Inositol Polyphosphate-mediated Repartitioning of

Vol. 264, No. 13, Issue of May 5, pp. 7349-7356,1989 Printed in U.S.A.

THE JOURNAL OF BIOLOGICAL CHEMISTRY

0 1989 by The American Society for Biochemistry and Molecular Biology, Inc

Inositol Polyphosphate-mediatedRepartitioning of Aldolasein Skeletal Muscle Triads and Myofibrils* (Received for publication, June 8, 1988)

Rolf ThieleczekS, Georg W. Mayr, and Neil R. Brandts From the Znstitut fur Physiologische Chemie, Abteilung fur Biochemie Supramolekularer Systeme, Ruhr- Uniuersitat, 04630 Bochum, Federal Republic of Germany and the §Department of Pharmacology, University of Miami School of Medicine, Miami, Florida 33101

comformation with a concomitant loss of enzymatic activity. Theeffects of inositol1,4,5-trisphosphate (Ins(1,4,5)P3),which has been hypothesized to be a These glycolytic proteins also bind to sarcolemma and sarcochemical transmitter in excitation-contraction cou- plasmic reticulum (SR)l vesicles prepared from ventricular pling in skeletal muscle, on aldolase bound to isolated muscle and to the lipids isolated from these membranes (5). triad junctions were investigated. Fructose- 1,6-bis- Aldolase and glyceraldehyde-3-phosphate dehydrogenase are phosphate aldolase was identified as the major specific present in fast skeletal muscle several-fold in excess of the binding protein for the Ins(l,4,6)P3 analogue glycolal- other glycolytic enzymes (7). A major binding reservoir of dehyde(2)-1-phospho-D-myo-inositol4,5-bisphos- these enzymes is actin (8, 9) and the troponin-tropomyosin phate which can form covalent bonds with protein complex (8,lO). Half of the total aldolase and glyceraldehydeamino groups by reduction of the Schiff’s base inter- 3-phosphate dehydrogenase is bound to thinfilament proteins mediate with [3H]NaCNBH3.This analogue, Ins(1,4,5) in resting muscle (11). The catalytic sites are involved in P3, and the inositol polyphosphates inositol 1,3,4,5tetrakisphosphate and inositol 1,4-bisphosphate were binding as theactivities are inhibited upon binding (12). The nearly equipotent in selectively releasing membrane free and bound enzymes are in dynamic equilibrium at physof about 3 PM. The rank iological ionic strength; aldolase and glyceraldehyde-3-phosbound aldolase with a order of the values was identical to the K I values phate dehydrogenase can be selectively released from the thin for inhibition of aldolase. Aldolase was also released filament proteins by metabolites in a concentration-dependby its substrate fructose1,6-bisphosphate and by 2,3- ent manner at physiologically relevant levels (8, 9). Aldolase bisphosphoglycerate. Ins(1,4,5)Pa-induced aldolase re- has been observed as a control point in glycolyticflux in lease did not disrupt the triad junction; glyceralde- several tissues (13, 14); even in skeletal muscle it has been hyde-3-phosphate dehydrogenase, a known junctional questioned whether aldolase acts as a true “equilibrium” enconstituent, was displaced only at much higher zyme (15). It has been proposed that aldolase could be a Ins(1,4,5)P3 concentrations. Ins(l,4,5)P3 was as effec- control point in the glycolytic pathway if its activity were tive as fructose 1,6-bisphosphate in releasing aldolase regulated by adsorption/desorption from other cellular confrom myofibrils. A finite number of binding sites for stituents (11). aldolase exist on triads (B,,, = 43-47 pmol of tetraThe site of excitation-contraction coupling in skeletal musmeric aldolase/mg of triad protein, KO = 2 3 nM). The cle is the triad junction where the invaginated transverse junctional foot protein was implicated as an aldolase (T-) tubule system is held in apposition tothe terminal binding siteby affinity chromatography with the junc- cisternae (TC) of the SR by junctional foot processes. Vesitional foot protein immobilized on Sepharose 4B. The cular complexes havebeen isolated with retention of morphopotential consequences of aldolase being bound in the logy from this cellular region (16) and current evidence indigap between the terminal cisternae and the transverse tubule toinositol polyphosphate and glycolytic metab- cates that thejunctional foot protein (JFP) originally defined as M, 300,000 is the ryanodine-sensitive Ca2+release channel olism in thatlocal region are discussed. of the SR (17). Brunschwig et al. (18) first reported the localization of aldolase and glyceraldehyde-3-phosphate dehydrogenase to the triadjunction fraction of skeletal muscle microsomes. Theydemonstrated thatthe M, 34,000 and The glycolytic enzymes have been historically considered 38,000 proteins, originally proposed by Campbell et al. (19) to as soluble constituents of the cell. Some of these proteins, be constituents of the junctional feet could be removed by however, are known to bind to particulate cellular compo- KC1 treatment without loss of triadic integrity. Caswell and nents. In particular, aldolase and glyceraldehyde-3-phosphate co-workers (4,20) subsequently reported that the M, 34,000 dehydrogenase, because of their cationic nature, bind to acidic band was glyceraldehyde-3-phosphate dehydrogenase and proteins such as theband 3 proteinof erythrocytes (1,2) and to calsequestrin (3, 4). In addition, they bind to phosphatiTheabbreviationsusedare: SR, sarcoplasmicreticulum; dylinositol liposomes (5, 6). Upon binding, aldolase changes (2,3)BPG, 2,3-bisphosphoglycerate;Fru(l,6)P~,fructose 1,6-bisphos* This work was supported by Grants GI 72/1-1, Konzell 4 (to R. T.) and Ma 989/1-1 (to G . W. M.) from the Deutsche Forschungsgemeinschaft andby a grant of the American Heart AssociatiLn, Florida Affiliate and Sun Coast Chapter N. (toR. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed.

phate;GcaPIns(4,5)P2, glycolaldehyde(2)-l-phospho-D-myo-inositol 4,5-bisphosphate; Ins(l,4)Pz, D-myo-inositol 1,4-bisphosphate; Ins(1,4,5)P3, D-myo-inositol 1,4,5-trisphosphate;Ins(1,3,4,5)P4, Dmyo-inositol 1,3,4,5-tetrakisphosphate;MOPS, 3-(N-morpho1ino)propanesulfonic acid; SDS, sodium dodecyl sulfate; PAGE,polyacrylamide gel electrophoresis; JFP, junctional foot protein;TC, terminal cisternae;TES, 2-( [2-hydroxy-l,l-bis(hydroxymethyl)ethyl] amino}ethanesulfonic acid HPLC, high pressure liquid chromatography; SV8, Staphylococcus aureus V8 protease.

