Surgar transport in animal cells: The passive hexose transfer system

ProO, Biophys. molec. Biol., MOll43, pp. 33~i9, 1984. Printed in Great Britain.All rightsreserved.

0079~i107/84 $0.00+.50 Copyright © 1984Pergamon Press Ltd

SUGAR TRANSPORT IN ANIMAL CELLS: THE PASSIVE HEXOSE TRANSFER SYSTEM A. CARRUTHERS Department of Biochemistry, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, Massachusetts 01605, U.S.A.

CONTENTS L INTRODUCTION

II.

BASIC FEATURES OF THE PASSIVE HEXOSE TRANSFER SYSTEM

1. 2. 3. 4. 5. 6. 7.

III.

33

General Transfer is Facilitated Transfer is Passive Transfer is Saturable Competition between Sugars for Transport Inhibitor Studies Conclusions

34 34 34 34 35 36 37 37 38 38 38 39 39 40 43 44 44 45 47

THE KINETICS OF HEXOSE TRANSFER

I. General 2. Enzyme and Transport Kinetics 3. Experimental Tests of Kinetic Schemes (a) Procedures (b) Interpretation of V=.~ and K~ values (c) Testing the transport model 4. Other Transport Models (a) Kinetics (b) Testing the simultaneous model 5. Conclusions IV. ISOLATION AND CHARACTERIZATIONOF THE HEXOSE TRANSPORTER

1. Identification of the Transporter (a) Modification of membrane protein composition (b) Binding studies 2. Isolation and Purification of the Transporter 3. Reconstitution of Transport Activity 4. Conclusions V. HEXOSE TRANSFER MODULATION AND REGULATION

1. Interactions Between the Sugar Transporter and its Lipid Environment 2. Hexose Transfer Regulation in Carbohydrate Homeostasis (a) The origin of functional carrier in adipose and muscle (b) The signals controlling hexose transfer in adipose and muscle (c) Hexose transfer regulation in other tissues 3. Conclusions ACKNOWLEDGEMENTS REFERENCES NOTE ADDED IN PROOF

47 47 48 48 50 51 52 53 53 56 57 58 62 64 65 65 69

I. I N T R O D U C T I O N Studies of the passive hexose transfer system of animal cells have progressed, in recent years, from phenomenological studies on whole cells to studies which begin to address the molecular basis of transmembrane hexose flux and its regulation by metabolic and hormonal factors. These studies include investigations of m e m b r a n e protein/lipid interaction and the mechanisms of m e m b r a n e transport, m e m b r a n e receptor function and transmembrane signaling. Our ability to ask these questions arises from the development of experimental procedures 33 JPB ~3:1-C

34

A. CARRUTHERS

which permit the identification and subsequent solubilization, purification and reconstitution of various membrane proteins into synthetic lipid membranes. These procedures are not without problems, however, and their utility is dependent on their ability to reconstitute transport systems into synthetic membranes in such a way as to preserve or reproduce native function. This paper will describe the various properties of the native and reconstituted passive hexose transfer system and, in addition, will consider the regulation of hexose transfer by metabolic and hormonal factors. The active, cation-dependent hexose transfer system typical of epithelial cells has been reviewed elsewhere (Crane, 1977).

II. BASIC FEATURES OF THE PASSIVE HEXOSE TRANSFER SYSTEM 1. General

Hexose transfer in eukaryotic cells is characterized by the passive, rapid entry of specific sugars into the cytosol from the external milieu. This process follows saturation kinetics and shows competitive inhibition of the rate of net transport between different sugars and by relatively low concentrations of phloretin or cytochalasin B. These features indicate that during the process of hexose transfer, sugars interact with a limited number of membrane sites. These sites are commonly referred to as "carriers" (Widdas, 1952). 2. Transfer is Facilitated Certain sugars penetrate cells at rates several orders of magnitude faster than would be predicted for the free diffusion of sugars through the lipid bilayer. For example, the estimated permeability coefficient for the transbilayer flux of D-glucose in protein-free artificial lipid bilayers is in the order of 2-4 x 10 - l ° cm sec -~ (Jung, 1971a; Lidgard and Jones, 1975; Carruthers and Melchior, 1983b). The permeability coefficients for D-glucose flux in animal cells, however, range from 5 x 10- s cm sec- 1 in frog sartorius muscle (Narahara and Ozand, 1963; see Carruthers, 1983) to 2 x 10-5 cm sec-1 in the human erythrocyte (Jung, 1971b). Other sugars, such as mannitol and L-glucose, penetrate cells almost as slowly as D-glucose transverses protein-fr~, artificial membranes. These findings are consistent with the view that sugar transport in animal cells is mediated by membrane components (presumably proteins; see Section IV) which catalyse, in a selective fashion, the rapid transbilayer flux of sugar. 3. Transfer is Passive

Hexose transfer in animal cells may be monitored using a variety of methodologies. These include measuring the loss or uptake of radiolabeled sugars by cells or using direct (Harris, 1963) or indirect (Taverna and Langdon, 1973) biochemical techniques to measure the total amount of sugar inside the cell (Widdas, 1980). Each of these methods has shown that both influx and efflux of sugar occur simultaneously. These are unidirectional sugar fluxes. The magnitude and direction of the net sugar flux is obtained by subtracting unidirectional fluxes (see Fig. 1). It was apparent early in studies with human erythrocytes (Bang and Orskov, 1937; Wilbrandt et al., 1947; Widdas, 1952) that the direction of net sugar transport was always from high to low sugar concentration. On injection of sugar-poor red cells into sugarrich medium, net sugar influx would proceed until the cytosolic sugar concentration was identical to that of the external milieu and unidirectional sugar influx and efflux were balanced. Thereafter no further net sugar flux would be observed. This behavior is common to all cells transporting sugars by the passive mechanism, although in some ceils, appreciable intrac~llular sugar metabolism may maintain very low cytosolic sugar concentrations and equilibrium may not be achieved. In such a case, a non-metabolizable but transported analogue of o-glucose must be used to allow transmembrane hexose equilibration (Narahara and Ozand, 1963; Craik and Elliot, 1979; Baker and Carruthers, 198 la). As this net flux from high to low sugar concentration resembles simple diffusion, the mechanism of hexose transport is often termed mediated or facilitated diffusion.

Sugar transport in animal cells: the passive hexose transfer system

35

bihlyer

ECF

CYTOSOL

s4

,0!ux0 ~
4. Transfer is Saturable

Hexose transfer in most animal cells is more complex than simple diffusion. The relationship between sugar flux and sugar concentration is linear only at very low sugar concentrations and can be seen to saturate at higher sugar levels (Le Fevre, 1948; Widdas, 1952; Elbrink and Bihler, 1975; Naftalin and Holman, 1977; Widdas, 1980). Figure 2 shows this relationship. Such kinetics (Michaelis-Menten) are characterized by two constants: Km and Vmax.I'm,, is the maximum rate of catalysis (in this case, transmembrane sugar flux) and Km is the substrate concentration where the rate offlux is 1/2 V,,a.. Vm~ is related to both the number of transporters and the translocational activity of individual transfer proteins. K,, is, indirectly, a measure of the affinity of the transporter for its substrate; the lower the Kin, the higher the affinity. Km and Vm,~values for sugar transport are sugar-specific in a single cell type and may vary from cell to cell for a single sugar.

Vm

X

:, Vrn U.

S i

Km CONCENTRATION FIO. 2. Saturation kinetics. Flux of sugar is expressed as a function of sugar concentration and takes the form, flux

V=

v®s K~+S

V,. is the maximum flux at saturating sugar levels and Km is that concentration of sugar producing a flux of 1/2 Vm.

36

A. CARRUTHERS 5. Competition between Sugars for Transport

The value of studies of competition between sugars for transport sites is two-fold. (1) Such studies indicate that during the process of transport, sugars interact with a limited number of transport sites, (2) Information regarding the stereochemical requirements for transport are obtained and, from this, attempts can be made to model the sugar binding sites of the transporter. This approach is not without difficulties (Rees and Holman, 1981). First, binding to the transport system and sugar translocation through the transporter may be a substrate-induced phenomenon. Thus, if more than one site on the sugar molecule is important for high affinity binding or transport it is not possible to determine whether this reflects the simultaneous, stereochemical requirements at the primary recognition site or sequential requirements at more distal sites as the sugar moves through the transporter. Second, changes in hydrogen bonding of hexose or protein to water may be important at some stages of transport and this may also affect the apparent affinity constants. In general, the possible hydration of substrate or transporter and its contribution to binding has not been considered. The most systematic analyses of the stereochemical requirements of hexose transfer systems have been carried out by Barnett et al. (1975) and Rees and Holman (1981). These studies examined the requirements of the human erythrocyte and rat adipocyte hexose-transfer systems respectively. Earlier studies had shown that L-glucose, raffinose and mannitol are not transported to any significant extent by most sugar transporting cells (Fisher and Lindsay, 1956; Battaglia and Randle, 1960) whereas o-glucose, 3-O-methylglucose and 2-deoxy-D-glucose are transported rapidly (Kipnish and Parish, 1965; Kohn and Clausen, 1971). These observations indicated that the transport system shows a high degree of specificity for the o-stereoisomers of hexose sugars in the pyranose ring form. Barnett et al. (1975) found that 6-O-alkyl derivatives of galactose and glucose inhibited the erythrocyte glucose transport system when in the outside medium but were ineffective when inside the cells. On the other hand, propyl-fl-D-glucopyranoside was an effective inhibitor from the interior but not the exterior of cells. Barnett et al. (1973) have also examined the effects of a number of derivatives of glucose and other hexoses on hexose-transfer. Replacement of hydroxyl groups with hydrogen alters the affinity of the transporter for sugar in a number of ways. An increase in affinity was observed with C-2 hydroxyl replacement but a decrease in affinity with C-3 replacement. Bonding of sugar with the transfer site was assumed to involve hydroxyls at C-I, C-3 and C-4 with some hydrophobic interactions involved at C-6. Barnett et al. (1975) have proposed that the glucose molecule approaching from the extracellular milieu, enters the reactive transport site with the C-1 end of the pyranose ring leading. The converse is predicted for sugar exit from cytosol to blood with the C-4, C-6 end of the molecule entering the transport site first (see Fig. 3).

R0

H0

outside

inside

FIG.3. Proposed modelfor sugartransport in humanerythrocytes(Barnettet al., 1975).The sugar, 6-O-propyl-D-glucose(R-- C3H7;R'-- H) can bindto the E2conformerofthe transporterbut cannot be transportedfor stericreasons.Propyl-fl-D-glucopyranoside(R= H; R'= C3H7)can bindto the E1 conformer of the transporter but is not translocated.D-Glucosebinds to both conformersand is translocatedby the conformationalchangeESI~.-~-ES2.(ModifiedfromBarnettet al., 1975;Widdas, 1980).

Sugar transport in animal cells: the passive hexose transfer system

37

In contrast, the insulin-activated adipocyte hexose-transfer system shows only a weak requirement for hydrogen-bonding at the C-4 and C-6 positions but strong requirements for hydrogen bonding at the ring oxygen, the fl-position at C-1, a hydroxyl in the equatorial position at C-3 and, to a lesser extent, a hydroxyl at C-6 (Rees and Holman, 1981). These studies showing significant differences in the stereochemical requirements of erythrocyte and adipocyte hexose transfer, have not yet been applied to other call types or their purified, reconstituted transport.

6. Inhibitor Studies

Passive hexose transfer is inhibited competitively in most cells by very low concentrations ofphloretin and cytochalasin B. These observations reinforce the view that a limited number of membrane sites are involved in hexose transfer. There is now a large body of data indicating that cytochalasin B acts at or near the binding site at the endofacial surface of the human erythrocyte bilayer (Widdas, 1980). Cytochalasin B also appears to act at the endofacial binding site in the rat adipocyte and avian erythrocyte hexose transfer systems (Czech, personal communication; Simons, 1983a). Phloretin, however, acts at the external sugar binding site of the erythrocyte (Krupka and Deves, 1981). These findings support the view that intracellular and extracellular sugar binding site stereochemistry may be markedly different yet the apparent affinity of each site for transport substrate may not differ. The model of Barnett et al. (1975, see above) can accommodate these observations readily. Cytochalasin B has become a powerful pharmacological tool in the isolation of the transporter and in the study of transport regulation by insulin and other agents (see Sections IV and V).

7. Conclusions

Hexose transfer in animal cells is mediated by a facilitated, passive, selective, saturable mechanism inhibitable by very low (< 1/zM) levels of cytochalasin B or phloretin. The last three features of this system establish that there are a limited number of sugar transport sites on each cell. The number of these sites can be estimated using two approaches. (1) By determination of the number of D-glucose-displaceable cytochalasin B binding sites on the membrane. (2) An indirect kinetic method. Table 1 shows the results of such determinations in a number of cell types. These methods will be discussed in more detail below but it must be emphasized that each is not without difficulties and the results must be taken to indicate only the range of transport sites.

TABLE 1. HEXOSE-TRANSFER PROTEIN NUMBERS IN VARIOUS CELL TYPES N / z m -2

Tissue H u m a n erythrocyte Frog sartorius muscle Barnacle muscle Isolated adipocyte Isolated hepatocyte Pigeon erythrocyte Giant nerve fibre of

Loligo

Kinetic 6x 1x 1.6 x 6.7 x 4× 3.2 x 5x 4×

104 104 104 10 2 (control) 10 3 (insulin-stimulated) 104 10 3 10 3

D-Glucose-sensitive cytochalsin-binding 1.9 x 10 3 -6 x 10 5 4 x 10 3 2.7 x 104 -not measurable

Where both kinetic and cytochalasin B binding studies have been made, the disparity between results is striking. O f particular interest is the carrier-density of the adipocyte assessed by cytochalasin B-binding. This is greater t h a n that of the highly permeable h u m a n erythrocyte.

38

A. CARRUTHERS III. THE KINETICS OF HEXOSE TRANSFER

1. General This section is intended as a simple introduction to the methodologies used in determining the kinetics of hexose transfer in any cell. The reader is directed step by step from theoretical analysis to the practical determination of meaningful parameters for transfer. Although by no means comprehensive, this section contains sufficient information for the resolution of most hexose transfer systems. The kinetic approach is to define simply and precisely the operational characteristics of transport as a series of kinetic formalisms. These formalisms may then provide the basis for a more comprehensive understanding of the process of transfer. However, the reader must exercise caution in accepting physical interpretations of kinetic schema. Most models imply some degree of physical transformation of the transporter during hexose translocation in spite of the fact that most kinetic schema ignore any such changes. The alternative approach has been to construct physical models whose kinetic properties mimic the operational properties of the transporter. Although such models may be successful functionally, they remain difficult to verify. This does not reduce the general utility of the kinetic approach, however, for it is clearly advantageous to be able to describe and predict accurately the behavior of the transfer system. Present practical and theoretical techniques used in the study of transport systems are derived for the most part from earlier studies of enzyme kinetics. This has resulted in the considerable simplification of transport data analysis and avoids the use of a variety of simplifying approximations. The reader is recommended the reviews by Lieb and Stein (1974), Stein and Lieb (1974) and Lieb (1982).

