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Junctional transverse tubules (TT) isolated from triads of rabbit skeletal muscle by centrifugation in an ion-free sucrose gradient were compared with membrane subfractions, predominantly derived from the free portion of TT, that had been purified from sarcoplasmic reticulum membrane contaminants by three different methods. The markers used were diagnostic membrane markers and the dihydropyridine (DHP) receptor, which is a specific marker of the junctional membrane of TT. Junctional TT have a high membrane density (Bmax. 60 pmol/mg of protein) of high-affinity (Kd 0.25 nM) DHP-binding sites using [3H]PN200-110 as the specific ligand. When analysed by SDS/PAGE under reducing conditions and by Western blot techniques, the TT were found to contain a concanavalin A-binding 150 kDa glycoprotein which probably corresponds to the alpha 2-subunit of the DHP receptor. This conclusion was supported by correlative immunoblot experiments with a specific antibody. Junctional TT are further distinguished from free TT by the presence of a high number (Bmax. 20 pmol/mg of protein) of [3H]cyclic AMP receptor sites, as determined by the Millipore filtration technique of Gill & Walton [(1974) Methods Enzymol. 38, 376-381]. Use of this method means that the number of receptors may have been underestimated. The TT-bound cyclic AMP receptor was identified as a 55 kDa protein by specific photoaffinity labelling with 8-N3-[3H]cyclic AMP, and had similar phosphorylation properties and apparent molecular mass to the RII form of the regulatory subunit of cyclic AMP-dependent protein kinase. Co-localization of the intrinsic cyclic AMP-dependent protein kinase and of the DHP receptor complex to the junctional membrane of TT supports the hypothesis that the 170 kDa alpha 1-subunit of the receptor is a substrate for the kinase.

Calsequestrin was isolated from chicken fast-twitch skeletal muscle, and partial amino terminal sequence was determined. The sequence (NH2) EEGLNFPTYDGKDRVIDLNE shows high identity with known mammalian calsequestrins contained in the Protein Identification Resource data bank (1). Most importantly, this 20 amino acid sequence shares complete identity with the amino terminus of aspartactin, a putative laminin-binding protein of the extracellular matrix (2, 3). The possible relationship of aspartactin to calsequestrin is discussed.

By using extraction in the presence of Ca2+ and Triton X-100 and then in the presence of EGTA without detergent, a set of Ca2+-dependent phospholipid binding proteins has been identified in the membranes of transverse tubules (T-tubules) and sarcoplasmic reticulum (SR), isolated from rabbit skeletal muscles. Longitudinal SR, junctional SR and T-tubule membranes yielded about 9, 14 and 3.3 micrograms of EGTA-soluble proteins per 1 mg of membrane protein, respectively. In the presence of 1 mM CaCl2, 68 and 33 kDa proteins of T-tubules and junctional SR as well as 30 kDa protein of T-tubules were shown to bind to liposomes made of 1:1 w/w mixtures of (i) phosphatidylcholine and (ii) phosphatidylserine, phosphatidic acid, or phosphatidyl ethanolamine. In the presence of EGTA, the above-mentioned proteins were mostly found in the supernatants. Binding of the proteins with liposomes consisting of pure phosphatidylcholine was negligible.

Various detergents can be used to dissolve phospholipids, resulting in very narrow 31PNMR resonances. The resonances are well resolved, allowing identification and quantitative analysis of phospholipids in a mixture. The chemical shift depends strongly on pH, reflecting changes in the state of ionization of the phospholipid headgroup moieties. Samples of phospholipids dissolved in aqueous detergents are conveniently prepared and give narrower 31P resonances than do phospholipids dissolved in organic solvents.

Membrane-bound phosphorylatable proteolipids were reported to play a role in the regulation of transmembrane Ca2+ fluxes by catecholamines. A generally applicable purification procedure is described by which such proteolipids as the cardiac sarcoplasmic reticulum phospholamban is purified by solvent extraction followed by high pressure liquid chromatography on microparticulate silica. Phospholamban is thereby purified with a yield of 3.37 mg from 100 mg of sarcoplasmic reticulum proteins, significantly higher than that obtained by any of the previously reported procedures. It appeared homogeneous upon dodecyl sulfate-polyacrylamide gel electrophoresis where it is stained by Coomassie blue and detected by autoradiography. The same procedure is applicable to cardiac sarcolemmal calciductin. Both proteolipids exhibit the same Mr 11 000 and pI 3.7 upon two-dimensional gel electrophoresis. Their amino acid compositions are very similar if not identical. This raises the intriguing possibility that phospholamban and calciductin are identical though they obviously belong to different membranes.

