The determination of the separate Ca2+ pump protein and phospholipid profile structures within reconstituted sarcoplasmic reticulum membranes via X-ray and neutron diffraction

We have previously compared the electron density profiles for several highly-functional reconstituted sarcoplasmic reticulum membranes with that for the isolated sarcoplasmic reticulum membrane (Herbette, L., Scarpa, A., Blasie, J.K., Wang, C.T., Saito, A. and Fleischer, S. (1981) Biophys. J. 36, 47–72). In this paper, we compare the separate calcium pump protein profile within these reconstituted sarcoplasmic reticulum membranes, as derived by X-ray and neutron diffraction methods, with that within isolated sarcoplasmic reticulum membranes. In addition, the time-average perturbation of the lipid bilayer by the incorporated calcium pump protein within these reconstituted sarcoplasmic reticulum membranes has been determined in some detail.     

The determination of the separate Ca2+ pump protein and phospholipid profile structures within reconstituted sarcoplasmic reticulum membranes via X-ray and neutron diffraction

We have previously compared the electron density profiles for several highly-functional reconstituted sarcoplasmic reticulum membranes with that for the isolated sarcoplasmic reticulum membrane (Herbette, L., Scarpa, A., Blasie, J.K., Wang, C.T., Saito, A. and Fleischer, S. (1981) Biophys. J. 36, 47-72). In this paper, we compare the separate calcium pump protein profile within these reconstituted sarcoplasmic reticulum membranes, as derived by X-ray and neutron diffraction methods, with that within isolated sarcoplasmic reticulum membranes. In addition, the time-average perturbation of the lipid bilayer by the incorporated calcium pump protein within these reconstituted sarcoplasmic reticulum membranes has been determined in some detail.     

Localization of the high affinity calcium binding protein and an intrinsic glycoprotein in sarcoplasmic reticulum membranes

Several proteins in sarcoplasmic reticulum preparations move in a band with a mobility, in sodium dodecyl sulfate-polyacrylamide gels (0.1 M phosphate buffer, pH 7.0), corresponding to a molecular mass of about 55,000 daltons. Only one of these proteins is the high affinity calcium binding protein. An intrinsic glycoprotein is also present in this band, and it is this glycoprotein which is found in vesicles reconstituted after dissolution of sarcoplasmic reticulum in deoxycholate. Both of these proteins are found in rather constant ratios with the ATPase in light, intermediate, and heavy sarcoplasmic reticulum vesicles. Transverse tubular vesicles can be isolated from the heavy sarcoplasmic reticulum vesicles after disruption of the membrane in a French pressure cell (Lau, Y.H., Caswell, A.H., and Brunschwig, J.P. (1977) J. Biol. Chem. 252, 5565-5574). These vesicles are enriched in their content of the high affinity calcium binding and depleted of the intrinsic glycoprotein. Cycloheptaamylose . fluorescamine complex (CFC) labels the intrinsic glycoprotein heavily indicating that it is at least partially exposed on the cytoplasmic surface of sarcoplasmic reticulum membranes. Since the carbohydrate component of the protein must lie in luminal spaces, it is inferred that the intrinsic glycoprotein is a transmembrane protein. The high affinity calcium binding protein is not labeled by CFC indicating that it is not exposed on the cytoplasmic surface of sarcotubular vesicles. The protein is also not affected by proteolytic digestion of sarcoplasmic reticulum vesicles and can be isolated intact from trypsin-digested vesicles. It is not removed from sarcoplasmic-reticulum vesicles by washing with buffers containing Chelex 100 or ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). These data show that the high affinity calcium binding protein is localized in the interior of the sarcotubular system and suggest that it might be common to both sarcoplasmic reticulum and transverse tubular membranes.     

