THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260. No. 11, Issue of June 10, pp. 68924898,198.5 Printed in U.S.A.

Interaction of Glyceraldehyde-3-phosphate Dehydrogenase with Isolated Microsomal Subfractions of Skeletal Muscle”

(Received for publication, August 2, 1984)

Anthony H. Caswell and Adrian M. Corbett From the Department of Pharmacology, University of Miami School of Medicine, Miami, Florida 33152

A protein of subunit M, 34,000 (corrected here and subsequently to 36,000) has been isolated from a mus- cle homogenate which catalyzes the formation of the triad junction from isolated transverse tubules and terminal cisternae. This protein is identified as glyc- eraldehyde-3-phosphate dehydrogenase on the basis of N-terminal amino acid sequence, amino acid composi- tion, and enzymic activity. The oxidation of glyceral- dehyde phosphate by the enzyme is slowly but progres- sively inhibited by terminal cisternae, longitudinal re- ticulum, and transverse tubules. Addition of Triton X- 100 to terminal cisternae markedly enhances and ac- celerates the inhibition. Two distinct constituents of microsomes exhibit a progressive inhibition. One com- ponent has been fractionated on a hydroxyapatite col- umn and was identified as calsequestrin. Calsequestrin reveals an immediate inhibition of glyceraldehyde phosphate oxidation which can be reversed by concen- trations of CaCL below millimolar levels or by high ionic strength. Isolated terminal cisternae contain glyceraldehyde-phosphate dehydrogenase which can be extracted by high ionic strength.

A normal preparation of skeletal muscle microsomes con- tains extrinsic proteins which may be eluted with KCI. Camp- bell et ul. (1) have described two such proteins with subunit molecular weights of 38,000 and 34,000 which associate spe- cifically with the terminal cisternae of sarcoplasmic reticulum. They have argued on the basis of electron microscopy that these proteins are the junctional processes which connect the terminal cisternae to transverse tubules. Protruding struc- tures were visible on the cytoplasmic side of terminal cisternae membranes which disappeared after extraction of these two proteins with KCI. We have observed that isolated terminal cisternae contain a thickened external leaflet which appeared to be associated on the cytoplasmic side to the attachment of electron-dense matter on the luminal side of the vesicle (2). KC1 treatment caused the loss of this thickened external leaflet while the electron-dense matter within the lumen of the vesicle became diffuse. I t is likely that this thickened membrane observed in our studies is similar to the protruding processes observed by Campbell et al. (1). It is not, however, clear at the present stage whether these protruding processes or thickening correspond to the organized array of junctional processes seen in the triad of intact muscle.

In a previous paper we have evaluated the dynamics and

* This work was supported by Grant AM 21601 and Training Grant HL 07188 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

structure of the triad junction from a different viewpoint. We have found in the past that isolated transverse (TI) tubules and terminal cisternae (TC) are capable of reassociating in the presence of certain specific salts. The most prominent of these is K cacodylate (3). We have found a physiological correlate of this process through the observation that triad reformation may occur in the presence of a soluble fraction from a homogenate of skeletal muscle. We have purified this factor to homogeneity and demonstrated that it is a protein of subunit molecular weight 34,000 and native molecular weight approximately 140,000.* Circumstantial evidence based on a comparison of their gel electrophoresis data with ours suggests that this protein is, in fact, identical to the extrinsic protein of subunit molecular weight 34,000 described originally by Campbell et ul. (1). Nevertheless, our isolation protocol has demonstrated that this protein is a major con- stituent of the intact muscle.

In this paper we identify this protein as glyceraldehyde-3- P dehydrogenase and demonstrate the degree and specificity of interaction of this enzyme with different microsomal mem- branes. Glyceraldehyde-3-P dehydrogenase has been de- scribed by several authors to exhibit a specific interaction with a number of different proteins either of the membrane or other subcellular structures (4-11). Extensive studies have been carried out on the interaction between the enzyme (band VI) and the anion channel of the erythrocyte (band 111) (4- 9). The interaction of the glyceraldehyde-3-P dehydrogenase with skeletal muscle microsomes bears some resemblance to its interaction with the anion channel of erythrocytes.

MATERIALS AND METHODS

Preparations of Vesicles-The preparations of TC/triads and lon- gitudinal reticulum from rabbit sacrospinalis muscle is as described by Lau et al. (12). The preparation of T-tubules and light and heavy TC is described by Brandt et al. (13).