Specific insulin binding sites in cardiac sarcolemmal (SL) and sarcoplasmic reticular (SR) membranes were examined. Under in vitro conditions, the specific insulin binding to SL was two to three times greater than that to SR isolated from myocardium of different species. Insulin binding to these membranes was rapid, saturable, and temperature dependent. Scatchard plots were nonlinear and revealed high-affinity sites with K1 of 1.2 +/- 0.1 and 2.3 +/- 0.4 nM and low-affinity sites with K2 of 34.0 +/- 11.6 and 87.7 +/- 18.7 nM for SR and SL membranes, respectively. Unlabeled insulin accelerated the dissociation of previously bound labeled insulin, reflecting negative cooperativity of site-to-site interaction in both membranes; however, the dissociation rate in SR was about twofold higher than that in SL. The optimum binding of insulin to SR showed a plateau between pH 6.5 and 7.5 in comparison with a sharp peak at pH 8.0 for SL. Insulin significantly enhanced ATP-dependent Ca2+ binding to SL and SR membranes by approximately 100 and 40%, respectively. The Na+-dependent Ca2+ uptake in SL and the oxalate-supported Ca2+ uptake in SR were also stimulated by insulin; the maximum increase in Ca2+ accumulation in these vesicles was approximately 150 and 25%, respectively. These studies indicate that specific insulin binding sites in SL are characteristically distinct from those in SR and that insulin is capable of stimulating Ca2+ pumps located in these membranes.

Membranes of sarcoplasmic reticulum were labelled with 1-fluoro-2,4-dinitro[3H]benzene at pH 6.5 and with 2,4,6-trinitrobenzenesulphonate at pH 9.2. Conditions were chosen to restrict reaction to amino groups, and the effect of blockings of these groups by methyl acetimidate was determined. All proteins were labelled to some extent by both reagents, but, whereas the trinitrophenylation of both lipid and protein amino groups was almost completely blocked by methyl acetimidate, the dinitrophenylation of the ATPase at pH 6.5 was much less affected. The seven amino groups on the ATPase that were labelled under these conditions did not react with methyl acetimidate. This reagent can therefore be used to enhance the specificity of fluorodinitrobenzene for amino groups in a hydrophobic environment. The amino groups on the minor proteins and on the phospholipids that reacted with fluorodinitrobenzene at pH 6.5 were probably in an aqueous environment, since the reaction was blocked by methyl acetimidate.

Monkfish (Lophius americanus) sarcoplasmic protein patterns were found to be polymorphic with respect to separations using isoelectric focusing. Reproducible protein pattern variations were not detected using cellulose acetate or polyacrylamide gel disc electrophoresis. Monkfish sarcoplasmic proteins separated on pH 3.5-9.5 Ampholine PAGplates or on pH 2.5-9.0 agarose IEF gels yielded similar patterns showing 3 distinct, reproducible variations. On close examination, the pH 3.5-9.5 Ampholine PAGplate patterns could be further subdivided into 3 additional variations. A high resolution pH 3.5-5.0 agarose IEF gel was able to resolve a total of 10 different monkfish pattern variations in a sample of 24 individuals. A model was proposed suggesting the existence of 16 distinct variations in the monkfish sarcoplasmic protein pattern, based on the various combinations of 4 protein bands. In identifying samples of monkfish meat, it is necessary to compare the unknown pattern with the possible variant patterns to effect a reliable identification. On recommendation by the Associate Referee, the method for fish species identification based on polyacrylamide gel isoelectric focusing, 18.A01-18.A04, has been adopted official final action with no restrictions as to the species that may be identified.

Using Raman spectroscopy, we found that the sarcoplasmic reticulum lipids of combined muscles from rabbit leg undergo at least two reversible temperature phase changes, centered at about -15 and 13 degrees C. Below the first transition, the lipid Raman CH st region is characteristic of the hexagonal lamellar gel phase. Above the second transition, the Raman CH stretch region is that of a "melted" lamellar phase, somewhat more rigid than a monophasic lipid system. The composition of the lipids was determined and the possibility of a relation between the major head group types and the phase transitions is discussed. Since SR Ca2+ATPase activity is enhanced at about 14-19 degrees C, the Raman studies suggest that ATPase activity is enhanced when the 13 degrees C transition is complete.