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Aldolase Repartitioning by Inositol Polyphosphates in Skeletal Muscle

that glyceraldehyde-3-phosphate dehydrogenase could catalyze the reformation of the triad junction from mechanically separated components. The confirmation that the M, 38,000 band was aldolase has appeared only in abstract form (3). We have previously demonstrated that theA form of aldolase (skeletal muscle isomer) binds with high affinity inositol polyphosphates (21). Ins(1,4,5)P3 (22) and possibly inositol 1,3,4,5-tetrakisphosphate(Ins(1,3,4,5)P4)(23)aresecond messengers in nonstriated muscle cells. The inositol polyphosphatesIns(1,4,5)P3,Ins(1,3,4,5)P4,andinositol1,4bisphosphate (Ins(1,4,)Pz) are potent competitive inhibitors of the glycolytic activity of aldolase A in the cleavage of fructose 1,6-bisphosphate (Fru(l,6)Pz).They induce a conformational change upon binding to the enzyme (21). We (24) have recently developed the glycolaldehyde derivative of Ins(1,4,5)P3substituted atthe 1 phosphateposition, glycolaldehyde(2)-l-phospho-~-myo-inositol4,5-bisphosphate(GcaPIns(4,5)Pz) to identifypotential Ins(1,4,5)P3 binding proteins. This ligand can form Schiffs bases with amino groups of binding proteins. This Schiffs base can be reduced by rHINaCNBH3 with the concomitant introduction of 3H into the resultant amine. GcaPIns(4,5)Pz bindsto and inhibits aldolase A with almost equal potency as Ins(1,4,5)P3 and thus canalso be used as an affinity label for this enzyme. Furthermore, GcaPIns(4,5)Pz acts as an agonist with equal potency for Ca2+release from permeabilized parotid acinar cells (24). We now report that aldolase on TC membranes is the major GcaPIns(4,5)Pz binding protein of the skeletal muscle triad.GcaPIns(4,5)Pz, inositol polyphosphates and other bisphophometabolites induce release of aldolase from the membrane sites. We compare the effects of Ins(1,4,5)P~ on triadic aldolase with the effect on aldolase bound to the myofibrils and present qualitative evidence that the JFP of the triad junction which is embedded in the TC membrane (25) itself may provide an aldolase binding site. The consequences of inositol polyphosphate induced repartitioning of aldolase are discussed in relation to inositol polyphosphate metabolism and toglycolysis occurring within the triadjunction. MATERIALS AND METHODS

Ins(1,4,5)P3,Ins(1,3,4,5)P4,and Ins(l,4)Pp were produced by standard procedures (21). Fru(l,G)P*, 2,3-bisphosphoglycerate ((2,3)BPG), and Staphylococcus aureus V8 protease (SV8) were purchased from Boehringer Mannheim. [3H]ouabain and [32P]H3POd wereobtained from Du Pont-New England Nuclear. GcaPIns(4,5)P2was produced semisynthetically by procedures recently reported (24). All other chemicals were of at least reagent grade. Preparations of Organelles TC/triads and longitudinal SR were prepared from rabbit back muscle as described by Caswell et al. (16). Transverse tubules and light and heavy TC were prepared by mechanical disruption of the triads in the French press and subsequent separation by isopycnic centrifugation as described by Brandt et al. (26). In some preparations, the T-tubulecomponent of the triad was labeled by the binding and entrapment of [3H]ouabain as described previously (16). Partition Assays

these samples different concentrations of inositol polyphosphates, GcaPIns(4,5)Pz, and other metabolites were added yielding a final suspension volume of 180 pl. After 5-min incubation a t room temperature, the reaction mixtures were centrifuged in a Beckman Airfuge (Beckman Instruments Inc.) for 15 min at 30 p. s. i. An aliquot of 100 pl was carefully taken from the supernatantfor determination of protein, aldolase, and glyceraldehyde-3-phosphate dehydrogenase activity. Aliquots were also subjected to SDS-PAGE on 12% polyacrylamide separating gels (27). Myofibrillar Binding Assays-Myofibrils were isolated from rabbit leg muscleand TritonX-100 washed essentially as described by Solaro et al. (28). Protein was determined according to Bradford (29). Triton X-100-treated myofibrils were washed (thoroughly vortexed and centrifuged for 3 min a t 7000 X g) three times in buffer M: 50 mM KC1, 20 mM Tris/HCl, 2 mMMgC12, 1 mM dithioerythritol, pH 7.5, and adjusted to a concentration of25 mg/ml. Aldolase A was dialyzed against 2 X 1 liter of 0.1 M KCl, 40 mM triethanolamine/HCl, 1 mM M&L, 1mM dithioerythritol, pH 7.3, and adjusted to a concentration of 1 mg/ml. The myofibril suspension was mixed with the aldolase solution at a volume ratio of 1:l.After an incubation for 30 min at 25 "C, 1-ml portions of the myofibrillar suspension were washed five times as above in initially 2 ml and then 1 ml of buffer M. After washings, the protein concentration was adjusted to 2.5 mg/ml by addition of buffer M. These aldolase-repleted myofibrils (40 pl) were mixed with 10 pl of a solution containing increasing concentrations of either Ins(1,4,5)P3, Fru(1,6)P2,or KC1 (for controlling ionic strength effects). After an incubation for 15 min at 25 "C,the samples were centrifuged in an Eppendorf centrifuge a t 7000 X g and 20 p1 of the supernatantswere assayed for aldolase activity. Enzymatic Assays

