THE JOURNAL OF BIOLOGICAL CAEMI~TRY Vol. 248, No. 24, Issue of December 25, pp. 8457-8464, 1973

Printed in U.S.A.

Specificity in the Association of Glyceraldehyde 3-Phosphate

Dehydrogenase with Isolated Human Erythrocyte Membranes*

(Received for publication, June 8, 1973)

JEFFREY A. KANTI AND THEODORE L. STECK~

From the Departments of Biochemistry and Medicine, The University of Chicago, Chicago, Illinois 60637

SUMMARY

We have characterized the binding of glyceraldehyde 3- phosphate dehydrogenase to isolated human erythrocyte membranes in an effort to establish whether this in vitro association has physiologic significance.

At pH 8 and p = 0.015 M, approximately 2 X lo6 glyceral- dehyde-3-P dehydrogenase molecules bound reversibly and homogeneously to the inner (but not the outer) membrane surface with an association constant of 1 X 10’ M-l. This binding capacity exceeded by roughly lo-fold the cell’s glyc- eraldehyde-3-P dehydrogenase content. Binding dimin- ished with increasing ionic strength and pH, suggesting a strong electrostatic component in the association. Milli- molar amounts of certain metabolites also released bound enzyme from the membrane.

In contrast, an “indifferent” strongly basic protein, cyto- chrome c, lacked specificity in its association with the erythro- cyte membrane. That is, binding was relatively weak, oc- curred at both membrane surfaces, and may have been limited in extent by close packing (i.e. monolayer formation) rather than by a tied number of binding sites. Cytochrome c showed much stronger association with carboxymethyl- cellulose than did glyceraldehyde-3-P dehydrogenase, but was more readily eluted from the membrane by electrolytes. Cytochrome c binding to membranes was not altered by any metabolite tested.

We hypothesize that a fraction of erythrocyte glyceralde- hyde-3-P dehydrogenase may be specifically bound to the inner surface of the membrane in viuo in a fashion which is responsive to metabolic and ionic transients.

The relationship of certain glycolytic enzymes to cell mem- branes is enigmatic. Glycolytic enzymes are considered to be

* This investigation was supported by Grant BC-95A from the American Cancer Society.

$ Recipient of a Medical Scientist Training Program Trainee- ship, Public Health Service Grant 5 TO 5 GM0 1939 from the National Institute of General Medical Sciences; the data are taken in part from a dissertation submitted to the University of Chicago in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

$ Recipient of a Schweppe Foundation fellowship; to whom correspondence should be addressed.

soluble cytoplasmic constituents, yet hexokinase is bound to isolated mitochondria (cj. 1, 2), and human (3-5) and bovine (6) glyceraldehyde-3-P dehydrogenase and aldolase are recovered on isolated erythrocyte ghosts. This association could merely represent artifactual electrostatic adsorption of proteins to membrane surfaces in low ionic strength media, since these enzymes are eluted from the isolated membranes by physiologic salt solutions (1, 4, 6, 7). Neither the occurrence of enzyme- membrane complexes at low ionic strength nor their reversal at high ionic strength provides adequate in vitro criteria for deducing the presence of these enzymes on membranes in viva.

Therefore, we have sought evidence for specificity in the association of glyceraldehyde-3-P dehydrogenase with the iso- lated human erythrocyte membrane. We postulated that a physiologic binding site might be confined to one membrane surface, while nonspecific adsorption could occur indiscriminately at both surfaces. Homogeneity and high affinity would also befit specific binding sites. Resistance to elution by electrolyte solutions might be greater for specifically bound than non- specifically bound proteins. Furthermore, physiologic binding might be responsive to relevant effecters, such as glycolytic intermediates. We studied models for nonspecific electro- static interactions, using another basic protein, cytochrome c, and a cation exchanger, CM-cellulose, to contrast with the dehydrogenase-membrane pair.

According to these criteria, glyceraldehyde-3-P dehydrogenase appears to interact specifically with the erythrocyte membrane. Some of this enzyme could be membrane-bound in viva in an association which may be sensitively regulated by local metabolic events.

EXPERIMENTAL PROCEDURE

Materials-NAD+, NADH, NADP+, glyceraldehyde-3-P (Ba2+ and ethanol-free), ATP, ADP, AMP, adenosine 3’, 5’- monophosphate, 2,3-di-P-glycerate, 3-P-glycerate (free of 2,3- di-P-glycerate) , bovine serum albumin (Fraction II), oxidized horse heart cytochrome c (90 to 100% pure), rabbit muscle glyceraldehyde-3-P dehydrogenase (68 units per mg of protein), and Triton X-100 were purchased from Sigma Chemical Co.; CM-cellulose was from Whatman. All other chemicals were reagent grade from Fisher, Mallinckrodt, and Baker.

Erythrocyte Membranes-Human erythrocyte membranes were prepared from freshly drawn or outdated bank blood; results were not affected by the source or blood type. Standard

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TABLE I

Orientation and glyceraldehyde-5-P dehydrogenase (GSPD) content of membrane preparations

Membrane preparation

Standard ghosts ........ Depleted ghosts ........ Resealed ghosts. ....... Inside-out vesicles from

standard ghosts. ..... Inside-out vesicles from

depleted ghosts. .....