The localization of Ca-accumulating structures in the longitudinal body wall muscle (LBWM) of the opisthobranch mollusc Dolabella auricularia and their role in the contraction-relaxation cycle were studied by fixing the LBWM fibers at rest and during mechanical response to 400 mM K or to 10(-4)--10(-3) M acetylcholine in a 1% OsO4 solution containing 2% K pyroantimonate. In the resting fibers, electron-opaque pyroantimonate precipitate was mostly localized at the peripheral structures, i.e., along the inner surface of the plasma membrane, at the membrane of the surface tubules, and at the sarcoplasmic reticulum. In the fibers fixed during mechanical activity, the precipitate was diffusely distributed in the myoplasm in the form of numerous particles with corresponding decrease in the amount of the precipitate at the peripheral structures. Electron-probe X-ray microanalysis showed the presence of Ca in the precipitate, indicating that the precipitate may serve as a measure of Ca localization. These results are in accord with the view that, in the LBWM, the Ca stored in the peripheral structures is released into the myoplasm to activate the contractile mechanism.

Two new proteins with apparent molecular masses of 53 kDa and 190 kDa have been identified in both sarcoplasmic reticulum and human blood platelets using a monoclonal antibody, FII1b5. The sarcoplasmic reticulum FII1b5 antigens were present in the terminal cisternae fraction, but were absent from light sarcoplasmic reticulum. The platelet and skeletal muscle proteins were not sensitive to digestion with endoglycosidase H under conditions that removed carbohydrate from the 53 kDa glycoprotein in sarcoplasmic reticulum or GPIIIa in platelet microsomes and did not bind 45Ca in a nitrocellulose overlay calcium-binding assay. These results distinguished the FII1b5 antigens from the 53 kDa glycoprotein and calsequestrin of sarcoplasmic reticulum. The 190 kDa platelet and sarcoplasmic reticulum proteins were extracted from membranes with high concentrations of NaCl, indicating that the high molecular mass FII1b5 antigens are peripherally associated with the bilayers. In contrast, the platelet and muscle 53 kDa proteins remained membrane-bound in the presence of high salt concentrations, suggesting that they are integral proteins.

Methods, applications and limitations of quantitative electron probe analysis, X-ray mapping, electron energy loss analysis and energy filtered imaging are described, with emphasis on the analysis of thin (less than 200nm) cryosections. Energy dispersion electron probe analysis can measure reliably 5 to 10mM/Kg of biologically prevalent elements in 50nm diameter areas of 100 to 150 nm thick cryo sections during 100-300 sec counts. The minimal detectable mass (MDM) with a conventional thermionic electron source is approximately 10(-19)g Fe (100 sec count) and can be reduced to 10(-20)g through the use of a field emission gun (FEG). A spatial resolution of 8.7nm is demonstrated in two-dimensional Fourier transforms of Mo X-ray maps of stained catalase crystals. Significant biological results of quantitative electron probe analysis include the measurement of total Ca released from the Mg and K taken up by the sarcoplasmic reticulum during muscle contraction, and the demonstration that mitochondria do not contribute to the physiological regulation of cytoplasmic free Ca levels in cardiac, vascular smooth and striated muscle. Electron energy loss analysis (EELS) promises a significant improvement in sensitivity for the measurement of Ca; based on statistical errors of the measurement, 250 microM/Kg Ca should be measureable with EELS in 250 sec. through the Ca L-edge loss. The use of a doubly corrected magnetic sector spectrometer as a transmission electron microscope imaging filter outside the microscope vacuum is illustrated, and the resolution of the iron core (7.5nm) and surrounding organic shell of single ferritin molecules is demonstrated in, respectively, iron M and carbon K loss images.

Amiodarone is a drug used in the treatment of cardiac arrhythmias and is believed to have a persistent interaction with cellular membranes. This study sought to examine the structure and location of amiodarone in a membrane bilayer. Amiodarone has a high membrane partition coefficient on the order of 10(6). Small angle x-ray diffraction was used to determine the position of the iodine atoms of amiodarone in dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under conditions of low temperature and hydration where the DPPC bilayer is in the gel state. The time-averaged position of the iodine atoms was determined to be approximately 6 A from the center (terminal methyl region) of the lipid bilayer. A dielectric constant of kappa = 2, which approximates that of the bilayer hydrocarbon core region, was used in calculating a minimum energy structure for membrane-bound amiodarone. This calculated structure when compared with the crystal structure of amiodarone demonstrated that amiodarone could assume a conformation in the bilayer significantly different from that in the crystal. The results reported here are an attempt to correlate the position of a membrane-active drug in a lipid bilayer with its time-averaged conformation. This type of analysis promises to be of great use in the design of drugs with greater potency and higher specificity.

