Smooth muscle cell sarcolemma. Purification and properties of plasma membranes from the rat uterus

A membrane fraction with sarcolemmal properties was purified from the smooth muscle layers (myometrium) of rat uterus by successive differential and equilibrium centrifugation in sucrose. The putative sarcolemmal fraction was identified by iodination with [^125I]iodosulfanilic acid, had an equilibrium density of 1.15, and was enriched in enzyme activities usually associated with the plasma membrane including 5′-nucleotidase (EC 3.1.3.5) and (Na⁺ + K⁺) ATPase (EC 3.6.1.3). These membranes were free of mitochondrial or nuclear membrane contamination, suggesting the relative enrichment of sarcolemmal membranes in the fraction. Proteins of the membranes were heterogeneous with respect to molecular weight, but only a few were labelled when intact muscle was radioiodinated. Uniform resistance of sarcolemmal proteins to trypsin digestion and salt extraction suggested many are tightly bound or intrinsic membrane proteins and was a further indication of the homogeneity of membranes in this fraction.     

Components of purified sarcolemma from porcine skeletal muscle

Sarcolemmal membranes were isolated from porcine skeletal muscle by modifications of a LiBr-extraction technique. Latency determinations of acetylcholinesterase, ouabain-sensitive p-nitrophenylphosphatase, [3H]ouabain binding, and (Na+ + K+)-ATPase activities indicated that 65-76% of the membranes were sealed inside-out vesicles. The preparations were enriched in cholesterol and phospholipid, and demonstrated adenylate cyclase activity and both cAMP and cGMP phosphodiesterase activities. An indication of the purity of this fraction was that the Ca2+-ATPase activity (0.13 mumol Pi mg-1 min-1 at 37 degrees C) was 3.8% of that of porcine skeletal muscle sarcoplasmic reticulum preparations. Pertussis toxin specifically catalyzed the ADP-ribosylation of a Mr 41,000 sarcolemmal protein, indicating the presence of the inhibitory guanine nucleotide regulatory protein of adenylate cyclase, Ni. An endogenous ADP-ribosyltransferase activity, with several membrane protein substrates, was also demonstrated. The addition of exogenous cAMP-dependent protein kinase or calmodulin promoted the phosphorylation of a number of sarcolemmal proteins. The calmodulin-dependent phosphorylation exhibited an approximate K 1/2 for Ca2+ of 0.5 microM, and an approximate K 1/2 for calmodulin of 0.1 microM. 125I-Calmodulin affinity labeling of the sarcolemma, using dithiobis(succinimidyl propionate), demonstrated the presence of Mr 160,000 and 280,000 calmodulin-binding components in these membranes. These results demonstrate that this porcine preparation will be valuable in the study of skeletal muscle sarcolemmal ion transport, protein and hormonal receptors, and protein kinase-catalyzed phosphorylation.     

Isolation of plasma membrane vesicles, derived from transverse tubules, by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media.

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. (Na+ + K+)-stimulated, Mg2+-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca2+-stimulated, Mg2+-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell segments in an amber low-speed (800 x g) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at 100,000 x g for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of (Na+ + K+)-stimulated ATPase compared with the other fractions, but it had essentially no Ca2+-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate. Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined bu a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basememt membrane, could not be detected in this fraction.     

Isolation and characterization of cardiac sarcolemma.

A procedure was developed for the isolation of cardiac sarcolemmal vesicles. These vesicles are enriched about ten-fold (with respect to the tissue homogenate) in K+-stimulated p-nitrophenylphosphatase, (Na+ + K+)-ATPase, 5'-nucleotidase activities and sialic acid content, all of which are believed to be components of the sarcolemma. The sarcolemma of tissue culture cardiac cells were radioiodinated and the distribution of this radioiodine paralleled the distribution of the other membrane markers above. There was very little contamination of the sarcolemmal fraction by sarcoplasmic reticulum (as judged by Ca2+-ATPase and glucose-6-phosphatase activities) or inner mitochondrial membranes (as judged by succinate dehydrogenase activity). There may, however, be some contamination by outer mitochondrial membranes (as judged by monoamine oxidase and rotenone-insensitive NADH cytochrome c reductase activities) which have rarely been monitored in cardiac sarcolemmal preparations. The purity of this preparation is good when compared with other cardiac sarcolemmal preparations. This preparation should be very useful in studying the roles of the cardiac sarcolemma (e.g. in excitation contraction coupling and Ca2+ binding).     