Phospholipid fatty acyl chain asymmetry in the membrane bilayer of isolated skeletal muscle sarcoplasmic reticulum

We previously showed [Herbette, L. G., Blasie, J. K., DeFoor, P., Fleischer, S., Bick, R. J., Van Winkle, W. B., Tate, C. A., & Entman, M. L. (1984) Arch. Biochem. Biophys. 234, 235-242; Herbette, L. G., DeFoor, P., Fleischer, S., Pascolini, D., Scarpa, A., & Blasie, J. K. (1985) Biochim. Biophys. Acta 817, 103-122] that the phospholipid head-group distribution in the membrane bilayer of isolated sarcoplasmic reticulum is asymmetric. From these studies, both the total number of phospholipid head groups and the total lipid, as well as the head-group species for these lipids, were found to be different for each monolayer of the membrane bilayer. In this paper, we demonstrate for the first time that there is significant asymmetry in the distribution of unsaturated fatty acids between the two monolayers; i.e., the outer monolayer of the sarcoplasmic reticulum contained more unsaturated and polyunsaturated chains when compared to the inner monolayer. X-ray diffraction measurements demonstrated that the time-averaged fatty acyl chain extension for the outer monolayer was approximately 20% less than for the inner monolayer. This is consistent with the concept that the greater degree of unsaturation in the outer monolayer may provide for a decreased average fatty acyl chain extension for that layer. This architecture for the bilayer may be related to both the "resting" state mass distribution of the calcium pump protein within the membrane bilayer and possible "conformational" states of the calcium pump protein during calcium transport by the sarcoplasmic reticulum.     

Functional characterization of junctional terminal cisternae from mammalian fast skeletal muscle sarcoplasmic reticulum

Junctional terminal cisternae are a recently isolated sarcoplasmic reticulum fraction containing two types of membranes, the junctional face membrane with morphologically intact "feet" structures and the calcium pump membrane [Saito, A., Seiler, S., Chu, A., & Fleischer, S. (1984) J. Cell Biol. 99, 875-885]. In this study, the Ca2+ fluxes of junctional terminal cisternae are characterized and compared with three other well-defined fractions derived from the sarcotubular system of fast-twitch skeletal muscle, including light and heavy sarcoplasmic reticulum, corresponding to longitudinal and terminal cisternae regions of the sarcoplasmic reticulum, and isolated triads. Functionally, junctional terminal cisternae have low net energized Ca2+ transport measured in the presence or absence of a Ca2+-trapping anion, as compared to light and heavy sarcoplasmic reticulum and triads. Ca2+ transport and Ca2+ pumping efficiency can be restored to values similar to those of light sarcoplasmic reticulum with ruthenium red or high [Mg2+]. In contrast to junctional terminal cisternae, heavy sarcoplasmic reticulum and triads have higher Ca2+ transport and are stimulated less by ruthenium red. Heavy sarcoplasmic reticulum appears to be derived from the nonjunctional portion of the terminal cisternae. Our studies indicate that the decreased Ca2+ transport is referable to the enhanced permeability to Ca2+, reflecting the predominant localization of Ca2+ release channels in junctional terminal cisternae. This conclusion is based on the following observations: The Ca2+, -Mg2+ -dependent ATPase activity of junctional terminal cisternae in the presence of a Ca2+ ionophore is comparable to that of light sarcoplasmic reticulum when normalized for the calcium pump protein content; i.e., the enhanced Ca2+ transport cannot be explained by a faster turnover of the pump. Ruthenium red or elevated [Mg2+] enhances energized Ca2+ transport and Ca2+ pumping efficiency in junctional terminal cisternae so that values approaching those of light sarcoplasmic reticulum are obtained. Rapid Ca2+ efflux in junctional terminal cisternae can be directly measured and is blocked by ruthenium red or high [Mg2+]. Ryanodine at pharmacologically significant concentrations blocks the ruthenium red stimulation of Ca2+ loading. Ryanodine binding in junctional terminal cisternae, which appears to titrate Ca2+ release channels, is 2 orders of magnitude lower than the concentration of the calcium pump protein. By contrast, light sarcoplasmic reticulum has a high Ca2+ loading rate and slow Ca2+ efflux that are not modulated by ruthenium red, ryanodine, or Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of the profile structures of isolated and reconstituted sarcoplasmic reticulum membranes

The profile structures of functional reconstituted sarcoplasmic reticulum (RSR) membranes were investigated as a function of the lipid/protein (L/P) ratio via x-ray diffraction studies of hydrated oriented multilayers of these membranes to a resolution of 10-15 A, and neutron diffraction studies on these multilayers to lower resolutions. Our results at this stage of investigation indicate that reconstitution of SR with variable amounts of Ca2+ pump protein for L/P ratios greater than 88 results in closed membraneous vesicles in which the Ca2+ pump protein is distributed asymmetrically in the membrane profile; a majority of the protein density is contained primarily in the extravesicular half of the membrane profile whereas a relatively lesser portion of the protein spans the hydrocarbon core of the RSR membranes. These RSR membranes are functionally similar and resemble isolated light sarcoplasmic reticulum in both profile structure and function at a comparable L/P ratio. Reconstitution with greater amounts of Ca2+ pump protein (e. g. L/P approximately 50-60) resulted in substantially less functional membranes with a dramatically thicker profile structure.     