Sarcoplasmic reticulum (SR) from winter flounder (Pseudopleuronectes americanus) of various ages was investigated with regard to fluorescent age pigment (FAP), potential of enzymic lipid peroxidation, fatty acid composition, protein pattern and Ca2+-sequestering ability. FAP accumulation, protein pattern and Ca2+-sequestering ability were also evaluated after the SR were incubated in vitro with the peroxidizing cofactors: NADH, ADP and Fe3+. No difference in FAP content was observed in SR isolated from fish of different ages. However, a higher accumulation of FAP in muscle tissue from old fish was observed. A slow but significant increase in FAP in SR was observed when the SR from both young and old fish were incubated with peroxidizing cofactors. It was demonstrated that 4-year-old fish had higher levels of NADH-dependent lipid peroxidation in their SR than 2-year-old fish. No differences in fatty acid composition were observed in the SR of fish aged from 2 to 8 years old. Two species of high-molecular-weight protein were found to increase in SR isolated from aging fish. These species resembled the polymerized proteins derived after incubation of the SR with the peroxidizing cofactors. Ca2+-uptake decreased when the SR was incubated with peroxidizing cofactors, but the results are difficult to interpret since Ca2+-uptake of SR was affected by captive stress.

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.

Herein we describe the distribution of G-proteins in canine cardiac sarcolemma (SL) and sarcoplasmic reticulum (SR) and in rabbit skeletal muscle SL, T-tubules, and junctional and longitudinal SR in comparison to G-proteins of human erythrocyte and bovine brain. G-proteins were unequivocally present in cardiac SL and SR and in skeletal T-tubules. Both cardiac fractions had two substrates specifically ADP-ribosylated by cholera toxin migrating on a sodium dodecyl sulfate-polyacrylamide gel at about 42 and 45 kDa. In skeletal muscle membranes, cholera toxi-labeled substrates migrated at about 42 and 62 kDa. Three substrates for pertussis toxin were resolved by sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis in cardiac SL at about 38, 40, and 43 kDa. Only the two higher molecular weight substrates were detected in cardiac SR and in any of several skeletal muscle membrane fractions. Comparison of G-proteins in muscle membrane fractions with G-proteins isolated from bovine brain and human erythrocyte as well as their reaction with antisera to either a common sequence of alpha subunits of G-proteins (G alpha common antibody) or to a unique sequence of the alpha subunit of Go (G alpha o antibody) indicated that the two lower molecular weight bands in cardiac SL are Go or Go-like, and therefore the upper band is probably Gi. These data demonstrate that pertussis toxin substrates are more heterogeneous than previously described and have implications for studies attempting to attribute physiological functions to G-protein isolates.

Sarcoplasmic reticulum (SR) terminal cisternae (TC) of frog (Rana esculenta) fast-twitch skeletal muscle have been purified by isopycnic sucrose density gradient centrifugation. Biochemical characteristics and Ca2+ release properties have been investigated and compared to those of the homologous fraction of rabbit skeletal muscle TC. The frog SR fraction obtained at the 38/45% sucrose interface appears to be derived from the terminal cisternae region as judged by: (a) thin section electron microscopy showing vesicles containing electron opaque material and squarelike (feet) projections at the outer surface; (b) protein composition (Ca2+-ATPase, calsequestrin, and high Mr proteins); (c) Ca2+ fluxes properties. The content of calsequestrin was higher in frog TC by 50% and the Ca2+ binding capacity (624 or 45 nmol of Ca2+/mg of TC protein, depending upon experimental conditions) was 3-4 times that of rabbit TC. Species-specific antigenic differences were found between junctional SR proteins of frog and rabbit TC. After active Ca2+ preloading in the presence of pyrophosphate (Palade, P. (1987) J. Biol. Chem. 262, 6135-6141), caffeine and doxorubicin elicited Ca2+ release from either TC fraction but with much faster rates in frog TC than in rabbit TC (14 versus 3 mumol of Ca2+/min/mg of protein). The present results provide new evidence for the existence of marked differences in Ca2+ release properties between TC of amphibian and mammalian fast-twitch muscle. Higher Ca2+ binding capacity and faster release rates in frog TC might compensate for the comparably greater diffusion distance being covered by the released Ca2+ from the Z-line to the actomyosin cross-bridges in the A-I overlap region.

