The effect of carnosine on Ca-channels in rabbit skeletal muscle sarcoplasmic reticulum]

Carnosine (beta-alanyl-L-histidine), which is present in millimolar concentrations in skeletal muscles, induces Ca2+ release from the heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum by activation ruthenium red-sensitive Ca-release channels. The effect of carnosine is dose-dependent, which indicates the presence of saturable carnosine-binding sites in the Ca-release channel molecule. The half-maximal Ca2+ release is observed in the presence of 8.7 mM carnosine. At the same time, carnosine addition to the medium increases the affinity of sarcoplasmic reticulum Ca-channels for the Ca-release activators, caffeine and adenine nucleotides. It is concluded that carnosine is an endogenous regulator of skeletal muscle sarcoplasmic reticulum Ca-channels which modulates the affinity of these channels for different ligands.     

Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmic reticulum. Comparative biochemical analysis of component activities.

Sarcolemmal and sarcoplasmic reticulum membrane vesicle fractions were isolated from cardiac microsomes. Separation of sarcolemmal and sarcoplasmic reticulum membrane markers was documented by a combination of correlative assay and centrifugation techniques. To facilitate the separation, the crude microsomes were incubated in the presence of ATP, Ca2+, and oxalate to increase the density of the sarcoplasmic reticulum vesicles. After sucrose gradient centrifugation, the densest subfraction (sarcoplasmic reticulum) contained the highest (K+,Ca2+)-ATPase activity and virtually no (Na2+,K+)-ATPase activity, even when latent (Na+,K+)-ATPase activity was unmasked. In addition, the sarcoplasmic reticulum fraction contained no significant sialic acid, beta receptor binding activity, or adenylate cyclase activity. Sarcolemmal membrane fractions were of low buoyant density. Preparations most enriched in sarcolemmal vesicles contained the highest level of all the other parameters and only about 10% of the (K+,Ca2+)-ATPase activity of the sarcoplasmic reticulum fraction. The results suggest that (Na+,K+)-ATPase, sialic acid, beta-adrenergic receptors, and adenylate cyclase can be entirely accounted for by the sarcolemmal content of cardiac microsomes. Gel electrophoresis of the sarcolemmal and sarcoplasmic reticulum membrane fractions showed distinct bands. Membrane proteins exclusive to each of the fractions were also demonstrated by phosphorylation. Cyclic AMP stimulated phosphorylation by [gamma-32P]ATP of two proteins of apparent Mr = 20,000 and 7,000 that were concentrated in sarcoplasmic reticulum, but the stimulation was markedly dependent on the presence of added soluble cyclic AMP-dependent protein kinase. Cyclic AMP also stimulated phosphorylation of membrane proteins in sarcolemma, but this phosphorylation was mediated by an endogenous protein kinase activity. The apparent molecular weights of these phosphorylated proteins were 165,000, 90,000, 56,000, 24,000, and 11,000. The results suggest that sarcolemma may contain an integral enzyme complex, not present in sarcoplasmic reticulum, that contains beta-adrenergic receptors, adenylate cyclase, cyclic AMP-dependent protein kinase, and several substrates of the protein kinase.     

Caffeine interaction with the Ca-release channels of heavy sarcoplasmic reticulum. Evidence that 170 kD Ca-binding protein is a caffeine receptor of the Ca-channels

The study of Ca2+- and caffeine-induced Ca2+ release from heavy sarcoplasmic reticulum vesicles under the different conditions suggests that Ca2+ and caffeine can interact with the common receptor of the Ca-release channels. The reticulum membranes were solubilized using nonionic detergent polyoxyethylene 9-lauryl ether, and affinity chromatography on reactive red 120-agarose was carried out. The 170 kD Ca-binding protein which is eluted by caffeine is the most probable candidate for the caffeine receptor of the Ca-channels.     

