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At least 19 recordsLinked to original sources
PubMed1976

Association of gylcogenolysis with cardiac sarcoplasmic reticulum.

Sarcoplasmic reticulum fragments isolated from dog cardiac muscle possess a calcium-accumulating system associated with a series of enzymes linked to glycogenolysis. These enzymes include: adenylate cyclase, cyclic AMP-dependent protein kinase, phosphorylase b kinase, phosphorylase (b/a, 30/1),"debrancher" enzyme, and glycogen (0.3 to 0.7 mg/mg of protein). The sarcoplasmic reticulum preparation produced glucose 1-phosphate and glucose from either endogenous or exogenous glycogen. Both the calcium-accumulating and glycogenolytic enzymes sediment in a single peak at 33% sucrose on a linear continous sucrose density gradient, and the complex remains intact throughout repeated washing. Glycogen particles appear to be associated with the sarcoplasmic reticulum in situ as well as in the isolated microsomal fraction. The sarcoplasmic reticulum-glycogenolytic complex, monitored by a linked enzyme spectrophotometric assay, shows several features: (a) activation of phosphorylase activity to peak rate occurs over a very rapid time course which cannot be duplicated using combinations of purified enzymes; (b) activation is inhibited by protein kinase inhibitor; (c) phosphorylase b functions as in the purified form with respect to AMP (Km, 0.3 mM); (d) in the presence of limiting amounts of glycogen, optimal phosphorylase b activity in the sarcoplasmic reticulum requires the presence of debrancher, and the activity is sensitive to inhibitors of that enzyme such as Tris, which suggests the possiblity that the enzymes bear a specific structual relationship to the glycogen present. Phosphorylase b leads to a activation in the sarcoplasmic reticulum was completely resistant to ethylene glycol bis(beta-aminoethyl either)-N,N'-tetraacetic acid (EGTA). Inhibition of calcium accumulation by or release of bound calcium from sarcoplasmic reticulum by X537A (RO 2-2985) did not alter the EGTA resistance. These results suggest that cardiac sarcoplasmic reticulum is a complex organelle containing functions that may be related to excitation-contraction coupling and intermediary metabolism.

Adenosine Monophosphate↗
PubMed1976

Synthesis of adenosine triphosphate during release of intravesicular and membrane-bound calcium ions from passively loaded sarcoplasmic reticulum.

Sarcoplasmic reticulum isolated from rabbit skeletal muscle and incubated in a medium containing Ca2+ in the absence of ATP retains intravesicular and/or membrane-bound Ca2+. The synthesis of ATP coupled with the release of intravesicular Ca2+ is totally inhibited by the ionophore X-537A. Release of the membrane-bound Ca2+, retained after short periods of incubation (10min) or after release of the intravesicular Ca2+ by ionophore X-537A, still supports some synthesis of ATP. The ratios of Ca2+ released to ATP synthesized are 2.5-3.2, when bound and intravesicular Ca2+ are released simultaneously, and 3.1-4.0, when only bound Ca2+ is released. The results show that the synthesis of ATP by sarcoplasmic reticulum during release of passively accumulated Ca2+ by EGTA [ethanedioxybis(ethylamine)tetra-acetic acid] is accompanied by a loss of membrane-bound Ca2+.

Adenosine Diphosphate↗
PubMed1977

Subunits of the calcium ion-pump system of sarcoplasmic reticulum.

Sarcoplasmic reticulum membranes with high content of Ca2+ -ATPase (80% of total protein) were dissolved in a non ionic medium and were submitted to isoelectric focusing in polyacrylamide gels. The membrane protein was resolved into six main bands whose isoelectric points range from 6 to 5. The mol. wt. of these peptides is about 100 000 as estimated by second dimension electrophoresis in sodium dodecyl sulfate-polyacrylamide system. The electrophoretic behaviour of the purified ATPase enzyme is similar to that of crude membranes.