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.     

Acid-induced uncoupling of sarcoplasmic reticulum membranes is reversed by reconstitution

Mild acid treatment of sarcoplasmic reticulum membranes results in a first order decline in calcium transport activity while calcium stimulated ATPase activity remains unimpaired (Berman, M.C., McIntosh, D.B. and Kench, K.E. (1977) J. Biol. Chem. $\text{252}$ 994–1001). This uncoupling is apparently irreversible. Acid inactivated sarcoplasmic reticulum membranes, solubilized with Triton X-100 and reconstituted by passage through a Bio-Bead column, reactivated 80% of the calcium transport activity when compared with untreated, control reconstituted SR vesicles. It is suggested that the inactivated conformation of the (Ca²⁺, Mg²⁺-)ATPase is constrained by lipid-protein interactions.     

The secondary structure of calcium pump protein in light sarcoplasmic reticulum and reconstituted in a single lipid component as determined by Raman spectroscopy

Raman spectra have been measured of the following samples: active calcium pump protein in light sarcoplasmic reticulum (SR) membranes, lipids extracted from light SR membranes, active calcium pump protein reconstituted in dielaidoylphosphatidylcholine (DEPC), and pure DEPC. The spectra of native SR lipids and of pure DEPC are different, and yet when these spectra are subtracted from the spectra of the respective protein-lipid complexes, the resulting amide I spectra of the calcium pump protein are the same, indicating that appropriate criteria have been chosen for subtraction of the spectrum of a lipid. This spectrum has been analyzed for secondary structure with the following results. The SR calcium pump protein contains 51 +/- 5% helix, in agreement with a prediction of secondary structure obtained from an analysis of the sequence, and 21 +/- 4% beta-strand. In addition, the presence of protein broadens and lowers the main melting transition of DEPC.     

Phosphorylation of a 22,000-dalton component of the cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase.

Cardiac microsomes were incubated with [gamma-32P]ATP and a cardiac adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase in the presence of ethylene glycol bis(bets-aminoethyl ether)-N,N'-tetraacetic acid. After solubilization in sodium dodecyl sulfate and fractionation by polyacrylamide gel electrophoresis, a single microsomal protein component of approximately 22,000 daltons was found to bind most of the 32P label. The 32P labeling of this component increased several fold when NaF was included in the incubation medium. No other component of cardiac microsomes, including sarcoplasmic reticulum ATPase protein, contained significant amounts of 32P label. This 22,000-dalton phosphoprotein formed by cyclic AMP-dependent protein kinase had stability characteristics of a phosphoester rather than an acyl phosphate. Washing of microsomes with buffered KCl did not decrease the amount of 32P labeling to the 22,000-dalton protein, suggesting that this protein is associated with the membranes of sarcoplasmic reticulum rather than being a contaminant from other soluble proteins. The 22,000-dalton protein was susceptible to trypsin. Brief digestion with trypsin in the presence of 1 M sucrose did not significantly affect microsomal calcium transport activity, but prevented both subsequent phosphorylation of the 22,000-dalton protein and stimulation of calcium uptake by cyclic AMP-dependent protein kinase, suggesting that this protein is a modulator of the calcium pump. These results are consistent with previous findings (Kirchberger, M.A., Tada, M., and Katz, A.M. (1974) J. Biol. Chem. 249, 6166-6173; Tada, M., Kirchberger, M.A., Repke, D.I., and Katz, A.M. (1974) J. Biol. Chem. 249, 6174-6180) that cyclic AMP-dependent protein kinase-catalyzed phosphorylation is associated with stimulation of calcium transport in the cardiac sarcoplasmic reticulum, and further indicate that this phosphorylation occurs at a component of low mass (22,000 daltons) of the cardiac sarcoplasmic reticulum which, while separable from the calcium transport ATPase protein (100,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has the ability to regulate calcium transport by the cardiac sarcoplasmic reticulum.     