Mechanism of the stimulation of Ca2+-dependent ATPase of skeletal muscle sarcoplasmic reticulum by protein kinase

Sarcoplasmic reticulum isolated from moderately fast rabbit skeletal muscle contains intrinsic adenosine 3',5'-monophosphate (cAMP)-independent protein kinase activity and a substrate of 100 000 Mr. Phosphorylation of skeletal sarcoplasmic reticulum by either endogenous membrane bound or exogenous cAMP-dependent protein kinase results in stimulation of the initial rates of Ca2+ transport and Ca2+-ATPase activity. To determine the molecular mechanism by which protein kinase-dependent phosphorylation regulates the calcium pump in skeletal sarcoplasmic reticulum, we examined the effects of protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Skeletal sarcoplasmic reticulum vesicles were preincubated with cAMP and cAMP-dependent protein kinase in the presence (phosphorylated sarcoplasmic reticulum) and absence (control sarcoplasmic reticulum) of adenosine 5'-triphosphate (ATP). Control and phosphorylated sarcoplasmic reticulum were subsequently assayed for formation (5-100 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E approximately P) of Ca2+-ATPase. Protein kinase mediated phosphorylation of skeletal sarcoplasmic reticulum resulted in pronounced stimulation of initial rates and levels of E approximately P in sarcoplasmic reticulum preincubated with either ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) prior to assay (Ca2+-free sarcoplasmic reticulum), or with calcium/EGTA buffer (Ca2+-bound sarcoplasmic reticulum). These effects were evident within a wide range of ionized Ca2+. Phosphorylation of skeletal sarcoplasmic reticulum by protein kinase also increased the initial rate of E approximately P decomposition. These findings suggest that protein kinase-dependent phosphorylation of skeletal sarcoplasmic reticulum regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the active calcium transport observed at steady state.     

Changes in the structure, composition and function of sarcoplasmic-reticulum membrane during development.

The structure, chemical composition and function of the microsomal fraction, isolated by differential centrifugation and purified on sucrose gradients, from muscle of fetal, newborn and young rabbits were characterized and compared with those of sarcoplasmic reticulum vesicles from adult muscle. Negative staining shows that the microsomal vesicles isolated from muscles of embryos and newborn animals are smooth, in contrast to vesicles obtained from adult muscle which contain 4-nm particles on their surface. The particles appear first in the microsomal vesicles from muscles of 5--8-day-old rabbits. Their number increases with the age of the animals. Ca2+-pump protein, with molecular weight about 100000, accounts for 10% of the total protein content in sarcoplasmic reticulum membrane, isolated at the earliest stages of development analysed. Its amount increases continuously with the rabbit's age to the adult value of about 70% of total sarcoplasmic reticulum protein. The low amount of 100000-dalton protein and lack of 4-nm surface particles in sarcoplasmic reticulum vesicles obtained from fetal and newborn rabbits are strictly correlated with the low activity of Ca2+-dependent ATPase and the ability to take up Ca2+. These activities rise in parallel with the age of the rabbits. On the other hand, Mg2+-dependent ATPase activity is very high at the early stages of development and declines continuously to a low value in sarcoplasmic reticulum from adult muscle. The sarcoplasmic reticulum membrane from fetal and newborn rabbits contains a higher amount of lipids as compared with the membrane present in the muscle of adult animals. The ratio of both phospholipid to protein and neutral lipid to protein decreases with the age of the rabbits. The composition of sarcoplasmic reticulum phospholipids also changes during development.     

The separate profile structures of the functional calcium pump protein and the phospholipid bilayer within isolated sarcoplasmic reticulum membranes determined by X-ray and neutron diffraction

The detailed profile structure of the isolated sarcoplasmic reticulum membrane was studied utilizing a combination of X-ray and neutron diffraction. The water and lipid profile structures within the sarcoplasmic reticulum membrane were determined at 28 A resolution directly by neutron diffraction and selective deuteration of the water and lipid components. The previously determined electron density profile structure of the sarcoplasmic reticulum membrane at 12 A resolution was subjected to model refinement analysis constrained by the neutron diffraction results, thereby providing unique higher resolution calculated lipid and protein profile structures. It was found that the lipid bilayer profile structure of the isolated sarcoplasmic reticulum membrane is asymmetric, primarily the result of more lipid residing in the inner versus the outer monolayer of the sarcoplasmic reticulum lipid bilayer. The asymmetry in the lipid composition was necessarily coincident with a complimentary asymmetry in the protein mass distribution between the two monolayers in order to preserve the overall cross-sectional area of lipid and protein throughout the lipid bilayer region of the sarcoplasmic reticulum membrane profile structure. Approximately 50% of the mass of the total protein was found to be localized externally to the sarcoplasmic reticulum membrane lipid bilayer protruding from the outer lipid monolayer into the extravesicular medium. The structural features of the protein protrusion appear to be rather variable depending upon the environment of the sarcoplasmic reticulum membrane. This highly asymmetric structural organization of the sarcoplasmic reticulum membrane profile is consistent with its primary function of unidirectional calcium transport.     