I Sidedness markers

otal WPD activity5 NADH

diaphoras&

1.83 105 <0.02 100 ND* 102

2.10 6

<0.02 17

101 99

1

124

121

0 Assayed following treatment with 0.1% Triton X-100. b Acetylcholine hydrolase (EC 3.1.1.7), a marker for the outer

surface of the erythrocyte membrane. Membranes f 0.1% Triton X-100 were assayed according to Steck and Kant (9). Final reagent concentrations in 0.8 ml: acetylthiocholine chloride, 0.78 mM; 5,5’-dithiobis(2-nitrobenzoic acid), 0.625 mM; sodium phos- phate buffer (pH 7.5), 85 mu (cf. 18).

c NADH-cytochrome c oxidoreductase (EC 1.6.99.3), a marker for the inner surface of the erythrocyte membrane. Membranes ZIZ 0.005% saponin were assayed according to Steck and Kant (9). Final reagent concentrations in 0.5 ml: oxidized cytochrome e, 0.5 mg per ml; NADH, 0.2 mix; sodium phosphate buffer (pH 8.0) containing 150 mM NaCl, 4.0 mM. Values > 100% accessibility indicate slight inhibition by saponin.

d Percentage of total activity accessible

enzyme activity minus detergent =

enzyme activity plus detergent x 100.

8 ND = not determined in this preparation; values from five other experiments range from 1.06 to 1.48.

hemoglobin-free unsealed ghosts1 were obtained by suspending erythrocytes which had been washed three times with 150 mM

NaCl-5 mM sodium phosphate, pH 8.0, in 40 volumes of 5 mM sodium phosphate, pH 8.0, pelleting the membranes, and re- suspending in 5Pg2 for two or three more wash cycles (9, 11). Resealed ghosts were prepared like unsealed ghosts except the hemolysis and subsequent washes were performed in 40 to 100 volumes of 5P8 containing 1 mM MgSO4. Sealed inside-out vesicles were generated by incubation of unsealed ghosts in 40 volumes of ice-cold 0.5 mM sodium phosphate (pH 8.0 to 8.5) for 30 min, followed by pelleting and homogenization (9, 10, 12). These were separated from unsealed contaminants on Dextran T-110 (Pharmacia) gradients (density = 1.005 to 1.070 g per ml) (9,lO). Ghosts and vesicles were stored in 5P8 for up to 2 weeks at O-5”. Intact erythrocytes for experiments at. low ionic strength were prepared by four washes of whole blood with 5 to 10 volumes of 300 mM sucrose in 5P8. All membrane prep- arations were assayed for orientation and permeability as de- scribed in Table I (cf. 8-10).

Depleted ghosts (i.e. ghosts devoid of glyceraldehyde-3-P dehydrogenase) were prepared by incubating unsealed ghosts

1 Standard ghosts and ghosts depleted of glyceraldehyde-3-P dehydrogenase activity are “unsealed” (i.e., permeable to a variety of probes and macromolecules); preparations of inside-out vesicles, resealed ghosts, and intact erythrocytes are, for the most part, impermeable even to small solutes (8-10).

2 The abbreviations used are: 5P8, 5 mM sodium phosphate (pH 8.0) ; PBS, 5P8 containing 150 rnM NaCl.

in 10 to 20 volumes of PBS for 20 min on ice (see Table I). Fol- lowing centrifugation (10 min at 15,000 rpm in a Sorvall SS-34 rotor), the supernatant solution was collected for preparation of a glyceraldehyde-3-P dehydrogenase concentrate (see below) and the pellet was freed of remaining dehydrogenase activity by two more washes with 40 volumes of PBS followed by two or three washes with 40 volumes of 5P8. Depleted inside-out vesicles were generated by substituting depleted ghosts for standard unsealed ghosts in the vesiculation protocol.

Erythrocyte Glyceraldehyde-S-P Dehyclrogenase Concentrate- The material eluted from standard unsealed ghosts by PBS (see above) was washed in an ultrafiltration cell (Diaflo model 52 plus PM-10 membrane, Amicon Corp., Lexington, Mass.) until its conductivity approximated that of the wash buffer (5 mu sodium phosphate, pH 8.0 or 7.0) and the protein con- centration was 0.2 to 0.7 mg per ml. Rabbit, muscle glyceralde- hyde-3-P dehydrogenase was freed of (NH&S04 in this fashion.

Assay of Glyceraldehyde-S-P Dehydrogenme Activity-The method of Cori (13) was adapted according to Steck and Kant (9). Under these conditions the increment in absorbance at 340 nm during the second minute after initiating the reaction with glyceraldehyde-3-P varied directly with the enzyme added.

Binding Assays--Conditions for the association of glyceralde- hyde-3-P dehydrogenase and cytochrome c with membranes or CM-cellulose are described in individual table and figure legends. Following incubation to equilibrium ( 2 1 hour), membrane samples were centrifuged at, 24” for 20 min at 30,000 rpm in a Spinco type 40 rotor; CM-cellulose and intact erythrocyte sam- ples were centrifuged for 20 min at 3,000 rpm in a Sorvall HL-8 rotor. Aliquots of the supernatant solutions (0.1 to 0.2 ml) were added to either (a) one-quarter volume of 5% bovine serum albumin in 5 mM Na2HP04 for assay of glyceraldehyde-3-P de- hydrogenase activity or (b) 0.3 ml of 5P8 for determination of cytochrome c at 530 nm (oxidized) and 550 nm (reduced). Bo- vine serum albumin curtailed the progressive inactivation of glyceraldehyde-3-P dehydrogenase (14), but did not otherwise affect the results.