2. Enzyme and Transport Kinetics Lieb and Stein (1974) have demonstrated the equivalence of enzyme and transport kinetics. Take the simplest kinetic scheme for the conversion of substrate S into product P by an enzyme, E. E + S~.-~-ES~.-~-E+ P

(1)

The simplest scheme for the transport of a permeant substrate in solution 1 (S 1) across a membrane to solution 2 ($2) by a transport protein E is also of the form. E + S I~.-~-ES~.-~-E+ $2

(2)

These two schemes are kinetically equivalent although physically different. In the former scheme, the molecule S is converted to product P whereas in the latter scheme, the permeant substrate is unchanged but has been transfered from solution 1 to solution 2. This scheme describes transport by a simple pore--a continuous passage through the membrane accessible simultaneously at both sides of the membrane but capable of binding only one molecule at a time. A more complex kinetic scheme for the conversion of substrate S into product P by an enzyme is E~ + S,-~-ES..~-E2+ P EI~,-~-E2

(3)

Here the enzyme can exist in two isomeric forms, Et and E2. Conversion of substrate into product by ES results in the conformational change of E1 to E 2. Before further substrate can be processed by the enzyme, E 2 must revert to Ex. The transport scheme analogous to this enzyme system is El + S 1,~ES~,~E2 + $2 EI~,-~-E2

(4)

Sugar transport in animal cells: the passive hexose transfer system

39

Transporter protein may exist in two conformational states El and E 2. E 1 may bind sugar in solution 1 only and E2 in solution 2. This results in the formation of ES which undergoes conformational change to release S into the opposite, trans solution and to form the opposite conformer of E. Net movement of S from one solution to the other may proceed only if the two free conformations E1 and E2 revert spontaneously. Lieb and Stein (1974) and Stein and Lieb (1974) have shown that the addition of further intermediate steps to each type of scheme does not result in kinetically different solutions. Indeed it is kinetically impossible to distinguish between a scheme such as (4) or a similar scheme with two or more intermediate states (ES', E S " . . . ) . This is illustrated concisely by Lieb (1982) and is implicit in the use of steady state methods for the resolution of enzyme kinetics (Cleland, 1963). Figure 4 and Table 2 demonstrate this point.

A

El

f

'

E2

c

aS1~

b~

52

ES outside

inside

B

EI .

g

II

E2

a 51 b

ES1.

e f S2 C

'

d

ES2

FIG.4. Simplecarrier modelsfor hexosetransfer.(A) One-complexsimplecarrier. E is the emptyform of the carrierexposedat either side I or 2 ofthe membrane.Sugar,S, may bind to El or E,. a-fare rate constants for the appropriate reactions. (B) Two-complex simple carrier model. This is the conventional carrier model. T A B L E 2. S T E A D Y - S T A T E S O L U T I O N S F O R O N E - A N D T w o - C O M P L E X F O R M S OF SIMPLE C A R R I E R

K 2 1 S 1 -t- S I S 2 V142

K12K 21Roo+ K21R12S1 + K12R21S2+ R©eSIS2 w h e r e Re, = R 12 -}- R21 -- Roo a n d

One complex I 1 n R12=~+~ n R21=~

1_}_1

i

Z 1

n Roo=r+~

1 1 n Ree=~+'B

r,~=~-({+~) K 21 -- - ~da +te~ ) Constraint abe = def

Two complexes ~+~i+~I e+d

I1

1

I;-t-~+~ 1 I

b+¢

i+]/

1 1 1 e+~ 1 b+c "8-I-¥-F'~ • + i l ~

~ (-~+~'+J ) ~ at~e ' !d +~!~_~ d ,' aceh = bdfg

3. Experimental Tests of Kinetic Schemes (a) Procedures Table 3 summarizes the procedures and nomenclature for the determination of the kinetic constants of the transport system. With influx, the cis solution is the extracellular solution and the trans solution is the intracellular solution. The converse is true for efflux. Flux of

40

A. CARRUTHERS TABLE 3. KINETIC PARAMETERSFOR 3-O-METHYGLUCOSETRANSPORT IN THE INTERNALLY DIALYZEDSQUID GIANTAxoN (FROMBAKERAND CARRUTHERS, 1981b)

Type of experiment

Conditions Blood[sugar] Axoplasm[sugar]

Zero-trans fluxes Uptake

Exit

goro

Characterizedby

Measuredvalues?

Ks uptake (K~t.1) Vm~ uptake (1~2t.1) Km exit (K~'~2) Vm,, exit (~'~2)

1.34 mM 1.99 pmol.cm-2"sec -l 5.5 mM 8.6 pmol-cm-2"sec -1

Ks external (K~.I) Vm,. exchange (V") K~, internal (K~.2) Vm~,exchange (V¢')

1.34 mM 1.99 pmol. cm- 2. sec- 1 5.5 mM 2.1 pmol. cm -2 .sec-1

Km internal (K~C~l) Vm,, net entry (W2'~l) K,, external (K~C~2) Vm,, net exit (V~I'.2)

6.0 mM 2.2 pmol"cm- 2. sec- 1 1.34 mM 8.6 pmol"cm- 2. see- 1

Ks exchange (Kee) Vma,exchange (V"e)

1.39 mu 2.0 pmol. cm. sec- 1

Infinite-trans fluxes

Uptake Exit Infinite-cis fluxes* Uptake

Exit Equilibrium-exchange Internal [sugar] = external [sugar] uptake and exit

* Inflnite-cis net fluxes were measured as the differencebetween unidirectional uptake and exit. ? Obtained by Lineweaver-Burk analysis of experimental data.

sugar is always measured in the direction cis to trans and is expressed as a function of either cis or trans sugar concentration. These basic procedures have been adopted (Lieb and Stein, 1972) to ensure the unequivocal determination of kinetic constants. F o r example, the infinite-trans procedure ensures that the trans-solution is always saturating and that variations in trans levels of sugar due to flux from cis to trans do not occur. In other words, the contribution of trans-sugar to the observable kinetic parameters will not vary with cis sugar levels. The maximal flux, Vmw and half-saturation concentration, Km, for a given procedure m a y be estimated in a number of ways. Most frequently, the initial rate of flux at a given hexose concentration is determined. With this method the time-course of uptake or efflux is measured at all sugar concentrations. If flux vs time is linear during the first n seconds then the initial rate of flux is obtained by measuring uptake or exit over a time interval of less than n seconds. These data may then be analyzed using a variety of methods. Most commonly, Lineweaver-Burk analysis is used where the reciprocal of flux is plotted as a function of the reciprocal of concentration. Regression analysis is used to determine the line of best fit to the data and the obtained y-intercept is the reciprocal of Vm,, and the x-intercept the reciprocal of Kin. This type of analysis suffers from the amplification of errors at low concentrations of sugar. This and alternative analytical procedures are shown in Table 4. Rather than measuring initial transfer rates, an alternative approach is to follow the time course of sugar uptake or loss then to determine Vmaz and K m using the integrated rate equation approach (Hankin et al., 1972; Baker and Naftalin, 1979; see Fig. 6). Sugar transport experiments have been performed using a variety of analytical procedures, but the equilibrium exchange procedure (where unidirectional fluxes are identical) and infinite-trans procedure must be examined using radiolabeled sugars for no net cis to trans flux will be observed. (b) Interpretation o f V ~ x and K,. values Table 5 summarizes the interpretation of Vm~, and Km values in terms of the basic observable parameters for the simple carrier. This table permits us to calculate the various

Sugar transport in animal cells: the passive hexose transfer system TABLE 4. ANALYSIS OF INITIAL RATE DATA

Type of plot

Lineweaver-Burk 1

K m

1

y-intercept

x-intercept

Slope

(1/v vs 1/S) 1

. . . . X -- + V Vmax S Vmax

1

- 1

Vmax

Km

Km/Vmax

Eadie

(S/v vs S) Km 1 --×S S/1)=Vmax+--~ Vmax nofstee (v vs v/S) 13 V= Vmax - ~

Km

-K m

Vmax

Km

[/max

Vmadgra

1 Vmax

Km

Eisemhal Cornish-Bowden Fro= Km 1 V S Hanes (1932) has drawn attention to the statistical dangers attending any linear transformation of the Michaelis-Menten equation. Of these methods, the Lineweaver-Burk function is the most widely used and least satisfactory. Merit judgements on the three methods are based o~a a number of desirable features such as I'm, and K= in numerator, S in the numerator and the dependent variable on only one side of the equation. The Eadie Plot is the only function satisfying two of these requirements and is preferred by some workers. The Hofstee Plot, however, has no reciprocals of Vm~ and Km. This is considered by some sufficient to warrant the use of this function. The most satisfactory analytical approach is that described by Eisenthal and Comish-Bowden (1974). For each observation of V at S there exists a straight line in Vma~Km space with intercepts - S on the K m axis and V on the Vm~ axis. This line relates all values of Vm~ and K m that satisfy the Eisenthal-Cornish-Bowden equation exactly for the particular Values of V and S.it follows that the coordinates of the point where the lines intersect provide the only values of Vm~ and K m satisfying the Eisenthal--Cornish-Bowden equation for each observation. Figure 5 illustrates such an analysis of the inhibition of sugar exit in the human erythrocyte by phloretin. Here, where observations may be subject to error, it may be questioned whether one is justified to give equal weight to all intersections in determining the best fit values Vm,~ and K m since they are not all equally precise. In practice, excellent results are obtained without weighting for the median of a sample, unlike the mean, is not greatly affected by weighting (Bowley, 1928; Eisenthal and Cornish-Bowden, 1974).

Imax

100

(~%)

IX UJ Z ,.J 14.

0

Y

-10

-8

-6

-4

-2

, ~Ki (o.si,m

0

-p.LOSErl'l FIG. 5. An Eisenthal-Cornish-Bowden plot of the inhibition of D-glucose efflux from the human erythrocyte by phloretin. Ordinate (inhibition ~), abscissa, phloretin concentration ~M). Data are plotted as - [phloretin'l on the abscissa and inhibition on the ordinate. The points are connected and extended into positive [phloretin] space. This is repeated for all points. The point of intersection of the lines provides the Ki and lm~ (maximum inhibition) for inhibition of exit by phloretin. (Carruthers and Melchior, unpublished).

41

42

A. CARRUTI-IERS

A exit

\

entry I

10mY lmin

B

0'051 I

0,3

~_T

"t

v-

/

0 r/tSo-St)0,01

/

)

6O

S/T

FIG. 6. Integrated rate equation procedure for determination of hexose-transfer kinetic parameters. (A) Time-course of sugar exit and entry determined by Turbidimetry. Red Cell ghosts were loaded with 50mM D-glucose and injected (0.5/~1) into 400/d of isotonic sugar-free solution for exit determinations. With influx measurements, 0.5/~1 of D-glucose free ghosts were injected into solution containing 100 mM D-glucose in osmotic excess to the cells. (Carruthers and Melchior, unpublished). (B). Tranformation of the time-course data to yield the kinetic constants for transfer. Ef/tux. The integrated rate equation of Karlish et al. (1976) in the form suggested by Baker and Naftalin (1979) was used as _

In St~So V~.2 = _ _

SO--S,

t K ~ 2 (So-S,)

1 1 K~'~z P

- - . +

where SOis the quantity of sugar in 1 I. of cells at zero-time and S, is the amount contained in the cell at time t. P is the osmolality of the internal and external osmotically-active membrane impermeant species (300 mosm). - l n S~/So/So-S,) is PlOtted as a function of t/(So-S,). The slope, = VZlt.2/gzll~2 =9.18 ___2.00 and the intercept= - 1/K~ z + 1/P= -0.0399 +0.0144. It follows therefore that K~'.z = 27.3_+9.9 mM and V~t.2 = 250_+ 24 mmol l-1 rain-1. Influx. The integrated rate equation of Hankin et al., (1972) was used as

In(I+N/P) K~2c.~+P+So N V2~1K2~ i© t P(P+So) t P(P+So) ln(l + N/P)/t is plotted as a function of N/t where N is the amount of sugar present in the cells at time t. So is the external sugar concentration. These data give a slope (K~2"~1+P+So)/(P(P+So)) of 0.00515_+0.00012 and y intercept (-V~t~t .Ki2¢~t/P(P+So)) of -0.07_+0.001 P = 198 mOsmol/1. K~,C~t = 5.41 _+0.91 mM; V~*.1= 77.8 __4.1 mmol- l min - I.

r e s i s t a n c e t e r m s ( R t 2 , R21, Roe, R,=) a n d affinity t e r m s ( g 1 2 , K21 ) for b o t h t y p e s o f m o d e l s y s t e m ( o n e - a n d t w o - c o m p l e x ) . If t h e r e s i s t a n c e t e r m R is m u l t i p l i e d b y n (the c o n c e n t r a t i o n of total transporters) the resultant has units of time and represents the turnover time or total t i m e r e q u i r e d for a t r a n s p o r t e r t o c o m p l e t e a single t r a n s p o r t cycle u n d e r t h e a p p r o p r i a t e experimental conditions. A l t e r n a t i v e l y w e c a n e s t i m a t e n f r o m m e m b r a n e g l u c o s e p e r m e a b i l i t y , P. P = Vm~/Km ( C a r r u t h e r s , 1983) a n d t h e t u r n o v e r n u m b e r T , = d / P w h e r e d - - m e m b r a n e thickness. It

Sugar transport in animal cells: the passive hexose transfer system

43

TABLE 5. INTERPRETATIONOF EXPERIMENTALDATA IN TERMSOF THE OBSERVABLEPARAMETERSFOR THE SIMPLECARRIER

Procedure zero-trans

Vm~ ~-2 = ~ ~L

Km

1

Kr-2 =

1

K21Roo R21

1 = -"

K I2R21 Ree K21R12 R., K21R12 Kilc-2 = - -

K~ t- x = -

K~t~2 =

1 R~ l'qc

1~2

RI 2

Vi~l = - -1

Re,

K~C.1= _K 1 2_R 2 1

R21

equilibrium-exchange

Roo

R,~--f--

R21

1= - -

infinite-tram

infinite-cis

K 12

V"°=

1

--

R,~

K12Roo

K~% 2 = -

-

K21Roo K~U1= - Ree

follows, therefore, that n = Vm,~ (molecules/cm2/s) Tn. here we assume that one molecule of hexose reacts with one transporter. We further assume a value for d. (c) Testing the transport m o d e l Normally one would begin with the simplest transport model then proceed to more complex models if, and only if, the simplest model must be rejected. The simplest model (the simple pore) predicts that the presence of sugar in the t r a n s - s o l u t i o n will inhibit flux from cis to trans. As this phenomenon is almost never observed for sugar transport systems (but see Baker and Carruthers, 1981a), this model must be rejected. In the human erythrocyte trans sugars accelerate flux from cis to trans (Levine et al., 1965). Sugar efflux (but not influx) in the squid axon is inhibited by trans sugar (Baker and Carruthers, 1981b, 1984). Previously (Lieb and Stein, 1974), the demonstration of acceleration of sugar flux from cis- to t r a n s - s o l u t i o n s by trans-sugar had been used as one criterion for acceptance of the simple cartier model. Although such a phenomenon is entirely consistent with the simple carrier, it has been described only for hexose transfer in the human red cell. With most other hexose transfer systems (adipocyte, hepatocyte, muscle, rabbit red cell, avian erythrocyte; Simons, 1983a) trans-sugar is without effect on sugar flux from cis to trans solution. Furthermore, Vm,~ and K m for zero-trans sugar entry in these cells are normally identical to Vmaxand Km for exit. Such systems are symmetrical and are readily compatible with the simple carrier model for transport. Asymmetry of sugar transport has been described in the human red cell (for review see Widdas, 1980), the frog sartorius muscle (Narahara and Ozand, 1963) and the squid giant axon (Baker and Carruthers, 1981b). Here the Km and Vm~ for zero trans entry are smaller than the corresponding values for exit. The squid axon hexose transfer system appears to be unequivocally asymmetric. Hankin et al. (1972) have developed unambiguous rejection criteria with which to test the asymmetric simple carrier and the data from the giant axon experiments satisfy these criteria (Carruthers, 1980; Baker and Carruthers, 1984). With frog sartorius, insufficient data exist to test these rejection criteria rigorously. Nevertheless, the simple cartier hypothesis requires that Kiam/K~t --- Vimaax/V°ut. This condition is not satisfied in frog sartorius (Narahara and Ozand, 1963). It is possible that this arises from experimental problems associated with flux determinations in intact muscle groups. Until this is verified this finding is sufficient to reject the asymmetric simple cartier model for hexose transfer in frog muscle. Intact red cell data are also incompatible with the simple asymmetric carrier model for the asymmetry criteria