Isolation of Endogenous Promoter Protein-The promotor protein was isolated according to the technique of Corbett et a1.’ A rabbit was killed by a blow on the head and bled, and the sacrospinalis muscles immediately were excised and homogenized for 1.5 min in a Waring blender which contained 150-170 ml/muscle (approximately 50 g) of 0.1 M KCI, 10 mM Tris CI, pH 7.0, and 0.5 mM Tris-EDTA, pH 7.0. The homogenate was centrifuged at 10,000 X g for 20 min and the supernatant was centrifuged at 100,000 X g for 1 h. The supernatant was fractionated using (NH,),SO, precipitation as described earlier? The fraction sedimenting between 75 and 100% (NH.),SO, saturation was retained and resuspended in 10 ml of a solution of 5 mM histidine, pH 7.0. This sample was then applied to a Bio-Rad Bio-Gel HTP hydroxyapatite column (2.5-cm diameter, 40-cm length) which had been preincubated in 5 mM histidine, pH 7.0. The column was eluted using a constant volume mixing chamber (550 ml) into which was infused 0.7 M Na2HP04, pH 7.0. The protein which eluted 250 ml

The abbreviations used are: T-tubules, transverse tubules; TC,

’ A. M. Corbett, A. H. Caswell, N. R. Brandt, and J.-P. Brunschwig, terminal cisternae.

submitted for publication.

6892

Glyceraldehyde-phosphate Dehydrogenase in Muscle Microsomes 6893

after the void volume was collected and concentrated by (NHMO4 precipitation as described previously? The concentrated protein was chromatographed on a Sephacryl S200 (Pharmacia) Column (2.5-cm diameter, 60-cm length) employing 5 mM histidine, pH 7.0. The protein containing sample from this column was employed directly in the subsequent experiments or was dialyzed against 4 liters of 5 mM histidine, pH 7.0.

Assay of Glyceraldehyde-3-phosphate Dehydrogenase Actioity-Me- dia employed for the enzyme assay are similar to those of Tsai et al. (8). Assays involving determination of the inhibition of enzyme activity by membranes or membrane fractions employed a medium containing 2 ml of 10 mM imidazole, 10 mM Na acetate, 0.1 mM Tris- EDTA, 0.1 mM Na2HAs04, 1 mM dithherythritol, 0.1 mM NAD+. The medium was adjusted to pH 7.0 with acetic acid. Temperature was 22 “C. The vesicle or protein was added as described in the figure and 30 s later the reaction was initiated by the addition of 0.1 mM glyceraldehyde 3-phosphate adjusted to pH 7.0 using NaOH. Produc- tion of NADH was monitored employing an Eppendorf fluorometer with excitation wavelength 365 nm and a 400 nm ultraviolet cutoff filter. The production of NADH was nonlinear and in most experi- ments the NADH formed in 135 s was employed as a measure of enzyme activity.

The medium for the assay of eluted extrinsic protein employed 30 mM imidazole acetate, pH 7.8, 1 mM dithioerythritol, 0.4 mM NazHAsO,, 1 mM NAD+, 0.1 M NaCl. Temperature was 22 “C. The reaction was started by the addition of 1 mM glyceraldehyde 3- phosphate, pH 7.0, and assayed fluorimetrically as described above.

Extraction and Fractionation of Terminal Cisternae Proteins-A preparation of TC/triads from a continuous sucrose density gradient (32 mg of protein/ml) was assayed for protein by the method of Bradford (14), and 2 mg of Triton X-lOO/mg of vesicle protein was added. The suspension was diluted with 0.25 M sucrose, 2 mM histi- dine, pH 7.0, and centrifuged at 125,000 X g for 60 min. The pellet was suspended in a small volume (0.5-1.0 ml) of 250 mM sucrose, 2 mM histidine, pH 7.0. Protein was estimated by the method of Bradford (14) and 2 mg of Zwittergent 3-14 (Calbiochem-Behring)/ mg of protein was added. The suspension was then adjusted to 0.3 M by addition of 2 M NaC1. The sample (10 mg of protein out of 40 mg total) was passed through a Bio-Rad Bio-Gel HTP hydroxyapatite column (0.9-cm diameter, 40-cm length). The column had been pre- equilibrated with a solution containing 0.2 M NaCl, 0.2% Lubrol PX (Sigma), 4 mM Tris-C1, pH 7.0. The column was developed employing a constant volume mixing chamber (125 ml) containing pre-equili- bration buffer into which was infused a solution of 1 M Na2HP0,, pH 7.0. Samples from the column were collected and dialyzed twice against 4 liters of 10 mM imidazole acetate, pH 7.0, for 12 h. The procedure was similar for fractionation of material which dissolved in Triton X-100 except that 2 ml (out of 50 ml) of Triton supernatant was applied directly to the hydroxyapatite column and eluted in the same way. When calsequestrin was isolated, the Triton-resistant TC/ triad fragments were dissolved and chromatographed on the hydrox- yapatite column above. The calsequestrin-containing fractions were pooled and dialyzed as described.