A plasma membrane-enriched preparation from giant barnacle muscle fibers

We describe a procedure for obtaining a highly enriched plasma membrane (sarcolemmal) preparation from muscle fibers of the giant barnacle (Balanus nubilus). The sarcolemmal-enriched portion migrated as a light fraction (F1) at the 10-24% sucrose interface. This fraction displayed saturable ouabain binding (Kd = 0.119 microM) that was enriched 10 times compared to that in the original homogenate. F1 was also prepared using muscle fibers previously labeled with 1,2-ditritio-1,2(2,2'-disulfo-4,4'-diisothiocyano)diphenylet hane, disodium salt [( 3H]-H2DIDS). F1 was enriched 25-fold in [3H]H2DIDS binding sites with respect to the homogenate. Ca2+-ATPase and succinic dehydrogenase-activities were low in F1, as was oxalate-supported Ca2+ uptake. Compared to membranes of sarcoplasmic reticulum origin, F1 was enriched in sarcolemma membranes by about 45-fold while it was enriched approximately 30-fold over mitochondrial membranes. Thus, F1 provides an extremely pure source of external muscle membranes.     

A study on the ouabain-sensitive (Na+ + K+)-ATPase activities of skeletal muscle and brain membrane fractions from genetically dystrophic mice (author's transl)]

The ouabain-sensitive (Na+ + K+)-ATPase activities of membrane fractions from hind-leg muscle and brain of normal and genetically dystrophic mice (C57BL/6J-dy strain) were studied, and the following results were obtained. 1) The ouabain-sensitive (Na+ + K+)-ATPase activity of frozen muscle sarcolemmal fraction from normal mice was several times higher than that of fresh one. 2) The ouabain-sensitive (Na+ + K+)-ATPase activity of frozen muscle sarcolemmal fraction from dystrophic mice was almost equal to that from normal one. But the muscle membrane yield from dystrophic mice was considerably low compared with the yield from normal one. 3) With brain membrane fractions, no differences were observed between normal and dystrophic mice in the ouabain-sensitive (Na+ + K+)-ATPase activity as well as in the yield of membrane fractions.     

Localization of sarcolemmal proteins to lipid rafts in the myocardium

The localization of sarcolemmal proteins within the membrane can have a dramatic effect on excitation-contraction coupling. We examine the localization of the Na+-Ca2+ exchanger, the dihydropyridine receptor, and other proteins involved in excitation-contraction coupling in rat heart using biochemical and immunolocalization techniques. Specifically, we assess the distribution of proteins within the lipid raft fraction of the sarcolemma. We find that the distribution of proteins in lipid raft fractions is very dependent on the solubilization technique. A common technique using sodium carbonate/pH 11 to solubilize non-lipid raft proteins was inappropriate for use with sarcolemmal membranes. Use of Triton X-100 was more efficacious as a solubilization agent. A large majority of the Na+-Ca2+ exchanger, Na+/K+-ATPase, and plasma membrane Ca2+ pump are not present in lipid rafts. In contrast, most adenosine A1 receptors and dihydropyridine receptors were in lipid raft fractions. Most of the adenosine A1 receptors could be co-immunoprecipitated with caveolin indicating a localization to caveolae (a subclass of lipid rafts). In contrast, the dihydropyridine receptors could not be co-immunoprecipitated with caveolin. Most biochemical data were confirmed by high resolution immunolocalization studies. Using correlation analysis, only a small fraction of the Na+-Ca2+ exchangers colocalized with caveolin whereas a substantial fraction of dihydropyridine and adenosine A1 receptors did colocalize with caveolin. The most pertinent findings are that the Na+-Ca2+ exchanger and the dihydropyridine receptor are in separate sarcolemmal subcompartments. These spatial relationships may be relevant for understanding excitation-contraction coupling.     