44

A. CARRUTHERS

are not satisfied (Hankin et al,, 1972). Moreover, numerous workers have demonstrated the operational presence of two sugar binding sites at the inner surface of the bilayer (Naftalin and Holman, 1977; Widdas, 1980). Carruthers and Melchoir (1983a) have shown that these findings arise from the complex interaction of the transporter with cytosolic modulatory factors (perhaps ATP; see Jacquez, 1983) and that erythryocyte hexose transfer is in fact symmetric. Nevertheless, the simple carrier model could not account for their experimental findings. 4. Other Models for Hexose Transfer (a) Kinetics The compatibility of hexose transfer data with one type of transport model does not constitute proof that hexose transfer is mediated by such a mechanism. It may be possible to derive fundamentally different kinetic schema which describe the operational characteristics of the system just as readily. Here we will consider a second class of transport model, the simultaneous or linear system. The simple carrier model describes a mechanism in which the sugar binding site of a transport protein may be present alternately at either the cis or trans side of the bilayer, but never at both sides simultaneously. With the simultaneous or linear model, the transporter contains two sugar binding sites; one at each end of the molecule (Fig. 7). Hence, the

inside

outside

$I

FIG.7. The simultaneous,linear carrier model.The carrier, shown here in a highlyschematicform, consistsoftwo halves,eachofwbichcan bind sugarwitha characteristicdissociationconstant.When sugar binds, a conformationchange occurs permittingsugar to exchangebetweeneach hall'of the carrier in a central, intramembranous"pool". The carrier then undergoes further eonformational change releasingthe sugar at the opposite side of the membrane.

transporter may have two binding sites exposed simultaneously at opposite sides of the membrane. Such a system may have four possible states: cis and trans-sites unoccupied, cis-site occupied by sugar and trans-site vacant, cis-site vacant and trans-site occupied, both cis and trans-sites occupied. Transport may proceed in the last three conditions. When sugar is absent from the extracellular fluid, efflux of sugar is proportional to Fx~.si" kl

(1)

where Fx. s~is the fractional occupation of internal sites by intracellular sugar and kl is a rate constant proportional to the rate of hexose transfer across the membrane. F~i.s~may be

Sugar transport in animal cells: the passivehexosetransfersystem

45

expressed in the usual way for enzyme type reactions and for a single sugar takes the form [Si]

=

1

fx~'s'= [ S i ] + K i

I+(K][SJ)

(2)

where Ki is the half-saturation constant for the internal sugar binding site. Efltux of sugar may therefore be given by 1

V,. o -----(-1 _k(KTff[Si])) • V~_.o

(3)

where V~..o is Vm~,for exit and is proportional to kx multiplied by the total number of carriers in the membrane. When sugar is present at both sides of the membrane, both eis and trans sites may be occupied and the unidirectional flux mediated by these carriers will be proportional to Fx,.s~'XoSo"kC

(4)

where F=i. s~. Xo.Sois the fraction of total carrier with cis and trans-sites occupied and k¢ is a rate constant, proportional to the rate of hexose transfer through a doubly occupied carrier. Efflux through the remaining carrier may be described as Fx~.s~ (1 - Fxo.So)-kl

(5)

Total unidirectional exit is therefore given by Fxi .si " (1-F~o .So)" kl + Fx~.sCXo.So • k ~

(6)

which in full is 1

~'/i-+0w~"{(1 -~-(K.t/l'Si])) (

1

+ {(1+ (1][Si]))(1

1

1.-{-(K-of['So'))} l V~t~o

(7)

1 -'~V'* + (Ko/[So]) ] )

where Ko is the half-saturation constant for the external binding site, i and o refer to intraeellular and extracellular and 1,'e is proportional to kc multiplied by the carrier concentration. ~L~, Ve', K~ and Ko are determined by the procedures outlined in Table 4. Unidirectional influx is obtained by interchanging i~-+o. Two important simplifying assumptions made in this derivation are: (1) the binding site is in rapid equilibrium with sugar in its immediate surroundings, (2) this equilibrium is effected many times more rapidly than the translocation of hexose.

(b) Testing the simultaneous model In tissues where hexose transfer is compatible with the asymmetric or symmetric simple carrier hypothesis, it is a simple matter to show that the simultaneous model predicts readily the observed operational properties of transfer. Here we have a situation where two fundamentally different models are consistent with the available experimental data. This problem is not beyond resolution. Baker and Widdas (1973), Widdas (1980) and Krupka and Deves (1981) have developed simple carrier and simultaneous carrier kinetics which describe the effects of competitive inhibitors on transport and permit the experimental distinction between these two models. The basis of the test is that the inhibition of hexose transfer in a one site system by inhibitors present at both sides of the membrane is different to that expected of a two site system.

46

A. CARRUTHERS

With the simple carrier model, flux in the presence of two competitive inhibitors acting at opposite sides of the membrane is given by V--

(VZti • [S°]/K~t-'i) - (V,_.o " • [S~]/KfLo) 1 + ([Io]/K~)+ ([Li]/K[ t) + ([So]/K~ti) (1 + ([L0/K~0)+ ([Si]/Kf'o) (1 + ([Io]/Ki'o))

+ [s,] [So]

(8)

zt it K i l o go-~i

where V and K have the meanings described in Table 3 (Krupka and Deves, 1981). Io and L~ are the inhibitors present at the outside and inside of the cell respectively. Kp means that the half-saturation constant for inhibition by I is determined in a zero-trans experiment and K i t m e a n s that the half saturation constant for inhibitor I is determined in an infinite-trans experiment where the trans-solution contains saturating sugar levels• The relationship between the inhibitions produced by each inhibitor alone and that with both inhibitors present is V 1 (~---~oLi-)=

V

V

(~--~-1)+ (~i-1)

(9)

where Vis the flux rate in the absence of inhibitor and VIo and VLi the rate in the presence of inhibitors Io and Li respectively. VIoL~ is the rate in the presence of both inhibitors. Alternatively, (V---~io-1) =

[I°] +[Li] (KIo)app (KLi)app

(10)

where (KIo)app and (KL0app are defined as the inhibitor concentrations required to reduce the flux rate by half at a given sugar concentration. These equations apply to zero-trans entry and exit experiments. Table 6 shows the values of (KIo)app and (KLi)app expressed in terms of experimental parameters.

TABLE6. VALUESFog (KIo)app AND (KLi)app IN ZERO-TRANSEXPERIMENTS Inhibitor

Zero-trans entry

Zero-trans exit 1 + [S,]/KfLo

(Klo)app (KL0app

r ~ (1 + [SoJ/Ko~_,) zt l + [ So]/Ko-~ I/K~t + it zt , ([S~_I/KL, Ko.i)

(1/K~)+ ([S,]/K~ K~'~o) KL~ (1 + [S~]/Kft-.*)

The expression apply to both the carrier and simultaneous models for transport.

With the simultaneous model, eqn (8) may be rewritten to give v-

(rxjl+(ru]

/uJ

\ ,.o \ r r J /

(11)

Here both inhibitor sites exist simultaneously and the assumption is made that substrate or inhibitor dissociation is rapid with respect to the rate of substrate translocation. If we assume that *~ ~,-,t_ r,-i, _ r,-itL (addition of two inhibitors or sugar and inhibitor at opposite I o - - j ~ . 1o - - j ~ . I o sides of the membrane occurs without interference) we obtain

Sugar transport in animal cells: the passivehexose transfer system

47

and // V '~ [Io] ELi] [Io] [Li] ~,]~-~oLi1 = (KIo)app - + (KLi)app + (KIo)app (KLi)app

(13)

(KIo)app and (KLi)app, expressed in terms of operational parameters, are shown in Table 6. The experimental procedure is to use two inhibitors, one a competitive inhibitor at the external site only and the other a competitive inhibitor at the internal site. (KIo)app and (KL0app are determined in zero-trans experiments in the absence of the other inhibitor, then sugar flux is measured in the absence of inhibitor and in the presence of a mixture of the inhibitors (LJIo = (KLi)app/(KIo)app). This is repeated over a range of inhibitor concentrations (keeping L.flo constant) and V/(VLilo) is plotted as a function of inhibitor concentration. Using this approach (with phloretin and cytochalasin B as external and internal inhibitor respectively), Krupka and Deves (1981) have shown that in the human erythrocyte, V/(VLiIo) vs inhibitor concentration is best described by eqn (9), not eqn (12). Their conclusion is, therefore, that the human erythrocyte hexose transfer system does not have simultaneous cis and trans-binding sites but a single site which is exposed alternately at cis and trans-sides of the bilayer. Such an approach has not yet been applied to other hexose transfer systems.

5. Conclusions This section has described methods for the unambiguous determination of the kinetic parameters for the simple carrier model for transport. With a minimum of simplifying assumptions these parameters may also be determined for the simultaneous transport model. Furthermore, it is possible to test whether the operational kinetic parameters best describe a one-site, simple carrier or a two-site, simultaneous carrier. Most hexose transfer systems are well described by both simple carrier and simultaneous carrier kinetics. The test between one-site and two-site models has been performed only in the human red cell. This, the most extensively studied hexose transfer system is not resolved. The human erythrocyte hexose transfer system is symmetric but appears to be incompatible with both the simple carrier hypothesis (Carruthers and Melchior, 1983a) and simultaneous transport-site models (Krupka and Deves, 1981). Here we have an example of a transport system apparently consistant with one-site kinetics yet more complex than the simple carrier model. Red cell hexose transfer is, however, atypical. Most other passive hexose transfer systems are symmetric but do not display the phenomenon of counter flow or acceleration of transfer by trans-sugar. It seems extremely unlikely that kinetic analyses will reveal the physical basis for hexose transfer. Nevertheless, their utility as descriptive, predictive formalisms is of value, particularly in studies of hexose transfer regulation by hormonal and metabolic factors (see Section V). IV. I S O L A T I O N AND C H A R A C T E R I Z A T I O N OF THE HEXOSE TRANSPORTER The kinetic properties of hexose transfer indicate that sugar transport is mediated by a limited number of selective membrane sites. These sites or transporters were thought to be proteins which span the bilayer (Widdas, 1952). A variety of methods have been used in their identification, isolation, (Jones and Nickson, 1981) purification and reconstitution into artificial lipid membranes. This section will review briefly these procedures.

1. Identification of the Transporter This problem has been approached in a number of ways. Historically, the first approach was to modify membrane polypeptide content or composition (in situ) then to determine the effects of this on hexose transfer. Such modifications included selective protein elution and proteolysis. Attempts were also made to label the transporters with site-directed affinity labels. A rather more sophisticated approach adopted was to disrupt membrane structure, usually by detergent action, to give protein-lipid-detergent complexes. These could then be purified using fractionation techniques. The protons in these fractions were then

48

A. CARRUTHERS

incorporated into model lipid membranes and the hexose transfer activities of the reassembled systems monitored. (a) Modification of membrane protein composition Due to the ready availability of human erythrocytes, much of this work was carried out with the red cell. Removal of cellular contents from red cells by hypotonic lysis results in "pink ghosts" which retain their transport activity (Jung et al., 1973; Carruthers and Melchior, 1983a). Inside-out-vesicles with stereospecific D-glucose transport can be prepared from these ghosts by incubation in dilute alkali medium followed by density gradient centrifugation (Zoccolli and Leinhard, 1977). These vesicles contain intrinsic polypeptides of band 3 and region 4.5 (see Steck, 1973) but are depleted of extrinsic polypeptides of bands 1, 2, 5 and 6 (see Fig. 8). These results suggest that band 3 and/or 4.5 proteins may be associated with sugar transport.

A

B

C

O

E __1

92 K 66K

2 3 /+,1 4.,2 band/+,5

/.,.5 K

5 --6

31 K

7

21 K FIG. 8. Polyacrylamidegel electrophoresis of red cell membrane proteins. A: Molecular weight markers. B: Intact red cell membranes. C: Salt washed, EDTA washed membranes. D: Washed membranes solubilized in 0.5% Triton X100. E: Cholate-solubilized washed membranes. Approximately 100/~gof protein was added to each track. Gel, 7%. The positions of the various protein bands of intact membranesare indicatedto the right of the gel. (Carruthers and Melchior, unpublished). Masaik and LeFevre (1977) observed that exposure oferythrocytes to trypsin was without effect on hexose transfer. When trypsin was incorporated into red cells by making ghosts, however, a progressive inhibition of hexose transfer was observed which correlated with the loss of spectrin located at the inner membrane surface. There was no apparent effect on protein band 3. These results suggest that band 3 proteins are not associated with hexose transfer. A rather more likely conclusion is that such studies lack sufficient resolution to dissociate band 3 proteins from bexose transfer function, (b) Bindin# studies In theory, it ought to be possible to identify the hexose transfer protein by determining which membrane protein binds D-glucose, the in vivo substrate. Unfortunately, the relatively

Sugar transport in animal cells: the passive hexose transfer system

49

low affinity of the transfer system for D-glucose (Kin = 10- a to 10- 2 M) renders such an approach of limited use and, as might be expected, numerous conflicting results have been reported (see Jones and Nickson, 1981). A rather better approach would be to determine which proteins bind either phloretin or cytochalasin B. These agents are competitive inhibitors of hexose transfer acting at sub-micromolar levels ( K i = 0 . 1 7 and 0.5#M respectively). Cytochalasin B appears to inhibit hexose transfer competitively in human erythrocytes by acting at the interior of the cell (Widdas, 1980). Trypsin inhibits cytochalasin B binding to red cell membranes when applied to the interior of red cells but not when applied to the exterior of the erythrocyte (Baldwin et al., 1980). These data suggest, therefore, that the inhibitor binds to the transporter at an endofacial site sensitive to proteolysis by trypsin. There are three classes of cytochalasin B binding sites in the erythrocyte membrane (Jung and Rampal, 1977). Of these three sites, only binding at the high affinity site (site I) is inhibited stoichiometrically by D-glucose. It is this site which displays the characteristics of the hexose-transfer system. Site I has been partially purified by Sogin and Hinkle (1978) and by Baldwin et al. (1979) and has been identified as protein band 4.5 with a molecular weight of 55,000. Furthermore, when this site is reconstituted into artificial lipid membranes, the permeability of the membrane to D-glucose is increased many-fold and hexose flux across the bilayer displays many of the characteristics of flux across erythrocyte membranes, e.g. saturation kinetics, inhibition by cytochalasin B, trypsin and high concentrations of phloretin (Kasahara and Hinkle, 1977; Goldin and Rhoden, 1978; Wheeler and Hinkle, 1981). Unfortunately these studies do not constitute proof that the major hexose transport proetin is indeed band 4.5. The proteins in the above studies were not purified to homogeneity and the properties of the reconstituted system are quantitatively different to those of the native system (Kin for reconstituted uptake ~ Km for native uptake; see Wheeler and Hinkle, 1981). Pessin et al. (1982) and Carter-Su et al. (1982) have attempted to circumvent this problem by photo-affinity labeling the transport protein in situ using [3H]cytochalasin B. Cytochalasin B becomes a highly reactive molecule upon high intensity ultraviolet irradiation and forms covalent bonds with neighboring molecules. Red cells and chicken embryo fibroblast membranes were incubated with [3H]cytochalasin B in the presence or absence of D-glucose. These were then exposed to ultraviolet irradiation resulting in the D-glucose-sensitive covalent labeling of a component that migrated as a single broad band on sodium dodecyl sulphate-polyacrylamide gels with a maximum at 54,000 (red cell) and 46,000 (chicken embryo fibroblasts). This covalent labeling was inhibited competitively by D-glucose suggesting strongly that the cytochalasin B binding component of the erythrocyte hexose transporter is band 4.5. There are, however, a number of reasons why this identification oftbe hexose transporter is not conclusive. There are 0.39 to 0.5 cytochalasin B binding sites per band 4.5 polypeptide chain (Baldwin et al., 1979; Sogin and Hinkle, 1978). These data must therefore imply that only one-half of band 4.5 proteins are associated with hexose transfer or that the transporter is a dimer. Secondly, only 5 ~o of the calculated total number of transport sites are photoaffinity-labeled by cytochalasin B (Carter-Su et al., 1982). Does this mean that only 5 ~o of band 4.5 is transporter or that only 5 ~o of binding sites can be readily labeled? Third, is the phloretin binding site also present on the protein containing the cytochalasin B binding site? As both agents inhibit transfer protein activity competitively, it is essential to confirm which proteins bind the inhibitors. Phloretin is only a very poor inhibitor of transport by reconstituted band 4.5 (Kasahara and Hinkle, 1977; Wheeler and Hinkle, 1981). Cytochalasin B, on the other hand, is a potent inhibitor. As intact cell studies show that both phloretin and cytoehalasin B act as potent inhibitors of transfer, it is apparent that the reconstituted system differs from the native transfer system. If different proteins bind these agents, we might conclude that more than one protein-type constitutes the native transport system. Other attempts to affinity label the transporter using [x4C]glucosyl isothiocyanate or maltosyl isothiocyanate have shown that D-glucose inhibited binding to band 3 proteins. These agents inhibit erythrocyte hexose transfer with fairly high affinity (Taverna and 5PB 43:1-D

50

A. CARRUTHERS

Langdon, 1973; Mullins and Langdon, 1980a,b). Here one must conclude that band 3 proteins constitute the transporters. Possible resolution of this problem may come from studies with phloretinyl-3-benzylazide (PBA) a phloretin analogue with an inhibitory potency some 6-20 fold greater than phloretin. In the dark, 10 #M PBA inhibits erythrocyte hexose transfer reversibly by some 90%. Exposure of treated cells to ultraviolet irradiation for some 12 min renders the PBA inhibition of transfer irreversible. Control transfer is unaffected (Fannin et al., 1982). Careful competition and binding studies with this high efficiency, high affinity probe may resolve this problem.