RESULTS

Identification of the Protein Which Promotes the Formation of the Triad Junction from Isolated Organelles as Glyceralde- hyde-3-phosphate Dehydrogenuse-The N-terminal sequence of the promotor protein has been identified by Edman deg- radation (15) as Val-Lys-Val-Gly-Val-Asn-Gly-Phe-Gly-Arg- Ile-Gly-Arg-Leu-Val-Phe-Arg-Ala-Phe. This sequence is identical to that of rabbit and porcine glyceraldehyde-3-P dehydrogenase. The amino acid composition of the promotor protein is shown in Table I. The amino acid composition determined for rabbit and porcine skeletal muscle glyceral- dehyde-3-P dehydrogenase is presented for comparison and within the limits of experimental error, the compositions of the two proteins may be considered to be identical. Glyceral- dehyde-3-P dehydrogenase forms a tight complex with NAD+ which is associated with a discrete absorbance band with a maximum at approximately 365 nm (18). A spectrophoto- metric scan indicates that this band is present in the promotor protein and is at least equal in intensity to that of an authentic

TABLE I Amino acid composition of the 34-kDapromotor protein

G3PDH, glyceraldehyde-3-P dehydrogenase.

Amino acid Promotor Rabbit Porcine protein GBPDH” G3PDHb

residueslmol

Cysteine 4.0 3.0 4 Aspartic acid 36.0 35.5 39 Threonine 18.2 21.6 22 Serine 16.6 19.7 17 Glutamic acid 19.0 18.7 19 Proline 10.2 11.9 13 Glycine 31.2 31.7 34 Alanine 30.9 32.6 33 Valine 29.1 31.2 30 Methionine 7.6 8.4 9 Isoleucine 16.1 15.3 18 Leucine 16.9 17.8 18 Tyrosine 8.0 8.6 9 Phenylalanine 13.0 12.9 14 Histidine 9.9 9.8 11 Lysine 22.8 23.9 27 Arginine 9.4 10.1 10

Based on data from Velick and Furfine (16) using a subunit M, - of 34,000.

unit M, of 36,000. Published values from Harris and Perham (17) assuming a sub-

“I

FIG. 1. Reduction of NAD by the promoter protein in the absence and presence of TC/triads. Incubation conditions for the enzyme are as describedunder “Materials and Methods.” The medium contained 40 pg of promotor protein ( S ) or glyceraldehyde-3-P de- hydrogenase (G3PDH) (Sigma). Where stated, TC/triads (240 pg of protein) and 1% Triton X-100 were added to the incubation medium.

sample of glyceraldehyde-3-P dehydrogenase from Sigma (data not shown). Fig. 1 shows an assay of enzymic activity of the promotor protein in comparison with that of an au- thentic sample of the enzyme. Again it is seen that the activity matches that of the authentic enzyme. The isolated protein appears as a single discrete band in sodium dodecyl sulfate- gel electrophoresis? The subunit and the native molecular weights of the promotor protein are nearly identical to those of glyceraldehyde-3-P dehydrogenase’ based on sodium do- decyl sulfate-polyacrylamide gel electrophoresis and equilib- rium analytical centrifugation. We have previously identified the protein by its subunit molecular weight at 34,000. How- ever, the molecular weight based on amino acid sequence is 36,000 (17) and we therefore correct our subunit M, subse- quently in this paper.

6894 Glyceraldehyde-phosphate Dehydrogenase in Muscle Microsomes

Fig. 1 also shows the influence on the enzymic activity of adding TC/triad vesicles. The intact vesicles cause a slight but progressive inhibition of NAD reduction. This progressive effect may cause complete inhibition in a period of 20 min. In contrast, Triton-treated TC/triads cause an immediate (less than 1 min) inhibition of glyceraldehyde-3-P dehydrogenase activity.