Quantitation-Ghosts and erythrocytes were counted in PBS in a model A Coulter counter (100-p aperture) and corrected for coincidence. Packed ghosts usually contain 5 to 7 x log ghosts per ml. Because the protein content of erythrocyte mem- branes varies with the trapping, adsorption, and desorption of polypeptide components, the tightly bound membrane acetyl- choline&erase activity (4) was adopted for purposes of com- parative quantitationP One membrane equivalent is equal to the acetylcholinesterase activity of one ghost or erythrocyte.

Protein was estimated by the method of Lowry et al. (15), using a bovine serum albumin standard.

RESULTS

Glyceraldehyde-S-P Dehydrogenase Content of Membrane Prep- arations-In keeping with the reports of other investigators (6, 16, 17), we found that 60 to 70% of the total red cell glyceralde- hyde-3-P dehydrogenase was associated with hemoglobin-free ghosts. Table I, left-hand column, indicates the level of the dehydrogenase in the various membrane preparations used in this study. The reduced specific activity of glyceraldehyde-3-P dehydrogenase in resealed ghosts can be ascribed to the entrap- ment of extraneous cytoplasm (primarily hemoglobin) while the higher specific activity in inside-out vesicles reflects the loss of certain major polypeptides during vesiculation (19).

3 J. A. Kant and T. L. Steck, unpublished observations.

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FIG. 1. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of ghosts and glyceraldehyde-3-P dehydrogenase. A, standard ghost,s, 40 pg of protein applied; B, depleted ghosts, 40 rg of protein applied; C, concentrate of glyceraldehyde-3-P de- hydrogenase eluted from standard ghosts, 18.2 /*g of protein ap- plied; D, rabbit muscle glyceraldehyde-3-P dehydrogenase, 18.2 pg of protein applied. TD, tracking dye. Preparation of samples and electrophoresis were performed according to Fairbanks et al. (6) as modified by Steck and Yu (20). Acrylamide concentration was 5.0%; sodium dodecyl sulfat’e was 0.2%. Gels were st’ained for nrotein with Coomassie blue.

Since our erythrocyte glyceraldehyde-3-P dehydrogenase (of 295% purity; vide infra) has a specific activity of 25 to 35 pmoles per min per mg of protein, we can calculate from Table I, left-hand column, that the membrane-bound enzyme represents 5 to 7% of the isolated ghost protein. This estimate agrees well with values obtained by quantitative densitometry of erythrocyte membrane proteins on polyacrylamide gels; the 35,000-dalton polypeptide (Band 6 in Fig. l), identified as the monomeric subunit of glyceraldehyde-3-P dehydrogenase (16)) accounts for 5% of the integrated staining profile of standard unsealed ghosts (11).

Glyceraldehyde-S-P Dehydrogenase Elution-We have con- firmed that glyceraldehyde-3-P dehydrogenase (4, 6, 16) and Band 6 (11, 16) are released from erythrocyte ghosts by salt solutions of p 2 0.15 M (see Table I, left-hand column, and Fig. 1). We have further verified the identity of Band 6 by demon- strating that it migrates as a single, approximately 8 S peak on

sucrose gradients, coincident with the enzymic activity of the 137,000 dalton glyceraldehyde-3-P dehydrogenase tetramer (14).4 Because Band 6 represents greater than 95% of the protein eluted by PBS (as determined by densitometry on scans of gels such as that shown in Fig. 1, Gel C), we felt justified in using these preparations as our source of human red cell glyceralde- hyde-3-P dehydrogenase in reassociation studies.

Xidedness of Bound Glyceraldehyde-S-P Dehydrogenase-It seems likely that firmly bound erythrocyte membrane enzyme activities are localized to one surface or the other (8, 9): This asymmetry provides an index of the permeability and orientation of membrane preparations. For example, the relative avail- ability of acetylcholinesterase to its substrates directly reflects the exposure of the outer membrane surface to the bathing medium (cf. 8-10). Similarly, NADH diaphorase can be used to measure the accessibility of the cytoplasmic side of the mem- brane (cf. S-10). Surfactants such as Triton X-100 or saponin disrupt the membrane permeability barrier and presumably render all of the enzyme accessible. The two right-hand col- umns of Table I illustrate how these criteria were used to char- acterize the membrane preparations used in this study. Stand- ard and PBS-washed ghosts offer free access of substrate to both inner and outer surface enzymes. In contrast, cytoplasmic surface NADH diaphorase is completely inaccessible in resealed ghosts, while acetylcholinesterase is nearly so in inside-out vesicles. Elution of glyceraldehyde-3-P dehydrogenase by PBS washes did not appear to affect the accessibility profile of ghosts (although, under special conditions, ghosts can be re- sealed by salt solutions (cf. 9)) or the ability of ghosts to give rise to sealed inside-out vesicles.

