMICAL

PROPERTIES

A (from Staph&wxus aweus) was added to each well. After 30 min the ‘l-labeled protein A solution was removed, and the plates were rinsed, dried, the wells were separated and the bound radioactivity in each well was determined using a gamma counter. Competition binding studies were done using antisera diluted with Buffer A to the appropriate titer value concentrations and then incubated 4 to 5 h at room temperature with varying concentrations of holoenzyme, Q or /3 subunits. The immunoprecipitate which formed was removed by centrifugation at lOO,OOOg., for 30 min in a Beckman Airfuge (Beckman, Palo Alto, Calif.). Aliquots of 100 ~1 of the antisera were then transferred into the microtiter plate wells previously coated with the test antigen (0.5 mg/ ml) as described above. The Protein A was iodinated by the method of Dorvall(19). Anti-ovalbumin serum, anti-actin serum (supplied by Dr. James Lessard, Division of Cell Biology Children’s Hospital Research Foundation, University of Cincinnati), anti-holoenzyme Na+,K+-ATPase and preimmune serum, as well as the subunit-directed antibodies, were used to test the specificity of antibody binding. Ovalbumin, bovine serum albumin, and cytochrome c served as control antigens for nonspecific binding. Assays for Na+,K+-ATPase a&&y. The Na+,K+ATPase activity was measured using a coupled enzyme spectrophotometric assay (20) in a medium containing 30 mM histidine, 2.5 mM NazATP (P-L Biochemicals), 5.0 mM MgClz, 95 mM NaCl, 10 mM KCI, 1.0 mM EGTA-Tris, 0.36 InM NADH, 2 mM phospho(enol)pyruvate, and 10 ~1 pyruvate kinasellactate dehydrogenase (Sigma), pH 7.2. The enzyme preparations used had specific activities of approximately 20 pmol of ATP hydrolyzed/mg protein/min at 3’7”C, bound 1.8-2.2 nmol of ouabain/mg protein. Less than 0.5% of the ATPase activity was ouabain insensitive. The antisera, preimmune sera, globulin fractions, and albumin solutions were extensively dialyzed against a 1 mM EGTA-Tris buffer, pH 7.2, before being used. They were then preincubated with 0.75-1.0 pg of Na+,K+-ATPase at 37°C for 15 min, and then ATP was added to initiate the reaction. The enzyme activity data are corrected for endogenous serum Na+,K+-ATPase activity, but not for the modest (lo15%) increase of activity observed with preimmune sera or the globulin fractions. Other measurements and procedures. Protein concentrations were determined by the method of Lowry et al. (21) with bovine serum albumin as standard. The purity of the enzyme, its subunits and immunoglobulin proteins were assessed by gel electrophoresis. Polyacrylamide gels (7.5 and 10%) containing 0.1% SDS were prepared according to Laemmli (22) and the gels were stained with Coomassie Brilliant Blue. The gels were also stained to detect the presence of carbohydrate using the periodic acid/Schiff reagent (PAS) (23). The specificity of the antibodies

OF

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Na+,K+-ATPase

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373

was tested by the double-diffusion method of Ouchterlony (24) and agar immunoelectrophoresis as described by Michael et al. (12). In addition, the lamb kidney enzyme was resolved on the 7.5% SDS-polyacrylamide gels, and 2-mm gel slices were placed into separate wells of the plastic Cooke plates. The gel proteins were then extracted into 100~pl volumes of 0.5 M Na&Os for 12 h. The gel slices were removed, the plates washed with Buffer A. The wells were then exposed to antiserum for 90 min and the antibody-antigen complexes formed were determined using ‘l-labeled protein A.