Inhibition of Glyceraldehyde-3-P Dehydrogenase by Micro- somal Subfractions-Fig. 2 shows the influence of a prepara- tion of TC/triads on the glyceraldehyde-3-P dehydrogenase enzymic activity as a function of concentration of vesicles, time of incubation, and presence or absence of detergent. The figure emphasizes that inhibition of enzymic activity is pro- gressive, being approximately three times higher after 3 min of incubation than after 30 s. Further inhibition occurs with increased periods of incubation. A very potent inhibition of enzymic activity is manifested if the vesicles are treated with Triton X-100. The extent of this inhibition is less dependent on time of incubation as shown in the figure after 30 s of incubation. A possible role of the detergent may be to expose sites or proteins to which the enzyme would not normally have access in the intact vesicle.

Fig. 3 shows a similar experiment carried out on the differ- ent microsomal subfractions of muscle, In the absence (panel A ) and presence (panel B ) of the detergent Triton X-100, all the particulate subfractions of muscle cause some degree of inhibition of the enzyme but the heavy TC conspicuously differs from the other fractions in initiating a substantial inhibition in the absence of detergent. This preparation has been prepared by passing TC/triads through a French press, reforming the junction with potassium cacodylate and passing through a French press a second time. It is, therefore, possible that the greater activity of these vesicles reflects a loss of membrane integrity, giving rise to greater accessibility of the enzyme to internal sites or proteins of interaction. Panel B shows a considerable variation in the potency of the different microsomal subfractions in inhibiting the enzyme in the pres- ence of Triton X-100. Heavy terminal cisternae are more potent than TCltriads and these are more potent in turn than

the other microsomal subfractions, longitudinal reticulum, light terminal cisternae, and T-tubules. Since the kinetics of the inhibition are complicated, we have not attempted to quantitate the kinetic parameters. It is possible that the light terminal cisternae have a small number of highly potent sites for inhibition since the inhibition is linear as a function of concentration of the vesicles in the presence and absence of Triton X-100.

Glyceraldehyde-3-P Dehydrogenase as an Extrinsic Mem- brane Protein-The presence in skeletal muscle microsomes of an extrinsic protein with subunit molecular weight 36,000 suggests the possibility that this protein may be identical to the glyceraldehyde-3-P dehydrogenase isolated from a muscle homogenate. Fig. 4 demonstrates that a variety of salts are able to effect the release of glyceraldehyde-3-P dehydrogenase from a preparation of TC/triads. The maximum releaseable enzyme activity corresponds to 37 pg of enzyme/mg of vesicle protein. The most potent salt (lowest effective concentration) is KSCN followed by potassium gluconate, KCl, and potas- sium cacodylate. KC1 and K gluconate both elicit the same maximum enzyme release. Potassium cacodylate is both less potent and less efficacious since the maximum activity re- leased with this salt is approximately 20 pg/mg of protein. Potassium cacodylate at low concentration also partly inhibits the intrinsic release of glyceraldehyde-3-P dehydrogenase ob- served without salt addition. KSCN appears to be ineffective at high concentrations. However, it is likely that this salt is eliciting the release not only of the extrinsic proteins but also of endogenous inhibitors of the enzyme. Therefore, the low activity observed at high concentrations of KSCN may not reflect the real release of enzyme but rather the concomitant release of both enzyme and inhibitor.

Fractionation of Inhibitors of Glyceraldehyde-3-P Dehydro- genase-Fig. 5 shows the influence on the activity of glycer- aldehyde-3-P dehydrogenase of sarcoplasmic reticulum junc- tional membranes which have been extracted and fractionated by hydroxyapatite chromatography. The junctional fragments were first prepared by treating a preparation of TC/triads with Triton X-100 to dissolve nonjunctional membrane pro-

FIG. 2. Inhibition of glyceralde- hyde-3-P dehydrogenase (G3PDH) activity by TC/triads. Incubation con- ditions are as in Fig. 1.34 pg of promotor protein was employed and, where stated, 1% Triton X-100 was included. Inhibi- tion was estimated from the slope of the curve of NADH production at the time stated and compared with that in the absence of vesicles.