Our initial experiments indicated that glyceraldehyde-3-P dehydrogenase, like NADH diaphorase, was fully available to its substrates in sealed inside-out vesicles, but was inaccessible in resealed ghosts and sealed right-side-out vesicles (8, 9).8 Moreover, in sealed right-side-out membrane preparations the fraction of total glyceraldehyde-3-P dehydrogenase activity exposed correlates closely with values for both the diaphorase and a cyclic adenosine 3’ : 5’-monophosphate-binding site which has been shown to be an inner surface component (21). Fur- thermore, the enzyme could be eluted by PBS from unsealed ghosts and sealed inside-out vesicles, but not from resealed ghosts. These data clearly indicate that the glyceraldehyde-3-P dehydrogenase is confined to the cytoplasmic surface of the ghosts, as previously proposed (8, 9, 17).

We must, however, distinguish between two possible causes for this asymmetrical enzyme distribution, namely (a) that glyceraldehyde-3-P dehydrogenase can only be bound at the cytoplasmic surface of the membrane, or (b) that enzyme can bind at both surfaces but happened to be confined to the inner surface of the membrane because that was the side of the mem- brane closest to the cytoplasm at the moment of hypotonic hemolysis. An approach to this question was to strip the mem- brane of glyceraldehyde-3-P dehydrogenase and measure the reassociation of this enzyme with the two membrane surfaces.

Glyceraldehyde-S-P Dehydrogenase-binding Sites on Erythrocyte Membranes-Ghosts depleted of glyceraldehyde-3-P dehydro- genase bound this enzyme in a manner suggestive of a mass law relationship (Fig. 2). Scatchard plots (22) of these data (inset to Fig. 2) showed an apparent affinity constant of K = 1.0 & 0.2 X 10’ M-I and a capacity of 2.1 f 0.6 (SD.) million binding sites per ghost in six experiments. The linearity of the

4 J. Yu and T. L. Steck, unpublished observations.

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lo-’ x MEMBRANE EQUIVALENTS

FIG. 2. The binding of glyceraldehyde -3 -P dehydro- genase (GSPD) to erythrocyte membranes. Glyceraldehyde- 3-P dehydrogenase was incubated with four different membrane preparations for 90 min on ice in 0.5 ml of 5P8 containing stabilizing solutes, as indicated below. The mem- branes were then pelleted and aliquots of the supernatant taken for enzyme assay. O--O, depleted inside-out vesicles plus 125 pmoles of glyceraldehyde-3-P dehydrogenase in 5P8; O---O, depleted ghosts plus 166 pmoles of glyceraldehyde-3-P dehydro- genase in 5P8; 0-0, resealed ghosts plus 166 pmoles of glycer- aldehyde-3-P dehydrogenase in 5P8-1 mM MgS04; m----m, intact erythrocytes plus 158 pmoles of glyceraldehyde-3-P dehydrogenase in 5P8-300 mM sucrose. Controls with depleted ghosts indicated that the stabilizing solutes are not the cause of the diminished binding observed with intact erythrocytes and resealed ghosts. Inset, Scatchard plot of binding data for depleted ghosts; R = attomoles of glyceraldehyde-3-P dehydrogenase bound per ghost; A = unbound glyceraldehyde-3-P dehydrogenase (M). Data points for <lOcG and >90% glyceraldehyde-3-P dehydrogenase binding were inaccurate, hence discarded.

Scatchard plot, a more stringent test of homogeneity than “re- ciprocal plots” (23), suggests a single type of binding site. Be- cause the data points at the extremes of the binding curve were rejected in constructing this plot, this analysis cannot rule out additional classes of sites. However, direct measurement of the binding capacity with saturating levels of added enzyme gave values similar to those obtained from the Scatchard plot, i.e. 1.6 to 1.9 million sites per membrane for both standard and depleted ghosts. Hence, there is no evidence for additional classes of binding sites. Another potential source of error in this analysis is variation in the self-association state of the glyceraldehyde-3-P dehydrogenase, i.e. dissociation of enzyme tetramers into subunits or aggregation into multienzyme com- plexes. In fact sucrose density gradient centrifugation studies showed that in glyceraldehyde-3-P dehydrogenase concen- trates, some enzyme activity sedimented faster than the pre- dicted 8 S peak.4 Therefore, the binding capacity could be overestimated, perhaps as much as 2-fold.

It can be estimated from the total activity of glyceraldehyde- 3-P dehydrogenase in hemolysates that there are 1.4 to 2.4 X lo5 enzyme molecules per erythrocyte, so that at most 7 to 24% of the 1 to 2 million binding sites could be occupied in viva.

The sidedness of the glyceraldehyde-3-P dehydrogenase bind- ing site was examined by reassociation studies (Fig. 2). We ob- serve that depleted inside-out vesicles and unsealed ghosts bind the enzyme almost identically. In two experiments, inside-out vesicles appeared to have 2.0 and 2.5 x lo6 binding sites per membrane equivalent and K values of 0.6 and 1.8 x 10’ M-‘.