Changes in the surface charge properties of isolated cardiac sarcolemmal vesicles measured by light scattering. I. Characteristics of rat and canine preparations

The cation-binding characteristics of isolated sarcolemmal vesicles from rat and canine cardiac muscle cells were investigated. To help elucidate the molecular properties involved in these interactions the cation-induced aggregation behavior of rat and canine cardiac sarcolemmal vesicles, sonicated unilamellar vesicles (SUVs) made from sarcolemmal lipid extracts, and SUVs generated from combinations of synthetic lipids similar to those found in the sarcolemmal membrane, as well as mitochondrial and sarcoplasmic reticulum enriched membrane fractions were examined. Our results indicate that cations, such as Ca2+, to indeed bind to the sarcolemmal membrane surface. They also suggest that two (or more) interacting sites are involved in the Ca2+-induced aggregation of the isolated sarcolemmal vesicles, and that sarcolemmal lipid components could be the primary binding sites. The modulating (secondary) sites on the other hand may be protein or carbohydrate in nature, or require specific lipid organizational properties. Finally, the results indicate that the interactions of cations, such as Ca2+, with the sarcolemmal surface are species specific, with the sarcolemmal membranes of both rat and canine preparations having different physico-chemical properties.     

Structure-function studies of canine cardiac sarcolemmal membranes. II. Structural organization of the sarcolemmal membrane as determined by electron microscopy and lamellar X-ray diffraction

The morphological and ultrastructural properties of highly purified canine cardiac sarcolemmal vesicles, prepared by a modification (Colvin, R.A., Ashavaid, T.F. and Herbette, L.G. (1985) Biochim. Biophys. Acta 812, 601-608) of the method of Jones et al. (Jones L.R., Madlock, S.W. and Besch, H.R. (1980) J. Biol. Chem. 255, 9971-9980), were examined by several techniques. Thin-section electron microscopy showed predominantly intact unilamellar vesicles with little staining beyond the lipid bilayer boundaries. Freeze-fracture electron microscopy demonstrated that the majority of particles are approx. 90 A diameter and present at a density of 780 +/- 190 micrometers-2 (+/- S.D.). If it is assumed that some of these particles represent the (Na+ + K+)-ATPase, the finding that they are largely confined to the convex fracture face suggests a predominant right-side-out orientation of these sarcolemmal vesicles that is consistent with biochemical assays. The sarcolemmal membrane width measured by electron microscopy (unhydrated membrane width of 50-70 A) is consistent with the unit cell dimensions of 56-77 A determined by lamellar X-ray diffraction (hydrated membrane width). A unit cell dimension of 56-62 A was also found by X-ray diffraction for sarcolemmal lipids extracted from these preparations, indicating that the isolated sarcolemmal preparations do not contain a significant surface coat (glycocalyx). As both cardiac and skeletal sarcoplasmic reticulum membranes have a 80-100 A membrane width, these findings demonstrate that the purified sarcolemmal membrane is structurally distinct from both cardiac and skeletal sarcoplasmic reticulum. In contrast to the protein-rich skeletal sarcoplasmic reticulum membrane, which contains a single essential protein responsible for the regulation of cytosolic Ca2+ concentration, the sarcolemma is a lipid-rich membrane that contains a variety of proteins associated with many regulatory functions served by this membrane in cardiac muscle.     

Subfractionation of cardiac sarcolemma with wheat-germ agglutinin.

The properties of highly purified bovine cardiac sarcolemma subfractionated with the lectin, wheat-germ agglutinin (WGA) were studied. Two different membrane subfractions were isolated, one which was agglutinated in the presence of 1.0 mg of WGA/mg of protein (WGA+ vesicles) and a second fraction which failed to agglutinate (WGA- vesicles). These two membrane fractions had quantitatively different rates of Na+/K+-dependent, ouabain-sensitive ATPase and Na+/Ca2+ exchange activities, yet a similar protein composition, which suggests that they were both derived from the plasma membrane. WGA- vesicles had a decreased number of [3H]quinuclidinyl benzilate-binding sites and no detectable [3H]nitrendipine-binding sites. Electron-microscopic and freeze-fracture analysis showed that the WGA+ fraction was composed of typical spherical sarcolemmal vesicles, whereas the WGA- fraction primarily contained elongated tubular structures suggestive of the T-tubule vesicles which were previously isolated from skeletal muscle. Assays of marker enzymes revealed that these fractions were neither sarcoplasmic reticulum nor plasma membrane from endothelial cells. Moreover, WGA agglutination did not result in the separation of right-side-out and inside-out vesicles. On the basis of these findings we propose that the WGA+ fraction corresponds to highly purified sarcolemma, whereas the WGA- fraction may be derived from T-tubule membranes.     