The sarcoplasmic calcium-binding proteins (SCP) from crayfish, lobster and shrimp myogen have been purified to homogeneity. These proteins exist as dimers and dissociate in the presence of sodium dodecyl sulfate or urea in subunits of 22000 molecular weight. During the last step of purification (DEAE-cellulose chromatography), SCP emerges in three peaks in the ratio of 14:1.5:1 for crayfish, of 7:2:1 for lobster and of 3:2:1 for shrimp. Gel electrophoresis and isoelectrofocusing experiments, under native and denaturing conditions, indicate that among the three SCP isotypes there are only two different polypeptide chains, alpha and beta, which appear in the form of three dimers: alpha 2, alpha beta and beta 2. The alpha and beta subunits differ slightly in polypeptide chain composition as found by amino acid analyses of the crayfish and lobster SCPs, and also by comparison of tryptic peptides for crayfish SCPs. The polymorphism observed in crustacean SCPs, which is increased by their ability to form dimers, contrasts with the situation prevailing among other invertebrate SCPs and vertebrate parvalbumins where only monomeric isotypes are found. Equilibrium binding studies show that all three SCP isotypes from both crayfish and lobster display the same metal-binding properties. They have in their dimeric form six Ca2+-binding sites: two calcium-specific sites, two Ca/Mg sites that interact with positive cooperativity and two Ca/Mg sites that interact with negative cooperativity. Interactions between the two subunits of SCP seem to result in cooperative binding of Ca2+, which in turn may control more efficiently Ca2+ fluxes in crustacean muscle.

Vesicles were isolated from membranes of the sarcoplasmic reticulum (SR) of rabbit slow-twitch muscle by differential and sucrose density gradient centrifugation after homogenization. In some experiments, the vesicles were further fractionated by loading them with calcium oxalate followed by centrifugation in a sucrose density gradient. Protein composition of the isolated vesicles was complex and differed from the protein composition of fast-twitch muscle vesicles. However, other protein components, which were also present in fast-twitch muscle SR vesicles, have been identified: Ca2 + -dependent ATPase, calsequestrin, 160 000 molecular weight glycoprotein and 53 000 molecular weight glycoprotein. The amount of the Ca2 + -dependent ATPase and calsequestrin was several times lower in the slow-twitch muscle SR vesicles. This has been observed in both the original and the loaded vesicles. The slow-twitch muscle SR vesicles showed active calcium transport, Ca2 + -dependent ATPase activity, and the formation of the phosphorylated intermediate under conditions similar to those established for fast-twitch muscle SR. However, these activities, when expressed per mg of total protein, were several times lower than the analogous activities in the SR vesicles isolated from fast-twitch skeletal muscle. When the same enzyme activities were expressed per mg of the 105 000 molecular weight ATPase, the values obtained were very similar in both kinds of vesicles. The results indicate that the slow rate of calcium transport, found in slow-twitch muscle SR vesicles, may be related to a low content of the calcium-transporting ATPase in the membrane.

Air/water interface films were obtained from human erythrocytes and rabbit sarcoplasmic reticulum membranes at 'zero surface pressure. according to Verger, R and Pattus, F. (Chem. Phys. Lipids (1976) 16, 285-291). The lipid and protein distribution of these membrane films suggest that the film composition is determined by the composition of the membrane and the mode of integration of its components. When kept at low surface pressure, slow film expansion occurred due to unfolding of proteins at the interface. This process can be stopped by compressing the films at a higher surface pressure than 15 dyn/cm. Acetylcholinesterase activity from human erythrocyte films is highly dependent on the condensation state of the film. Ca2+-ATPase from sarcoplasmic reticulum films was still activable by Ca2+. Freeze-fracture studies on erythrocyte membrane films suggest the such films are monolayers in which proteins are randomly distributed.

1. The myogen protein patterns of lateral muscle of Liza ramada (Risso) and Chelon labrosus (Risso), revealed by isoelectric focusing, are reported. Specific differences are noted both in the white and in the red muscle (where they are more evident). 2. Red muscle shows the presence of a chromoprotein, found to be myoglobin, with a pI characteristic for the species. 3. Blood haemoglobins were examined with the same technique and also found to be species specific.

Frozen sections (10 micrometer) were cut from fast, slow and cardiac muscles of rainbow trout, frog, and rat. Rates of 45Ca2+-uptake by thin sections were compared at each animal's normal body temperature. Initial rates of Ca2+-uptake were 1.8 and 2.4 times higher, respectively, in trout and rat fast than slow muscles. In spite of a lower body temperature (10 degrees C) rates of Ca2+-uptake by trout fast muscles were 2.8 times higher than for rat Extensor digitorum longus at 37 degrees C. It is suggested that the high functional capacity of fish sarcoplasmic reticulum (SR) is related to adaptations associated with the need for rapid cycles of contraction and relaxation during high speed swimming. The biochemical characteristics of SR isolated from trout fast and slow muscles has also been investigated. The ratio of Ca2+-ATPase activity between fast and slow fibres (2:1) was similar to that obtained for Ca2+-uptake by whole muscle sections. No evidence was obtained for modulation of calcium transport by cAMP dependent protein kinases. The protein composition of highly purified trout SR was investigated by polyacrylamide gel electrophoresis. The concentration of 95 000 to 100 000 dalton (calcium pump protein) was significantly reduced in slow compared to fast muscle SR. Slow muscle SR contains a high proportion of additional protein bands of 48 000 and 31 000 molecular weight.