Membrane localization of myocardial type II cyclic AMP-dependent protein kinase activity

Crude cardiac membrane vesicles were separated into subfractions of sarcolemma and sarcoplasmic reticulum. The subfractions were used to determine the origin and type of cyclic AMP-dependent protein kinase activity present in myocardial membranes. A cyclic AMP-binding protein of molecular weight 55,000 was covalently labeled with the photoaffinity probe 8-azido adenosine 3',5'-mono[32P]phosphate, and found to copurify with the (Na+ + K+)-ATPase activity of sarcolemma, and away from the (Ca2+ + K+)-ATPase activity of sarcoplasmic reticulum. Endogenous cyclic AMP-dependent protein kinase activity also copurified with sarcolemma. Protein substrates phosphorylated by cyclic AMP-dependent protein kinase activity had apparent molecular weights of 21,000 and 8000 and were present in both sarcolemma and sarcoplasmic reticulum. However, while addition of cyclic AMP alone resulted in phosphorylation of sarcolemma proteins, both cyclic AMP and exogenous, soluble cyclic AMP-dependent kinase were required for phosphorylation of sarcoplasmic reticulum proteins. Addition of the calcium-binding protein, calmodulin, to either sarcolemma or sarcoplasmic reticulum resulted in phosphorylation of the 21,000 and 8000-dalton proteins, as well. The results suggest that cardiac sarcolemma contains an intrinsic type II cyclic AMP-dependent protein kinase activity that is not present in sarcoplasmic reticulum. On the other hand, Ca2+- and calmodulin-dependent protein kinase activity is present in both sarcolemma and sarcoplasmic reticulum.     

A study of passive Ca2+ flow through the sarcoplasmic reticulum of skeletal muscles. I. Passive Ca2+ efflux from vesicle membranes

The same level of passively loaded Ca2+ was observed both in the heavy (enriched in terminal cisternae) and light (enriched in longitudinal reticulum) sarcoplasmic reticulum (SR) fractions. The level of passively loaded Ca2+ of the both SR fractions decreased in the presence of 150 mM K+. However the rate and extent of Ca2+ release was greater from heavy SR fraction. The rate of Ca2+ release under conditions of antiport of K+, Na+, choline+ and gluconate-, Cl-, SCH- increased proportion with their permeability through the SR membrane. The initial rate of Ca2+ release also became higher under equal concentration of monovalent cation chloride both inside and outside the SR vesicles. Apparently, in this case Ca2+ release occurs through Ca-channels which are open at a membrane potential.     

Comparison of the (Ca2+ + Mg2+)-ATPase proteins from normal and dystrophic chicken sarcoplasmic reticulum.

The involvement of membrane protein in dystrophic chicken fragmented sarcoplasmic reticulum alterations has been examined. A purified preparation of the (Ca2+ + Mg2+)-ATPase protein from dystrophic fragmented sarcoplasmic reticulum was found to have a reduced calcium-sensitive ATPase activity and phosphoenzyme level, in agreement with alterations found in dystrophic chicken fragmented sarcoplasmic reticulum. An amino acid analysis of the ATPase preparations showed no difference in the normal and dystrophic (Ca2+ + Mg2+)-ATPase. The (Ca2+ + Mg2+)-ATPase was investigated further by isoelectric focusing and proteolytic digestion of the fragmented sarcoplasmic reticulum. Neither of these methods indicated any alteration in the composition of the dystrophic (Ca2+ + Mg2+)-ATPase. We have concluded that the alterations observed in dystrophic fragmented sarcoplasmic reticulum are not due to increased amounts of non-(Ca2+ + Mg2+)-ATPase protein, and that the normal and dystrophic (Ca2+ + Mg2+)-ATPase protein are not detectably different.     