2. Isolation and Purification of the Transporter A number of methodologies have been applied to the isolation and purification of the human erythrocyte hexose transporter. Figure 9 illustrates the method of Kasahara and

native

[

bilayer

salt w a s h

0.5 Triton XIO0

DEAE cellulose

~ band 4.5 protein in lipid triton micelles

FIG. 9. Purification of the erythrocyte hexose transfer protein. Native membranes are salt washed and EDTA washed to elute peripheral proteins (see Fig. 8 and Kasahara and Hinkle, 1977). The membranes are then solubilized in 0.5 % Triton X 100. This produces mixed lipid, Triton X 100, protein micelles. This solution is then applied to a DEAE cellulose column from which band 4.5 proteins and lipids emerge in the void volume. The bound proteins may then be eluted with IM NaC1.

Hinkle (1977). Peripheral membrane proteins are removed by alkaline EDTA treatment with NaC1 washes. These protein-depleted membranes are then incubated with 0.5 % Triton X 100, a non-ionic detergent, for 20 rain. After centrifugation, the supernatent is applied to a DEAE-cellulose column equilibrated with 0.5 % Triton X 100. The column is washed with the same (low ionic strength) solution and the flow-through fraction collected. The remaining proteins are eluted with 1 i NaC1. The flow-through fraction alone contains transport activity and consists of 96 % band 4.5 and a small amount of band 7. Triton X100 may then be removed by treatment with Biobeads SM-2 and the protein reconstituted into artificial membranes. Other methodologies have been described elsewhere (see Jones and Nickson, 1981). Partial purification of transporter has been achieved using affinity chromatography on wheat germ lectin-sepharose (Froman et al., 1981) although the major component obtained is band 3 protein. The choice of detergent is an important consideration although their application is largely an empirical exercise (for review see Maddy, 1982). Non-ionic detergents such as Triton X 100 appear to solubilize more transporter than ionic detergents such as cholate. Non-ionic detergents are, however, more difficult to remove than ionic detergents following solubilization of the transporter. For example, Triton X100 can be removed by treatment

Sugar transport in animal cells: the passive hexosetransfersystem

51

with Biobeads SM-2. This procedure, results in the loss of 50% protein unless exogenous lipid is added (Baldwin et al., 1979). Triton X100 may also be removed by gel filtration with Sephadex-G50 (Yu and Steck, 1975; Yu et al., 1973; Jones and Nickson, 1978). Again, this procedure results in the loss of protein by adsorption to the gel unless exogenous lipids are added. Cholate, on the other hand, may be removed by simple dialysis (Goldin and Rhoden, 1978). Our laboratory obtains a higher yield of transporter with Triton X100 which is now our choice of detergent. 3. Reconstitution of Transport Activity Two types of reconstitution procedures have been described. The proteins are reconstituted either into liposomes (lipid vesicles) or into planar membranes of known lipid composition. Liposomes form spontaneously when lipids are mixed with aqueous buffer. These structures, which may be very large (1-2 # m diameter), are multilamellar and are unsuitable for transport studies due to their multicompartmental nature. A significant improvement is obtained if these structures are sonicated then subjected to a rapid freeze/thaw cycle followed by further sonication (Kasahara and Hinkle, 1977). This produces unilamellar liposomes (single-walled vesicles) of average diameter 0.1 pm. Reconstitution of the transporter is achieved by simple addition of the Triton-free protein solution to the liposomes prior to the freeze/thaw cycle (0.3 mg protein to 7.5 mg lipid). The optimal sonication times are low (5-10 sec) and sonication is in a bath type sonicator (for details, see Kasahara and Hinkle, 1977). This method works well in spite of its empirical nature. Another method of forming large vesicles (unilamellar liposomes) is to solubilize lipids in aqueous solution using an ionic detergent such as cholate. This forms a mixed micelle solution (Carey and Small, 1970) which is then dialyzed continuously against cholate-free solution resulting in the formation of Large Unilamellar Vesicles (LUVs). Vesicle size depends upon micelle size prior to dialysis and upon the rate of detergent removal during dialysis (Zunbuehl and Weder, 1981). The size of the detergent/lipid micelle increases with increasing ratios of lipid to cholate. We routinely use a lipid to cholate molar ratio of 1 and a dialysis rate of 2 ml/min for 24 hr (lipid/detergent mixture volume, 2 ml dialyzed against a constant volume of 2 1.). This leads to the formation of LUVs from egg phosphatidylcholine with a mean diameter of 0.36 #m and a size range of 0.14).5 #m. Reconstitution of transport activity is effected by adding the cholate-solubilized protein to the mixed micelle system prior to dialysis or by adding the Triton X 100-free protein suspension to the mixed micdles prior to dialysis (04).3 mg protein added to 7.5 mg lipid). Care must be taken to maintain the lipid/cholate ratio close to unity on addition of protein. Figure 10 illustrates this methodology. The LUVs may then be concentrated by centrifugation or by vacuum dialysis. It is extremely important to ensure that the vesicle size population is homogeneous. Attempts to determine initial rates of hexose transport in a population of LUVs of widely varying sizes yield kinetically meaningless results. It is for this reason that the above reconstitution methodoligies are recommended. Other methodologies, for example vesicle formation by applying mixed micelle systems to Sephadex G-50 columns, provide vesicles with a wider population of sizes. Bearing this in mind, reconstitution of the transport proteins into planar bilayers has certain advantages for sugar flux determinations. Flux is measured over a single bilayer into large pools. Insertion of the transporter into the bilayer is achieved by addition of protein to one buffer pool. Measurements of membrane electrical resistance show a permanent decrease in resistance on protein addition indicating reaction of the proteins with the bilayer (Cherry et al., 1971; Lossen et al., 1973). Band 4.5 protein addition also increases bilayer permeability to D-glucose by some 40-fold (Jones and Nickson, 1981). This effect is specific for D-glucose (L-glucose flux was unaffected) indicating that reconstitution is feasible with this system. One major problem associated with the use of planar bilayers is that they are formed using lipids dissolved in organic solvent. As not all traces of solvent are removed on bilayer formation, this could influence protein activity markedly. Moreover, there is no convenient method to quantitate how much protein penetrates the planar bilayer. This is important

52

A. CARRUTHERS surface

vlew

tong, section

o~,

ch0tate/tipid

"x~P , p.ro/t~In ij

micettesadded

chorale oved

vesicte

FIG. 10. Reconstitutionby dialysis.Membrane integral proteins and iipids are solubilizedin 30 mg ml-1 chelate. Exogenouslipid is added in the form of mixed lipid/chelate micelles. Subsequent removalofchelateby dialysisformsvesicleswhosebilayerscontain integral proteins. Bothschematic surface viewsand longitudinal sectionsof the protein/lipid/chelate mixed micellesare shown. when expressing fluxes (for example flux/mg protein) and comparing and contrasting different experiments. However, it is only appropriate to mention that reconstitution experiments with vesicles have not demonstrated the quantity of protein inserted into the bilayers. Indeed, we observe that such vesicles swell when mixed with "isotonic" buffer indicating that some quantity of protein is free to exert an oncotic effect inside the vesicles. This presumably arises from the incomplete insertion of protein into the membrane. 4. Conclusions

The membrane spanning proteins of the human erythrocyte which catalyze the selective transbilayer flux ofhexose have been identified tentatively as polypeptides of 55,000 mol. wt. These proteins migrate on sodium dodecyl sulphate polyacrylamide gels with the characteristics of band 4.5 protein. Estimates from inhibitor binding studies indicate that only half this protein is associated with hexose transfer. Furthermore, reliable kinetic estimates indicate that this protein fraction mediates hexose transfer quantitatively differently to the native system. Band 4.5 protein accounts for some 10% of red cell membrane polypeptide. This is a broad band consisting of multiple glycoproteins. The transporter isolated by Sogin and Hinkle (1978) has an apparent mol. wt. of 55,000, is a glycoprotein and contains 17% by weight carbohydrate (5 % neutral sugars, 7 % glucosamine and 5 % sialic acid). Treatment of the protein with endo-fl-galactosidase lowers the reel. wt. from 55,000 to 45,000. It is not known what effect this has on the kinetics of reconstituted hexose transfer. Only half of the protein binds to lectin agarose gels (Gorga et al., 1979; Meuller et al., 1979). These proteins demonstrate a tendency to aggregate (41 mol % of the amino acid composition is apolar). Sogin and Hinkle (1977) indicate that freeze-fracture electron microscopy shows particles of diameter 62 A corresponding to a mol. wt. of 110,000, thus implying that the transporter is a dimer. These findings should be interpreted cautiously, however, for freeze fracture E.M. also demonstrates the presence of similar particles (70 A) in protein-free, artificial membranes (de Kruijff et al., 1979). Radiation inactivation techniques suggest that the native transporter may be a tetramic assembly of 50,000 mol. wt. monomers (Jung et al., 1980). These conclusions ignore the apparently significant transport activity of band 3 proteins. It

Sugar transport in animal cells: the passivehexose transfer system

53

is important that these contradictory findings be resolved. It is by no means inconceivable that proteins from bands 3 and 4.5 are associated with hexose transfer. Indeed, the results of Mullins and Langdon (1980a,b) showing that the non-permeant inhibitor of transfer, maltosyl isothiocyanate, binds to band 3 in a maltose and cytochalasin B-sensitive fashion, suggest strongly that band 3 proteins are associated with the external sugar binding site of the transporter. Competition studies are required to confirm this. An association of band 3 and 4.5 proteins may constitute the native transporter. Each protein may transport sugar, but in a quantitatively different fashion to the native system. Without direct evidence, speculation along these lines is, however, unwarranted. A further troublesome point could be that the conditions of reconstitution (e.g. lipid species) may be so unphysiological that they support activity in one protein type but not another. A complete study of the lipid requirements of reconstitution (e.g. lipid species) would be of value here. In summary, the advances made in the identification and purification of the erythrocyte hexose transfer proteins have been considerable. Nevertheless the nature of the native transporter remains uncertain. This field requires the positive identification of the native system before the mechanisms of hexose translocation by bilayer spanning protein(s) can be examined further. V. HEXOSE TRANSFER M O D U L A T I O N AND R E G U L A T I O N It is beyond the scope of this review to discuss in whole the detailed aspects of hexose transfer regulation in eukaryotic cells. The reader is referred to recent reviews by Clausen, (1975, 1977, 1980), Elbrink and Bihler, (1975) and Czech (1977, 1980). Two aspects of sugar transport regulation will be examined here. The first is of a more fundamental nature and relates to the interactions between membrane proteins and lipids in the plane of the bilayer. Such studies may provide some insight into the mechanism of hexose transfer. The second will deal with the integration of hexose transfer and metabolism in carbohydrate homeostasis. 1. Interactions Between the Suoar Transporter and its Lipid Environment Studies using ionophores in model membrane systems have demonstrated that the temperature dependence of facilitated transfer systems reflects, to some degree, the underlying transport mechanisms. Gramicidin is thought to catalyse transbilayer cation flux by forming fixed channel structures which span the membrane. Valinomycin, on the other hand, is thought to increase K + permeability by acting as a mobile K + carrier (Grell et al., 1975). In studies of black lipid (planar) membranes, the effects of gramicidin on cation flux are relatively insensitive to temperature changes and changes in the physical state of the membrane (changes from a crystalline, to fluid structure). The effects of valinomycin are, however, extremely sensitive to temperature and bilayer physical state (Krasne et al., 1971). With valinomycin, the K + permeability of glyceroyl dipalmitate:glyceroyl distearate (1:1 by weight) membranes is negligible at temperatures where the bilayer is solid (below 40°C). However, as the temperature is raised above 40°C the bilayer melts and valinomycin-mediated K + permeability increases by more than three orders of magnitude. No such behavior is observed with the channel-former, gramicidin. The implication is that the activity of a channel is affected little by the physical state of surrounding bilayer but a mobile carrier (which requires either cortformational changes or diffusional activity for effect) is sensitive to the surrounding bilayer physical state and requires fluid bilayer for optimal activity. Such a hypothesis is impossible to test in native eukaryotic plasma membranes where their high cholesterol content suppresses any lipid phase transition (crystalline to fluid transition) which might otherwise occur (Melchior, 1982). Hence, as temperature is altered, there is little change in the physical state (phase behavior) of the bilayer. However, the membranes of many prokaryotes undergo a crystalline to fluid phase transition (Melchior 1982) and the effects of such transitions on hexose transfer in these organisms have been investigated extensively (Thilo et al., 1977; McElhaney, 1982).

54

A. CARRUTHERS

Working with E. coil, Thilo et al. (1977) were able to show that transport activity is increased as temperature and membrane fluidity are increased (Fig. 11). Furthermore, by supplementing the growth medium with high or low melting point fatty acids, they were able to modify the onset temperature (Tin) of the bilayer phase transition. Under these conditions hexose transfer activity was shown to parallel the modified phase behavior of the bilayers. These results support the view that prokaryotic hexose transfer requires fluid bilayer and involves either some conformational modification of the transfer protein during the process of hexose translocation and/or positional translocation. Such conformational changes may be prevented while the transfer protein is situated in crystalline (solid) bilayer. 3.1 '

3,2 ~

3.3 ~

~'

30"

3./, 'v

3.5

zo •

3.6 10%

I qJ

Z

< I1/

1

0

. . . . . . . . .

t"- - - -

"-t . . . .

i

!

: I

I

I

I

K >>1

I

1 I I

1 K~I

I I

K<< I T,>T>T I

T>Th

I ; I t i

T
100

~ (n s0 !

.

I

E I

i

'

2 Th137o Ttl30 ° I

i •1

I

!I 4

T1,120OC- ' ~

'

II 3.4 (TEMPERATURE) -I , 103 [°K'1 ]

3.2

I

3.3

I 3.5

3.s

FIG. 11. The distribution of carrier proteins between fluid- and ordered-membraneregions. The course of the membrane transition in E. coli fatty acid auxotroph T105 supplemented with trans-Ag-16:1 fatty acidsis shown at the top of the figureas the ratio of fluidto total membranearea. The solid curves at the bottom of the figureare calculated fl-glucosidetransport rates for different distribution constants,K, ofcarrier proteinspartitioningbetweenfluidand crystallinebilayerregions (shown schematicallyin the center of the figure).The open circlesin the lowerpart of the figureshow the experimentally determined in vivo temperature dependence of p-nitrophenyl fl-D-glucopyranoside(NphGlu)hydrolysis.The best fit betweentheory and experimentwas obtained for K = 15. (From Thilo et al., 1977).