Aldolose-Fructose-l,6-bisphosphate aldolase A was isolated from rabbit skeletal muscle according to Penhoet and Rutter(30). Aldolase activity was determined as described (21). A substrate concentration of 0.5 mM Fru(l,6)P2 (300-fold above K,) was employed in order to obtain maximal enzyme activity in the presence of competitive inhibitors. This concentration of substrate will be shown to be also sufficient to completely dissociate aldolase from membranes or myofibrils. Glyceraldehyde-3-phosphate Dehydrogenase-Glyceraldehyde-3phosphate dehydrogenase was assayed at 25 "C in a buffer composed of 0.1 M triethanolamine/HCl, 2 mM dithioerythritol, 0.6 mM NAD, 2 mM potassium phosphate, 40 p~ glyceraldehyde-3-phosphate,pH 7.4. A 2 0 4 aliquot of the supernatant from a sedimentation experiment was mixed with 780 pl of this buffer, and the absorbance was followed at 340 nm. Ins(1,4,5)P3-5-phosphat~e-Radioactiveinositol phosphates were produced from human erythrocyte ghosts using [32P]Pifollowing the method of Hawkins et al. (31). Radioactive Ins(1,4,5)P3was isolated by HPLC (32) using the retention time for the unlabeled Ins(1,4,5)P3 standard. About 90% of the radioactivity was incorporated into Ins(1,4,5)P3.The pooled fraction containing 4.4 mM Ins(1,4,5)P3with a specific radioactivity of 37 GBq/mol was used as substrate for the phosphatase assay. TC/triads (1mg/ml) were incubated with varying concentrations of radioactive substrate in 525 pl of an assay buffer containing an ATP-regenerating system (33). At selected times, 110pl aliquots were mixed with an equal volume of 1M HClO,. After 15 min on ice, the samples were centrifuged, and 200 p1 of the supernatant were mixed with 40 pl of 2.5 M KHC03 and centrifuged. The supernatant was quantitatively removed, lyophilized and then resuspended in 500 pl H20. The remaining Ins(1,4,5)P3 was quantitated by scintillation counting after separation by HPLC. The maximum Ins(1,4,5)P3 degrading activity under these reaction conditions was 15.8 nmol/min/mg triads with a KD of 520 pM. This V,, is 4-fold higher than that of the heavy SR fraction reported by Milani et al. (34) and comparable to thatof their T-tubule fraction. The KDunder their conditions was 15.5 pM.

Partitioning on Isopycnic Sucrose Gradient-Freshly isolated TC/ Cross-linkingof GcaPIns(4,5)P2 to Skeletal Muscle Organelks triads were suspended at 10 mg/ml in 1 ml of triad buffer (250 mM GcaPIns(4,5)P2 was prepared semisynthetically as described presucrose, 30 mM potassium gluconate, 1 mM MgClz, 3 mM histidine, 10 mM TES, pH 7.3). To these mixtures either 50 p~ Ins(1,4,5)P3or viously (24). TC/triads (1 mg/ml), longitudinal SR (1 mg/ml), or aldolase (0.5 mg/ml) were suspended in cross-linking buffer (50 mM 1 mM KC1, as a control to compensate for change in ionic strength was added. The samples wereloaced onto linear sucrose density KCl, 0.5 mM EDTA, 0.5 mM dithioerythreitol, 5 mM TES, pH 7.0). gradients (15-50% sucrose) in a SW 28 swinging bucket rotor and The reaction was started by adding 10 p~ GcaPIns(4,5)P~immedirecentrifuged at 70,000 X g overnight. The gradients were fractionated ately followed by an addition of 1 mM [3H]NaCNBH3 (Amersham and assayed for protein, entrapped [3H]ouabain,and aldolase activity. Corp., specific radioactivity 40.7 TBq/mol, pH 7.5) yielding a final Airfuge Sedimentation-Freshly prepared TC/triads were sus- volume of 100 pl. After an incubation for 1 h a t room temperature, ice-cold 30% pended in 160 pl of triad buffer at either 1 or 5 mg protein/ml. TO the reaction was stopped by the addition of27plof

Aldolase Repartitioning by

Inositol Polyphosphates in Skeletal Muscle

trichloroacetic acid. Following a 10-min incubation on ice, the samples were centrifuged and the pellets washed with 200 pl of ice-cold 8% trichloroacetic acid. The pellets were dissolved in 50 pl of Laemmli sample buffer (27) with 4% SDS and subjected to SDS-PAGE on 10% polyacrylamide separating gels. 3H-labeled polypeptides were visualized by autoradiography following soaking of the Coomassie Blue-stained gel for 30 min ina fluorographic reagent (Amplify, Amersham Corp.). A Kodak X-Omat AR-5 film was exposed to the dried gel for two weeks a t -70 "C.

1 la

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Peptide Mapping Peptide mapping was performed with S. aureus V8 protease following the method of Cleveland et al. (35) as modified by Mayr and Heilmeyer (36). The M,40,000 band was isolated from 10-mg triads on 10% separating gels by cutting out theslightly stained band and electroeluting the protein in a Biotrap (Schleicher and Schuell) a t 200 V a t 4 "C overnight according to Ref. 37. Digestion was carried out on the M, 40,000 band and on pure aldolase a t 800 pg/ml with 80 pg/ml protease a t 37 "C for 30 min. Reaction products were separated on 15%acrylamide separating gels. Junctional Foot Protein Affinity Chromatography The JFP ( M , 300,000) was isolated from TC/triads dissolved in 1 M NaCI, 2 mg of Zwittergent 3-I4/mg protein by immunoaffinity chromatography as described by Kawamoto et al. (38). Followingtheir procedure, the entire yield from one preparation (200-400 pg) was coupled to 1 ml of CNBr-activated Sepharose 4B (Sigma). After blockingunreacted siteswith 0.2 M Tris-CI, the gel matrix was washed with gluconate/MOPS buffer (20 mM potassium gluconate, 20 mM Tris-MOPS, pH 7.0) to permit partial renaturation of the crosslinked JFP. The column was incubated for 1 h a t room temperature with either 1.5 mgof aldolase or 2.2 mg ofglyceraldehyde-3-phosphate dehydrogenase (Sigma) in gluconate/MOPS buffer and then washed extensively until the absorbance measured a t 254 nm had returned to background level. Subsequently, the column was eluted with 3 ml of gluconate/MOPS buffer containing 10p~ Ins(1,4,5)P3.The column was then washed with 10 ml of gluconate/MOPS buffer when A 2 s had returned to background. Thereafter, a second elution with 3 ml of gluconate/MOPS buffer containing 50 p~ (2,3)BPG was carried out followed again by a wash with gluconate/MOPS buffer. Finally the column was eluted with 1 M NaCl in gluconate/MOPS buffer. Each of the eluates was pooled, concentrated, and subjected to SDSPAGE. ,," ,