Peripheral membrane proteins of sarcoplasmic and endoplasmic reticulum. Comparison of carboxyl-terminal amino acid sequences

Peripheral endoplasmic reticulum membrane proteins residing in the lumen of the endoplasmic reticulum occupy the same space as other secreted proteins. The presence of a four amino acid salvage or retention signal (KDEL-COOH = Lys-Asp-Glu-Leu-COOH) at the carboxyl-terminal end of peripheral membrane proteins has been shown to represent a signal or an essential part of a signal for their retention within the endoplasmic reticulum membrane. In heart and skeletal muscle, a number of sarcoplasmic reticulum proteins have recently been identified which are peripheral membrane proteins. The high-affinity calcium-binding protein (55 kilodaltons (kDa] appears to conform to the above described mechanisms and contains the KDEL carboxyl-terminal tetrapeptide. Thyroid hormone binding protein is present in the sarcoplasmic reticulum, in addition to its endoplasmic reticulum location, and has a modified but related tetrapeptide sequence (RDEL = Arg-Asp-Glu-Leu), which also probably functions as the retention signal. Calsequestrin and a 53-kDa glycoprotein, two other peripheral membrane proteins residing in the lumen of the sarcoplasmic reticulum, do not contain the KDEL retention signal. The sarcoplasmic reticulum may have developed a unique retention mechanism(s) for these muscle-specific proteins.     

Subcellular location of adenylate cyclase in rat cardiac muscle

Crude homogenates of rat cardiac muscle were fractionated in order to examine the subcellular location of adenylate cyclase in this tissue. The fractionation procedure employed differential centrifugation of homonized material, followed by collagenase treatment, centrifugation on a discontinuous sucrose density gradient and extraction with 1 M KCl. The particulate fraction obtained by this procedure contained a high specific activity and yield of adenylate cyclase, moderate levels of mitochondria and low levels of sarcoplasmic reticulum and contractile protein as judged by marker enzyme activities. Adenylate cyclase was purified 20-fold with a 33% yield from the crude homogenate, while mitochondrial, sarcoplasmic reticulum and contractile protein yields were 5, 0.4 and 0.7% respectively. The membrane fractions prepared in this manner were examined by sodium dodecyl sulfate · gel electrophoresis.Adenylate cyclase copurified with ouabain-sensitive (Na⁺ + K⁺)-ATPase, a plasma membrane marker enzyme, and not with Ca²⁺-accumulating activity, which is associated with the sarcoplasmic reticulum. The distribution of marker enzyme activities indicates that heart adenylate cyclase is not located in the sarcoplasmic reticulum but is localized predominantly, if not exclusively, in the plasma membrane.     

Immunological and biochemical properties of transverse tubule membranes isolated from rabbit skeletal muscle

A new method for isolating transverse tubule membranes from rabbit skeletal muscle has been developed. This procedure has the advantage of being mild, fast, and producing with good yields a purified membrane fraction. The transverse tubule membranes are purified by a discontinuous sucrose density centrifugation after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. Immunofluorescence staining of cryostat sections of rabbit psoas muscle with purified goat antibodies directed against the purified membranes shows that the reacting antigens are distributed at the boundary of the A and I bands of the myofibrils where transverse tubules are localized in mammalian muscle. The purified antibodies showed no cross-reactivity with sarcoplasmic reticulum, nor did they show any fluorescence staining of the muscle plasma membrane, indicating that the isolated membranes indeed originate from the transverse tubules. The transverse tubule fraction has a characteristic protein composition distinguishable from that of sarcoplasmic reticulum, a much higher cholesterol content than that of the crude microsomes, plasma membrane, and sarcoplasmic reticulum, and a phospholipid content about twice as high as that of sarcoplasmic reticulum and plasma membrane. The purified transverse tubule membrane has a distinct phospholipid composition with high contents of sphingomyelin and phosphatidylserine. A Mg2+-activated ATPase characteristic of the transverse tubule fraction undergoes a 20-30-fold increase in specific activity during purification. The levels of Ca2+-ATPase activity present in the purified transverse tubule fraction remain comparable to those of sarcoplasmic reticulum even after extensive removal of the latter.     