In contrast, intact erythrocytes and resealed ghosts bound this enzyme very poorly; meaningful Scatchard plots could not be

constructed. We have found, in addition, that rabbit muscle glyceraldehyde-3-P dehydrogenase binds nearly as well as the human erythrocyte enzyme to depleted ghosts and just as poorly to intact erythrocytes and resealed ghosts. Thus it seems that a homogeneous population of high affinity glyceraldehyde-3-P dehydrogenase binding sites is confined to the cytoplasmic sur- face of the red cell membrane.

Since most of polypeptides 1, 2, and 6 (Fig. 1) are lost from the membrane during vesicle formation (19), this material (originally termed ape&in (24)) does not appear to represent the locus of glyceraldehyde-3-P dehydrogenase attachment to the membrane. Further support of this conclusion resides in the observation that all of polypeptides 1,6, and 6 can be eluted from these membranes without the release of any glyceralde- hyde-3-P dehydrogenase monomer, Band 6 (11; CJ also Ref- erence 17).

To minimize nonspecific electrostatic associations the above binding studies were conducted at pH 8.0. Similar binding of glyceraldehyde-3-P dehydrogenase to membranes was also ob- served at pH 7.0, which more closely approximates the bulk intracellular pH. In three such experiments, depleted ghosts bound a maximum of 1.8 to 2.3 X 106 molecules of glyceralde- hyde-3-P dehydrogenase with a K of 1 to 4 x 10’ ~-1; however, unlike pH 8.0, high levels of enzyme caused an inexplicable inhibition of binding. As at pH 8.0, resealed ghosts bound little or no glyceraldehyde-3-P dehydrogenase.

Cytochrome c Binding to Erythrocyte Membranes-Several characteristics of cytochrome c made it interesting to compare with glyceraldehyde-3-P dehydrogenase. It is a strongly basic protein (25) which might be expected to bind avidly to poly- anionic membrane surfaces. Furthermore, it can be reversibly eluted from mitochondrial membranes by raising the ionic strength to physiologic levels (26,27). There are also indications that it functions physiologically on only one side of the inner mitochondrial membrane (28-30), although it can bind to both surfaces (28, 29). Cytochrome c is not found on the isolated red cell membrane,3 suggesting its utility as a nonspecific ligand in our binding studies.

Fig. 3 shows that oxidized cytochrome c bound equally well to standard and glyceraldehyde-3-P dehydrogenase-depleted ghosts. A capacity of 26 f 3 x lo6 molecules per ghost (five experiments) and an affinity constant of K = 2 to 3 X lo5 M-’

were determined. Reduction of cytochrome c with an excess of NaBH4 or NazSz04 did not alter this binding.

In four of five experiments, resealed ghosts were saturated by 7.6 to 16.2 X lo6 molecules of cytochrome c per ghost, ap- proximately half of the 26 x lo6 molecules bound to unsealed ghosts. A typical experiment is shown in Fig. 3. In contrast to the behavior of glyceraldehyde-3-P dehydrogenase, cyto- chrome c thus appears to bind to both sides of the erythrocyte membrane.

E$ect of Ionic Strength on Glyceraldehyde-S-P Dehydrogenase and Cytochrome c Binding to Membranes-While it was known that the association of glyceraldehyde-3-P dehydrogenase with the erythrocyte membrane is highly dependent on ionic strength (4, 6, 16), this relationship had not been systematically inves- tigated. Fig. 4 depicts the release of glyceraldehyde-3-P de- hydrogenase from ghosts as a function of pH and ionic strength; parallel rebinding studies give very similar results. The ionic strength dependence of elution is sigmoid in form at all pH values tested. There is a rightward shift of the elution profile with decreasing pH. It is noteworthy that near physiologic ionic strength (p = 0.15 M) the membrane binding of glyceraldehyde-

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lo- x GHOSTS IONIC STRENGTH (M)

FIG. 3 (left). The association of cytochrome c with ghosts. Cytochrome c (72.6 nmoles) was incubated with standard ghosts (O-O), depleted ghosts (O-O), and resealed ghosts (O--O) for 120 min on ice in 0.5 ml of 5P8. The membranes were then pelleted and supernatant aliquots taken for determina- tion of unbound cytochrome c. The resealed ghost incubations contained 1 mm MgS04 as a stabilizing solute; controls showed that this level of MgSOa did not significantly affect binding to standard or depleted ghosts.

FIG. 4 (center). The effect of ionic strength and pH on the binding of glyceraldehyde-3-P dehydrogenase (GSPD) to ghosts. Standard ghosts were incubated for 90 min on ice in 0.5 ml of 5 mM sodium phosphate buffers, pH 6.0 to 8.0, adjusted with NaCl to the ionic strength shown. The membranes were then pelleted

3-P dehydrogenase is negligible above pH 7.0, but becomes significant between pH 7.0 and 6.0.

The ionic strength dependence of cytochrome c binding to ghosts differed significantly from that of glyceraldehyde-3-P dehydrogenase. Its ionic strength profile was hyperbolic, not sigmoid (Fig. 5). Consequently, 50% of the cytochrome was free at p = 0.045 M while only 2% of glyceraldehyde-3-P-de- hydrogenase was released at this ionic strength. In contrast to the pH dependence of the glyceraldehyde-3-P dehydrogenase elution profile (Fig. 4), the curves for cytochrome c at pH 8.0 and 7.0 were identical.