What of interactions between lipids and the hexose transporter in eukaryotic cells? Many workers have attempted to modify the physical state of eukaryotic cell membranes by pharmacological means (e.g. addition of exogenous fatty acid, exposure to detergents, anesthetics and by cholesterol removal using liposomes). Unfortunately, the effects of such manipulations on hexose transfer are complex and result, most probably, from multiple site actions. In addition, none of these studies documented rigorously any changes in membrane

Sugar transport in animal cells: the passive hexose transfer system

55

state. A rather more direct approach to this problem was taken by Melchior and Czech (1979). These workers isolated adipocyte hexose transport proteins (protein bands 3, 4.5 and 7) and reconstituted them into model membranes of known composition and physical state. When the transporters were reconstituted into vesicles formed from a 1:1 mixture of egg lecithin and egg phosphatidylethanolamine, cytochalasin B-sensitive hexose transport was absent at 0°C where the bilayer is crystalline but increased as the temperature was raised and the bilayer became progressively more fluid. This parallel dependence of bilayer fluidity and hexose transfer activity was observed when the phase transition was shifted up in temperature by choice of higher melting point lipids. These data suggest strongly that eukaryotic hexose transfer proteins also require fluid bilayer for optimum activity. Unfortunately, the wide range of vesicle sizes produced by their reconstitution procedure did not permit the kinetic analysis of hexose transfer hence, direct evidence in support of their hypothesis was lacking. We have repeated similar experiments with the erythrocyte hexose transfer proteins using more uniform vesicle sizes for reconstitution and the light scattering method for hexose flux determinations (Carruthers and Melchior, 1983a; Carruthers and Melchior, unpublished). Figure 12 summarizes the

A ....,"/ .,....- - " '

o

P

/

o

]

ml LL

''X

I-.

! , !

,..

<

;

i!

f ! -'%

,

"kf o "O o

0

o c

60

B L

2]

DPL : El=` ~

Hl

O""CI: "°'''''~''''-'¢e'" O| ...... ---- ~ / _ _ . , . 0 t8 20

../...'."" EPI EPC ../"

1

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

TEMPERATURE

.

.

.

30

.

40

.

50

C

FIG. 12. Effects of bilayer physical state on V=,~for hexose transfer mediated by human erythrocyte sugar transport proteins. (A) Differential Scanning Calorimetry Thermogram of dipalmitoyl lecithin: egg lecithin (25:75 ratio by weight) vesicles incorporating the Triton-X100 solubilized proteins of salt-washed erythroeyte membranes. A broad endotherm is observed between 10 and 45°C and corresponds to a bilayer phase transition from the hquid-crystaUine to fluid state. (B) Vm~ for protein-mediated D-glucose exit in egg PC vesicles and 25: 75 (ratio by weight) DPL: Egg PC vesicles vs temperature. Protein-mediated exit is defined as the cytochalasin-B sensitive flux (>98%). Vm= values were obtained by integration of the volume changes of the glucose (20 mM) loaded vesicles on injection into glucose free buffer. The dashed line represents the absence of detectable transport (Vine=O). Transport is absent in DPL:Egg PC vesicles at temperatures where the bilayer is predominantly crystalline. The red cell proteins used were those shown in Fig. 8, track D. Protein to lipid ratio 5:100. (Carruthers and Melchior, unpublished).

56

A. CARRUTHERS

results of our kinetic analyses. In dipalmitoyl lecithin: egg lecithin (25: 75) bilayers showing a broad melt between 10 and 45°C, hexose transfer activity is absent below 20°C. As the temperature is increased and the bilayer becomes more fluid, Vm,~for sugar efflux from the vesicles increases. Vmaxfor efflux from egg lecithin vesicles (these bilayers are fully fluid above 10°C) also shows a strong dependence on temperature but in contrast with the former case, significant activity is measurable below 20°C. These results demonstrate that hexose transfer protein activity is influenced strongly by the physical state of the surrounding bilayer and, by analogy with model systems, that some diffusional or gross conformational change occurs during translocation. Studies with different lipid classes have yet to be carried out. Indeed, the transporter may have specific lipid requirements for it is known that erythrocyte glycophorin associates preferentially with phosphatidylethanolamine (Taraschi et al., 1982). These studies point to the influence of the surrounding lipid on hexose transfer protein activity. If lipid domains exist in native bilayers (i.e. lipids are heterogeneously dispersed to produce "pockets" or local regions of lipid of different composition to the overall composition of the bilayer) this could provide the basis for hexose transfer modulation. Activity could be increased by release of transporter from crystalline to fluid lipid domains. Moreover, hexose transfer protein activity might be useful as an indicator of the physical state of the surrounding lipid bilayer. Information regarding the nature of the translocation-conformational change might be obtained, from studies of the effect of D-glucose on the tryptophan fluorescence of the purified, reconstituted transporter. The cytochalasin-B binding component of the transporter (band 4.5) contains about seven tryptophan residues (Baldwin and Lienhard, see Gorga and Lienhard, 1982). Tryptophan residues in nonpolar environments emit maximally at about 330 nm and in polar environments at 340-350 nm (Burstein et al., 1973). The maximum fluorescence of the reconstituted transporter is observed at 336 nm (Gorga and Lienhard, 1982) indicating that the spectrum is composed of residues in both polar and non polar environments. On addition of D-glucose or cytochalasin B, the wavelength of maximum emission is shifted to the blue (reduced) by some 3 nm and fluorescence at longer wavelengths is quenched. One interpretation of these data is that ligand binding induces a protein conformational change which brings quenching groups into proximity with tryptophan residues exposed to the aqueous phase. Conversely, this might be a direct effect of ligand binding. If such an effect were to persist in transporters reconstituted into crystalline bilayer (where transport activity is absent) then we may conclude that this is a binding phenomenon per se and is unrelated to sugar translocation. If, however, the effect is abolished, we may be able to investigate the phenomenon further to provide information about the lipid species/physical state requirements of hexose translocation and its associated protein conformers. -

2. Hexose Transfer Regulation in Carbohydrate Homeostasis

Table 7 summarizes the properties of glucose transport and metabolism in a variety of vertebrate tissues. In tissues where hexose transfer rates are higher than rates of hexose metabolism, transport is not rate-limiting for metabolism and appears not to be regulated. In tissues where hexose transfer rates are very low (e.g. muscle, fat) and metabolic rates are variable, hexose transport is often rate-limiting for metabolism and is under some form of TABLE 7. PROPERTIESOF GLUCOSEAND METABOLISMIN TYPICALVERTEBRATETISSUES Transport Rate Rapid

Type Non-regulated

Variable, rate- Regulated limiting Very rapid Non-regulated

Intracellular glucose level High Very low High

Metabolism Slow, stable, breakdown only Variable, breakdown and storage Variable, breakdown, storage, synthesis

Tissues Mammalian erythrocyte, lens of eye, placenta, bone Muscle, adipose, Nucleated erythrocytes Liver

Sugar transport in animal cells: the passive hexosetransfersystem

57

metabolic and/or hormonal control. Thus in muscle, hexose transfer is stimulated reversibly during exercise, anoxia or exposure to insulin. In fat, hexose transport is stimulated by insulin alone whereas in avian erythrocytes, transfer is stimulated during metabolic depletion. Analysis of the kinetics of hexose transfer in the stimulated state shows that in most tissues studied the capacity (Vmax)of the system is increased whereas Km remains unaltered (Elbrink and Bihler, 1975; Baker and Carruthers, 1983). This has been interpreted in two ways: (1) the number of active transport proteins in the membrane is increased, (2) the rate of protein-mediated transfer (catalytic rate constant of functional transfer proteins) is increased. A third possibility is, of course, a combination of these two effects. A number of different methods have been used to distinguish experimentally between these possibilities. Olefsky (1978) demonstrated in adipocytes isolated from white fat, that when Vmaxof control and insulin-stimulated hexose transfer are measured as a function of temperature, parallel Arrhenius plots are obtained with equal activation energies. Since the energetics of transfer are unaltered, Olefsky argued that the number of transporters, rather than the activity of existing carriers, is increased by insulin. A rather more direct approach has been adopted by Cushman and Wardzala (1980) who have demonstrated that insulin produces an increase in the number of o-glucose-sensitive cytochalasin B binding sites in adipocyte plasma membranes. These data support the view that the density of functional transport protein in the insulin-treated adipocyte plasma membrane is increased. Such experiments are difficult to perform with vertebrate muscle (where isolated cell preparations are not readily available) but have been carried out with isolated invertebrate giant muscle cells with essentially identical findings (Baker and Carruthers, 1983). Two questions arise from these studies. (1) How is the increase in carrier density effected (are the transporters already in situ but inactive or are they of extra-plasma membrane origin)? (2) What is the nature of the signal(s) leading to the activation of transport? Both questions remain incompletely answered. (a) The origin of functional carrier in adipose and muscle There are two schools of thought regarding the origin of the extra carriers in insulin-stimulated muscle and fat. One school suggests that the carriers are already present in the bilayer but are inactive. Insulin would then activate these latent carriers (Cartcr-Su and Czech, 1980). The second school has provided compelling evidence in support of the view that carriers exist within intracellular microsomes or membranes. On treatment of muscle and fat with insulin, hexose carriers are translocated from cytosol to plasma membrane thus increasing carrier density (Wardzala and Jeanrenaud, 1981; Cushman and Wardzala, 1980; Suzuki and Kono, 1980). Indeed, all features of hexose transfer activation by insulin (e.g. ATP-dependcnce, temperature dependence, independence of protein synthesis) are seen with insulin-induced carrier translocation from cystosol to plasma membrane (Kono et al., 1981, 1980). The existence of cytosolic hexose carriers has been confirmed using two techniques. Cytosolic membranes have been isolated and intrinsic hexose transfer protein activity determined (Suzuki and Kono, 1980) or D-glucose sensitive cytochalasin B binding assessed (Cushman and Wardzala, 1980). Both techniques show that the onset of insulin-stimulation of hexose transfer is associated with an apparent loss of carriers from cytosol to plasma membrane and that recovery from insulin treatment is associated with carrier transfer back to the cytosol. The time course of these effects parallels the time course of effects of insulin on hexose transfer in intact cells (Kono 1982). Three series of experiments argue against the translocation hypothesis. Carter-Su and Czech (1980) have reported that total sugar transfer activity isolated and reconstituted from fat cells, is increased by insulin. This is incompatible with the simple quantitative transfer of carriers from cytosol to membrane. Resh (1982) has shown that the insulin activation of the (Na +, K +)-ATPase in adipocytes is not associated with a transfer of sodium pumps from cytosolic to plasma membranes. Rather, the activation of the (Na +, K +)-ATPase by insulin seems to result from activation of existing pumps. The third argument against the translocation hypothesis is more indirect (Oppenheimer et al., 1983). With intact adipocytes,

58

A. CARRUTHERS

insulin increases the affinity of the insulin-like growth factor II (IGF-II) receptor for its ligand with no apparent change in the number of cell surface binding sites. It can be shown that when adipocytes are homogenized, plasma membrane and microsomal (cytosolic) membranes isolated and IGF-II binding assayed, prior treatment of the intact fat cells with insulin causes an increase in the number of IGF-II binding sites in the plasma membrane fraction with a concomitant decrease in sites in the microsomal fraction. These data contrast with those from intact cell studies and suggest that insulin induces a redistribution of IGF-II receptors between plasma membrane and microsomal fractions upon homogenization and preparation of membranes. Such an effect could account for the apparent translocation of carriers from cytosol to membrane. However, it is not clear how internalization of IGF-II may have affected the Scatchard analysis of binding to intact cells. Studies at lower temperatures may help here. Until the number of plasma membrane specific transport sites can be detected accurately (cytochalasin B, which penetrates cells readily, also labels cytosolic membrane sites) this possibility will remain unresolved. Electron microscopic studies with antibodies to the hexose transfer protein may be of value here. Morphological studies have shown that the plasma membrane of adipocytes has many microvesicular invaginations of uniform size and distribution (Williamson, 1964; see Fig. 13). Insulin has no effect on the size, number or relationship of these microvesicles with the plasma membrane (Chlapowski et al., 1983). Moreover, insulin is without effect on the pinocytotic activity of the adipocyte plasmalemma (Jarett and Smith, 1975; Novikoff et al., 1980; Chlapowski et al., 1983). These findings are in apparent contradiction with suggestions that insulin induces membrane cycling in the adipocyte (Suzuki and Kono, 1980; Cushman and Wardzala, 1980). The (Na ÷, K ÷)-ATPase data of Resh (1982) indicate that the induction of carrier translocation from cytosol to cell surface by insulin, if real, may be hexose transfer protein-specific rather than a general phenomenon. The observation of insulin-induced carrier translocation originates from biochemical studies with insulin-treated adipocytes. Only two such studies have been reported with insulin-treated muscle (Wardzala and Jeanrenaud, 1981, 1983). It is uncertain, therefore, whether recycling is an insulin-specific effect or whether it also occurs with the metabolic regulation of hexose transfer such as is seen in muscle. Studies along these lines could tell us whether insulin regulation of hexose transfer is intrinsically similar to metabolic regulation of transfer. Furthermore, if carrier translocation is an artifact induced by homogenization of insulin-treated cells, studies of this system may provide a key to the molecular basis of insulin action. (b) The signals controlling hexose transfer in adipose and muscle This aspect of hexose transfer regulation has been discussed in detail elsewhere (Elbrink and Bihler, 1975; Clausen, 1975, 1977, 1980; Czech, 1977, 1980; Baker and Carruthers, 1983a). As yet these signals remain unidentified. The major target tissues for insulin action in mammals are muscle and, to a lesser extent, adipose. Hexose transfer in muscle may also be stimulated during exercise, anoxia or ATP-depletion. Metabolic depletion per se is without effect on hexose transfer in adipose although it does abolish insulin activation of transport and recovery from insulin activation of transport in fat. As metabolic depletion appears to be without effect on insulin binding to adipocytes (Kono et al., 1977) these results suggest that some metabolic intermediate (e.g. ATP) is involved in the mechanism of transport stimulation or insulin receptor action in adipose. Some evidence for the latter has been obtained recently (Kasuga et al., 1982; Kasuga et al., 1983) showing that insulin induces the ATP-dependent phosphorylation of the insulin receptor. The ATP or energy requirement for inactivation of adipose hexose transfer argues strongly for an ATP or energy dependent step distal to the insulin receptor. This step must act as an antagonist to insulin receptor action. This situation is quite different to that in muscle where the effects of metabolic depletion and insulin on hexose transfer are additive (Elbrink and Bihler, 1975). The variety of other (pharmacological) agents which act on hexose transfer in adipose and muscle is great and tends to confuse this issue (Czech, 1977). A number of agents have been proposed to be responsible for mediating these various

Sugar transport in animal cells: the passive hexose transfer system

FIG. 13. Electron-micrographs of thin sections of isolated adipocytes showing microvesicles (arrows) which appear to open to the surface, m, mitochondria; L, lipid. The bar at the bottom right of the micrograph corresponds to 0.5 gm length. These micrographs were kindly supplied by Dr. Francis Chlapowski.