Membrane proteins involved in potassium shifts during muscle activity and fatigue

Muscle activity is associated with potassium displacements, which may cause fatigue. It was reported previously that the density of the large-conductance Ca2+-dependent K+ (BK(Ca)) channel is higher in the T tubule membrane than in the sarcolemmal membrane and that the opposite is the case for the ATP-sensitive K+ (K(ATP)) channel. In the present experiments, we investigated the subcellular localizations of the strong inward rectifier 2.1 K+ (Kir2.1) channel and the Na+-K+-2Cl- (NKCC)1 cotransporter with Western blot analysis of different muscle fractions. Furthermore, muscle function was studied while trying to manipulate the opening probability or transport capacity of these proteins during electrical stimulation of isolated soleus muscles. All experiments were made with excised muscle from male Wistar rats. Kir2.1 channels were almost undetectable in the sarcolemmal membrane but present in the T tubule membrane, whereas NKCC1 cotransporters were present in the sarcolemmal membrane. For muscles incubated in a buffer containing pinacidil, NS1619, Ba2+, or bumetanide, there was a faster reduction in peak force (P < 0.05). Furthermore, bumetanide incubation reduced the peak force at the onset of electrical stimulation (P < 0.05). Thus the effects on muscle force indicate that these drugs can affect K+-transporting proteins and thereby influence K+ accumulation, especially in the T tubules, suggesting that K(ATP) and BK(Ca) channels are responsible for K+ release and decrease in force during repeated muscle contractions, whereas Kir2.1 and NKCC1 may have a role in K+ reuptake.     

Two major antigens of heart sarcolemma are Ca2(+)-binding glycoproteins that copurify with the dihydropyridine receptor.

Ca2+ binding has been studied in isolated heart sarcolemmal membranes using the 45Ca overlay technique. 45Ca bound to two sarcolemmal polypeptides of 125 kDa and 97 kDa in preparations from dog, rabbit, cow and pig. During fractionation on DEAE ion-exchange and wheat-germ lectin affinity columns, the two Ca2(+)-binding polypeptides copurified with the dihydropyridine receptor associated with the voltage gated Ca2+ channel. These polypeptides were the major proteins in the isolated fraction as judged by silver staining in SDS-PAGE. Antisera raised against purified dog heart, sarcolemma indicated that the 125 and 97 kDa polypeptides were highly antigenic components of this membrane. The antisera cross-reacted with similar polypeptides in cardiac sarcolemmal preparations from rabbit, cow and pig, but not sarcoplasmic reticulum membranes. Purified antibodies against the 125 kDa polypeptide did not cross-react with the 97 kDa polypeptide, while antibodies against the 97 kDa polypeptide did not cross-react with the 125 kDa polypeptide. Both the 125 kDa and 97 kDa polypeptides bound wheat-germ lectin, suggesting both were glycoproteins. It is unlikely that these Ca2+ binding glycoproteins represent subunits of the dihydropyridine receptor-Ca2+ channel in this membrane.     

Dihydropyridine binding to the L-type Ca2+ channel in rabbit heart sarcolemma and skeletal muscle transverse-tubules: role of disulfide, sulfhydryl and phosphate groups

The dihydropyridine receptor is associated with the L-type Ca2+ channel in the cell membrane. In this study we have examined the effects of group-specific modification on dihydropyridine binding in heart sarcolemmal membranes isolated from the rabbit. Specifically, dithiothreitol and glutathione were employed to assess the possible role of disulfide (-SS-) bonds in the binding of [3H]dihydropyridines. NEM, PCMS and iodoacetamide were employed to examine the effect of blocking free sulfhydryl groups (-SH) on the binding of [3H]dihydropyridines to their receptor in heart sarcolemma. Glutathione inhibited [3H]PN200-110 binding to sarcolemmal membranes 100%, with an IC50 value of 50 microM, while DTT inhibited maximally by 75% with an IC50 value in the millimolar range. Alkylation of free sulfhydryl groups by NEM or iodoacetamide inhibited binding of [3H]PN200-110 binding in cardiac sarcolemma approx. 40-60%. Blocking of free sulfhydryl groups by PCMS completely inhibited [3H]PN200-110 binding to their receptor in sarcolemmal membranes in a dose-dependent manner with an IC50 value of 20 microM. These results suggest the involvement of disulfide bonds and free sulfhydryl groups in DHP binding to the L-type Ca2+ channel in heart muscle. We also examined the effect of membrane phosphorylation on the specific binding of the dihydropyridine [3H]nitrendipine to its receptor. Phosphorylation was studied in cardiac sarcolemmal as well as skeletal muscle transverse-tubule membranes. Phosphorylation due to endogenous protein kinase and cAMP-dependent protein kinase was without effect on [3H]nitrendipine binding in both cardiac sarcolemmal and skeletal muscle membranes. Addition of exogenous calmodulin under conditions known to promote Ca2+/calmodulin-dependent phosphorylation increased [3H]nitrendipine binding 20% with no alteration in KD in both types of membrane preparation. These results suggest a role for calmodylin in dihydropyridine binding to L-type Ca2+ channels.     