From these data, it seemed likely that the binding of glyceral- dehyde-3-P dehydrogenase to the erythrocyte membrane in- volved stronger electrostatic interactions than did cytochrome c. To test whether this difference merely represented the ion ex- change behavior of the two proteins or reflected specificity of the membrane for the dehydrogenase, we examined the binding of these proteins to an indifferent cation exchanger. CM-cellulose was charged with one protein or the other and then incubated in salt solutions of increasing ionic strength. Fig. 5 shows that glyceraldehyde-3-P dehydrogenase was released from CM- cellulose at lower salt concentrations than it was from ghosts. Conversely, cytochrome c required much higher ionic strengths for elution from the cation exchanger than from the ghosts. Similar results were obtained at pH 7.0. Thus, the enhanced binding of glyceraldehyde-3-P dehydrogenase to the red cell membrane apparently reflects more than nonspecific electro- static adsorption.

Effect of Metabolites on Association of Glyceraldehyde-S-P Dehydrogenase with Membranes-We searched for conditions which might foster glyceraldehyde-3-P dehydrogenase binding to the membrane at physiologic ionic strength. However, none of the following agents showed evidence of promoting reassocia- tion: MgS04; Kf substituted for Na+; 1 to 3 mM 3-P-glycerate, 2,3-di-P-glycerate, adenosine 3’: 5’-monophosphate, AMP, ADP, or ATP (either alone or in the presence of 1 to 3 mM MgSOS;

IONIC STRENGTH (MI

and aliquots of supernatant taken for determination of unbound enzyme. o--o, pH 8.0; O-O, pH 7.0; m----U, pH 6.5; o---o, pH 6.0.

FIG. 5 (right). The effect of ionic strength on the association of glyceraldehyde-3-P dehydrogenase (GZPD) and cytochrome c with ghosts and CM-cellulose. Standard ghosts (O-O), standard ghosts plus cytochrome c (A-A), CM-cellulose plus glyceraldehyde-3-P dehydrogenase (O-O), and CM-cellulose plus cytochrome c (A-A) were allowed to equilibrate for 120 min on ice in 5P8 adjusted with NaCl to the ionic strength shown. Incubation volumes were 0.5 ml for experiments with ghosts, 0.6 ml for experiments employing CM-cellulose. Following centrif- ugation, the supernatant activity of these proteins was measured.

B

0 1 2 3 1 2 3

METABOLITE CONCENTRATION (mM)

FIG. 6. Release of erythrocyte membrane glyceraldehyde-3-P dehydrogenase by metabolites. Standard ghosts, 75 pl, were in- cubated for 90 min on ice in 0.5 ml of 5P8 containing increasing concentrations of different metabolites. Following centrifuga- tion, supernatant activity was determined. Metabolites were made up in 5 mM sodium phosphate and adjusted with NaOH to pH 8.0. A, m---m, glyceraldehyde-3-P; O----O, NAD+; O-O, NADH; A-A, NAD+ plus 0.1 mM glyceraldehyde-3-P; A-A, NADH plus 0.1 mM glyceraldehyde-3-P. B, O-O, NADP+; O-0, NADPH; A-A, NADP+ plus 0.1 mM glyceraldehyde-3-P; A-A, NADPH plus 0.1 mM glyceralde- hyde-3-P.

millimolar levels of glyceraldehyde-3-P, NAD+, or NADP+ (individually or in combination) ; concentrated ghost-free hemolysates, or incubation temperatures of 20” and 37”. In fact, experiments at intermediate ionic strengths (E.C = 0.055 to 0.090 M) demonstrated that submillimolar levels of certain metabolites released significant amounts of bound enzyme.

We further investigated the elution of glyceraldehyde-3-P dehydrogenase from ghosts as a function of metabolite con- centration in 5P8 (Fig. 6). (Under these conditions, the ionic

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strength increment from the added metabolites should have caused no more than 5% of the enzyme elution observed. Of the metabolites examined, only ATP (see below) affected the activity of glyceraldehyde-3-P dehydrogenase in our assay.)

Glyceraldehyde-3-P in concentrations up to 2 mu eluted no enzyme from the membrane by itself, but profoundly in- fluenced the effect of certain other metabolites (Fig. 6A).

The NAD+ elution curve of glyceraldehyde-3-P dehydrogenase was comprised of two components which met at approximately 0.2 mu. Glyceraldehyde-3-P, 0.1 mM, markedly potentiated enzyme release by NAD+ and caused the elution curve to be- come more regularly hyperbolic (Fig. 6A).

The effect of NADH resembled that of NAD+ plus glyceralde- hyde-3-P. It was more potent than NAD+ alone; full elution was attained at 1 mu. The presence of 0.1 mM glyceral- dehyde-3-P further potentiated elution by NADH (Fig. 6A).

NADP+ and NADPH exhibited sigmoid curves for glyceralde- hyde-3-P dehydrogenase elution (Fig. 6B). They were far less potent than NADH or low concentrations of NAD+. The addition of 0.1 MM glyceraldehyde-3-P, furthermore, caused no significant change in elution profile.