RESULTS

The purified lamb kidney Na+,K+ATPase preparations appear as two bands when analyzed by SDS-polyacrylamide gel electrophoresis. These bands have apparent molecular weights of 95,000 and 44,000 (16). The isolated subunits migrate as single bands when analyzed by gel electrophoresis (16, 25). Only the 0 subunit gave a positive (PAS) reaction for the presence of carbohydrates. The specificities of the antibodies raised in rabbits to the isolated (Yand /3 subunits were tested using doublediffusion immunoprecipitation and agar gel immunoelectrophoresis (Fig. 1). Single precipitin arcs between the antisera and the immunizing antigen were observed with no antibody cross-reactivity toward the other enzyme subunit. Rabbit preimmune sera did not form any precipitin arcs with the antigens, and the immune sera did not react with any lamb preimmune serum components. In addition, neither holoenzyme- nor subunit-directed antiserum gave precipitin arcs when initially tested against the insoluble, membranous, holoenzyme. Sodium dodecyl sulfate and deoxycholate solubilization of the holoenzyme proved to be unsatisfactory for the immunodiffusion studies. However, the use of 5% polyoxyethylene-9-lauryl ether to “solubilize” the enzyme made it possible to obtain precipitin lines on ouchterlony plates between Na+,K+-ATPase and the holoenzymeand a-subunit-directed antibodies, but not with the P-directed antisera. When immunoelectrophoresis of the solubilized holoenzyme was done, two precipitin lines were observed (not shown) when using the holoenzyme- or a-subunit-

FIG. 1. Specificity of the subunit-directed antibodies. (A) Characterization by double immunodiffusion in 0.8% agarose gels. (I) The center well contained the a subunit with the outer wells containing antisera: (1) anti-cY-subunit antiserum (designated a-l); (2) anti-holoenzyme antiserum (NKA-1); (3) anti-psubunit antiserum (B-1); (4) anti-o-subunit antiserum (p-2); (5) preimmune serum; (6) anti-a-subunit antiserum (a-3). (II) The center well contained fl subunit while the outer wells l-6 contained antiserum as designated in (A). (III) The center well contained Na+,K+-ATPase, solubilized in 5% polyoxyethylene-9lauryl ether, while the outer wells (l-6) contained antisera as designated above. The center well samples contained approximately 15 pg and the outer wells contained approximately 400 pg of total serum protein. Antiserum designation numbers are for particular rabbits. (B) Agar immunoelectrophoresis: Plate 1. The wells contained: (a) B subunit; (b) cx subunit; (c) p subunit; and (d) (Y subunit. The troughs contained (e) anti-a antiserum (a-3); (f) fi antiserum (B-1); (g) anti-b antiserum (p-2).

IMMUNOCHEMICAL

PROPERTIES

OF

FIG. 2. Determination of antibody binding specificity to SDS-polyacrylamide gel resolved lamb kidney Na+,K+-ATPase. The dotted line represents the Coomassie blue staining pattern for the enzyme (10 pg sample). The open circle line (0) represents the profile of cY-subunit-specific serum binding to the resolved holoenzyme (40 pg sample) and the open triangle line (A) the binding of P-subunit-specific serum to the enzyme (40 pg sample), The purified enzyme was subjected to electrophoresis (7.5% gels) and the separated peptides were detected by Coomassie blue staining or by radioactivity after antibody binding to the proteins with ‘l-labeled protein A.

directed antibodies. Apparently, the enzyme preparation was “solubilized” into two electrophoretically different components.

THE

Na+,K+-ATPase

375

SUBUNITS

The specificity of the antisera was further demonstrated by using an indirect binding assay that we have used previously to study antibodies raised to the holoenzyme (26). The purified enzyme was resolved on SDS-polyacrylamide gel and then slices of the gels were placed into separate wells of plastic microtiter plates. The protein in the slices was extracted from the gel with a portion of the protein binding to the plate wells. The plate-adsorbed proteins were then exposed to the antisera and the bound antibody-antigen complexes were detected by ‘%I-protein A. Figure 2 illustrates the monospecific nature of the antisera binding to the (Yand /3 subunits of Na+,K+-ATPase. This same indirect binding assay was used to determine maximum antibody binding levels and the titer values, or 50% binding dilutions, of the subunit-specific antibodies. These values are comparable to those observed for the holoenzyme antibodies. Figure 3A shows a typical titer curve for a sample of cx subunit-directed immune serum. Similar titer values were obtained for antibody binding to both the (Y subunit and the holoenzyme, and there is a low affinity (-15%) cross-reactivity to the /3 subunit. While there was considerable variability in the relative maximal levels of antibody binding, in general, holoenzyme binding was about 40-60% and A

1004 100 (

I

I

!