Adenosine Triphosphatases↗
PubMed1976

Comparative study of the lipid composition of rabbit and lobster sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) membranes isolated from rabbit and lobster muscles have similar phospholipid classes, but they differ in plasmalogen content. The plasmalogenic species are mostly distributed among phosphatidylethanolamines (PE's) and make up about 62% of the total in rabbit SR and about 46% in lobster membranes. Lobster SR phospholipids contain large amounts of polyunsaturated fatty acids which are present in low amounts in rabbit membranes. The total unsaturated fatty acids of phosphatidylcholines (PC's) represent about 53% and 73% of the total fatty chains for rabbit and lobster SR, respectively. The values found for PE's were about 56% and 64%, respectively. Furthermore, lobster membranes contain significant amounts of PC and PE molecular species with unsaturated fatty acids in positions 1 and 2, whereas rabbit SR contain low amounts.

Adenosine Triphosphatases↗
PubMed1977

Assembly of the sarcoplasmic reticulum. Localization by immunofluorescence of sarcoplasmic reticulum proteins in differentiating rat skeletal muscle cell cultures.

Immunofluorescent staining techniques were used to study the distribution of the Ca(2) + Mg(2+)-dependent ATPase and calsequestrin in primary cultures of differentiating rat skeletal muscle cells, grown for different periods of time under various culture conditions. In mononucleated myoblasts calsequestrin was detected after 45 h in culture whereas the ATPase was not detected until 60 h. After cell fusion began, both proteins could be identified in all multinucleated cells. Myoblasts grown for longer than 60 h in low Ca(2+) medium contained calsequestrin and the ATPase, even though they were unable to fuse. These studies at the cellular level confirm biochemical findings on the biosynthesis of calsequestrin and the ATPase. Immunofluorescent staining of myoblasts showed that calsequestrin first appears in a well-defined region of the cell near one end of the nucleus. At later times, the staining occupied progressively larger regions adjacent to the nucleus and took on a fibrous appearance. This suggests that calsequestrin first accumulates in the Golgi region and then gradually spreads throughout the cell. In contrast, the ATPase appeared to be concentrated in many small patches or foci throughout the cytoplasm and was never confined to one particular region, although some parts of the cell often stained more intensely than others. In multinucleated cells, alternating dark and fluorescent strands parallel to the longitudinal axis of the cells were evident.

Adenosine Triphosphatases↗
PubMed1976

Calcium transport ATPase of canine cardiac sarcoplasmic reticulum. A comparison with that of rabbit fast skeletal muscle sarcoplasmic reticulum.

To define the mechanism responsible for the slow rate of calcium transport by cardiac sarcoplasmic reticulum, the kinetic properties of the Ca2+-dependent ATPase of canine cardiac microsomes were characterized and compared with those of a comparable preparation from rabbit fast skeletal muscle. A phosphoprotein intermediate (E approximately P), which has the stability characteristics of an acyl phosphate, is formed during ATP hydrolysis by cardiac microsomes. Ca2+ is required for the E approximately P formation, and Mg2+ accelerates its decomposition. The Ca2+ concentration required for half-maximal activation of the ATPase is 4.7 +/- 0.2 muM for cardiac microsomes and 1.3 +/- 0.1 muM for skeletal microsomes at pH 6.8 and 0 degrees. The ATPase activities at saturating concentrations of ionized Ca2+ and pH 6.8, expressed as ATP hydrolysis per mg of protein, are 3 to 6 times lower for cardiac microsomes than for skeletal microsomes under a variety of conditions tested. The apparent Km value for MgATP at high concentrations in the presence of saturating concentrations of ionized Ca2+ is 0.18 +/- 0.03 ms at pH 6.8 and 25 degrees. The maximum velocity of ATPase activity under these conditions is 0.45 +/- 0.05 mumol per mg per min for cardiac microsomes and 1.60 +/- 0.05 mumol per mg per min for skeletal microsomes. The maximum steady state level of E approximately P for cardiac microsomes, 1.3 +/- 0.1 nmol per mg, is significantly less than the value of 4.9 +/- 0.2 nmol per mg for skeletal microsomes, so that the turnover number of the Ca2+-dependent ATPase of cardiac microsomes, calculated as the ratio of ATPase activity to the E approximately P level is similar to that of the skeletal ATPase. These findings indicate that the relatively slow rate of calcium transport by cardiac microsomes, whem compared to that of skeletal microsomes, reflects a lower density of calcium pumping sites and lower Ca2+ affinity for these sites, rather than a lower turnover rate.