Functional characterization of reconstituted sarcoplasmic reticulum vesicles

A detailed functional characterization of reconstituted sarcoplasmic reticulum (SR) vesicles with similar lipid content as normal SR was obtained by studies of ATPase activity and calcium transport in transient state, steady state, and equilibrium conditions. For this purpose, enzyme phosphorylation with ATP, hydrolytic activity, calcium transport, phosphorylation with Pi, and ATP synthesis by reversal of the pump were measured, and utilized to demonstrate function and orientation of catalytic sites. The preparations used in these studies displayed the highest activity reported for reconstituted sarcoplasmic reticulum systems. The rates of phosphoenzyme formation from ATP and hydrolysis as well as steady state levels matched the values obtained with normal SR vesicles. Calcium transport and repeated cycles of ATP synthesis by reversal of the pump were also obtained. However, the efficiency of transport and ATP synthesis from a Ca2+ gradient was approximately three times lower than in native vesicles. This deficiency could not be attributed to passive calcium leak from the reconstituted vesicles but, in part, can be explained by the bidirectional alignment of the calcium pump in reconstituted SR. It is suggested that vectorial transport requires a more complex level of protein structure than that for sustaining simple ATPase activity. Time resolution of the phosphorylation reaction by rapid quench methods can be used to estimate the orientation of the calcium pump in the membrane. Such studies indicate that the calcium pump protein is largely bidirectionally oriented in reconstituted SR vesicles.     

Regulation of calcium transport by protein phosphatase activity associated with cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium-calmodulin-dependent protein kinase on a 22,000 proteolipid, called phospholamban. Phosphorylation by the calcium-calmodulin-dependent protein kinase is associated with stimulation of the initial rates of calcium transport (Davis, B. A., Schwartz, A., Samaha, F. J., and Kranias, E. G. (1983) J. Biol. Chem. 258, 13587-13591). The present study shows that protein phosphatase activity, associated with canine cardiac sarcoplasmic reticulum vesicles, can catalyze dephosphorylation of the calcium-calmodulin-dependent sites on phospholamban. The activity was maximally stimulated by manganese; fluoride was inhibitory, but its effect was reversible. Dephosphorylation of phospholamban, which was prephosphorylated by calcium-calmodulin-dependent protein kinase, resulted in a reduction of the stimulation on calcium transport rates, particularly at submaximal calcium concentrations. The decrease in calcium transport was associated with a statistically significant decrease in the apparent affinity (EC50) for calcium. Rephosphorylation of phospholamban by the endogenous calcium-calmodulin-dependent protein kinase caused full recovery of the stimulation on calcium transport rates and reversal of the effects mediated by the protein phosphatase. Thus, the calcium pump in cardiac sarcoplasmic reticulum appears to be under reversible regulation mediated by endogenous calcium-calmodulin-dependent protein kinase and protein phosphatase. Such regulation may represent an important control mechanism for the myocardium.     

Fourier transform infrared spectroscopic studies of lipid-protein interaction in native and reconstituted sarcoplasmic reticulum

Fourier transform infrared spectroscopy has been used to monitor lipid-protein interaction and protein secondary structure in native and reconstituted sarcoplasmic reticulum vesicles. Studies of the temperature dependence of the CH₂ symmetric stretching frequency reveal no cooperative phase transitions in purified sarcoplasmic reticulum or in vesicles reconstituted with dioleoylphosphatidylcholine, although a continuous introduction of disorder into the lipid acyl chains is observed as the temperature is raised. In addition, temperature-dependent changes are observed in the Amide I and Amide II vibrations arising from protein peptide bonds. A comparison of lipid order in native sarcoplasmic reticulum and its lipid extract showed that the introduction of protein is accompanied by a slight increase in lipid order. Reconstitution of Ca²⁺-ATPase from sarcoplasmic reticulum with dipalmitoylphosphatidylcholine (lipid/protein ratio 30:1), reveals a perturbed lipid melting event broadened and reduced in midpoint temperature from multilamellar lipid vesicles. The onset of melting (27–28°C) correlates well with the onset of ATPase activity and confirms a suggestion (Hesketh, T.R., Smith G.A., Houslay M.D., McGill, K.A., Birdsall, N.J.M., Metcalfe, J.C. and Warren, G.B. (1976) Biochemistry 15, 4145–4151) that a liquid crystalline environment is a requirement for optimal protein function. Finally, Ca²⁺-ATPase has been reconstituted into binary lipid mixtures of DOPC and acyl-chain perdeuterated DPPC. The effect of protein on the structure and melting behavior of each lipid component was monitored. The protein appears to preferentially interact with the DOPC component.     