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FIG. 1. Identification of proteins in longitudinal SR and triads which covalently bind GcaPIns(4,5)Pz.Aldolase (lanes I and l a ) , longitudinal SR (lanes 2 and Sa), and triads(lanes3 and 3a) were incubated with GcaPIns(4,5)P*and [3H]NaCNBH3as described under "Materials and Methods." Aldolase (0.5 pg) and membranes (50 pg)were electrophoresed ona Laemmli SDSslab gel (10% separating gel) which was then stained with Coomassie Blue (lanes 1-3). The corresponding autoradiograms from these polypeptide patterns are shown in lanes l a , 2a, and 3a, respectively. The position of the aldolase band is marked by an arrow.

only band which was eliminated by addition of a 100-fold excess of Ins(1,4,5)P3 or (2,3)BPG to the labeling reaction (not shown). Proteolytic digestion on the M , 40,000 band of TC/triads RESULTS was performed with SV8 protease (Fig. 2). Lanes 1 and 2 show Potential inositol polyphosphate binding sites were identi- the electrophoresispatterns of pure aldolase A and TC/triads fied in the longitudinal SR and TC/triadsby covalent labeling on 15% acrylamide separating gels. Lane 3 contains the prowith GcaPIns(4,5)P*.Fig. 1shows the electrophoreticpatterns tease. The M , 40,000 band, electroeluted from the original for pure aldolase (lane 1), longitudinal SR (lane 2), and TC/ triad electropherogram and re-electrophoresedwithout treattriads (lune 3 ) stained with Coomassie Blue.The companion ment or after a 30-min digestion with the protease SV8 are lanes ( l a , 2a, and 3a) show the autoradiograms after impreg- shown in lanes 4 and 5, respectively. Lane 6 shows the SV8 nation of the gel with a fluorographic reagent. The lanes digestion products of aldolase. Aldolasewas more completely so that minor digested than the electroeluted material under the reaction containing membranes were overloaded (50 pg) proteins could be readily visualized. Thus, the SR Ca2+AT- conditions as indicated by the lower content of material at M , Pase (Mr 102,000), which accounts for >90% of the longitu- 40,000. Only those peptides seen in the aldolase pattern are dinal SR protein, and 45% of the TCprotein and calsequestrin present in the digestion products from the M , 40,000 band of (MI 66,000), which accounts for 45% of the TC protein, are TC/triads. not quantitatively stained. Junction breakage and reformation has been proposedas a Aldolase A binds GcaPIns(4,5)P2with high affinity (24); a dynamic event in excitation-contraction coupling in skeletal high content of radioactivity introduced by [3H]NaCNBH3 muscle (39). To testfor the effects of Ins(1,4,5)P3on the triad reduction is found at the position of aldolase (lanesI and l a ) . junction, [3H]ouabain-labeledtriads were mixed with 50 p~ A faint degree of radioactivity is found at the position of the Ins(1,4,5)P3 and then centrifuged on an isopycnicsucrose Ca2+ATPase in the longitudinal SR (lanes 2 and 2a). This gradient. Fig. 3 shows the protein distribution patterns for may simply be an artifact because of the high content of the untreated (open circles) and Ins(l,4,5)Ps-treated (filled cirM , 102,000 protein. Similarly, faint radioactive bands are cles) triads. Less than 5%of the protein is removed from the present at the positions of the major TC proteins at M , triad band by Ins(1,4,5)P3treatment; thisprotein now appears 102,000 and M , 66,000 (lanes 3 and 3a). In addition, the TC/ in the soluble protein region above the gradient. The distritriad preparation shows minor bands not present in the lon- bution patterns for the [3H]ouabainmarker for T-tubules in gitudinal SR in the M , 70,000-80,000 region and above M , the untreated and Ins( 1,4,5)P&eated triads were identical 200,000. The major radioactiveband in the TC/triadmigrated (data not shown). No radioactivity was detected at the isowith M , 40,000 and co-electrophoresed with aldolase. This pycnic point of the free T-tubules (22-28% sucrose). In conband was not seen in the longitudinal SR lane and was the trast, almost all of the aldolase activity comigrating with the

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FIG. 3. Distribution patterns forprotein and aldolase activity after centrifugation of TC/triads on isopycnic sucrose gradients. Isolated TC/triads (10 mg) were incubated with 50 pM Ins(1,4,5)P3 and recentrifuged on a linear sucrose density gradient (X). The fractions were assayed for protein (0, control; 0, Ins(1,4,5)P3) and aldolase activity (0, control; W, Ins(1,4,5)P3). The distribution of [3H]ouabain, a marker for the T-tubule component of the triad, matched the distribution of protein for the untreated triads when both the control and Ins(1,4,5)P~-treated organelles were recentrifuged (data not shown for clarity).

0

FIG.2. Cleveland peptide analysis of the M,40,000 band in skeletal muscle triads. Aldolase and the M,40,000 band electroeluted from triads were digested with S. aureus V8 protease for 30 min and the reaction products subjected to SDS-PAGE on a 15% separating gel. The gel lanes are asfollows: lane l , aldolase (5 pg); lane 2, TC/triad (20pg); lane 3, SV8 protease (2 pg); lane 4, M,40,000 band electroeluted from TC/triads (20 pg); lane 5, products of the SV8 digestion of the electroeluted M,40,000 band (20 pg); and lane 6, products of the SV8 digestion of aldolase (20 pg). The top of the separating gel and thedye front are marked by arrows.