I I

A

2 0 4 0 60 mg protein/mg G3PDH

Glyceraldehyde-phosphate Dehydrogenase in Muscle Microsomes 6895

7- - 1 A

10 2 0 mg protoln/mg G3PDH

FIG. 3. Influence of different microsomal subfractions on glyceraldehyde-3-P dehydrogenase (G3PDH) activity. Micro- somal subfractions were prepared as described before (11, 12) and resuspended in 250 mM sucrose, 2 mM histidine, pH 7.0. Heavy TC was the fraction obtained after rejoined triads were broken in the French press the second time (12). The incubation medium contained 40 pg of promotor protein and in panel B 1% Triton X-100. LTC, light terminal cisternae; HTC, heavy terminal cisternae, LR, longi- tudinal reticulum, TT, transverse tubules.

teins such as the calcium pump. The precipitate was dissolved by treating it with the neutral detergent, Zwittergent 3-14, in the presence of NaCl. The sample was then applied to a hydroxyapatite column and eluted employing a continuous phosphate gradient. This permits excellent separation of the constituents of the triad. The protein scan shows that a number of more or less separated protein bands may be resolved on the column. Glyceraldehyde-3-P dehydrogenase activity in the presence of dialyzed fractions from the column shows that one major band from the column caused inhibition of the enzyme. Extensive inhibition is conferred by a protein which is eluted with high phosphate concentration. The gel electrophoretic pattern shows sequential elution of proteins of M, values 38,000,55,000, 45,000,80,000, 34,000 and 62,000. The following evidence indicates that 62,000 protein is cal- sequestrin. l) The protein migrates electrophoretically with the major protein band of terminal cisternae. 2) The Coomas- sie staining from the column indicates a major protein. Cal- sequestrin is a major protein of TC and from TC fragments resistant to Triton X-100 dissolution. 3) The band from the

column is strongly attached to hydroxyapatite. Stewart and MacLennan (19) have previously demonstrated a similar elu- tion of calsequestrin from the column. Our gel pattern indi- cates that the calsequestrin obtained from the column is essentially pure. The inhibition of enzymic activity is almost complete in the peak calsequestrin fractions from the column. The interaction of glyceraldehyde-3-P dehydrogenase with the TC/triad material which is solubilized by Triton X-100 is shown in Fig. 6. The material was chromatographed on a hydroxyapatite column and eluted under identical conditions to Fig. 5. The major protein peak which elutes first from the gel is associated with inhibitory activity. This inhibition dif- fers from that of calsequestrin in being slow in onset. Thus, little inhibition was observed in the first minute after incu- bation but, by 5 min, inhibition was nearly complete. The inhibition is not caused by an action of a protease on the enzyme since subsequent addition of 0.2 M KC1 prevents the inhibition. This resembles the pattern observed in intact organelles but differs from that observed with Triton-treated vesicles. Gel electrophoresis of this sample (data not shown) indicated that a number of protein constituents are present and we have not attempted to purify the inhibitor of glycer- aldehyde-3-P dehydrogenase from this sample.

Fig. 7 shows the inhibition of glyceraldehyde-3-P dehydro- genase, as a function of calsequestrin concentration, in the absence and in the presence of CaC12. In the absence of CaC12 the curve demonstrates a nonlinearity of inhibition at low concentrations of calsequestrin which suggests positive coop- erativity. The strength of the inhibition is demonstrated by the sharp curvature of the plot at higher concentrations of calsequestrin where inhibition is almost complete. If one assumes that at the lower concentrations calsequestrin is completely bound to the enzyme, then extrapolation of this curve to complete inhibition (dotted line) gives a stoichiometry of interaction of 0.36 mg of calsequestrin/mg of enzyme. Assuming the native molecular weight of calsequestrin is 62,000 and glyceraldehyde-3-P dehydrogenase is 140,000, the stoichiometry of interaction is 0.77 mole of calsequestrin/mol of glyceraldehyde-3-P dehydrogenase. The curve of the inhi- bition is clearly complex and is not simply described by a single site interaction. The presence of Ca2+, which is known to form a complex with calsequestrin, diminishes the potency of inhibition induced by calsequestrin. Again the curve is complex but the positive cooperativity seen in the absence of CaC12 is not seen when CaC12 is added. The dash l i n e shows the influence of varying the concentration of CaClz on the inhibition of the enzyme elicited by a single concentration of calsequestrin. The release of inhibition caused by CaCIz is linear in the range from 0 to 500 p~ CaC12. The subsequent nonlinearity could be attributed either to the fact that most of the inhibition has already been relieved at these concentra- tions of Ca2+ or to a possible saturation of the binding of CaCI2 to the calsequestrin.