A Procedure for Purification of the Ryanodine Receptor from Skeletal Muscle

In this paper, we describe a simple and reproducible method for purifying large quantities of ryanodine receptor from skeletal muscle membranes. The procedure involves the use of ion exchange chromatography and sucrose gradient centrifugation to purify the protein which has been identified as the calcium release protein of the sarcoplasmic reticulum (Imagawa, T., Smith, J., Coronado, R. and Campbell, K. (1987) J. Biol. Chem. 262:16,636–16,643). Addition of micromolar quantities of unlabeled ryanodine prior to solubilization and throughout the isolation procedure appears to stabilize the tetrameric structure of the ryanodine receptor. The purified receptor, consisting predominantly of a 400K polypeptide on SDS-PAGE, binds [³H]ryanodine with a binding affinity similar to that in membranes. Overall recovery of ryanodine binding activity was 21% of the initial activity with a 30-fold purification of the receptor.     

Reconstitution of an active calcium pump in sarcoplasmic reticulum.

Recovery of calcium transport and calcium-activated ATPase activity was studied in relation to the retention of protein components in sarcoplasmic reticulum reconstituted after solubilization with deoxycholate and centrifugation, followed by removal of the detergent from the supernatant by dialysis. Control sarcoplasmic reticulum was similarly treated except for omission of deoxycholate. Maximum capacity for oxalate- and phosphate-supported calcium uptake was increased 2- to 3-fold in reconstituted sarcoplasmic reticulum compared to original and control. Calcium uptake velocity of the reconstituted sarcoplasmic reticulum was approximately 80% that of original and 90% of control sarcoplasmic reticulum. Calcium uptake/ATP hydrolysis ratio was approximately 2 in the original sarcoplasmic reticulum and decreased to approximately 1 in the control and reconstituted sarcoplasmic reticulum. Calcium storage in the absence of calcium-precipitating anion was approximately 85% in control and 70% in reconstituted sarcoplasmic reticulum, compared to the original sarcoplasmic reticulum. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid-induced calcium release after phosphate-supported calcium uptake was slower in reconstituted sarcoplasmic reticulum than in original or control sarcoplasmic reticulum. Polyacrylamide gel electrophoresis of original and control sarcoplasmic reticulum showed similar amounts of protein components of approximately 93,000, 59,000, 50,000, 30,000 to 37,000, and 20,000 to 26,000 daltons. Reconstituted sarcoplasmic reticulum, however, lost over 85% of the 50,000- and 20,000- to 26,000-dalton proteins while retaining most of its calcium transport functions.     

Cytoplasmic residues of phospholamban interact with membrane surfaces in the presence of SERCA: A new role for phospholipids in the regulation of cardiac calcium cycling?

The 52-amino acid transmembrane protein phospholamban (PLB) regulates calcium cycling in cardiac cells by forming a complex with the sarco(endo)plasmic reticulum calcium ATPase (SERCA) and reversibly diminishing the rate of calcium uptake by the sarcoplasmic reticulum. The N-terminal cytoplasmic domain of PLB interacts with the cytoplasmic domain of SERCA, but, in the absence of the enzyme, can also associate with the surface of anionic phospholipid membranes. This work investigates whether the cytoplasmic domain of PLB can also associate with membrane surfaces in the presence of SERCA, and whether such interactions could influence the regulation of the enzyme. It is shown using solid-state NMR and isothermal titration calorimetry (ITC) that an N-terminally acetylated peptide representing the first 23 N-terminal amino acids of PLB (PLB_1-23) interacts with membranes composed of zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids in the absence and presence of SERCA. Functional measurements of SERCA in sarcoplasmic reticulum (SR) vesicles, planar SR membranes and reconstituted into PC/PG membranes indicate that PLB_1-23 lowers the maximal rate of ATP hydrolysis by acting at the cytoplasmic face of the enzyme. A small, but statistically significant, reduction in the inhibitory effect of the peptide is observed for SERCA reconstituted into PC/PG membranes compared to SERCA in membranes of PC alone. It is suggested that interactions between the cytoplasmic domain of PLB and negatively charged phospholipids might play a role in moderating the regulation of SERCA, with implications for cardiac muscle contractility.     