59

60

A. CARRUTHERS

effects on hexose transfer. They include cyclic nucleotides, C a 2 + ions, and ATP (Clausen, 1975; Czech, 1977). These agents must act as cytosolic messengers. An alternative control system could reside within the plane of the bilayer with no primary requirement for cytosolic messengers (such a system is involved in the regulation of adenylate cyclase activity; see Londos et al., 1981). Insulin reduces cAMP and increases cGMP levels in adipocytes and invertebrate skeletal muscle (Torres et al., 1978; Illiano et al., 1973; Baker and Carruthers, 1983). The hormone is without effect, however, on steady-state cyclic nucleotide levels in rat diaphragm (Tarui et al., 1976; Walaas et al., 1977). Other vertebrate muscle types have not been studied. These results argue against a role for cyclic nucleotides in insulin action in vertebrate muscle. Nevertheless, cAMP has been demonstrated to inhibit hexose transfer in invertebrate muscle (Baker and Carruthers, 1980; 1983) and a reduction in cAMP levels could mediate, in part, insulin's action in this tissue. Cyclic AMP may reduce the affinity of the insulin receptor for insulin in adipose (Pessin et al., 1983) and may thus be involved in regulating the action of insulin on adipose hexose transfer. The possible involvement of cyclic nucleotides in insulin action in vertebrate muscle is implied from studies showing that insulin stimulates cAMP-dependent phosphodiesterase in heart and skeletal muscle (Senft et al., 1967; Drummond et al., 1972; Woo and Manery, 1973). A role for Ca 2 ÷ ions in the regulation ofhexose transfer has been suggested by the results of a number of studies (for review see Clausen, 1980). In almost all cases, the activation of hexose transfer by physiological and pharmacological agents or conditions produces parallel alterations in Ca 2 ÷ flux in the tissue studied. This could, of course, be simply a permissive action and indeed a number of studies argue strongly that Ca 2 ÷ ions are not involved directly in the activation of hexose transfer by insulin (Baker and Carruthers, 1980,i983a; see Czech, 1977, 1980). Nevertheless Ca 2 ÷ ions have been shown to accelerate hexose transfer in an ATP dependent fashion in invertebrate muscle (Baker and Carruthers, 1980, 1983a) and this could account for the stimulation of hexose transfer in exercising skeletal muscle where cytosolic Ca 2 ÷ levels are raised (for review see Clausen, 1974). In vertebrate muscle following exercise, cytosolic Ca 2 ÷ levels fall to normal rest values yet hexose transfer can remain activated for many hours (Holloszy and Narahara, 1965). Here, the relationship between hexose transfer rates and cytosolic Ca 2 ÷ levels (if any) is less obvious. It is possible that Ca 2 ÷ ions activate intermediates which then act on hexose transfer. When Ca 2 ÷ returns to normal, pre-contraction levels, these intermediates may remain active and revert, only slowly, to their inactive form. The involvement of ATP in hexose transfer regulation was first described in detail by Randle and Smith (1958). These workers found that skeletal muscle hexose transfer was accelerated during metabolic depletion. They suggested that, under normal conditions, the membrane was phosphorylated and hexose transfer inhibited. Dephosphorylation of the membrane, therefore, leads to hexose transfer activation. Numerous studies have failed, however, to demonstrate that insulin induces dephosphorylation of adipocyte membrane proteins (see Czech, 1977). Rather, the opposite appears to occur. Insulin exposure leads to the phosphorylation of specific membrane proteins in adipocytes (Belsham et al., 1980). Similarly, Walaas et al. (1979) have shown that insulin activates the ATP-dependent phosphorylation of a 16,000 dalton peptide in diaphragm sarcolemma and that this effect is potentiated by GTP and low concentrations of the non-metabolizable GTP analogue 5'-guanylylimidodiphosphate (Gpp(NH)p). Recently, Baker and Carruthers (1983) have shown that stimulation of invertebrate muscle hexose transfer by insulin or Ca 2 ÷ ions and inhibition of transfer by cAMP requires both ATP and GTP hydrolysis. It should be emphasized, however, that thorough phosphorylation/dephosphorylation studies require, absolutely, the ability to purify the proteins of interest. Until recently, most studies have examined the phosphorylation of total membrane proteins and as such, are extremely difficult to interpret both quantitatively and qualitatively. Insulin is known to activate a protein kinase that catalyzes the phosphorylation of the insulin receptor in intact ceils (Kasuga et al., 1982a,b; PetruzzeUi et al., 1982) and in preparations of purified insulin receptor (Kasuga et al., 1983). In vitro, the insulin receptor

Sugar transport in animal cells: the passive hexose transfer system

61

derived from cultured 3T3-L1 adipocytes and human placenta is phosphorylated on tyrosine residues in the 90,000 dalton subunit of the receptor (Kasuga et al., 1982b; Avruch et al., 1982). This insulin-dependent tyrosine kinase activity copurifies with the receptor to near homogeneity suggesting that the insulin receptor is itself the protein kinase. Furthermore, the phosphorylated insulin receptor catalyzes the phosphorylation of exogenous protein and peptide (Rosen et al., 1983). Following insulin removal, the receptor remains phosphorylated and its kinase activity is intact. Enzymatic dephosphorylation of the receptor is presumably required to terminate its kinase activity. This kinase activity of the phosphorylated insulin receptor may be central to the activity of a variety of insulin-mimetic agents. Ortho-vanadate, which accelerates hexose transfer in adipocytes and muscle (Dubyak and Kleinzeller, 1980; Clausen et al., 1981) has been shown to stimulate to phosphorylation of the insulin receptor (Tamura et al., 1983). It is interesting to speculate that exercise and anoxia in muscle may also accelerate hexose transfer by activating the kinase. Possible schemes for hexose transfer regulation in adipose and muscle are shown in Fig. 14. At the present, these schemes are for the most part supposition being based upon indirect experimental evidence. In adipose, insulin-insulin receptor interaction leads to the ATP-dependent phosphorylation of the insulin receptor. This, in turn, directly or indirectly activates (by ATP-dependent phosphorylation) a phosphatase which acts on the transporters (or mechanisms controlling the partitioning of transporter between plasma membrane and cytosolic membrane) leading to hexose transfer activation. The transporters or their a (active)

ATP

C

~

-

CP

~

"~

G

~

GP

"

ATP /

~

RP

~-

R

. . .

/

ATP

+~ INSULIN

(inhibited)

b {ac~-ive)

OTP

~

I /I

~cP

~

÷

t

(inhibited)

~6P

/

~

R ~

t 5TP

+I

cAMP

INSULIN

1" L

(Ca ++ , c6MP) -4

FIG. 14. Possible mechanisms for hexose transfer regulation in adipose and muscle. (a)

Adipose:

interaction of the insulin receptor (R) with insulin leads to the ATP-dependent phosphorylation of the receptor to form RP which activates a phosphatase G by phosphorylation of G to GP. This in turn dephosphorylates the hexose transfer protein, C (which is normally phosphorylated by action of a phosphorylase) leading to transport activation. This model can accommodate the recruitment hypothesis if the mechanism leading to transport protein translocation from cytosoi to plasma membrane is inactive when intermediates are phosphorylated. GP would then dephosphorylate the system allowing protein translocation to proceed. On removal of insulin, the phosphorylase leads to the phosphorylation of C thus inhibiting transport. (b) Muscle: this scheme, which is analogous to that for adipose, has rather more specific nucleotide requirements. For example, GTP hydrolysis has been suggested to be important in both insulin activation of transport and cAMP-dependent inhibition of transport (Baker and Carruthers, 1983a).

62

A. CARRUTHERS

control system have either endogenous phosphorylase activity or are phosphorylated by an extrinsic phosphorylase. The phosphorylated system is inactive. Insulin, by dephosphorylating the system activates transport; this effect is ATP dependent. On removal of insulin, the system becomes rephosphorylated and, therefore, inactive. Such a model accounts for both the ATP-dependence of onset and removal of insulins' effects on hexose transfer but is only one of many possibilities. With muscle, the situation is more complex. Baker and Carruthers (1983a) have suggested that ATP depletion activates hexose transfer by removal of tonic inhibition by cAMP. Insulin and Ca 2 ÷ ions may act on transfer through the GTP dependent activation of a phosphatase which dephosphorylates the (inhibited) carriers thus activating hexose transfer. Future studies will, no doubt, concentrate on these possible aspects of hexose transfer regulation in adipose and muscle. The most interesting studies may determine whether activation of hexose transfer is associated with phosphorylation/dephosphorylation of the transfer proteins. This requires, however, the unequivocal identification of the native transporter. (c) Hexose transfer reoulation in other tissues Hexose transfer rates are also variable in avian erythrocytes and mammalian and invertebrate neurones (Wood and Morgan, 1969; Whitfield and Morgan, 1973; Simons, 1983a,b; Baker and Carruthers, 1981a,b). With the former tissue, hexose transfer is accelerated during metabolic depletion. Hexose transfer in nerve, however, is inhibited during depletion. Hexose transfer regulation in avian erythrocytes has been investigated extensively by Simons (1983a, b). Cytochalasin B-sensitive, saturable sugar transport is virtually absent in freshly drawn pigeon red cells. Treatment of these cells with the metabolic poison, cyanide, leads to the activation of transport. This effect is seen as an increase in Vm~,for transfer. Cyanide-induced metabolic depletion results in reduced cytosolic ATP levels (Carruthers and Simons, 1978) and increased Ca 2 + levels in other tissues (Blaustein and Hodgkin, 1969; Clausen, 1975; Baker, 1972). Simons (1983b) has evaluated the possibility that altered cytosolic Ca 2+ metabolism may mediate, in part, this effect on avian red cell hexose transport. Using a variety of independent but complementary techniques, he was able to demonstrate, unequivocally, that Ca 2+ ions were not directly involved in avian erythrocyte hexose transfer regulation. Metabolic depletion seems not to increase ionized Ca 2+ levels and, when the possibility of an increase in Ca 2 + levels is prevented by preloading cells with the Ca 2+ chelating agent, methyl-BAPTA, metabolic depletion continues to accelerate transport. The stimulation of transport by Ca 2+ ions and Ca 2 + ionophore, A 23187 seems to result from metabolic depletion with a further inhibitory pharmacological action of the Ca-A 23187 complex possibly independent of its effect on membrane Ca 2 + permeability. These experiments point to the involvement of metabolic intermediates (possibly ATP) in avian erythrocyte hexose transfer regulation (Simons, 1983b). ATP may also be involved in hexose transfer regulation in nerves. The uptake of 2-deoxy-D-giucose by rat cerebral cortex synaptosomes is reduced by some 50% in the presence of the metabolic poisons cyanide, 2,4-dinitrophenol or iodoacetate (Diamond and Fishman, 1973a). Similarly, both glucose uptake and 3-O-methyl glucose efflux in squid giant axons are reduced by cyanide (Baker and Carruthers, 1981a). This effect on axonal hexose transfer is seen as an increased Km for uptake with little or no change in the capacity (Vmax)of the transport system. Baker and Carruthers (1981b) investigated this phenomenon more closely using the internally dialysed giant axon. Here, it is possible to control the solute composition of the cytosol and measure the permeability of the plasma membrane to sugar simultaneously in a continuous fashion (for further details see Brinley and Mullins, 1967; Baker and Carruthers, 1981b). Using this technique they were able to demonstrate that altered cytosolic levels of Na +, Ca 2+, GTP, ADP, AMP, glucose-6-phosphate or fructose-6-phosphate were without effect on 3-O-methyl-glucose uptake. ATP, however, influenced transport markedly (Fig. 15). The Km for 3-O-methylglucose uptake was reduced from 4.1 mM in the nominal absence of ATP to 1.4 mM in the presence of 5 m u ATP. Vmax was

63

Sugar transport in animal cells: the passive hexose transfer system A ATPi mM 0 3OMGoma 10 J 5 115 I

I

s

1

1151

I 5 I

0

~0

I

I

B -- 2

~,n 2

7 O

E

o. v

o=E o 0 co

i~

I

0

8

I

I

0

I 20

[30MG]o (raM)

Time (h)

FIG. 15. Effect of ATPa on kinetics of 3-O-methylglucose uptake in squid nerves. A, ordinate 3-O-methylglueose uptake in pmol-em-2 s-1. Abscissa: time in hr. External 3-O-methylglucose and internal ATP concentrations are shown above the points. Temperature, 15°C; axon diameter, 860 #M, Era, - 5 0 inV. B, figure drawn from A. Ordinate: 3-O-methylglucose uptake. Abscissa: external 3-O-methylglueose concentration (raM). Both curves are rectangular hyperbolae with the followin~ constants; uptake in the presence of 5 mM --ATP~ (Q): apparent KIn, 1.4 raM; V ~ , 2.1 pmol. cm-2 s - t ; uptake in the absence of ATP~ (©): Apparent K=, 4.1 mM; V=a,, 2.2 pmol. cm -2 s -1. (From Baker and Carruthers, 1981b).

unaffected. Physiological cAMP levels (1/AM) w e r e without effect on transport. ATP hydrolysis seems to be involved in the regulation of the affinity of the transport system for its substrate. Only hydrolyzable ATP analogues mimic the action of ATP on transfer whereas non-hydrolyzable ATP analogues decrease the rate of sugar uptake, indicating that they may compete with ATP for interaction with the regulatory site(s). This interaction may result in the phosphorylation of these sites. Baker and Carruthers (1981b) have proposed a model which can account for these findings. Carrier C reacts with ATP to form C" ATP. Both types of carrier can bind sugar(s) and effect translocation, but binding to C. ATP occurs with higher affinity than to C. This is illustrated below. C+S

,. r~ •

+ ATP

C'ATP+S

C'S"

v

,

+ ATP

.

r~

~

C'ATPS

v -,

K I , g 2 , Ka, and K4 are dissociation constants ( K I > K a ) and V is a rate constant proportional to the rate of hexose transfer across the axolemma. Making the assumption that all reactions are rapid in comparison to V, it can be shown that

J T

V([S]/K1) + V([S][ATP]/K2K3)] 1 + ([S]/KI) + (fATP]/K2) + ([S][ATP]/K2K 3)

where J is the flux of sugar and T is a constant proportional to the number of carriers per unit membrane. V- T = Vma, for transport. As [ATP] is increased the apparent Km for transport approaches K3 but Vma, is unaffected. This model is an oversimplification in that the reaction of the transport system with ATP is likely to be more complex. The model does, however, predict the behavior of the axolemmal transfer system.

64

A. CARRUTHERS

Hexose transfer rates in the giant axon are unaffected by altered membrane potential or increased bioelectric activity (Baker and Carruthers, 1981a). Glucose metabolism is however, stimulated during activity--an effect which can be prevented by inhibition of the sodium pump using ouabain. The rate of glucose metabolism in rat cerebral cortex synaptosomes is also related to sodium pump activity (Diamond and Fishman, 1973b) indicating that certain features of axonal metabolism as well as transport are shared by m a m m a l i a n and invertebrate nerve. 3. C o n c l u s i o n s

Hexose transfer regulation is observed in most cells where the rate of sugar transport is rate limiting for sugar metabolism. The mechanism by which transport is regulated in most tissues is unknown. Indeed it is unclear whether a single mechanism is shared by all tissues in which transfer is regulated or whether each tissue effects regulation differently. Further it is by no means certain that hexose transfer activation in muscle by insulin or exercise share common pathways. These problems have arisen, for the most part, from technical difficulties associated with the tissue of study. These difficulties are reflected in the often conflicting findings in muscle and adipose transport studies (see Table 8). In tissues where transport rates may be determined readily and accurately, rather more precise conclusions may be drawn. For example, we now know that Ca 2 ÷ ions assume no direct role in hexose transfer regulation in avian erythrocytes (Simons, 1983b). ATP and G T P hydrolysis appear to be involved in the mechanism of insulin action in invertebrate muscle (Baker and Carruthers, 1983). With nerve, ATP hydrolysis leads to the increased affinity of the transfer system for its substrate. (Baker and Carruthers, 1981b). Nerve is uncharacteristic of other tissues where transfer is regulated. Here, transport seems not to be rate limiting for metabolism. Indeed, when metabolic activity is increased during the conduction of action potentials, hexose transfer rates remain unaltered. However, when the tissue is poisoned by arresting the oxidative phosphorylation of hexose (e.g. by cyanide), the requirement for hexose is increased, yet hexose transfer rates are reduced. The physiological significance of this is not obvious. We may expect rather more information regarding the nature of the signals controlling TABLE 8. EFFECTS OF INSULIN ON SUGAR UPTAKE lN MUSCLE AND ADIPOSE

Control Tissue Rat hemidiaphragm (Glucose) Rat Soleus (Glucose) Perfused rat heart (Glucose) (L-arabinose) Chick heart (Glucose) Frog Sartorius (3-0methylglucose) Balanus (3-0methylglucose) Fat pad (Glucose) Isolated adipocyte (3-0methylglucose) (2 deoxy-o-glucose) Brown fat adipocytes (3O-methylglucose)

(mM)

~mol g-1 hr-t)

+ Insulin (mM) ~molg -1 hr -1)

22

46

9

76

83

28

83

9

102

28

500

0.05

133

13

29

1.9 8

46

1580 31

Refs. Norman et al. (1959) Chaudry and Gould (1979) Post et al. (1961) Fisher and Gilbert (1970) Guidotti et al. (1966) Narahara and Ozand (1963) Carruthers (1983)

4.2

3.1

3.6

9.1

4.2

17.2"

4.5

41.6"

15~"

7

15t

8.6 x 10a~: 7.3 x 10a~ 20~

7

2.4 4

73 x 10a~: Vinten (1978) 28.4 x 10a:~ Olefsky (1978) 40~ Czech et al. (1974)

90 4.5 2.5 4

* pmol cm-2 sec-1. #tool/10 mg fat free dry weight per 3 hr. :1;pmol/106 cells per min.