.Ol Antibody

I .005 Dilution

I Ratio

I

I

I .ool

I 0

I .I0 Antibody

.05 Dilution

I .Ol

Ratio

FIG. 3. Determination of antibody binding to antigens as a function of antiserum concentration. The open circles (0) represent antibody binding to Na+,K+-ATPase, solid circles (0) binding to the (Y subunit, and the open triangles (A) binding to the p subunit. Bound antibody was detected using 1251-protein A. Values are given as the percentage of antibody binding relative to the maximum amount bound to each particular antigen (50 mg/ml) absorbed to the plastic microtiter plates. (A) Dilution curve for an immune serum sample directed against the (Y subunit (designated 01-4~). (B) Dilution curve for an immune serum sample directed against the @ subunit (designated @-lz).

! 0

376

BALL

ET AL.

/I binding 15-40’S of that to the (Ysubunit (Table I). The titer values and relative binding levels of the p-subunit-directed antibodies to the @ subunit, the holoenzyme, and the (Ysubunit were similarly determined. These antibodies exhibited a high affinity for the /3 subunit, with little cross-reactivity toward the holoenzyme (Fig. 3B). Maximum binding levels to the holoenzyme were less than 10% of that to the p subunit, and the titer values were correspondingly low. Maximal antibody binding levels to the LYsubunit varied and ranged from 10-25s with titer values about 10-20s of that for the /? subunit (Table I). These data suggest that, although there is a similarity between the antigenic sites of the isolated a subunit and the antigenic sites of the CYsubunit in the holoenzyme, the same is not true for B subunit. Direct competition binding studies were carried out in order to more fully determine the extent to which the antigenic sites of the isolated (Y subunit are exposed in the holoenzyme. In this study the diluted antisera were first exposed to varying

amounts of holoenzyme or CYsubunit. The precipitable antibody-antigen complexes were then removed, and the ability of the unbound antibodies to bind to plate-adsorbed (Ysubunit was determined. The undenatured holoenzyme competes for about 80% of the a-subunit-specific antibodies (Fig. 4). This compares well with the 95% inhibition of binding observed when using the (Y subunit as competitor. The concentration of protein needed to achieve a 50% inhibition of binding is used as a measure of relative antibody affinity. The data show that approximately 1 pmol of denatured (Y subunit and 6.8 pmol of holoenzyme (containing 4.6 pmol of CX)are required for a 50% decrease in the binding to plate adsorbed (Y. Therefore, the affinity of the (Ysubunit-specific antibodies toward the isolated (Y subunit appears to be about fivefold higher than toward the holoenzyme, but the majority of the antigenic sites are still recognized as holoenzyme sites. The concentration of alpha contained within the holoenzyme was calculated assuming that the holoenzyme is an IX.,& dimer with a iW, of 278,000 (27,28) containing two an-

TABLE DETERMINATION

OF ANTIBODY

I

TITEFG AND MAXIMAL

BINDING

RATIOS

Antigen Holoenzyme

Antiserum

Titer

a-Subunit (Y-13 a-3, (Y-41 (Y-43

specific

D-Subunit B-12 B-b 8-4 8-26

specific

1:800 1240 1:226 1:550

-

(Y Subunit

Percentage binding

40 102 36 38

6.5 7.0 6.0 7.0

Titer

1:670 1:667 1:400 1:620

160 1:45 1:91 -

fi Subunit

Percentage binding

Titer

Percentage binding

100 100 100 100

1:71 1% 123 1%

21 33 52 39

26 16 25 18

1480 1550 1600 1556

100 100 100 100

Note. Titer values are given as the dilution values for half-maximal binding. Titer values are a function of both antibody avidity and quantity. Maximal binding values are given as percentage binding relative to that for the immunizing antigen. The binding is quantitated using =I-protein A as described under Materials and Methods and each determination is done in duplicate. Antiserum numbers are designation numbers for particular rabbits while the subscript number indicates bleed number.