Other metabolites were tested (Table II). 3-P-glycerate, adenosine 3’) 5’-monophosphate, AMP, and ADP caused little or no elution (-10 %), while ATP and 2,3-di-P-glycerate showed strong elution (-50%). The presence of 0.1 mru glyceralde- hyde-3-P markedly potentiated elution by AMP, ADP, and ATP, had a small effect on adenosine 3’, 5’-monophosphate, and no effect on 3-P-glycerate and 2,3-di-P-glycerate action. Since ATP both elutes and inhibits glyceraldehyde-3-P dehydrogenase activity (17) by dissociating the tetramers into subunits (31), the percentages of enzyme released by ATP are probably under- estimated.

To examine the effects of these metabolites on cytochrome c binding, standard ghosts were incubated with an excess of cyto- chrome c and washed free of the unbound cytochrome. Levels of NAD+, NADH, and NADP+ which eluted nearly all of the glyceraldehyde-3-P dehydrogenase released less than 10 y0 of the cytochrome.

We also investigated the effects of metabolites on glyceralde- hyde-3-P dehydrogenase and cytochrome c binding to CM-cel-

TABLE II

Elution of erythrocyte membrane glyceraldehyde-3-P dehydrogenase by metabolites

Standard ghosts, 75 ~1, were incubated for 90 min on ice in 0.5 ml of 5P8 containing various metabolites (1 mM) in the presence or absence of 0.1 mM glyceraldehyde-3-P. The membranes were then pelleted and aliquots of supernatant assayed for enzyme activity. Metabolite stocks were made up in 5 mM sodium phos- nhate and adiusted with NaOH to pH 8.0.

Metabolite

Percentage of glyceraldebyde-3-P dehydrogenase soluble

- Glyc$dehyde- + Glycy-idehyde-

None ....................... 3-P-glycerate .............. Adenosine 3’,5’-monophos-

phate ..................... AMP ....................... ADP ...................... ATP ....................... 2,3-di-p-glycerate . ........

0 0

9 17 3 26 6 54

52 86 56 51

0 0

lulose. NAD+ or NADH, 0.1 to 0.3 111~, eluted >80% of glyc- eraldehyde-3-P dehydrogenase but no cytochrome c from CM- cellulose. These data suggest that the metabolites eluted the enzyme from ghosts by interacting with the enzyme rather than the membrane. The desorption of glyceraldehyde-3-P dehy- drogenasefrom CM-cellulose by small amountsof certain effecters suggests that selected metabolites might be useful in the puri- fication of this and other enzymes from tissue extracts on cation exchange columns.

DISCUSSION

We are confronted with the question of whether glyceralde- hyde-3-P dehydrogenase has a meaningful physiologic association with the erythrocyte membrane. Its strong association at low ionic strength is not, of itself, impelling evidence for in viva bind- ing, since physiologic salt solutions readily elute the enzyme from the isolated membrane. However, other indices examined here suggest the presence of specific glyceraldehyde-3-P dehy- drogenase binding sites on the erythrocyte membrane. Com- parison with the binding of cytochrome c strengthens this hy- pothesis. These points should be considered:

Soluble glyceraldehyde-3-P dehydrogenase was specifically bound at the inner surface of the membrane (Fig. 2), in contrast to the bilateral uptake of cytochrome c (Fig. 3). The presence of the dehydrogenase on the inner surface of the membranes as isolated was not the result of the soluble cytoplasmic enzyme becoming adsorbed to the nearest surface available, but reflects an asymmetry in the apportionment of binding sites between the two faces of the membrane. The small apparent binding of glyceraldehyde-3-P dehydrogenase to intact red cells and re- sealed ghosts could reflect (a) a commensurate accessibility of the inner membrane surface; (b) adsorption at the outer surface; or (c) an unspecified loss of 0 to 10% of supernatant enzyme activity without binding.

The binding of glyceraldehyde-3-P dehydrogenase to the inner membrane surface in 5P8 gave linear Scatchard plots (Fig. 2 inset), suggesting a single class of equivalent and independent sites with a high affinity (1 X 10’ M-I) for the enzyme. Since saturation studies yield similar capacity values, it seems unlikely that we are overlooking other, low affinity binding sites.

The binding of glyceraldehyde-3-P dehydrogenase and cyto- chrome c to both erythrocyte ghosts and CM-cellulose appears to involve electrostatic interactions, as judged by their de- pendence on ionic strength (Fig. 5). The binding of glyceralde- hyde-3-P dehydrogenase to CM-cellulose is significantly weaker that that of cytochrome c; the binding of glyceraldehyde-3-P dehydrogenase to ghosts is significantly stronger than that of cytochrome c. We interpret this contrast in behavior to indicate that the enzyme-membrane association involves more than nonspecific coulombic interactions.

It can be calculated from the dimensions of glyceraldehyde-3-P dehydrogenase5 that the 1 to 2 x lo6 molecules bound per ghost at saturation would project an area of -37 to 74 square p or 28 to 55% of the roughly 132 square p of inner membrane sur- face (34). Saturating levels of cytochrome c (i.e. 26 x lo6 molecules) would project an areas of -190 square p or -72% of the roughly 264 square p of surface available at the two sides of the membrane. That the area covered by cytochrome c at

6 A radius of 35 A was estimated for this roughly spherical mole- cule from both x-ray crystallographic (32) and partial specific volume (33) measurements. A radius of 15 A for cytochrome c could also be estimated from x-ray crystallographic (35) and par- tial specific volume data (25).