Crofford and Renold (1975)

Sugar transport in animal cells: the passive hexose transfer system

65

adipose and skeletal muscle hexose transfer when the kinetics of transport in these tissues can be examined more rigorously (for example see Rees and Holman, 1981; Simons, 1983b). ACKNOWLEDGEMENTS T h e a u t h o r w o u l d like t o e x p r e s s his g r a t i t u d e t o D r s . D. L. M e l c h i o r , T. W. H o n e y m a n a n d P. J. R y a n for t h e i r c o n s t r u c t i v e c r i t i c i s m o f t h e m a n u s c r i p t . T h e a u t h o r g r a t e f u l l y a c k n o w l e d g e s t h e receipt o f a W e l l c o m e T r u s t T r a v e l G r a n t . T h i s w o r k w a s f u n d e d in p a r t b y the receipt of a Research Fellowship from the Muscular Dystrophy Association. REFERENCES AVRUCH,J., NEMENOFE,A., BLACKSHEAR,P. J., PIERCE, M. W. and OSATHANONDH,R. (1982) Insulin-stimulated tyrosine phosphorylation of the insulin receptor in detergent extracts of human placental membranes. Comparison to epidermal growth factor-stimulated phosphorylation. J. biol. Chem. 257, 15162-15166. BAKER,G. F. and WIDDAS,W. F. (1973) The asymmetry of the facilitated transfer system for hexose in the human red cell and the simple kinetics of a two component model. J. Physiol. 231, 143-165. BAKER,G. F. and NAFTALIN,R. J. (1979) Evidence of multiple operational affinities for D-glucose inside the human erythrocyte membrane. Biochim. biophys. ,4cta 550, 474-484. BAKER,P. F. (1972) Transport and metabolism of ¢,alcium ions in nerve. Prog. Biophys. molec. Biol. 24, 177-223. BAKER,P. F. and CARRUTHERS,A. (1980) Insulin stimulates sugar transport in giant muscle fibres of the barnacle. Nature (London) 286, 276-279. BAKER, P. F. and CARRUTHERS,A. (1981a). Sugar transport in giant axons of Loligo. J. Physiol. 316, 481-502. BAKER,P. F. and CARRUTHERS,A. 098 lb) 3-O-methylglucose transport in internally dialysed giant axons of Loligo. J. Physiol. 316, 503-525. BAKER, P. F. and CARRUTHERS,A. (1983). Insulin regulation of sugar transport in giant muscle fibres of the barnacle. J. Physiol. 336, 397-43 I. BAKER, P. F. and CARRUTHERS,A. (1984) Transport of sugars and amino adds. In The Squid Axon. Current Topics in Membranes and Transport 22, 91-130. BALDWIN, S. A., BALDWIN,J. M., GORGA, F. R. and LIENHARD,G. E. (1979) Purification of the cytochalasin B binding component of the human erythrocyte monosaccharide transport system. Biochim. biophys. Acta 552, 183-188. BALDWIN,J. H., BALDWIN,S. A., CAUGHEY,J. H. and L1ENHARD,G. E. (1980) The monosaccharide transporter from the human erythrocyte. Transport activity on reconstitution. Fedn Proc. 39, 1813. BANG,O. and ORSKOV,S. L. (1937) Variations in the permeability of red blood cells in man with particular reference to the conditions obtaining in pernicious anaemia. J. din. Invest. 16, 279-288. BARNETT,J. E. G., HOLMAN,G. O. and MUNDAY,K. A. (1973) An explanation of the asymmetric binding of sugars to the human erythrocyte sugar transport system. Biochem. J. 135, 539-541. BARNETT,J. E. G., HOLMAN,G. D., CHALKEY,R. A. and MUNDAY,K. A. (1975). Evidence for two asymmetric conformational states in the human erythrocyte sugar-transport system. Biochem. J. t45, 417-429. BATTAGLIA,F. C. and RANDLE, P. J. (1960) Regulation of glucose uptake by muscle. IV. The specificity of monosaccharide transport systems in rat diaphragm muscle. Biochem. J. 75, 408-416. BELSnAM,G. J., DENTON,R. M. and TANNER, M. J. A. (1980) Use of a novel rapid preparation of fat cell plasma membranes employing percoll to investigate the effects of insulin and adrenaline on membrane phosphorylation within intact fat cells. Biochem. J. 192, 457-467. BLAUSTEIN,M. P. and HOI~KIN, A. L. (1969) The effect of cyanide on the efflux of calcium from squid axons. J. Physiol. 200, 497-527. BOWLEr, A. A. (1928) F. Y. Edgeworth's Contributions to Mathematical Statistics, p. 101, Royal Statistical Society, London. BRINLEV,F. J. JR. and MULLINS,L. J. (1967) Sodium extrusion by internally dialysed squid axons. J. gen. Physiol. 50, 2302-233 I. BURSTEIN,E. A., VEDENKINA,N. S. and IVKOVA,M. N. (1973) Fluorescence and the location of tryptophan residues in protein molecules. Photochem. Photobiol. 18, 263 279. CAREV,M. C. and SMALL,D. M. (1970) The characteristics of mixed miccllar solutions with particular reference to bile. ,4m. J. Med. 49, 590-608. CARRUTHERS,A. (1980) Sugar transport in large nerve and muscle fibres. Ph.D. Thesis. University of London. CAm~.UTHERS,A. (1983) Sugar transport in giant barnacle muscle fibres. J. Physiol. 336, 377-396. CARRUTHERS,A. and SIMONS,T. J. B. 0978) Calcium and ionophore A 23187 stimulate sugar transport in pigeon red cells. J. Physiol. 284, 49P. CARRUTHERS~A. and MELCHIOR, O. L. (1983a) Asymmetric or symmetric? Cytosolic modulation of human erythrocyte hexos¢ transfer. Biochim. biophys. ,4cta 728, 254-266. CARRUTHERS,A. and MELCHIOR,D. L. (1983b) Studies of the relationship between bilayer water permeability and bilayer physical state. Biochemistry 22, 5797-5807. CARTER-Su,C. and CZECH,M. P. (1980) Reconstitution of sugar transport activity from cytoplasmic membranes. J. biol. Chem. 255, 10382-10386. CARTER-Su,C., Pr~SSIN,J. E., MOVA,R., Gn'OMER,W. and CZECH,M. P. (1982) Photoaffinity labeling of the human erythrocyte D-glucose transporter. J. biol. Chem. 257, 5419-5425. CHAUDRV,I. H. and GOULD, M. K. (1979) Kinetics of glucose uptake in isolated soleus muscle. Biochim. biophys. ,4cta 177, 527-536. C~ERRV,R. J., BEGGER,K. U. and C'~APMAN,D. ( 197 l) Interaction oferythrocyte apoprotein with bimolecular lipid membranes. Biochem. biophys. Res. Commun. 44, 644-652.

JPB 43:1-E

66

A. CARROTr~RS

CSLAVOWSKI,F. J., BERTRAND,B. K., PESSIN,J. E., YOSHITOMO,O. and CZECS, M. P. (1983) The relationship of microvesicles to the plasma lemma of rat adipocytes. Eur. J. Cell Biol. 32, 24-30. CLAUSEN,T. (1975) The effect of insulin on glucose transport in muscle cells. Curr. Top. Memb. Transp. 6, 169-226. CLAUSEN,T. (1979) Calcium, glucose transport and insulin action. In Biochemistry o f Membrane Transport, FEBS Symposium No. 42, (eds. G. SARRENZAand E. CARAFOLI),pp. 481-499, Springer Verlag, Berlin. CL^US~N, T. (1980) The role of Ca in the activation of the glucose transport system. Cell Calcium 1, 31 !-325. CL^USEN, T,, ANDERSEN,T. L., SXURUP-JOHANSE~,M. and PET~:OVA,O. (1981) The relationship between the transport of glucose and cations across cell membranes in isolated tissues. XI. The effect of vanadate on 45Caefflux and sugar transport in adipose tissue and skeletal muscle. Biochim. biophys. Acta 646, 261-267. CLELAND,W. W. (1983) The kinetics of enzyme-catalysed reactions with two or more substrates or products. I. Nomenclature and rate equations. Biochim. biophys. Acta 67, 104-137. CRAIK, J. D. and ELLIOT, K. R. F. (1979) Kinetics of 3-O-methylglucose transport in isolated rat hepatocytes. Biochem. J. 182, 503-508. CRANE, R. K. (1977) The sodium gradient hypothesis and other models of carrier-mediated active transport. Rev. Physiol. Biochem. Pharmac. 78, 99-159. CRO~'ORD, O. B. and RESOLD, A. E. (1975) Glucose uptake by incubated rat epididymal adipose tissue. Characteristics of the glucose transport system and action of insulin, J. biol. Chem. 240, 3237-3244. CUSHMAN,S. W. and WARDZALA,L. J. (1980) Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. J. biol. Chem. 255, 4755-4762. CZECH, M. P. (1977) Molecular basis of insulin action. A. Rev. Biochem. 46, 359-384. CZECH, M. P. (1980) Insulin action and the regulation of hexose transport. Diabetes 29, 399-409. CZECH, M. P., LAWRENCE,J. C. JR. and LYNN, W. S. (1974) Hexose transport in isolated brown fat cells. A model system for investigation insulin action on membrane transport. J. biol. Chem. 249, 5421-5427. DIA~IOND,I. and FISHMAN,R. A. (1973a). High affinity transport and phosphorylation of 2-deoxy-D-glucose in synaptosomes. J. Neurochem. 20, 1533-1542. DIAMOND,I. and FISHMAN,R. A. (1973b) Development of Na stimulated glucose oxidation in synaptosomes. J. Neurochem. 21, 1043-1050. I~tJMMOND, G. I., SEVERSON,D. L, and SULAKHE,P. V. (1972) Nature of hormone and fluoride stimulation of adenylat¢ cyclase in muscle, In Insulin Action, (ed. I. B. FRIXZ), p. 227, Academic Press, New York. DOaYAK, G. R. and KLEINZELLER,A. (1980) The insulin-mimetic effects of vanadate in isolated rat adipocytes. Dissociation from effects of vanadate as a (Na+-K+)ATPase inhibitor. J. biol. Chem. 255, 5306-5312, EISENrr~L, R. and CORNIsH-BOWDEN,A. (1974) The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem. J. 139, 715-720. MCELHA~,~Y,R. N. (1982) Effect of membrane iipids on transport and enzyme activities. Curr. Top. Memb. Transp. 17, 317-380. FANNtN, F. F., EVANS,J. O. and DmDglCn, D. F. (1982) Phloretinyl-3-benzylazide: a high affinity probe for the sugar transporter in human erythrocytes. II. Irreversible transport inhibition is induced by photolysis. Biochim. biophys. Acta 684, 228-232. I.~FEV~, P. G. (1948) Evidence of active transfer of certain non-electroytes across the human red cell membrane. J. yen. Physiol. 31, 405. FtSl-IF.a,R. B. and LINOSAY,D. B. (1956) The action of insulin on the penetration of sugars into the perfused heart. J. Physiol. 131, 526-541. F1srml~, R. B. and GILBERT,J. C. (1970) The effect of insulin on the kinetics of pentose permeation of the rat heart. J. Physiol. 210, 297-323. Frtowa~N, G., LUDHAL,P. and ACEVEDO,F. (1981) Partial purification of the D-glucose transport protein from human erythrocyte membranes by affinity chromatography on wheat germ lectin-sepharose. FEBS Left. 129, 100-104. GOLDIN, S. M. and RnODEN, V. (1978) Reconstitution and "Transport Specificity Fractionation" of the human erythrocyte glucose transport system. J. biol. Chem. 253, 2575-2583. GORGA, F. R., BALDWIN, S. A. and L1ENHARO,G. E. (1979) The monosaccharide transporter from human erythrocytes is heterogeneously glycosylated. Biochem. biophys. Res. Commun. 91, 955-961. GORGA, F. R. and LIENHARD,G. E. (1982) Changes in the intrinsic fluorescence of the human erythrocyte monosaccharide transporter upon ligand binding. Biochemistry 21, 1905-1908. GRELL, E., FUNCK,T. and EthERS, F. (1975) In Membranes (ed. G. EISENMAN),Vol. 3, pp. 1-126, Marcel Dekker, New York. GultxYrn, G. G., LOPa~TI,L., GAJA,G. and FOA,P. P. (1966) Glucose uptake in developing chick embryo heart. Am. J. Physiol. 211, 981-989. HANES,C. S. (1932) The effect of starch concentration upon the velocity of hydrolysis by the amylase of germinated barley. Biochem. J. 26, 1406-1421. HANKIN, B. L., LmB, W. R. and S~IN, W. D. (1972) Rejection criteria for the asymmetric carrier and their application to glucose transport in the human red blood cell. Biochim. biophys. Acta 288, i 14-126. HARRIS,E. J. (I 983) An analytical study of the kinetics of glucose movement in human erythrocytes. J. Physiol. 173, 344-353. HOLLOSZV,J. O. and NA~rt~V,A, H. T. (1975) Studies on tissue permeability. X. Changes in permeability to 3-0methylglucose associated with contraction in frog muscle. J. biol. Chem. 240, 3493-355. ILLIANO,T., TALL,G. P. E., SIEGEL,M. I. and CtJATt~CASAS,P. (1973) Guanosine 3:5 monophosphate and the action of insulin and acetylcholine. Proc. natn. Acad. Sci. U.S.A. 70, 2443-2447. JACQUEZ,J. A. (1983) Modulation of glucose transport in human red blood ceils by ATP. Biochim. biophys. Acta 727, 367-378. JARRET, L. and SMITH, R. M. (1975) Ultrastructural localization of insulin receptors on adipocytes. Proc. natn. Acad. Sci. U.S.A. 72, 3526-3530. JONES,M. N. and NlCKSON,J. K. (1978) Electrical properties and glucose permeability of bilayer lipid membranes on incorporation of erythrocyte membrane extracts. Biochim. biophys. Acta 509, 260-271.