DISCUSSION

The finding that the isolated protein, which promotes the formation of the triad junction from the isolated organelles, is in fact glyceraldehyde-3-P dehydrogenase places the prior observations on the role of the 36,000 protein of sarcoplasmic reticulum in a new perspective. Glyceraldehyde-3-P dehydro- genase has been studied by a number of workers for its binding to both membrane proteins and cytoplasmic structural pro- teins. The current evidence suggests the enzyme has a specific affinity for highly acidic regions of certain proteins (7). The binding of glyceraldehyde-3-P dehydrogenase to proteins is associated with an inhibition of the enzymic activity (8).

6896 Glyceraldehyde-phosphate Dehydrogenase in Muscle Microsomes

0.1 0 . 2 0.3 0.4

M K anion + FIG. 4. Release by salts of glyceraldehyde-3-P dehydrogenase (C3PDH) from terminal cisternae

vesicles. Heavy TC were prepared as in Fig. 3. Vesicles (0.6 ml) in 0.2 ml of 250 mM sucrose, 2 mM histidine, pH 7, were centrifuged for 30 min a t 30 p.s.i. in an A 100 rotor of a Beckman Airfuge in the presence of the salt buffered at pH 7.0. The assay medium is described under "Materials and Methods." 50 pl of the supernatant from the centrifugation was employed. The G3PDH activity in the assay was compared under identical incubation conditions with that of an authentic sample from Sigma of rabbit muscle origin. 1 unit of activity corresponds to the activity of 1 pg of pure enzyme assayed under identical conditions.

M Phosphate FIG. 5. Hydroxyapatite fractiona&n of extracted junctional proteins and their influence on glyc-

eraldehyde-3-P dehydrogenase (GSPDH) activity. The preparation of TC/triad fragments and separation by hydroxyapatite chromatography are described under "Materials and Methods." The incubation medium was as described. 500 pl from each dialyzed fraction was added to the medium. Protein was assayed by the folin method to which was added 0.01% sodium dodecyl sulfate. Gel electrophoresis was by the method of Laemmli (20) using 8% acrylamide and 75 pl of protein samples.

Both the binding and the inhibition of enzymic activity can unidentified constituent. The second constituent differs from be reversed by high ionic strength and in acid media (6). Our calsequestrin in exhibiting a very slow but progressive action own observations are that glyceraldehyde-3-P dehydrogenase on the enzyme. In this regard it resembles the inhibition shows a specific association with calsequestrin and a second elicited by intact sarcoplasmic reticulum or T-tubule vesicles.

Glyceraldehyde-phosphate Dehydrogenase in Muscle Microsomes

FIG. 6. Hydroxyapatite fraction- ation of TC/triad samples which dis- solve in Triton X-100. The assay con- ditions are described under “Materials and Methods” and Fig. 5. The enzyme assay was commenced 30 s after addition of fraction to the glyceraldehyde-3-P de- hydrogenase (G3PDH) assay medium, and activity during 135s assay was mea- sured.

FIG. 7. Effect of calsequestrin on glyceraldehyde-3-P dehydrogenase (G3PDH) activity. Assay conditions are as described under “Materials and Methods.” 60 pg of promotor protein was employed and, where stated, 500 pM CaC1, or 20 gg of calsequestrin was in- cluded. The solid truces are plots of ac- tivity against calsequestrin concentra- tion while the dashed plot is against CaC12. The dotted trace is an extrapola- tion of the data.

L

I I I

I ’ 0 - * = - w

e .

-

‘. a

I , ‘. m . ”“ ”. .__ - ._ -.-.-.-.‘c‘

0.1 0.2 0.3 0.4 M phosphate

pM CaCI2

200 400 600 800 I 1 I I

I 1 I 0.5 1 .o 1.5 mg cal8equartr~n/mg G3PDH

Two questions may be raised concerning the interaction of glyceraldehyde-3-P dehydrogenase with calsequestrin. First, does the enzyme which is considered to be cytoplasmic have access in vivo to calsequestrin which is considered to be luminal? Second, does the interaction which may occur in low ionic strength environments also occur in the physiological milieu?