untreated triads (open squares) appears inthe soluble protein fraction after Ins(1,4,5)P3 treatment (filled squares). When TC vesicles, isolated by mechanical disruption of the triads followedby component separation on isopycnic gradients, were treated with Ins(1,4,5)P3, the distribution patterns for protein and aldolase activity were identical to those shown for intact triads inFig. 3. The redistribution of aldolase and glyceraldehyde-3-phosphate dehydrogenase associated with the TC/triadswas more quantitatively investigated using the Airfuge separation technique. Both glycolytic enzymes werereleased from the membranes to the supernatant in an Ins(1,4,5)P3 concentrationdependent manner (Fig. 4). In the absence of Ins(1,4,5)P3, about 64 nmol/min/ml aldolase activity (circles) was spontaneously released from triads suspended at 0.88 mg protein/ ml. Treatment with 20 pM Ins(1,4,5)P3 completely released aldolase to a final supernatant concentration of 125 nmol/ min/ml. The half-maximal effect was obtained at 3.4 p ~ . In contrast,glyceraldehyde-3-phosphatedehydrogenase activity (squares) is released from the triadic membranes when the Ins(1,4,5)P3 concentration exceeds about 20 p ~ The . glyceraldehyde-3-phosphate dehydrogenase activity released by 100 pM Ins(1,4,5)P3 was 18 nmol/min/ml which corresponds to only 0.6% of the total glyceraldehyde-3-phosphate dehydrogenase activity releaseable from triads by 0.2 M KC1 (4, 40). This relative insensitivity of glyceraldehyde-3-phos-

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I

I

0

20

40 60 Ins (I4,.51 P3

80

100

(uMI

FIG.4. Repartitioning of aldolase and glyceraldehyde-3phosphate dehydrogenase on TC/triads by Ins(1,4,5)Ps. TC/ triads (0.88 mg/ml) were incubated with increasing concentrations of Ins(1,4,5)P3and thenpelleted by Airfuge centrifugation. The aldolase (0)and glyceraldehyde-3-phosphate dehydrogenase (0)activities in the supernatants were then assayed as described under “Materials and Methods.”

phate dehydrogenase to Ins(1,4,5)P3 compared to aldolase was also demonstrated when the supernatants andpellets from a partitioning experiment at 4.44mg triad protein/ml were analyzed by SDS-PAGE (Fig. 5). To prevent overloading of the gel lanes, only 1.5% of the totalpellet protein was utilized. In contrast, 10% of the entire supernant was loaded on the corresponding gel lanes. These differences must be taken into account in viewing the electropherogram. In theabsence of Ins( 1,4,5)P3treatment ( l a n e 3),Coomassie Blue staining bands appear at M , 26,000, 36,000 (glyceraldehyde-3-phosphate dehydrogenase), 40,000 (aldolase), 50,000, and 70,000 in the supernatant. Glyceraldehyde-3-phosphate dehydrogenase and aldolase bands can be readily visualized of the supernatantsfrom in the pellet (lane4 ) . In scanning all the Ins(l,4,5)P3-treated TC/triads (lanes5, 7, 9, 11, and 13), the content of the M , 26,000, 50,000, and 70,000 bands does not appear to change with increasing [Ins(1,4,5)P3]. An in-


1

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5

6

7

8

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FIG.5. SDS-PAGE patternsfor the supernatants and pellets from Ins(l,4,5)P3-treatedTC/triads. TC/ triads (4.44 mg/ml) were treated with various concentrations of Ins(1,4,5)P3 and then partitioned by Airfuge centrifugation. Pellets were resuspendedin 200 pl of Laemmli buffer. Aliquots from the supernatants (16 pl) and pellets (3 pl) were then electrophoresed on a 10%separating gel. Lane 1 , TC/triads (14 pg), lane 2, aldolase (4 pg) andglyceraldehyde-3-phosphatedehydrogenase (4 pg). Lanes 3-14, supernatants and pellets, respectively, from TC/triads treated with 0 (lanes 3 and 4), 0.3 p~ (lanes 5 and 6 ) ,1 pM (lanes 7 and 8 ) , 3 pM (lanes 9 and l o ) , 10 p~ (lanes 11 and 12), and 30 pM (lanes 13 and 14) Ins(1,4,5)P3.

creased stainingintensity of the M, 40,000 band can be observed in the supernatant from the TC/triads treatedwith 1p~ Ins(1,4,5)P3(lune 7); this appears to be maximized after treatment with 10 pM Ins(1,4,5)P3 (lane 11) in agreement with the activity measurements (Figs. 4 and 6). The identity of this M , 40,000 band released by Ins(1,4,5)P3 as aldolase was confirmed by Cleveland peptide mapping (not shown). A concomitant decrease in the aldolase band of the pellets (lanes 6, 8, 10, 12, and 14) with increasing [Ins(1,4,5)P3] can be visualized. Depletion of the aldolase band appears to be almost complete at 30 p~ (lane 14). This nearly complete depletion was observed when even 500 pg of the pellet were loaded on a gel lane (not shown). In contrast, the increased staining intensity of the M, 36,000 band was first barely discernable in the supernant at10 pM Ins(1,4,5)P3 (lane 11) and clearly seen at 30 p~ Ins(1,4,5)P3 (lune 13). The pellet from the TC/ triads treated with 30 p~ Ins(1,4,5)P3 (lane 14), however, does not appear to be depleted in glyceraldehyde-3-phosphate dehydrogenase compared to the membranes incubated without Ins(1,4,5)P3 (lane 4 ) . These slight changesin supernatant content of the M , 36,000 band are in agreement with the glyceraldehyde-3-phosphate dehydrogenase activity measurements shown in Fig. 4. Fig. 6 compares the potencies of several inositol polyphosphates andrelated compounds for releasing aldolase from the triadic membranes. The half-maximal effective concentrations of inositol polyphosphates in aldolase release were in rank order: Ins(1,3,4,5)P4 (1.2 p M ) , Ins(1,4,5)P3 (2.5 p M ) , GcaPIns(4,5)Pz(2.8 pM), and Ins(l,4)P2(3.4 p ~ ) Measure. ments of the time course of Ins(1,4,5)P3 degradation by the intrinsic Ins(1,4,5)P35-phosphatase indicatedthat about 17% of the totalIns(1,4,5)P3could be hydrolyzed by the endof the 5-minincubation period. A comparable decrease could be calculated using the activity reported by Milani et ul. (34) in the absence of an ATP-regenerating system. Thus, the final

“ h . , . , . , . , . ,

1 -7 -6 -5 -4 -3 -2 log [agonistconcentration)

FIG.6. Partitioning of aldolase activity after treatment of TC/triadswithinositolpolyphosphates,Fru(l,G)Pz, (2,3)BPG, and Pi. Triads (4.4 mg/ml) were incubated withincreasing concentrations of (A) Ins(1,4,5)P3 ( 0 , Ins(l,4)Pz (A), Ins(1,3,4,5)P4 (O), (2,3)BPG (0),and ( B ) Fru(l,6)P2 (A), GcaPIns(4,5)P2 (B), and Pi (:) and then sedimentedin the Beckman Airfuge. The aldolase activities in the supernatants are plotted as a function of log (initial ligand concentration).The ordinate is the same on both panels.