Functional characteristics of reconstituted sarcoplasmic reticulum membranes as a function of the lipid-to-protein ratio

The ATP-induced Ca2+ accumulation efficiency and rates of Ca2+ uptake of the reconstituted sarcoplasmic reticulum (RSR) model membrane system were measured over an extended range of lipid-to-protein (L/P) molar ratios and were compared to those of isolated light sarcoplasmic reticulum (LSR). Highly purified sarcoplasmic reticulum (SR), dissociated in the presence of deoxycholate, was reconstituted for several L/P ratios, according to the same procedure, forming closed membranes vesicles composed of greater than 95% Ca2+ pump protein and SR lipids which were capable of ATP-induced Ca2+ accumulation in the absence of oxalate or other Ca2+ precipitating agents. This suggests that dissociation of SR and reconstitution to form RSR does not significantly affect the ability of the Ca2+ pump protein incorporated into the SR lipid bilayer to establish Ca2+ gradients. Electron micrographs of fixed and stained dispersions of RSR revealed a structural organization of the membrane that was dependent upon the L/P molar ratio. RSR with L/P greater than 88 were composed of closed vesicles whose membranes stained asymmetrically, similar to that observed for LSR. Closed vesicles of RSR with L/P less than 88 were composed of membrane that stained symmetrically. In addition, reconstituted SR preparations with well-defined L/P molar ratios greater than 88 possess a functional behavior similar to that of LSR (in the absence of oxalate, energy efficiencies are 60-70% and apparent initial uptake rates are 80% that of isolated LSR controls); RSR preparations with L/P less than 88 are characterized by significantly depressed values of the energy efficiencies and apparent initial uptake rates especially at low L/P ratios. Thus, we are the first to report a reconstituted SR model membrane system capable of attaining rates of Ca2+ uptake comparable to isolated LSR controls at comparable L/P ratios in the absence of oxalate or other Ca2+ precipitating agents.     

Isolation of highly active preparations of sarcoplasmic reticulum and Ca2-dependent ATPase from cardiac muscle]

A microsomal preparation with a high ability for Ca2+ uptake has been isolated from pigeon heart. A method of further purification of Ca2+-accumulating system of heart, based on the ability of sarcoplasmic reticulum for the energy-dependent Ca2+ accumulation in the presence of oxalate, has been developed. Upon centrifugation in the gradient of sucrose and KCl concentration the fragments of sarcoplasmic reticulum, rendered "heavy" by calcium oxalate, can be separated from foreign cell membranes. The main component of heart "calcium pump" is Ca2+-dependent ATPase (making up to about 50% of all proteins of the purified reticulum), having a molecular weight of 100.000--105.000. Specific activity of heart Ca2+-ATPase as well as the ability of purified heart sarcoplasmic reticulum for Ca2+ uptake are only slightly less than those of the skeletal muscle reticulum. The data obtained suggest that heart sarcoplasmic reticulum may be efficient for providing heart muscle relaxation.     

Isolation and characterization of transverse tubule from normal and dystrophic mice

I have recently reported the isolation and characterization of sarcoplasmic reticulum from normal and dystrophic mice. These sarcoplasmic reticulum fractions were similar in calcium pump function, calcium release properties, and lipid composition. In this report, I describe the isolation of mouse muscle transverse tubule membranes using a calcium phosphate-loading technique. When the relative purity of normal and dystrophic preparations was considered, transverse tubule from normal and dystrophic mice were similar in calcium-insensitive ATPase activity, cholesterol content, and membrane microviscosity (as estimated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene); transverse tubule yield from dystrophic muscle, however, was twice that from normal muscle, while sarcoplasmic reticulum yield from these same dystrophic muscles was only 60% that from normal muscle. This result may reflect a difference in the relative quantities of these membranes in situ.     

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.