Sugar transport in animal cells: the passive hexose transfer system

67

JONES, M, N. and NICKSON,J. K. (1981) M onosaccharide transport proteins of the human erythrocyte membrane. Biochim. biophys. Acta (:~)0, 1-20. JUNG, C. Y. (1971 a) Permeability of bimolecular membranes made from lipid extracts of human red cell ghosts to sugars. J. Memb. Biol. 5, 2300-214. JUNG, C. Y. 1971b) Evidence of high stability of the glucose carrier function in human red cell ghosts extensively washed in various media. Arch. Biochim. Biophys. 146, 213-226. JUNG, C. Y., CARLSON,L. M. and BALZER,C. J. (1973) Effect of proteolytic digestion on glucose transport carrier of human erythrocyte ghosts. Biochim. biophys. Acta 289, 108-114. JUNG, C. Y., Hsu, T. L., J. S., CHA, C. and HAAS,M. N. (1980) Glucose transport carrier of human erythrocytes. Radiation-target size of glucose-sensitive cytochalasin B binding protein. J. biol. Chem. 225, 361-364. KASAHARA,M. and HINKLE,P. C. (1977) Reconstitution and purification of the D-glucose transporter form human erythrocytes. J. biol. Chem. 253, 7384-7390. KASUGA,M., KARLSSON,F. A. and KAHN,C. R. (1982) Insulin stimulates the phosphorylation of the 95,000-Dalton subunit of its own receptor. Science 215, 185-187. KASUGA,M., ZICK, Y., BLITHE,D. L., KARI.KSON,F. A., BARING,H. U. and KAnN, C. R. (1982) Insulin stimulation of phosphorylation of the/~ subunit of the insulin receptor. J. biol. Chem. 257, 9891-9894. KASUGA, M., FUJITI-YAMAGUCHI,Y., BLITHE, D. L. and KAHN, C. R. (1983) Tyrosine- specific protein kinase activity is associated with the purified insulin receptor. Proc. hath. Acad. Sci. U.S.A. 80, 2137-2141. KaPNIS, D. M. and PARISH,J. E. (1965) Roles of Na + and K + in sugar (2-deoxy-D-glucose) and amino acid (aaminoisobutyric acid) transport in striated muscle. Fedn Proc. 24, 1051. Koh~, P. G. and CLAUSEN,T. (1971) The relationship between the transport of glucose and cations across cell membranes in isolated tissues VI. The effect of insulin, ouabain and metabolic inhibitors on the transport of 3O-methylglucose and glucose in rat soleus. Biochim. biophys. Acta 225, 277-290. KONO, T. (1982) Recycling of the insulin-sensitive glucose transport mechanism in fat-cells. Biochem. Soc. Trans. 10, 9-10. KONO, T., ROBINSON,F. W., SARVER,J. A., VEGA,F. V. and POINTER,R. H. (1977) Actions of insulin in fat cells. Effects of low temperature, uncouplers of oxidative phosphorylation and respiratory inhibitors. J. biol. Chem. 252, 2226-2233. KoNo, T., SUZUKI,K., DANSEV,L. E., ROaINSON,F. W. and BLEVINS,T. L. (1981) Energy-dependent and protein synthesis-independent recycling of the insulin sensitive glucose transport mechanism in fat cells. J. biol. Chem. 256, 6400-6407. Kg,~NE, S., EiSENMAN,G. and SZAnO,G. (1971) Freezing and melting of lipid bilayers and the mode of action of nonactin, valinomycin and gramicidin. Science 174, 412~,15. DE KRUIJFF,S., VERKLEY,A. J., VAN ECHTELD,C, J. A., GERRITSEN,W. J., MOMBER$,C., NOORDAM,P. C. and DEGIER,J. (1979) The occurrence of lipidic particles in lipid bilayers as seen by 3t P-NMR and freeze-fracture electron-microscopy. Biochim. biophys. Acta 5.~3, 200-209. KRUPKA, R. M. and DEVES,R. (1981) An experimental test for cyclic versus linear transport models. The mechanism of glucose and choline transport in erythrocytes. J. biol. Chem. 256, 5410-5416. LEVINE,M., OXENDER,D. L. and STEIlq,W. D. (1965) The substrate facilitated transfer of the glucose carrier across the human erythrocyte membrane. Biochim. biophys. Acta 109, 151-163. LIDGARO,G. P. and JONES, M. N. (1975) D-Glucose permeability of black lipid membranes modified by human erythrocyte membrane fractions, J. Memb. Biol. 21, 1-10. LIEB, W. R. (1982) A kinetic approach to transport studies. In Red Cell Membranes. A Methodological Approach (eds. J. C. ELLORYand J. D. YOUNG), pp. 135-164. Academic Press. LIES,W. R. and STEIN,W. D. (1972) Carrier and non-carrier models for sugar transport in the human red blood cell. Biochim. biophys. Acta 265, 187-207. LmB, W. R. and STEIN,W. D. (1974) Testing and characterizing the simple carrier. Biochim. biophys. Acta 373, 178-196. LONDOS,C., COOPER,D. i . F. and ROOBELL,M. (1981) Receptor-mediated stimulation and inhibition ofadenylate cyclases: the fat cell as a model system. Adv. cyclic Nacleotide Res. 14, 163-171. LOSSEN, O., BRENNECKE,R. and SCHUBERT,D. (1973) Electrical properties of black membranes from oxidized cholesterol and a strongly bound protein fraction of human erythrocyte membranes. Biochim. biophys. Acta 330, 132-140. MADDY, A. H. (1982) The solubilization of erythrocyte membrane proteins. In Red Cell Membranes. A Methodolooical Approach (eds. J. C. ELLORYand J. D. YOUNG),pp. 43M6. Academic Press. MASAIK,S. J. and LEFEVRE,P. G. (1977) Glucose transport inhibition by proteolytic degradation of the human erythrocyte membrane inner surface. Biochim. biophys. Acta 465, 371-377. MELCHIOR, n. L. (1982) Lipid phase transitions and regulation of membrane fluidity in prokaryotes. Curr. Top. Memb. Transp. 17, 263-316. MELCHIOR,n. L. and CZECH, M. P. (1979) Sensitivity of the adipocyte transport system to membrane fluidity in reconstituted vesicles. J biol. Chem. 254, 8744-8747. MUELLER,T. J., LI, YU. T. and MORmSON,M. (1979) Effect of Endo-fl-galactosidase on intact human erythrocytes. J. biol. Chem. 254, 8103-8106. MULLINS,R. E. and LANGDON,R. G. (1980a) Maltosyl isothiocyanate: An affinity iahel for the glucose transporter of the human erythrocyte membrane. 1, Inhibition of glucose transport. Biochemistry 19, 1199-1205. MULLINS,g. E. and LANGDON,R. G. (1980b) Maltosyl isothiocyanate: An affinity label for the glucose transporter of the human erythrocyte membrane. 2. Identification of the transporter. Biochemistry 19, 1205-1212. NAFTALIN,R. J. and HOLMAN,G. D. (1977) Transport of sugar in human red cells. In Membrane Tranaport in Red Cells (eds. J. C. ELLORYand V. L. LEw), pp. 257-300, Academic Press, New York. NARAHARA,H. T. and OZAND,P. (1963) Studies of tissue permeability, IX. The effect of insulin on the penetration of 3-O-methylglucose-3H in frog muscle. J. biol. Chem. 238, 40-49. NORMAN,D., MENOZZI,P., REID,D., LESTER,G. and HECHTER,O. L. (I 959) Action of insulin on sugar permeability in rat diaphragm muscle. J. yen. Physiol. 42, 1277.

68

A. CARRUTHERS

NOVIKOFF,A. B., NOV1KOFF,P. M., ROSEN,O. M. and ROBIN,C. S. (1980) Organelle relationships in cultured 3T3L1 preadipocytes. J. Cell Biol. 87, 180-196. OLEESKY,J. M. (1978) Mechanisms of the ability of insulin to activate the glucose transport system in rat adipocytes. Biochem. J. 172, 137-145. OPPENHEIMER,C. L., PESSlN,J. E., MASSAGUE,J., GITOMER,W. and CZECH, M. P. (1983) Insulin action rapidly modulates the affinity of the insulin-like growth factor II receptor. J. biol. Chem. 258, 4824-4830. PESSIN, J. E., TILLOTSEN,L. G., YAMADA,K., GITOMER,W., CARTER2Su, C., MORA, R., ISSELBACHER,K. J. and CZECH, M. P. (1982) Identification of the stereospecific hexose transporter from starved and fed chicken embryo fibroblasts. Proc. ham. Acad. Sci. U.S.A. 79, 2286-2290. PESSlN, J. E., GITOMER,W., OKA, Y., OPPENHEIMER,C. L. and CZECH, M. P. (1983)/~-Adrenergic regulation of insulin and epidermal growth factor receptors in rat adipocytes. J. biol. Chem. 258, 7386--7394. PETRUZELLI,L. M., GANGOLY,S., SMITH,C. J., COBB,M. H., ROBIN,C. S. and ROSEN,O. M. (1982) Insulin activates a tyrosine-speeific protein kinase in extracts of 3T3-L1 adipocytes and human placenta. Proc. natn. Acad. Sci. U.S.A. 79, 6792-6796. POST, R. L., MORGAN,H. E. and PARK, C. R. (1961) Regulation of glucose uptake in muscle. II The interaction of membrane transport and phosphorylation in the control of glucose uptake. J. biol. Chem. 236, 269-272. RANDLE, P. J. and SMITH,G. H. (1958) Regulation of glucose uptake by muscle. Biochem. J. 70, 501-509. REEs, C. G. and HOLMAN,G. D. (1981) Hydrogen bonding requirements for the insulin-sensitive sugar transport system of rat adipocytes. Biochim. biophys. Acta 646, 251-260, RESH, M. D. (1982) Quantitation and characterization of the (Na ÷, K+)-adenosine triphosphatase in the rat adipoeyte membrane. J. biol. Chem. 257, 11946-11952. ROSEN,O. M., HERRERA,R., OLOWE,Y., PETRUZZELLI,L. M. and COBB,M. H. (1983) Phosphorylation activates the insulin receptor tyrosine protein kinase. Proc. Bath, Acad. Sci. U.S,A. 80, 3237-3240. SENrr, G., SCHOLrZ, G., MUNSKE, K. and HOFFMANN, M. (1968) Influence of insulin on cyclic, 3:5-AMP phosphodiesterase activity in liver, skeletal muscle, adipose tissue and kidney. Diabetologia 4, 322-329. SIMONS,T. J. B. (1983a) Characterization of sugar transport in the pigeon red blood cell. J. Physiol. 338, 477-500. SIMONS,T. J. B. (1983b) The role of calcium in the regulation of sugar transport in the pigeon red blood cell. J. Physiol. 338, 501-526. SOGIN, D. C. and HXNKLE,P. C. (1978) Characterization of the glucose transporter from human erythrocytes. J. supramolec. Struct. 8, 447-453. STECK, T. L. (1974) The organization of proteins in the human red blood cell membrane. J. cell. Biol. 62, 1-19. STI~N,W. D. and LIEn,W. R. (I 974) Testing and characterizing the simple pore. Biochim. biophys. Acta 373, 165-177. SUZUKI, K. and KONO, T. (1980) Evidence that insulin causes transloeation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc. Bath. Acad. Sci. U,S.A. 77, 2542-2545. TAMURA,S., BROWN,T. A., DUr~LER,R. E. and LARNER,J. (1983) Insulin-like effect of vanadate on adipocyte glycogen synthase and on phosphorylation of 95,000 dalton subunit of insulin receptor. Biochem. biophys. Res. Commun. 113, 80-86. TARASCm, T. F., DE KRUUF, B., VERKLEIJ,A. and VAN ECHTELD,C. J. A. (1982) Effect of glycophorin on lipid pholymorphism. A 3tp-NMR Study. Biochim. biophys. Acta 685, 153-161. TARUI, S., SAITO,T., FUJIMOTO,M. and OKABAYASm,T. (1976) Effects of insulin on diaphragm muscle independent of the variations of tissue cAMP and cGMP. Arch. Biochim. Biophys. 174, 192-198. TAVERNA,R. D. and LANt3OON,R. G. (1973) A new method for measuring glucose translocation through biological membranes and its application to human erythrocyte ghosts. Biochim. biophys. Acta 298, 412-421. THILO, L., TRAUnLE, H. and OVERATH,P. (1977). Mechanistic interpretation of the influence of lipid phase transitions on transport functions. Biochemistry 16, 1283-1290. TORRES, H. N., FLAWIA,M. M., MEDRANO,H. and C'tJATRECASAS,P. (1978). Effects of insulin on the adenylyl cyclase activity of isolated fat cell membranes. J. Memb. Biol. 43, 1-18. VINTEN, J. (1978) Cytochalasin B inhibition and temperature dependence of 3-O-methylglucose transport in adipocytes. Biochim. biophys. Acta 511, 259-273. WALAAS,O., WALAAS,E., LYSTAD,E., ALBERTSEN,A. R. and I-tARO,R. S. and POSSUM,S. (1977), A stimulatory effect of insulin on phosphorylation of a peptide in a sarcolemma-enriched membrane preparation from rat skeletal muscle. FEBS Lett. 80, 417-422. V~/ALAAS,O., WALAAS,E., LYSTAD,E., ALBERTSEN,A. R. and HORN,R. S. (1979). The effect of insulin and guanosine nucleotides on protein phosphorylations by sarcolemmal membrane from skeletal muscle. Molec. cell. Endocr. 16, 45-55. WARDZALA,L. J. and JEANRENAUD,B. (1981) Potential mechanism of insulin action on glucose transport in the isolated rat diaphragm..I, biol. Chem. 256, 7090-7093. WARDZALA,L. J. and JEANRENAUD,B. (1983) Identification of the D-glucosc-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes. Biochim. biophys. Acta 730, 49-56. WHEELER,T. J. and HINKLE,P. C. (1981) Kinetic properties of the reconstituted glucose transporter from human erythrocytes. J. biol. Chem, 256, 8907-8914. WHITFIELO,C. F. and MORGAN,J. E. (1983) Effect of anoxia on sugar transport in avian erythrocytes. Biochim. biophys. Acta 307, 181-196. WIODAS,W. F. (1952) Inability of diffusion to account for placental glucose transfer in the sheep and consideration of the kinetics of a possible carrier transfer. J. Physiol. 118, 23-29. WIODAS,W. F. (1980) The asymmetry of the hexose transfer system in the human red cell membrane. Curr. Top. Memb. Transp. 14, 165-223. WILBRANDT,E. M. GUENSBERG,E. and LArOENER,J. (1947). Heir. Physiol. Acta 5, C20. WILLIAMSON,J. R. (1974) Adipose tissue. Morphological changes associated with lipid mobilization. J. Cell Biol. 20, 57-74. Woo, Y. T. and MANERY,J. F. (1973) Cyclic AMP phosphodiesterase activity at the external surface of intact skeletal muscle and stimulation of the enzyme by insulin. Arch. biochim. Biophys. 154, 510-519.

Sugar transport in animal cells: the passive hexose transfer system

69

WooD, R. E. and MORGAN,H. E. (1969) Regulation of sugar transport in avian erythrocytes. J. biol. Chem. 244, 1451-1410. Yu, J., FISCHMAN,D. A. and STECK,T. L. (1983) Selective solubilization of proteins and phospholipids from red blood cell membranes by non-ionic detergents. J. Supramolec. Struct. 1, 233-247. Yu, J. and STECK,T. L. (1975) Isolation and characterization of band 3, the predominant polypcptide of the human erythrocyte membrane. J. biol. Chem. 250, 9170-9175. ZOCCOLI,M. A. and LEIm-IARD,G. E. (1977) Monosaccharide transport in protein-depleted vesicle from erythrocyte membranes. J. biol. Chem. 252, 3131-3135. ZUMBOEHL,O. and WEDER, H. G. (1981) Liposomes of controllable size in the range of 40 to 180 nm by defined dialysis of lipid/detergent mixed mieelles. Biochim. biophys. Acta 640, 252-262. NOTE ADDED

IN PROOF

Nature of the Human Erythrocyte H exose Transfer Protein. Recently, Shelton and Langdon (1983) and Carruthers and Melchior (1984) have reported that both purified protein band 3 and band 4.5 confer stereospecific hexose transfer activity to synthetic membranes. Moreover, Shelton and Langdon report that the diffuse band 4.5 protein is absent in membranes protected against endogenous proteolytic activity. This is in keeping with the findings of Mullins and Langdon (1980b) indicating that band 3 covalently labeled with [t4C-IMITC is converted to labeled material with band 4.5 mobility as a result of endogenous red cell membrane proteas¢ activity. These results suggest strongly that, in spite of band 4.5's hexose transfer activity, the native hexose transfer protein is a component of band 3.

SHELTON,R. L. and LANGDON,R. G. (1983) Reconstitution of glucose transport using human erythrocyte band 3. Biochim. biophys. Acta 733, 25-33. CARRUTHERS,A. and MELCHIOR,D. L. (1984) A rapid method of reconstituting human erythrocyte sugar transport proteins. Biochemistry 24, 4816J,821.