Ins(1,4,5)P3 concentration would be less than theinitial concentration asindicated in Fig. 6. The ability of the phosphatase to hydrolyze Ins(1,3,4,5)P4 and GcaPIns(4,S)Pz has not been tested. Fru(l,6)Pz, the substrate for aldolase, released , (2,3)BPG, which is the enzyme with a K0.5 of 5.6 p ~whereas a weak inhibitor of aldolase activity, was estimated to be half. of the Fru(l,6)P2 maximally effective at about 80 p ~ Part will be cleaved during the incubation period approaching an equilibrium with the triose phosphates. Thus, the true KOS value for Fru(1,6)Pzwill be lowerthan suggested by Fig. 6. In contrast, inorganic phosphate (Pi) concentrations for halfmaximal effects were above 0.7 mM. Millimolar concentrations of Pi and ATP4- are required to release aldolase from the contractile proteins (9). In thisexperiment (Fig. 6) where the TC/triadswere resus-

7354

in Skeletal Muscle

Aldolase Repartitioning by Inositol Polyphosphates

pended at a final concentration of 4.44 mg/ml, only 30% of the activity (170 nmol/min/ml) was dissociated upon centrifugation in the absence of ligand addition. The average maximum aldolase activity solubilized by all ligandswas 570 nmol/ min/ml. The average protein content of the supernants was 0.1 mg/ml; as seen in Fig. 5, the M , 40,000 comprised about a specific activity 30% of that protein. From these estimates, for aldolase of 3000 mol of Fru(l,6)P2 hydrolyzed/mol of aldolase tetramer/min can be calculated. This estimate is in 0 good agreement with thereported value of 2900 mol of m O , , , , Fru(1,6)P2/molof aldolase/min (41). 0 20 40 60 80 100 Using the calculated specific activity, the activities in the Ins 11. 4,51 P, (DM) of supernatants from Figs. 4 and 6 can be quantified in terms FIG. 7. Repartitioning of aldolase from aldolase-repleted concentrations of aldolase tetramers released. At 0.88 mg myofibrils by Ins(1,4,5)Ps. Washed myofibrils were repleted with triads/ml (Fig. 4), 21 nM aldolase appeared in the supernatant aldolase andthenincubatedwith increasing concentrations of without Ins(1,4,5)P3 treatment, and the maximal concentra- Ins(1,4,5)P3. The myofibrils were then pelleted inanEppendorf tion released was 42 nM. A t 4.44 mg triad/ml (Fig. 6), 57 and microfuge and the supernatantsassayed for aldolase activity. 190 nM were the minimum and maximum, respectively. Using these partition ratios, the maximum number of aldolase bindis in the range of43-47 pmol tetramer/mg ing sites (BmaX) 4 triad protein and the KD can be calculated as 23 nM.' This value is near to the KD reported for aldolase binding to the band 3 protein of erythrocytes (1).The B,,, is consistent with previous estimates of the monomer comprising1%of the triad protein (20,42). For comparative purposes the effectiveness of Ins(1,4,5)P3 in releasing aldolase fromits major binding site onmyofibrils was tested (Fig. 7). In the absence of ligands, only 10 nmol/ min/ml activity was solubilized in the centrifugation assay, whereas a maximum of 250 nmol/min/ml was released by 100 p~ Ins(1,4,5)P3. A half-maximal effective concentration of Ins( 1,4,5)P3 was estimated at 15 pM. With Fru(l,6)Pza similar sigmoidal curve as for the Ins(174,5)P3-dependentaldolase dissociation was obtained; the was also estimatedto be 15 p~ (data not shown),which is in good agreement with the K O sreported for the release of aldolase by its substrate from actin (9). JFP Kawamoto et al. (38) havepreviously reported that the immobilized on Sepharoseselectively extracted proteinsof M , 34,000-40,000 from T-tubules disolved in low salt and Zwittergent. To test the possibility that the JFP itself binds d

5

'The apparent affinity constant of triads for aldolase was estimated from the experimental data as follows. The actual aldolase content, [AldTR], of an individual triad preparation was expressed as a fraction of the total aldolase binding capacity, [Ald,], of triads: [AldTR] = p.[Aldt], where p (0 5 p 5 1) representstheactual saturation of thetriadic aldolase bindingsites. The endogenous aldolase content is obtained from the measured maximum aldolase activity, actmax,of the supernatants (complete dissociation of bound aldolase) using the calculated specific activity actAldof 3000 mol Fru( 1,6)P2/mol tetramer/min: [AldTR] = actms./actAld. The apparent binding reaction canbe written as

,I

J(

73

1

1

3

2

1

.-

FIG. 8. Eluates from a junctional foot protein-Sepharose column loaded with aldolase. Protein pattern aftergel electrophoresis of the material that was successively eluted from the column with 10 pM Ins(1,4,5)P~( l a n e 3), 50 pM (2,3)BPG ( l a n e 4 ) , and 1 M NaCl (lane 5).respectively. The lanes I and 2 show aldolase fromthe break volume and a blank lane, respectively. The arrows mark the top of the separating gel and the dye front.