Low molecular weight GTP-binding proteins in cardiac muscle. Association with a 32-kDa component related to connexins.

Low molecular weight GTP-binding proteins and their cellular interactions were examined in cardiac muscle. Heart homogenate was separated into various subcellular fractions by differential and sucrose density gradient centrifugation. Various fractions were separated by sodium dodecyl sulfate-gel electrophoresis, blotted to nitrocellulose, and GTP-binding proteins detected by incubating with [alpha-32]GTP. Three polypeptides of M(r) 23,000, 26,000, and 29,000 were specifically labeled with [alpha-32P]GTP in all the fractions examined and enriched in sarcolemmal membranes. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 26- and 29-kDa polypeptides. A polypeptide of M(r) 40,000 was weakly labeled with [alpha-32P]GTP in the sarcolemmal membrane and tentatively identified as Gi alpha by immunostaining with anti-Gi alpha antibodies. Cytosolic GTP-binding proteins were labeled with [alpha-32P]GTP and their potential sites of interaction investigated using the blot overlay approach. A polypeptide of 32 kDa present in sarcolemmal membranes, intercalated discs, and enriched in heart gap junctions was identified as a major site of interaction. The low molecular weight GTP-binding proteins associated with the 32-kDa polypeptide through a complex involving cytosolic components of M(r) 56,000, 36,000, 26,000, 23,000, and 12,000. A monoclonal antibody against connexin 32 from liver strongly recognized the 32-kDa polypeptide in heart gap junctions, whereas polyclonal antibodies only weakly reacted with this polypeptide. The low molecular weight GTP-binding proteins associated with a 32-kDa polypeptide in liver membranes that was also immunologically related to connexin 32. These results indicate the presence of a subset of low molecular weight GTP-binding proteins in a membrane-associated and a cytoplasmic pool in cardiac muscle. Their association with a 32-kDa component that is related to the connexins suggests that these polypeptides may be uniquely situated to modulate communication at the cell membrane.     

The Ca2+-pumping ATPase and the major substrates of the cGMP-dependent protein kinase in smooth muscle sarcolemma are distinct entities

It has been proposed that the plasma membrane Ca2+ pump of smooth muscle tissues may be regulated by cGMP-dependent phosphorylation [Popescu, L. M., Panoiu, C., Hinescu, M. & Nutu, O. (1985) Eur. J. Pharmacol. 107, 393-394; Furukawa, K. & Nakamura, H. (1987) J. Biochem. (Tokyo) 101, 287-290]. This hypothesis has been tested on a smooth muscle sarcolemma preparation from pig thoracic aorta. The actomyosin-extracted membranes showed ATP-dependent Ca2+ uptake as well as cGMP-dependent protein kinase (G-kinase) activity. The molecular masses of the major protein substrates of the G-kinase (G1) and that of the Ca2+ pump were compared. Electrophoretic analysis of the phosphorylated intermediate of the sarcolemmal Ca2+-ATPase and the G1 phosphoprotein showed that these two proteins are not identical. The results were confirmed by using a 125I-calmodulin overlay technique and an antibody against human erythrocyte Ca2+-ATPase. Ca2+-uptake experiments with prephosphorylated membrane vesicles were carried out to elucidate possible effects of cGMP-dependent phosphorylation of membrane proteins on the activity of the Ca2+ pump. The cGMP-dependent phosphorylation was found to be extremely sensitive to temperature leading to very low steady-state phosphorylation levels at 37 degrees C. The difficulty was overcome by ATP[gamma S], which produced full and stable thiophosphorylation of G1 during the Ca2+-uptake experiments at 37 degrees C. However, the cGMP-dependent thiophosphorylation failed to influence the Ca2+-uptake properties of sarcolemmal vesicles. The results show that the Ca2+ pump of smooth muscle plasma membrane is not a direct target of the cGMP-dependent protein kinase and is not regulated by the cGMP-dependent phosphorylation of membrane proteins.     