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.     

Target size of calcium pump protein from skeletal muscle sarcoplasmic reticulum

The oligomeric size of calcium pump protein (CPP) in fast skeletal muscle sarcoplasmic reticulum membrane was determined using target theory analysis of radiation inactivation data. There was a parallel decrease of Ca2+-ATPase and calcium pumping activities with increasing radiation dose. The loss of staining intensity of the CPP band, observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, also correlated directly with the loss of activity. The target size molecular weight of the CPP in the normal sarcoplasmic reticulum membrane ranged between 210,000 and 250,000, which is consistent with a dimeric structure. Essentially the same size is obtained for the non-phosphorylated CPP or for the phosphoenzyme form generated from either ATP (E1 state) or inorganic phosphate (E2 state). Hence, the oligomeric state of the pump does not appear to change during the catalytic cycle. Similar results were obtained with reconstituted sarcoplasmic reticulum membrane vesicles with different lipid to protein ratios. We conclude that the CPP is a dimer in both native and reconstituted sarcoplasmic reticulum membranes. The target size of the calcium-binding protein (calsequestrin) was found to be 50,000 daltons, approximating a monomer.     

Stereological analysis of the ultrastructural organization of the cardiomyocytes in the red-cheeked suslik in different seasons of the year

The myocardium of red-cheeked sousliks was studied at different seasons with the use of stereological analysis. The volume and surface densities of the myofibrils, mitochondria, T system, sarcoplasmic reticulum as well as the surface-volume ratios of the main organelles were measured. Pronounced seasonal changes in physiological activity of the heterothermal animals were accompanied by marked reorganization of the spatial ultrastructure of the cardiomyocytes. The seasonal regression of the heart weight and diminution of the cardiomyocyte diameter were recorded during hibernation. The authors believe that the increased volume ratio of the cytoplasmic organelles to the myofibrils forms the basis for a rapid adaptive reaction of the heart during hibernation and waking up.     

Studies on the heterogeneity of sarcoplasmic reticulum vesicles.

Isolated sarcoplasmic reticulum vesicles from rabbit white muscle were separated into a light (15--20% of total microsomes) and a heavy (80--85%) fraction by density gradient centifugation. The ultrastructure, chemical composition, enzymic activities and localization of membrane components in the vesicles of both fractions were investigated. From the following results it was concluded that both fractions are derived from the membranes of the sarcoplasmic reticulum system of the muscle: (i) The protein pattern of both fractions is essentially the same, except for different ratios of acidic, Ca2+-binding proteins. (ii) The 105000 dalton protein of the light fraction cross-reacts immunologically with the Ca2+-dependent ATPase of the heavy fraction. (iii) Ca2+-dependent ATPase, although of different specific activity, is found in both fractions. After rendering the vesicles leaky, specific activities in both fractions reach the same value. The light fraction was found to consist of "inside-out" vesicles by the following criteria: (i) No Ca2+ accumulation can be measured and the Ca2+-dependent ATPase activity is low and variable. (ii) The rate of trypsin digestion is lower and, compared to the heavy microsomes, a different ratio of degradation products is obtained. (iii) The sarcoplasmic reticulum membrane has a highly asymmetrical lipid distribution. This distribution of aminophospholipids is opposite to that in vesicles of heavy fraction. The light sarcoplasmic reticulum fraction has a higher phospholipid to protein ratio than the heavy one. This is consistent with the possibility that the two fractions derive from different parts of the sarcoplasmic reticulum system.     

Solubilization of sarcoplasmic reticulum membranes by sodium dodecylsulphate. A Fourier-transform infrared spectroscopic study

In order to improve our understanding of membrane protein solubilization by sodium dodecylsulphate, sarcoplasmic reticulum vesicles have been treated with this surfactant at different detergent: protein mole ratios. Effects on Ca2(+)-ATPase activity, membrane protein solubilization, and protein conformation have been independently monitored, and correlations among the various parameters have been observed. The thermal denaturation of sarcoplasmic reticulum proteins in the presence of sodium dodecylsulphate has also been characterized spectroscopically.     