glycolytic proteins, one JFP-Sepharose column was incubated with aldolase in low salt medium. After washing away all where [Aldt,..],[TRt,,], and [TRJ represent the equilibrium con- unbound protein, thegel was eluted with 10 p~ Ins(1,4,5)P3. centrations during the partitioning assay of free aldolase, freebinding Fig. 8 shows that the only Coomassie Blue staining material sites, and occupied binding sites, respectively, and K is the apparent from this concentrated Ins(1,4,5)P3 eluate (lane3) co-electroaffinity constant of triads for aldolase. [Aldrm.] can be derived from phoresed with the original aldolase (lane 1). A subsequent the aldolase activity of the supernatant,act,,, measured in theabsenqe of agonist: [Aldf,..] = act,,/actAld. In the assumed1:l binding reaction, challenge with 50 PM (2,3)BPG, which caused elution of the columns nottreated with the free binding sites are given by [TRr,,] = [Aldr,] + [Aldt]. (1 - M , 40,000 bandinseparate p) and the occupied aldolase binding sites of the triad in theequilib- Ins(1,4,5)P3, here did not release additional protein (lane 4 ) . rium reaction are given by [ T L , ] = [AldTR] - [Aldrre.]. The apparent A small amount of protein could be released by subsequent affinity constant K , which depends on the assumed saturation 0, is addition of 1 M NaCl (lane 5 ) . On parallel columns, bound then given by glyceraldehyde-3-phosphate dehydrogenase could not be eluted by the polyphosphates at the concentrations tested. K = [TLcl/([Aldrreel. [TRrreel) The preparationof the JFP by immunoaffinity chromatog= aCtAdaCtmax- act,,)/(act,,(acto + actmex.(l- P ) / P ) ) raphy employs high levels of Zwittergent and NaSCN. These A reasonable estimate for is 1 (see"'Discussion" and Footnote 3). agents cause denaturation of the protein. Furthermore, quan[Aldrreel+ [TRa..]

K * [TR,,]


7355

lipid associations ( 5 ) . Two proteins of the triad, glyceraldehyde-3-phosphatedehydrogenaseandcalsequestrin,are known to bind aldolase in uitro (40 and 4,respectively). An association between aldolase and thejunctional foot has been suggested by several previous studies (18, 19, 38); Kawamoto et al. (38) reported that the JFP bound to Sepharose selectively extracted an M , 40,000 band from detergent-dissolved membranes. The JFP-Sepharose affinity chromatography experiment reported here indicates that the JFP retained a binding site for aldolase even in itspartially denatured state. Furthermore, aldolase was eluted by Ins( 1,4,5)P3at thesame concentration which dissociated it from the triadmembranes. DISCUSSION The associations of glyceraldehyde-3-phosphate dehydrogenase and aldolase to the JFP and other proteins of the triad Aldolase has been localized to the I band of fast skeletal are presently being investigated. muscle by hktocytochemical techniques (43). The major Half of the total aldolase (20 nmol/g) is bound to the thin structures in thisregion are the thinfilaments of the contracfilaments in resting muscle (11).The actin-aldolase complex tile apparatus and the SR/T-tubule triadjunctions. Both an uitro can be disrupted by Fru(1,6)Pz and other metabolites MI40,000 polypeptide which co-electrophoreses with the al- in (11).We have shown that Ins(1,4,5)P3 is equally effective as dolase monomer (18-20, 42) and aldolase activity (3) have Fru(l,6)PZ in releasing aldolase from myofibrils. Under our been detected in preparationsof isolated triads. The MI40,000 assay conditions, both compounds were slightly more potent band is preferentially located on the TCcomponent (20). We have now demonstrated that theM,40,000 band of the in releasing aldolase from the isolated triads. Whether this TCftriads is aldolase. The enzyme can be selectively disso- reflects a greater responsiveness of the membrane site to ciated from the membrane by the inositol polyphosphates changes in metabolite levels in uiuo remains to be shown. The concept of the gap between the T-tubule and TC Ins(1,4,5)P3, Ins(1,3,4,5)P4, and Ins(l,4)P2 as well as by its membranes being a restricted diffusional space for small substrate Fru( l,6)Pz at physiologically relevant concentramolecules has been invoked in models for excitation-contractions (7,44) without disruption of the triad junction. Glyceraldehyde-3-phosphate dehydrogenase, a functional constitu- tion coupling. The consequences of fixed CaZ+binding sites ent of the junction (4,20),was not dissociated by physiological near the SR Ca2+release channel (thejunctional foot protein) concentrations of Ins(1,4,5)P3.Furthermore, aldolase was the upon the Ca2' transient have been modeled by Cannel1 and only protein detected in the TC/triads capable of forming a Allen (48). A large step increase in the local Ins(1,4,5)P3 covalent bond with the probe GcaPIns(4,5)Pz. Thesefindings concentration upon T-tubule depolarization has been postudo not exclude the existence of other inositol polyphosphate lated by proponents of the Ins(1,4,5)Pa as a signal substance binding sites in the triad junction. Such sites could be masked hypothesis (49, 50). Aldolase bound to the foot protein or by the presence of aldolase. Furthermore, those Ins(1,4,5)P3 other proteins in the junctional gap would provide 'fixed Ins(1,4,5)P3. However, changes in the receptors, which lack the critical amino group near the binding sitesfor Ins(1,4,5)P3 concentration could cause repartitioning of the Ins(1,4,5)P3 binding site, cannot form the Schiffs base with GcaPIns(4,5)Pz and hence would not become radioactively bound aldolase which must be in equilibrium with the total aldolase of the cell, because the junctional gap is accessible to labeled during the reaction with [3H]NaCNBH3. A B,, of43-47 pmol of aldolase tetramer/mg of triad exogenous proteins (51). The free Ins(1,4,5)P3 concentration protein with a KOof 23 nM was estimated from the partition- in thejunctional gap willdepend not only upon the partitioning of aldolase between bound and free forms at two triad ing of aldolase and the total Ins(1,4,5)P3 concentration but protein concentrations. The amount bound is in reasonable also upon the concentrations of other inositol polyphosphates agreement with the estimatethat theM, 40,000 band (aldolase and Fru(l,6)Pz. Enzymes of the inositol metabolism, which monomer) comprises 1% of the total triadic protein (42). This are presently known to be associated with triadjunction calculation assumed 1) the aldolase binding sites were satu- components, have the capacity neither to produce a step rated in uiuo, 2) the sites remained saturated in the isolated change in Ins(1,4,5)Pa (52)nor to remove the signal substance triad preparation, and 3) only one monomer of the holoen- rapidly (34). The aldolase-Ins(l,4,5)P~complex could, howzyme is required for attachment. In these partitioningexper- ever, act as a pre-existing pool of Ins(1,4,5)Pa available for iments, 13-24 pmol of tetrameric aldolase/mg of triad protein Ins( 1,4,5)P3receptors which are sensitized by depolarization were released in the absence of test ligands. This value is (53). This poolwouldbe discharged by the increase in comparable to the 22 nmol of aldolase/g of muscle which is Fru(l,6)P2, a situation which is always met by increased not bound to actin in the intact cell (11).The total concen- glycolysis when the muscle is active, and would be refilled tration of triadic aldolase binding sites in the cell is less than after muscle activation during the decay of Fru(l,6)P2. : these sites are most likely saturated in uiuo. 0.1 p ~ thus, A membrane-associated glycolyticcomplex has been inAldolase is probably interacting with protein constituents of voked in other cell types (54) including ventricular myocytes the junction. Saturable binding would not have been detected (55) to provide local ATP for the regulation of ion pumps and if aldolase were associating with the acidic lipid head groups: channels. The inositol polyphosphates may indirectly activate phosphoinositides alone would provide about 50 nmol of sites/ phosphofructokinase, one of the key regulatory enzymes of mgof TC protein (45). Furthermore, the medium employed glycolysis, through competitive (non rate-limiting) inhibition here was of an ionic strength sufficient to preclude aldolase, of aldolase which affords feedback activation by Fru(1,6)P2 (21, 56). Recent studies indicate that the inositol polyphosThis concentration is estimated from the mean of 45 pmol phates can also directly modulate phosphofructokinase (57). of aldolase binding sites/mg of triad protein using a lipid to protein ratio of 0.64 pmol of phospholipid/mg of TC/triads (45),a packing of Small cumulative changes in inositol polyphosphate levels in parallel with muscle activlo6phospholipid molecules/pm2 membrane (46) and a surface to cell and acute changes in Fru(l,6)P~ volume ratio of 0.54 @m" (47). ity will cause aldolase repartitioning. This complex interplay