Na+, K+, H+ and Cl- permeability properties of rabbit skeletal muscle sarcolemmal vesicles

The ion permeability properties of rabbit skeletal muscle sarcolemmal vesicles were investigated by means of radioisotope flux, membrane potential, and light-scattering measurements. An enriched sarcolemmal fraction was obtained from the 22-27% region of sucrose gradients after isopycnic centrifugation. The presence of contaminating sarcoplasmic reticulum was assessed with the use of a purified sarcoplasmic reticulum vesicle fraction. 22Na+, 86Rb+, 36Cl-, and [3H]sucrose flux measurements indicated that the sarcolemmal fraction possessed isotope spaces ranging between 1.5 and 4 microliters/mg protein. Membrane potential measurements using the voltage-sensitive fluorescent probe 3,3'-dipentyl-2,2'-oxadicarbocyanine iodide (diO-C5-(3)) indicated that sarcolemmal vesicles were impermeable to H+ and Na+ but that 10-15% of the vesicles were permeable to K+. Light-scattering measurements indicated a small fraction of sarcolemmal vesicles were permeable to both K+ and Cl-. Whether the low permeability of sarcolemmal vesicles to Na+, K+, and Cl- is the result of a low concentration of ion channels or the inactivation of these channels during isolation is at present uncertain.     

Inhibition of Na+/Ca2+ exchanger activity in cardiac and skeletal muscle sarcolemmal vesicles by monoclonal antibody 44D7

Monoclonal antibodies 44D7 and 4F2 inhibited specifically the Na+-dependent Ca2+ fluxes characteristic of the Na+/Ca2+ exchanger in cardiac and skeletal muscle sarcolemmal vesicles. Preincubation of membrane vesicles with monoclonal antibody 44D7 inhibited 90% of the Na+-dependent Ca2+ uptake measured in the first 10 s of the reaction and 50% of that measured after 60 s. Ca2+/calmodulin-dependent ATPase activity and ATP-dependent Ca2+ uptake by sarcolemmal vesicles were not affected by monoclonal antibody 44D7 whereas the Na+-dependent release of accumulated Ca2+ was inhibited. In the presence of the 44D7 antigen isolated from human kidney, monoclonal antibody 44D7 could no longer inhibit Na+-dependent Ca2+ fluxes. The distribution of 4F2 antigenic activity in the isolated muscle membrane fractions correlated with that of Na+/Ca2+ exchanger activity; cardiac and skeletal muscle sarcolemmal vesicles expressed higher levels of the antigen than skeletal muscle transverse tubule membrane, while no antigen could be detected in sarcoplasmic reticulum membranes. Our results suggest that monoclonal antibodies 44D7 and 4F2 interact either directly with the Na+/Ca2+ exchanger molecules or with some other protein(s) responsible for the regulation of this activity in the heart and skeletal muscle.     

Protein analysis of cardiac sarcolemma: effects of membrane-perturbing agents on membrane proteins and calcium transport.