Calcium release from vesicles of heavy sarcoplasmic reticulum of rabbit skeletal muscles

The release of Ca2+ from vesicles of heavy sarcoplasmic reticulum after its accumulation due to hydrolysis of ATP, GTP, CTP, UTP or ITP has been studied using Antipyrylazo III, a metal-chromic Ca-indicator. All the studied substrates of the Ca-pump provide Ca2+ accumulation inside the heavy sarcoplasmic reticulum vesicles, the spontaneous Ca2+ outflux rate being different for different nucleoside triphosphates. It is only ATP that provides Ca-(caffeine)-induced Ca2+ release, however AMP, ADP, beta, gamma-methylene-ATP induce Ca2+ ejection in the presence of nonadenylic nucleotides. The ruthenium red (10(-7M) inhibits the induced ejection of Ca2+ from vesicles of the heavy sarcoplasmic reticulum, but does not prevent the spontaneous release of Ca2+ in the same concentrations. A conclusion is drawn that besides Ca-channels sensitive to Ca2+ and caffeine in the presence of ATP (or to AMP, ADP, beta, gamma-methylene-ATP in the presence of nonadenylic nucleotides) and possessing high sensitivity to the ruthenium red there is another pathway for Ca2+ in the heavy reticulum membranes along which its spontaneous release occurs after the substrate exhaustion. It is supposed that this release is provided by the presence of the Ca-ATPase protein.     

Interaction of different nucleotides with Ca-release channels from heavy sarcoplasmic reticulum

Calcium accumulation and release from the heavy fraction of sarcoplasmic reticulum vesicles have been studied in the presence of different nucleotides with the use of the Ca-sensitive dye antipyrylazo III for monitoring of the free Ca2+ concentration. The calcium- and caffeine-induced Ca2+ release is observed only with ATP but not with any of nonadenine nucleotides used as substrates for the Ca-pump. Adenine nucleotides provide for a rapid ruthenium red sensitive Ca2+ release from the vesicles, when nonadenine nucleotides are used as energy sources for Ca2+ uptake. A comparison of the nucleotides interaction with Ca-channels and Ca-ATPase supports the hypothesis that Ca-ATPase is involved in the operation of Ca-channels.     

Analysis of the arrangement of protein components in the sarcomplasmic reticulum of rat skeletal muscle.

The nature of the protein components and their location in the sarcoplasmic reticulum membrane were studied using sarcoplasmic reticulum vesicles isolated from rat skeletal muscle and purified by a density gradient centrifugation system. On the basis of analysis by means of sodium dodecyl sulfate gel electrophoresis, the protein components appear to be similar if not identical with those reported by others for rabbit sarcoplasmic reticulum, and the relative amount of each component is also similar to that found with rabbit sarcoplasmic reticulum. Evidence is presented that radioiodine-labeled diazotized diiodosulfanilic acid is a nonpermeant labeling agent of the protein components of sarcoplasmic reticulum vesicles; this agent minimally disturbs the functional activities of these membranes. By means of this labeling agent and perturbing agents, it is concluded that the protein components with molecular weights greater than 120,000 and the (Ca2+ + Mg2+)-adenosine triphosphatase partially or totally reside on or at the external surface of the sarcoplasmic reticulum vesicles. In the case of the adenosine triphosphatase, highly controlled trypsin treatment cleaves the molecule into two products, a 65,000 molecular weight fragment and a 56,000 molecular weight fragment. The evidence indicates that the 65,000 molecular weight component of the (Ca2+ + Mg2+)-adenosine triphosphatase is located in a more exposed fashion on the external surface of the vesicles than the 56,000 molecular weight compoenet and that some adenosine triphosphatase molecules have a more exposed position on the external surface of the vesicle than others. The protein components designated by MacLennan (MacLennan, D. H. (1975) Can. J. Biochem. 53, 251-261) as "calsequestrin" and "high affinity Ca2+ binding protein" are shown not to be on the external surface of the rat sarcoplasmic reticulum vesicle but rather to reside either within the core of the membrane or on the inside surface of the vesicle. The results of this study are in agreement with the model for the organization of the protein components of the sarcoplasmic reticulum membrene recently proposed by MacLennan (MacLennan, D. H. (1975) Can. J. Biochem. 53, 251-261).