IMMUNOCHEMICAL

PROPERTIES

OF

THE

Na+,K+-ATPase

SUBUNITS

377

FIG. 4. Determination of a-specific antibody binding to boloenzyme. The upper, open circle (0) curve depicts the decrease in a-specific antibody binding to plate-absorbed a subunit after prior exposure to antiserum to concentrations of increasing holoenzyme. The solid circle (0) curve is the decrease in antibody binding to peak absorbed a subunit after exposure of antiserum to increasing a concentrations. Antiserum sample was diluted 1:300 to insure an excess of plate absorbed antigen and 350~~1 antiserum samples were incubated for 4 h with either holoenzyme or (Y subunit. Samples were then centrifuged at 100,000~ for 30 min to remove any precipitate. 100 rl/well samples were then applied to plate wells already preabsorbed with the a subunit (0.5 mg/ml). All points were done in triplicate and the results are given as the percentage of binding relative to controls. Protein concentrations are given as pmol/lOO ~1 sample.

varying concentrations of the (Y or @ subunits, and then the ability of the unbound antibodies to cross-react with plate absorbed antigen was determined. Figure 6A shows that the fraction of the a-subunitraised antibodies which bind to the /3 subunit bind both the purified /3 and the (Y subunit. In fact, it is clear that the antibodies which bind to or cross-react with /3 actually have a higher affinity for the cx subunit. A 50% decrease in binding to the p subunit was attained by the addition of approximately 0.4 pmol (Y subunit versus 7.0 pmol of the /3 subunit. Similarly, Fig. 6B shows that the P-subunit-raised antibodies which bind to the CYsubunit can be totally removed by the @subunit, while the (Y subunit is only poorly competitive. A portion of the antibodies (-20%) have a high affinity for the cxsubunit but beyond that the concentrations of the (Y subunit which are needed to compete for the antibody binding are so high that they interfere with the plate assay and an increase in antibody binding was observed. Figure 6B shows that a 50% decrease in the binding to cr is obtained with approx-

tigenically identical LYsubunits (M, 95,000). In contrast to the (Y subunit-directed antibodies, the b-directed antibodies binding to the holoenzyme had an affinity too low to perform competition binding studies similar to those with the a antibodies. The effects of the LY-and B-directed antiserum populations on the Na+,K+-ATPase activity of the purified holoenzyme were also determined (Fig. 5). The anti-a-subunit immunoglobulins maximally inhibited 50-70’S of the enzyme activity, which is similar to the effects of the holoenzymedirected antibodies. The anti-/3 immunoglobulins had no detectable effect on enzyme activity, which is consistent with our observations (Fig. 1 and Table I) that these antibodies bind poorly to the holoenzyme. Additional competition binding studies also provided a means of analyzing the apparent cross-reactivity of the subunit-directed antibodies that was detected by the indirect binding assay. In these experiments the antisera were again exposed to

FIG. 5. Effects of varying concentrations of immunoglobulins on the ATPase activity of Na+,K+ATPase. The open triangles (A) represent the effects of two different &subunit-directed immunoglobulin fractions on ATPase activity (B-1 [broken line] and b-2 [solid line]). The solid circles (0) represent the effects of two a-subunit-directed immunoglobulin fractions (a-l [broken line] and a-4 [solid line]). The open circle (0) depicts the effects of holoenzyme-directed immunoglobulins on enzyme activity. All points were done in duplicate and values are given as percentage of activity relative to that in absence of immunoglobulins.

378

BALL

ET AL.