The location of calsequestrin in the sarcoplasmic reticulum has been debated over a number of years. MacLennan and Wong (20) originally proposed that the protein was hydrogen- bonded to the interior of the sarcoplasmic reticulum mem- brane. Thorley-Lawson and Green (21) demonstrated that calsequestrin could be labeled with iodine employing lacto- peroxidase as a catalyst. Morrison (22) has argued that iodi- nation induced by lactoperoxidase occurred at exposed sites in the membrane surface. Thorley-Lawson and Green have argued, on the basis of these experiments and the finding that calsequestrin can be extracted from the sarcoplasmic reticu- lum with EDTA, that calsequestrin is localized on the external side of the membrane leaflet. Jorgensen et al. (23) have demonstrated, employing antibodies directed against calse- questrin, that calsequestrin is located in the lumen of the sarcoplasmic reticulum. Meissner demonstrated that the in-

ternal electron-dense matter of isolated TC was composed of calsequestrin. Thus, the preponderant evidence is that calse- questrin resides, at least predominantly, in the lumen of the sarcoplasmic reticulum. However, this does not preclude a disposition of a portion of the calsequestrin within the mem- brane of the vesicle. Campbell et al. (1) and Brunschwig et al. (2) have observed that the electron-dense matter within iso- lated sarcoplasmic reticulum may be condensed near the membrane surface, implying that calsequestrin is not present within the vesicle in solution in the aqueous lumen. This view is further supported by the fact that vesicles treated with neutral detergent lose their membrane integrity while calse- questrin is not extracted. Attachment of calsequestrin to the lumenal side of the sarcoplasmic reticulum membrane is as- sociated with a thickening of the external region of the mem- brane. Both membrane thickening and calsequestrin attach- ment are destroyed by KC1 treatment which dissolves the M, 36,000 protein (1, 2). This implies that the organization associated with the attachment of calsequestrin to the mem- brane extends to the outer leaflet and gives circumstantial support to the possibility that a portion of calsequestrin, which is attached to the membrane, may interact with a cytoplasmic protein. Both glyceraldehyde-3-P dehydrogenase

6898 Glyceraldehyde-phosphate Dehydrogenase in Muscle Microsomes

and calsequestrin are hydrophilic water-soluble proteins and it is doubtful whether either of these proteins could exist spanning the biomolecular lipid leaflet. Thus, calsequestrin is a highly acidic protein and is readily extracted from the membrane. A transmembrane disposition would imply that the protein was an intrinsic one. An alternative disposition is that an intrinsic protein of the junctional region serves as a port or pore which permits the hydrophilic amino acids of calsequestrin to reside within the membrane. A transmem- brane disposition of calsequestrin might have provocative consequences in terms of a possible mode of passage of Ca2+ across the membrane while complexed with calsequestrin.

In this paper we have demonstrated that glyceraldehyde-3- P dehydrogenase binds to isolated calsequestrin or to calse- questrin exposed to the protein after treatment of TC with Triton X-100. Clearly, the bulk of calsequestrin is not acces- sible to glyceraldehyde-3-P dehydrogenase in the intact vesi- cle. This, however, is not surprising since most of the calse- questrin presumably exists as an aggregate within the vesicle lumen. Since glyceraldehyde-3-P dehydrogenase interacts with other membrane constituents, we are not able to con- clude from the inhibition of glyceraldehyde-3-P dehydrogen- ase activity by intact vesicles that this interaction is taking place directly with calsequestrin. The glyceraldehyde-3-P de- hydrogenase of isolated sarcoplasmic reticulum resides pre- dominantly in the TC region with significantly lower content in the longitudinal reticulum. Since this is the distribution of calsequestrin, the data are consistent with an in vivo attach- ment of the enzyme of calsequestrin.

The physiological significance of the binding of glyceral- dehyde-3-P dehydrogenase to membrane proteins has been extensively debated. Kliman and Steck (7) have indicated that glyceraldehyde-3-P dehydrogenase binds to erythrocyte membranes in vivo while Simpson et al. (9), employing cen- trifugation of packed erythrocyte lysates, found that binding was minimal. We have found that both high ionic strength and the presence of low concentrations of Ca2' inhibit binding of glyceraldehyde-3-P dehydrogenase to calsequestrin. Since the inhibition is complex, we have not evaluated the influence of ionic strength on intact vesicles. Nevertheless, isotonic KC1 causes considerable extraction of glyceraldehyde-3-P dehy- drogenase from a preparation of TC. Further evaluation of the extent of binding of glyceraldehyde-3-P dehydrogenase to sarcoplasmic reticulum is in progress.