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saturation exceeds that available at either membrane surface constitutes further evidence for its association with both sides of the membrane.

So large a fraction of surface covered raises the possibility that it is steric hindrance due to close packing (i.e. monolayer formation) rather than a fixed number of specific sites which limits the binding of these two ligands. Certainly the dense binding of cytochrome c is compatible with the other evidence for its nonspecific adsorption. In the case of glyceraldehyde-3-P dehydrogenase, existing albeit indirect evidence favors specific binding rather than monolayer formation. Its saturation density falls short of blanket coverage. Furthermore, in mono- layer formation we would expect bound molecules to interfere with further binding as saturation is approached, thus leading to a loss of apparent binding site equivalence, hence mid-zone curvature of the Scatchard plot (22, 23). However, the Scatchard plot is linear (Fig. 2, inset).

Preliminary experiments in this laboratory4 indicate that glyceraldehyde-3-P dehydrogenase is bound to Band S, the predominate polypeptide species in the erythrocyte membrane (cf. Fig. 1 and Reference 11). Triton X-100 extracts of ghosts contain both Bands S and 6 in a form which sediments in sucrose gradients more rapidly than expected for either component alone (36). This putative complex is not seen in the presence of 0.15 M NaCl, which is known to release the enzyme from its membrane-binding site. The estimate of 2 X lo6 binding sites per ghost for glyceraldehyde-3-P dehydrogenase agrees well with the 1.2 x lo6 Band S polypeptides per ghost estimated previously (ll), considering the inaccuracies inherent in each measurement and our observation that enzyme aggregation may have caused up to a a-fold overestimate of attachment sites (see “Results”). In fact, recent experiments using purified components have indicated a 1:1 stoichiometry between the enzyme molecule and the Band S polypeptideP The require- ment that the glyceraldehyde-3-P dehydrogenase binding locus be present only at the membrane’s inner face is satisfied by Band S, which appears to span the membrane thickness asymmetrically (cf. 8,37). If, in fact, Component S specifically binds glyceralde- hyde-3-P dehydrogenase, it fulfills the prediction of a homo- geneous, high density ligand site at the cytoplasmic side of the membrane and obviates the argument for monolayer formation.

The binding of glyceraldehyde-3-P dehydrogenase, but not cytochrome c, to ghosts was profoundly influenced by low con- centrations of certain metabolites. The fact that these effecters are either substrates or products of this enzyme or are structurally related compounds suggests that they could act by competing for the enzyme’s catalytic site with a complementary (i.e. active site-directed) receptor on the membrane. Alternatively, these elution effects might result from conformational changes in the enzyme, since NAD+ and glyceraldehyde-3-P cause structural changes upon binding to soluble rabbit muscle glyceralde- hyde-3-P dehydrogenase (38-40), and similar effects have been inferred for AMP, ADP, and ATP binding to the yeast enzyme (41). Specificity is apparent in the profound difference in potency between 2,3-di-P-glycerate and 3-P-glycerate (Table II), and between adenine nucleotides lacking and possessing a phosphate in the 2’ position. NADP+ and NADPH are dis- tinguished from the other nucleotides, furthermore, by their sigmoid elution profiles and the lack of potentiation by glyceraldehyde-3-P.

Despite evidence for specificity of association, we must still ponder how glyceraldehyde-3-P dehydrogenase could bind to the membrane in GUO, given that cytoplasmic ionic strength

and metabolite levels promote its dissociation in vitro (as shown in Figs. 4 and 6 and Table II). Indeed, no known physiologic requirement demands the association of glyceraldehyde-3-P dehydrogenase with membranes. However, cytochrome c clearly performs its function while membrane-bound, yet it too is quantitatively desorbed from the inner mitochondrial mem- brane by solutions of p = 0.15 M (26, 27). Mere desorption by physiologic salt solutions in titro clearly does not exclude meaningful protein-membrane association in viva. We may well be ignorant of factors which foster association in the cell. For example, in wivo glycolytic activity in the submembrane space could lower the local pH and thus significantly augment glyceraldehyde-3-P dehydrogenase binding (cf. Fig. 4). Our dissociation studies were also biased by mass law effects toward desorption, since elution was performed in a volume of medium roughly 10 times greater than that in the cytoplasmic space. Alternatively, the physiologic mandate may be such that only a small fraction of the glyceraldehyde-3-P dehydrogenase mole- cules should be bound at any instant.

Our data support the hypothesis that the glyceraldehyde-3-P dehydrogenase of the human erythrocyte may partition between the cytoplasm and specific membrane sites in a manner which is responsive to local variations in pH, ionic strength, and metabo- lite concentrations. The “purposes” which might be served by the binding of glycolytic enzymes to the membrane (such as a proximal energy source for active transport) have been the sub- ject of previous speculation (3, 6, 42), but remain to be experi- mentally defined.

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Jeffrey A. Kant and Theodore L. SteckIsolated Human Erythrocyte Membranes

Specificity in the Association of Glyceraldehyde 3-Phosphate Dehydrogenase with

1973, 248:8457-8464.J. Biol. Chem. 

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