Protein composition of cardiac sarcolemmal membranes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Membranes were observed to contain about 20 polypeptide bands ranging from 18000 to 200 000 dalton mass. Out of these, six bands were prominent and together comprised 57% of the membrane protein. When sarcolemmal membranes, phosphorylated by [gamma-(32)P] ATP in the presence of Ca(2+) or Na+ with and without K+, were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 2.4, the band III region (Mr 105 000) of gels was found to contain active sites of monomeric Ca-ATPase and (Na,K)ATPase. Bands I (Mr greater than 200 000), II (Mr 150 000), III (Mr 105 000), and VI (Mr 47 000) were accesible to trypsin; the extent of proteolysis was dependent on the time of exposure to, and the concentration of, trypsin (i.e, ratio of sarcolemmal protein/trypsin). Addition of molar sucrose protected sarcolemmal proteins from the tryptic proteolysis. Calcium transport was reduced by the action of trypsin; the degree of reduction was influenced by the time of exposure of membranes to trypsin as well as the concentration of trypsin. (Mg,Ca)ATPase activity, on the other hand, was elevated moderately at lower concentration and reduced at higher concentration of trypsin. Treatment with phospholipase C cium transport and (Mg,Ca)ATPase activity; electrophoretic patterns were unaffected by this treatment. Addition of lecithin to phospholipase C treated membranes produced a moderate increase in calcium transport. Exposure to Triton X-100 (1%) specifically solubilized three protein bands (Mr90 000, 67 000, and 57 000), whereas exposure to deoxycholate (1%) preferentially solubilized high-molecular-weight proteins, including band III (Mr 105 000); Lubrol-PX (1%) caused nonspecific solubilization of proteins, although the extent of solubilization with Lubrol-PX was considerably less than with either Triton or deoxycholate.     

Subcellular fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2+ + Mg2+)-ATPase activity in plasmalemma and endoplasmic reticulum

Isolated membrane vesicles from pig stomach smooth muscle (antral part) were subfractionated by a density gradient procedure modified in order to obtain an efficient extraction of extrinsic proteins. By using this method in combination with digitonin-treatment, an endoplasmic reticulum fraction contaminated with maximally 10 to 20% of plasma membranes was isolated, together with a plasma membrane fraction containing at most 30% endoplasmic reticulum. The endoplasmic reticulum and plasma membrane fractions differed in protein composition, reaction to digitonin, binding of wheat germ agglutinin, activities of marker enzymes and in the characteristics of the Ca2+ uptake. The Ca2+ uptake by the endoplasmic reticulum was much more stimulated by oxalate than the uptake by plasma membranes. Both fractions showed a (Ca2+ + Mg2+)-ATPase activity, but the largest amount of this enzyme was present in the plasma membranes. The study of the phosphorylated intermediates of the (Ca2+ + Mg2+)-ATPase by polyacrylamide gel electrophoresis revealed two phosphoproteins one of 130 kDa and one of 100 kDa (Wuytack, F., Raeymaekers, L., De Schutter, G. and Casteels, R. (1982) Biochim. Biophys. Acta 693, 45-52). The 130 kDa enzyme was predominant in the fraction enriched in plasma membrane whereas the distribution of the 100 kDa polypeptide correlated with the endoplasmic reticulum markers. The 130 kDa ATPase was the main 125I-calmodulin binding protein detected on nitrocellulose blots of proteins separated by gel electrophoresis. The (Ca2+ + Mg2+)-ATPase activity of the plasma membranes was higher than the (Na+ + K+)-ATPase activity, suggesting that the Ca2+ extrusion from these cells depends much more on the activity of the (Ca2+ + Mg2+)-ATPase than on Na+-Ca2+ exchange.     

Na+−Ca2+ exchange and Ca2+ channel characteristics in bovine aorta and coronary artery smooth muscle sarcolemmal membranes

Tension generation and Ca²⁺ flux in smooth muscle varies depending upon the diameter of a vessel and its location. The purpose of the present investigation was to determine if the biochemical characteristics of the Na⁺–Ca²⁺ exchanger and the Ca²⁺ channel differ in sarcolemmal membrane preparations isolated from a large conduit vessel (thoracic aorta) or from large and small coronary arteries. We also investigated the possibility of differences between sarcolemmal membranes isolated from coronary arteries dissected from the right and left ventricles. The purification of the sarcolemmal membranes was of a similar magnitude amongst the different groups. Contamination of the sarcolemmal membranes with other membranous organelles was negligible and similar amongst the groups. The K_m and V_max of Na⁺-dependent Ca²⁺ uptake in sarcolemmal vesicles was similar amongst the groups. Calcium channel characteristics were examined by measuring [³H]PN200-110 binding to sarcolemmal vesicles. The right coronary artery membranes from both large and small caliber vessels exhibited a higher K_d and the small right coronary artery sarcolemmal preparation had a lower maximal binding density for [³H] PN200-110. The results suggest that the right coronary artery, and in particular the small diameter right coronary artery, possesses altered Ca²⁺ channel characteristics in isolated sarcolemmal membranes.