Potassium pyroantimonate has been employed in this study to localize calcium in the vascular smooth muscle of the thoracic aorta of the rabbit. The pyroantimonate ion precipitates sodium, magnesium and calcium. Incubation of theisolated thoracic aorta in a high potassium bathing medium which does not contain sodium, magnesium or calcium depletes the tissue of sodium. Addition of 10.8 mM CaC12 to the incubation medium results in well-localized depositions of reaction product, presumably that of calcium pyroantimonate, in mitochondria, sarcoplasmic reticulum, and at the plasma membrane. Some or all of these organelles may, therefore, play a vital role in the contraction-relaxation cycle of vascular smooth muscle.

Studies were carried out of temperature relationship of dansylchloride, N-3-pyrenylmaleinimide fluorescence, SR membranes, self-luminescence caused by Ca-ATPase tryptophane - provided fluorescence and of pyrene excimerization in membrane preparations of sarcoplasmic reticulum (SR) of rabbit skeletal muscles. Temperature relationship of fluorescence intensity of dansylchloride and N-3-pyrenylmaleimide in Arrhenius coordinates has bends at 15 and 35 degrees. Selffluorescence of protein samples linearly depends on temperature. Temperature relationship of the ratio between the intensities of exsimeric and monomeric forms of pyrene Fa/Fm in Arrhenius coordinates has the bend at 20-22 degrees. Hence only the latter relationship coincides with the shape of Arrhenius graph for enzymatic activity of SR Ca-ATPase.

Calsequestrin (Mr = 40,000) is a calcium-binding protein (Kd = 1 mM, 50 sites/molecule) located within the terminal cisternae of the sarcoplasmic reticulum of skeletal muscle cells. The interaction of terbium, a calcium analog, with rabbit skeletal muscle calsequestrin was studied by fluorescence and circular dichroism spectroscopy. Direct measurement of terbium binding using a fluorescence assay for terbium revealed that calsequestrin bound approx. 30 mol of terbium per mol of protein with an affinity of approx. 7 microM. The fluorescence of terbium measured at 545 nm was enhanced dramatically upon binding to calsequestrin, reaching a maximum value at a terbium to protein ratio of 28. The excitation spectrum of protein-bound terbium and chemical modification studies revealed that energy transfer occurred between aromatic residues, including tryptophan and bound terbium. Terbium bound to calsequestrin could be removed by EGTA, or displaced by Ca2+ or La3+. In the presence of Ca2+ or La3+ terbium bound to calsequestrin with a higher apparent affinity and lower capacity. 0.1 M KCl or 5 mM MgCl2 had little effect on terbium binding. Terbium increased the intrinsic fluorescence of calsequestrin 2-fold, and increased the alpha-helical content of calsequestrin from 16 to 33%. Terbium binding induces the same conformational changes in calsequestrin as does calcium, confirming that terbium is a useful calcium analog in this system.

The cardiac high molecular weight proteins/ryanodine receptors were purified to homogeneity from junctional sarcoplasmic reticulum membranes and shown to exhibit large conductance calcium channel activity. High molecular weight proteins were solubilized from junctional sarcoplasmic reticulum in zwitterionic detergent and purified by size-exclusion chromatography followed by sucrose density gradient centrifugation. The purified proteins exhibited an apparent Mr = 400,000-350,000, and bound [3H]ryanodine with a Kd of 4.6 nM and a Bmax of 140-280 pmol/mg protein. High molecular weight proteins demonstrated divalent cation channel activity after incorporation into planar lipid bilayers. Two channel types were identified. Large conductance channels had a slope conductance of 96 +/- 13 pS and a Erev of 42 +/- 9 mV (n = 5); small conductance channels had a slope conductance of 5.5 +/- 1 pS [1.0 microM cis CaCl2; 50 mM trans Ba(OH)2]. Reducing cis calcium from 1 microM to 1 nM reduced the large conductance channel open time from 7 +/- 1% to 0.1% (holding potential, -100 mV). Adding ATP (1 mM) to the cis chamber increased channel open time from 6 +/- 1% to 52 +/- 4% (holding potential, -100 mV); 10 nM ryanodine increased and 100 microM ryanodine decreased percent of open time of the 96 pS channel, without altering unitary channel conductance. The large conductance channel was similar to the calcium release channel detected in native canine cardiac junctional sarcoplasmic reticulum vesicles. Our data suggest that the ryanodine receptor, the calcium-release channel, and the high molecular weight proteins are all identical proteins containing allosteric regulatory sites for calcium, ATP, and ryanodine.