titation of the amount of active protein finally bound to the CNBr-Sepharose is not as yet possible. Therefore, the experiments with the JFP-Sepharose columns can only be interpreted qualitatively with the caveate that nonspecific electrostatic protein-protein interactions will appear artifactually. However, the aldolase retained by the JFP-Sepharose was almost completely released by 10 PM Ins(1,4,5)P3, a concentration which was nearly saturating in the triad release experiments (Figs. 4-6). This correlation suggests that theJFP hasretained an inositol polyphosphate-sensitive aldolase binding site.

7356

Aldolase Repartitioning by Inositol Polyphosphutes in Skeletal Muscle

will tune local glycolytic activity in the junctional gap and, on the other hand, lead to changes in the free Ins(1,4,5)P3 concentration without the requirement of de nouo Ins(1,4,5)P3 production. Acknowledgments-Wewish to thank Prof. L. M. G. Heilmeyer, Jr. for stimulating discussions. The expert technical assistance of Monika Cochu, Ulrich Siemen, and Friedhelm Vogel is gratefully acknowledged. REFERENCES 1. Solti, M., and Friedrich, P. (1975) Mol. Cell. Biochem. 10, 145152 2. Yu, J., and Steck, T. L. (1975) J . Biol. Chem. 250,9176-9184 3. Corbett, A. M., Casewll,A. H., Kawamoto, R. M., Lugo-Gutierrez, F., and Brunschwig, J.-P. (1985) Biophys. J. 47,450a 4. Caswell, A. H., and Corbett, A. M. (1985) J. Biol. Chem. 2 6 0 , 6892-6898 5. Pierce, G . N., and Philipson, K.D. (1985) J. Biol.Chem. 260, 6862-6870 6. Gutowicz, J.. and Koimider-Schmidt,. A. (1987) . - BioDhvs. . - Chem. 27,97-102 7. Srivastava. D.K.. and Bernhard. S. A. (1986) . , Curr. TOO.Cell. Regul. 28, 1-68’ 8. Kuter, M. R., Masters, C. J., Walsh, T. P., and Winzor, D. J. (1981)Arch. Biochem. Biophys. 2 1 2 , 306-310 9. Arnold, H., and Pette, D. (1968) Eur. J. Biochem. 6,163-171 10. Clarke, F. M., and Masters, C. J. (1974) Biochim. Biophys. Acta 358,193-207 11. Clarke, F. M., Shaw, F. D., and Morton, D. J. (1980) Biochem.J . 186,105-109 12. Harris, S. J., and Winzor, D. J. (1987) Biochim. Biophys. Acta 911,121-126 13. Masters, C. J. (1985) Trends Biochem. Sci. 5, 189 14. Akkerman, J. W. N. (1985) Trends Biochem. Sci. 5, 187-188 15. Newsholme, E. A., and Crabtree, B. (1976) Biochem. SOC. Symp. 41,61-109 16. Caswell, A. H., Lau, Y. H., and Brunschwig, J.-P. (1976) Arch. Biochem. Biophys. 176, 417-430 17. Lai, F. A,, Erickson, H. P., Rousseau, E., Liu, Q.Y., and Meissner, G. (1988) Nature 3 3 1 , 315-319 18. Brunschwig, J.-P., Brandt, N. R., Caswell, A. H., and Lukeman, D. S. (1982) J. Cell. Biol. 93,533-542 19. Campbell, K. P., Franzini-Armstrong, C., and Shamoo, A. E. (1980) Biochim. Biophys. Acta602,97-116 20. Corbett, A. M., Caswell, A. H., Brandt, N. R., and Brunschwig, J.-P. (1985) J. Membr. Biol. 86, 267-276 21. Koppitz, B., Vogel, F., and Mayr, G. W. (1986) Eur. J. Biochem. 161,421-433 22. Berridge, M. J., and Irvine, R. F. (1984) Nature 312,315-321 23. Michell, R. (1986) Nature 324,613 24. Henne, V., Mayr, G. W., Grabowski, B., Koppitz, B., and Soling, H.-D. (1988) Eur. J . Biochem. 174,95-101 25. Kawamoto, R. M., Brunschwig, J.-P., and Caswell, A. H. (1988)

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