Campbell et al. (1) have proposed that proteins of subunit M, = 34,000 and 38,000 are constituents of the junctional feet and are identical to foot processes which are observed by electron microscopy in isolated TC. We have argued previ- ously (2) that the processes observed by tannic acid mordant- ing represent a thickening of the membrane which is distinct from the individual foot processes. The unit size of the glyc- eraldehyde-3-P dehydrogenase tetramer obtained from crys- tallography is 75 X 70 X 80 A (24). Since the normally

evaluated size of the juntional feet is 150 x 150 X 120 A, it would be necessary for several (tetrameric) molecules to com- pose a single foot. The ready release of glyceraldehyde-3-P dehydrogenase from membranes at high ionic strength may mitigate against this protein being the structural unit of the foot. Nevertheless, we originally isolated this enzyme through its property of promoting the formation of the triad junction from isolated organelles and consider it likely that an intimate association with the junction is occurring. We are currently employing immunocytological approaches to elucidate the distribution of the enzyme.

Acknowledgments-We particularly wish to thank Dr. Keith Brew for running the amino-terminal sequence and amino acid composition and for assisting us in the protein identification. We thank Norma Jacobs for typing the manuscript.

REFERENCES 1. Campbell, K. P., Franzini-Armstrong, C., and Shamoo, A. E.

2. Brunschwig, J.-P., Brandt, N. R., Caswell, A. H., and Lukeman,

3. Caswell, A. H., Lau, Y. H., Garcia, M., and Bmnschwig, J. P.

4. Schrier, S. L. (1963) J. Clin. invest. 42, 756-766 5. Shin, B. C., and Carraway, K. L. (1973) J. Biol. Chern. 248,

6. Kant, J. A., and Steck, T. L. (1973) J. Biol. Chem. 248 , 8457-

7. Kliman, H. J., and Steck, T. L. (1980) J. Biol. Chem. 255,6314-

(1980) Biochem. Biophys. Acta 6 0 2 , 97-116

D. S. (1982) J. Cell Biol. 9 3 , 533-542

(1979) J. Bwl. Chern. 264,202-208

1436-1444

8464

6321 8.

9.

10. 11.

12.

13.

14.

Tsz;I.-H., Murthy, S. N. P., and Steck, T . L. (1982) J. Biol.

Simpson, R. J., Brindle, K. M., and Campbell, I. D. (1983)

Masters, C. J. (1978) Trends Biochem. Sci. 3, 206-208 Lenstra, J. A., van Raaij, A. J. M., and Bloemendal, H. (1982)

Lau, Y. H., Caswell, A. H., and Brunschwig, J.-P. (1977) J. Biol.

Brandt, N. R., Caswell, A. H., and Brunschwig, J.-P. (1980) J.

Bradford. M. M. (1976) Anal. Biochern. 72, 74R-2.54

Chem. 2 5 7 , 1438-1442

Bwchern. Biophys. Acta 758,187-190

FEBS Lett. 148,263-266

Chern. 252,5565-5574

Biol. Chern. 256,6290-6298

15. Edman, P., and Begg, G. (1967) Eur. J. Biochern. 1, 80-97 16. Velick, S . F., and Furfine, C. (1963) in The Enzymes (Boyer, P.

D., Landry, H., and Myrback, K., eds) Vol. 7, pp. 243-273, Academic Press, New York

17. Harris, J. I., and Perham, R. N. (1968) Nature (Lond.) 219 ,

18. Racker, E., and Krimsky, I. (1952) J. Biol. Chem. 198 , 731-743 19. Stewart, P. S., and MacLennan, D. H. (1974) J. Biol. Chem. 2 4 9 ,

20. MacLennan, D. H., and Wong, P. K. P. (1971) Proc. Natl. Acad.

21. Thorley-Lawson, D. A., and Green, N. M. (1973) Eur. J. Biochem.

22. Morrison, M. (1974) Methods Enzyrnol. 32, 103-109 23. Jorgensen, A. O., Shen, Y.-C., Campbell, K. P., and MacLennan,

24. Watson, H. C., and Banaszak, L. J. (1974) Nature (Lond.) 204,

"- " -

1025-1028

985-993

Sci. U. 5'. A. 6 8 , 1231-1235

40,403-413

D. H. (1983) J. Cell Biol. 97,1573-1581

918-920