0

p moles I Antiserum Sample

2

4

6

8

10

P moles I Antiserum Sample

FIG. 6. Determination of the binding specificity for the cross-reacting antibodies. (A) The open circles (0) show the decrease in antibody binding to plate-absorbed a subunit after pretreatment of antiserum with (Y subunit. The open triangles (A) show the decreased binding with j3 subunit. The B-directed antiserum sample (B-2) was diluted 1:306 to ensure antigen excess. (B) The open triangles (A) show the decrease in antibody binding to plate-absorbed @ with increasing @ concentrations. The open circle (0) shows the decrease in binding with (Ysubunit. The a-subunit specific antiserum (o-4) sample was diluted 1:200. All points were done in triplicate and values are given as percentage

of control.

imately 1.1 pmol of p, and no more than a 30% decrease is achieved with 4 pmol of (Y subunit. These data show that the observed cross-reactivity of the antisera is not caused by simple contamination of each antigen preparation with the other subunit. It is probable that the antibodies are monospecific, and that there are weak but detectable similarities between some of the antigenic sites of the (Yand B subunits. DISCUSSION

The binding of antibodies to the antigenie sites on the constituent proteins of the-Na+,K+-ATPase can serve as a probe of the structure, function, and organization of the holoenzyme. A number of investigators have raised antibodies to various preparations of the enzyme and also to the isolated subunits (8-15). The results of these studies, however, have been widely divergent with respect to the ability of these antibodies to affect various functions of the holoenzyme. This is not surprising since there is a multiplicity of antigenic sites located on this enzyme, and there is no a priom’ reason for any animal to produce antibodies to any particular set of these sites. The major problem with the

previous studies has been that without detailed characterization of the antibodies obtained any specific interpretation of the antibody effects on holoenzyme function is impossible. Additional immunochemical methods, however, now make it possible to gain more information about the basic antigenic properties of Na+,K+-ATPase. In our previous studies, utilizing the surface adsorption technique (26, 27), we found that polyclonal antibodies raised in rabbits against the holoenzyme are directed against both the (Yand the /3 subunits of the enzyme. Antibodies directed against the lipid components of the enzyme (which can be as high as 1.0 pmol of lipid phosphate/mg protein (16)) cannot be detected, but a low-molecular-weight y or proteolipid component, which can be isolated from the enzyme preparation was found to be antigenically active. The majority of holoenzyme-directed antibodies bind to the (Ysubunit, and about 95% of the a-directed antibodies bind to the holoenzyme. In contrast, only about 60% of the P-subunit-directed antibodies also bind to the holoenzyme. These data suggest that the p subunit is less antigenic than the (Y subunit and that some modification or alteration of its antigenic nature may occur

IMMUNOCHEMICAL

PROPERTIES

upon its purification. However, most of the holoenzyme-specific antibodies raised bind to both the holoenzyme and to the SDSsolubilized, delipidated, and purified subunits. These results contrast with those reported by Larraga et al. (30) where the (Y, /3, and y subunits of the bacterial (Microccus lysodeckticus) membrane FiATPase which had been prepared by acrylamide gel electrophoresis, exhibited a lOO- to 500-fold reduction in their reactivitywith the holoenzyme-directed antibodies. In this report we have extended our previous studies and have raised antibodies to the isolated and denatured (Yand @subunits of the Na+,K+-ATPase. Double-diffusion immunoprecipitation and polyacrylamide gel electrophoresis studies have demonstrated that these antisera are monospecific toward the immunizing antigen. An indirect binding assay was used to determine the titer values of apparent antibody affinities. The cu-subunit-specific antibodies were found to have similar titer values for both the isolated (Ysubunit and for the holoenzyme, with approximately 80% of these antibodies directed to the holoenzyme sites. These results support our original conclusion that there are few changes in the antigenic sites of the (Ysubunit as a result of its isolation and delipidation, and that most of the antigenic sites of the (Y subunit are exposed in the membrane-embedded holoenzyme. This could suggest that there is little change in the conformational state of the (Ysubunit upon isolation or, more likely, that most of the antigenic sites of the (Ysubunit are exposed primary amino acid sequences and that the expected conformational changes in the protein do not alter antibody binding to these sequences. In contrast, although the p subunit is highly antigenic, few of the antigenic sites of the denatured protein can be detected in the holoenzyme. Since our previous studies with the holoenzyme-directed antibodies showed that about 60% of those P-subunit antigenic sites remained unaltered by its denaturation, it appears that a substantial number of antigenic sites that are expressed on the denatured p subunit are either se-