We have isolated two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. One, PL-II, is identical to the proteolipid previously obtained by others using organic solvent extraction. The other, PL-I, has an amino acid composition very similar to those of proteolipids we previously isolated from canine cardiac SR and lamb kidney (Na,K)-ATPase.

Muscles of invertebrate species contain abundant quantities of soluble, sarcoplasmic, high affinity Ca2+-binding proteins (SCBPs). The SCBPs belong to the calmodulin superfamily and contain four homologous domains (I-IV) which arose by reduplication of a gene for a small ancestral protein. We have determined the amino acid sequence of the SCBP from the sandworm Nereis diversicolor. This protein is the only SCBP which has been crystallized in a form suitable for three-dimensional structure determination by high-resolution x-ray analysis (Babu, Y. S., Cox, J. A., and Cook, W. J. (1987) J. Biol. Chem. 262, 11184-11185). N. diversicolor SCBP is a single polypeptide chain of 174 amino acids, including single residues of glutamine and histidine, 2 tyrosines, and 3 tryptophans. It is devoid of cysteine and has an acetylated amino terminus, a calculated Mr of 19,485, and a net charge of -13 at neutral pH. There was no evidence for heterogeneity in the sequence. Probable Ca2+-binding sites were recognized in domains I, III, and IV. Comparison with other available invertebrate SCBP sequences shows an unusually high degree of variability among these proteins, with only 9 residues common to all species.

It has been suspected that the cause of malignant hyperthermia (MH) is an abnormality in the sarcoplasmic reticulum of skeletal muscle. We isolated the sarcoplasmic reticulum from malignant hyperthermia-susceptible (MHS) patients and controls and analysed the protein composition with sodium dodecyl sulfate polyacrylamide gel electrophoresis. There were no remarkable changes in the sarcoplasmic reticulum protein composition profile of the scanned gel of the patients. Quantitative measurement of the relative proportion of each band in the gel, however, revealed a slight decrease in calsequestrin and a slight increase in protein of molecular weight 23,000. (Ca2+ -Mg2+)ATPase had no altered subfragments in MHS patients. Crude mitochondrial proteins and myoplasmic proteins showed minor alterations in composition in some patients. The data supported the thesis that malignant hyperthermia is due to defects in several different cell membranes including the sarcoplasmic reticulum and the mitochondria.

The requirement of calcium (Ca2+) in the excitation-contraction coupling of both skeletal and cardiac muscle is well established. However, the exact location of the intracellular storage sites of Ca2+ is not firmly established. We report here on the ultrastructural ultrastructural distribution of Ca2+ in white and red skeletal muscle and in cardiac muscle of the rat using combined phosphate-pyroantimonate (PPA) and oxalate-pyroantimonate (OPA) procedures. The methods are based on (a) stabilization and/or trapping of Ca2+ during the primary fixation step in glutaraldehyde by potassium phosphate or oxalate; (b) subsequent wash-out of all non-trapped cations such as Na+ and Mg2+ in potassium phosphate or oxalate; (c) conversion of the complexed or trapped Ca2+ into an electron-dense calcium pyroantimonate salt in 100 micron-thick tissue sections; and (d) wash-out of the excess potassium pyroantimonate at alkaline pH. With the OPA procedure, mitochondria of all muscle types showed little precipitate. The junctional sarcoplasmic reticulum was strongly reactive in relaxed white skeletal muscle, negative in contracted white fibres and negative in red skeletal and cardiac muscle, independent of the state of relaxation-contraction. Other organelles were essentially free of deposits. With the PPA method, the precipitate was almost exclusively confined to the sarcolemma and its T-tubular invaginations in cardiac and slow skeletal muscle, and was absent in fast skeletal muscle. Apart from occasional deposits in mitochondria, all other organelles were free of muscle. Apart from occasional deposits in mitochondria, all other organelles were free of precipitate. The sarcolemma-associated deposits were clearly confined to the inner leaflet of the lipid bilayer. The amount of precipitate varied within the contraction cycle, relaxed cells possessing the highest density. Exposure of the tissue to La3+ resulted in the complete absence of sarcolemma-bound precipitate suggesting that the Ca2+ is exchangeable. Furthermore, these cytological data suggest a basic difference in Ca2+ storage between white skeletal muscle on the one hand, and red skeletal and cardiac muscle on the other.