OF

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379

questered within the membrane bilayer, hidden by its association with the (Y subunit, or masked by its carbohydrates. These sites, when exposed, are substantially more antigenic than those which are exposed on the holoenzyme. Clearly, the antibodies raised to the isolated @subunit cannot be used to ascertain possible functional roles for the subunit as a component of the holoenzyme since they do not bind to it. An important problem with the previous studies using antibodies directed against the isolated subunits has been that demonstrations of the specificities of the antibodies and their ability to bind to the holoenzyme have not been reported. Therefore, meaningful interpretations of the results of their effects on enzyme functions are not possible. In previous studies, a-subunit-directed antibodies have been found to either inhibit enzyme activity (10,ll) or to have no effect (13, 31). Similarly, P-subunit-directed antibodies have been found either to inhibit enzyme activity (11) or to have no effect (10, 32). Our present study suggests that, although antibodies raised to the (Ysubunit bind to the holoenzyme and affect enzyme function, there appears to be little chance of generating antibodies to the isolated 6 subunit which will bind functionally important holoenzyme sites. One explanation for the results of Rhee and Hokin (ll), who observed inhibition of the Na+,K+-ATPase activity by P-directed antibodies, is that the antigen was contaminated with some (Y subunit. The very sensitive indirect binding assay we used, however, reveals a low-affinity cross-reactivity for the (Y- and B-subunit-directed antibodies that is not observed using agar immunoprecipitation techniques. The simplest explanation for this cross-reactivity is that there is contamination of each subunit by the other. The low affinities of these antibodies could then result from the low concentration of the contaminant making it a poor antigen. It would be possible for the purified @subunit to contain fragments of the larger (Y subunit which might comigrate with it and, therefore, not be apparent on SDS-poly-

380

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acrylamide gels. It is more difficult to envision a contaminating fragment of the /3 subunit which comigrates with the considerably higher-molecular-weight (Y subunit. Indeed, contrary to what might be expected if the cross-reactivity were due to contamination, it is the antiserum fractions to the (Y subunit which show the stronger cross-reactivity. Alternatively, the results of our competition binding studies could indicate that there are some partially homologous antigenic sites on the two subunits. ACKNOWLEDGMENTS We thank Diane Parry and Chuck Loftice for their excellent technical assistance. This work was supported by a grant from the Southwestern Ohio Chapter of the American Heart Association and by NIH Grants R23-HL-24941 (W.J.B.), ROl-AM-20875 (J.H.C.), ROl-HL-25545 (L.K.L.), and POl-HL-2261905 (A.S.). REFERENCES 1. SCHWARTZ, A., LINDENMAYER, G. E., AND ALLEN, J. C. (1975) Pharm Rev. 27,3-85. 2. GLYNN, I. M., AND KARLISH, S. J. D. (1975) Anna Rev. Ph.@oL 37.13~55,397-419. 3. WALLICK, E. T., LANE, L. K., AND SCHWARTZ, A. (1979) Annu Rev. Physiol 37, 13-55. 4. FORBUSH, B., KAPLAN, J. H., AND HOFFMAN, J. F. (1978) Biochemistry 17.3667-3676. 5. REEVES, A. S., COLLINS, J. H., AND SCHWARTZ, A. (1980) Biochern Biophys Res. Commun 95, 1591-1598. 6. ROGERS, T. B., AND LA~IXJNSKI, M. (1979) FEBS Lett S&373-376. 7. COLLINS, J. H., FORBUSH, B., III, LANE, L. K., LIC, E., SCHWARTZ, A., AND ZOT (REEVES), A. (1982) B&him Biophys. Acta 686,7-12. 8. AVERDUCK, R., GUNTHER, T., DORN, F., AND ZIMMERMAN, U. (1969) 2. Naturfosch. 24B. 693698. 9. ASKARI, A. (1974) Ann N. Y: Ad Sci 242,372388. 10. JEAN, D. H., AND ALBERS, R. W. (1977) J. Bid them 262.2450-2451. 11. RHEE, H. M., AND HOKIN, L. E. (1975) Biochem Biophgs Rea Commun 63,1139-1145.

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