Treatment of cardiac or skeletal muscle sarcoplasmic reticulum vesicles with 0.1 M sodium carbonate selectively extracts both the Ca2+-binding protein calsequestrin and the two "intrinsic glycoproteins," while leaving the Ca2+-dependent ATPase membrane bound. Phenyl-Sepharose chromatography in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and high salt (0.5 M NaCl) readily fractionates these solubilized proteins into a Ca2+-elutable fraction, which contains purified calsequestrin, and a low ionic strength elutable fraction, which contains one of the two intrinsic glycoproteins. Elution of calsequestrin from phenyl-Sepharose occurs near 1 mM Ca2+. Copurifying with calsequestrin are an homologous set of high molecular weight proteins, which like calsequestrin stain blue with Stains-All. These proteins are present in trace amounts and do not correspond to any sarcoplasmic reticulum proteins previously identified. Elution of calsequestrin from phenyl-Sepharose is consistent with the Ca2+-binding protein losing its hydrophobic character in the presence of millimolar Ca2+. This behavior is converse to that observed for several calmodulin-like proteins, which are eluted from hydrophobic gels in the presence of EGTA. The high yield and purity of calsequestrin prepared by this method makes possible a unique system for studying what may be a distinct class of Ca2+-binding proteins.

Phospholamban, originally described as a cardiac sarcoplasmic reticulum protein, was localized in cryostat sections of three adult canine skeletal muscles (gracilis, extensor carpi radialis, and superficial digitalis flexor) by immunofluorescence labeling with highly specific phospholamban antibodies. Only some myofibers were strongly labeled with phospholamban antibodies. The labeling of myofibers with phospholamban antibodies was compared to the distribution of Type I (slow) and Type II (fast) myofibers as determined by staining adjacent sections cytochemically for the alkali-stable myosin ATPase, a specific marker for Type II myofibers. All the skeletal myofibers labeled for phospholamban above background levels corresponded to Type I (slow) myofibers. The presence of phospholamban in microsomal fractions isolated from canine superficial digitalis flexor (89 +/- 3% Type I) and extensor carpi radialis skeletal muscle (14 +/- 6% Type I) was confirmed by immunoblotting. Antiserum to cardiac phospholamban bound to proteins of apparent Mr values of 25,000 (oligomeric phospholamban) and 5,000-6,000 (monomeric phospholamban) in sarcoplasmic reticulum vesicles from both muscles. Quantification of phospholamban in sarcoplasmic reticulum vesicles from cardic, slow, and fast skeletal muscle tissues following phosphorylation with [gamma-32P] ATP suggested that superficial digitalis flexor and extensor carpi radialis skeletal muscle contained about 16 and 3%, respectively, as much phospholamban as cardiac muscle per unit of sarcoplasmic reticulum. The presence of phospholamban in both Type I (slow) and cardiac muscle fibers supports the possibility that the Ca2+ fluxes across the sarcoplasmic reticulum in both fiber types are similarly regulated, and is consistent with the idea that the relaxant effect of catecholamines on slow skeletal muscle is mediated in part by phosphorylation of phospholamban.

Alterations in the phospholipid component of membranes were studied in acute myocardial ischemia with respect to sarcoplasmic reticulum (SR) and mitochondria (Mt) in the canine heart and compared with changes in the phospholipid composition of intact membrane treated with exogenous phospholipases (PLases) A2 and C, in order to examine the mechanism of ischemic degradation. As early as 30 min after coronary ligation, the total phospholipid content of SR and Mt decreased significantly, 16.0% and 5.6%, respectively. The patterns of SR and Mt phospholipids from the ischemic myocardia did not differ on the chromatograms from those of the non-ischemic myocardia, and no significant increases in lysophospholipids were found for up to 3 hrs. Among the components of phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) decreased mainly during ischemia, and depletion of PC exceeded that of PE in SR. PLase C hydrolysed phospholipids yielded no lysophospholipids, compared to the production of a large amount of lysophospholipids by PLase A2. It was concluded that degradation of membrane phospholipids occurs in the early stage of myocardial ischemia mainly in PC and PE, which are the major components of membrane phospholipids. This may be an expression of irreversible changes, and the activation of PLase C was considered to play an important role in their degradation.

Crystals of sarcoplasmic calcium-binding proteins from Nereis diversicolor have been grown from solutions of ammonium sulfate. The crystals are monoclinic, space group P2(1); the axes are a = 43.65 (1), b = 56.05 (1), c = 65.77 (1) A, and beta = 92.58 (2) degrees. The crystals are quite stable to x-rays and diffract beyond 2.5 A resolution. The asymmetric unit contains two protein molecules.