Adenosine Triphosphatases↗
PubMedDate not supplied

[Effect of psychotropic preparations on the activity of Ca- and Mg-dependent ATPase of the sarcoplasmic reticulum].

In sarcoplasmic reticulum of rabbit skeletal muscles the activity of Ca2+, Mg2+- dependent ATPase was distinctly inhibited under effect of neuroleptic drugs - derivatives of phenothiazine and butyrophenone. The effect of tricyclic antidepressants was less pronounced. Tranquilizers (derivatives of 1,4-benzodiazepine) inhibited the enzyme, but trioxazin was only slightly active. High concentrations of lithium salts and of psychostimulants caffeine and corasole were found to stimulate the Ca2+, Mg2+-ATPase activity; low concentrations of the substances slightly inhibited the enzyme. The blocking effect of psychotropic drugs was more distinct, if the enzyme preparations were previously treated with ATP.

Adenosine Triphosphatases↗
PubMed1976

The effect of La3+ on the characteristics of fragmented sarcoplasmic reticulum.

Fragmented sarcoplasmic reticulum (FSR) was prepared from the white muscles of the catfish (Amiurus nebulosus). The effect of La3+ on the functional characteristics of FSR was studied. La3+ in a concentration higher than 10(-4) M was found to decrease or arrest Ca2+ accumulation, cholinesterase activity and the activation of ATPase by Ca2+. La3+ added after the elimination of membrane-bound Ca2+ of FSR (1 mM EGTA, pH 7.1) does not substitute Ca2+ in its functions. The cholinesterase activity of FSR solubilized by deoxycholate and purified by gelfiltration is inhibited by La3+ present in a concentration higher than 10(-4) M and simultaneously with inhibition, the absorbance at 280 nm is increased. Ca2+ gives rise to similar changes only in concentrations higher than 10(-2) M.

Animals↗
PubMed1977

The cross-linking of rabbit skeletal muscle sarcoplasmic reticulum protein.

Sarcoplasmic reticulum proteins have been cross-linked in situ with two reagents, the disulphide-bridged bifunctional imido ester, dimethyl-3,3'-dithiobispropionimidate dihydrochloride and the mild oxidant cupric phenanthroline. Analysis of proteins so cross-linked by electrophoresis on agarose/acrylamide gels reveals that a series of new polypeptides, up to a molecular weight of 900 000, are formed. These have molecular weights which are multiples of 100 000. Further analysis of samples by electrophoresis in a second dimensions containing a reducing agent revealed the monomeric polypeptides from which the cross-linked polypeptides were formed. With dimethyl 3,3'-dithiobispropionimidate dihydrochloride homopolymers of the Ca2+-stimulated ATPase, calsequestrin and/or calcium binding protein were formed. With cupric phenanthroline only the Ca2+-stimulated ATPase was involved in polymer formation. It has been confirmed on another gel system that these two proteins which are involved in Ca2+ binding are not cross-linked intermolecularly with this latter reagent. We conclude that the 100 000 dalton Ca2+-stimulated ATPase polypeptides are within 2 A of each other in the membrane while calsequestrin and/or calcium binding protein are within 11 A of each other. Although there appears to be no limit to the extent of cross-linking of any of these polypeptides there is not indication of heteropolymer associations between them.

Adenosine Triphosphatases↗
PubMed1976

Chemical modification of the Ca2+-dependent ATPase of sarcoplasmic reticulum from skeletal muscle. I. Binding of N-ethylmaleimide to sarcoplasmic reticulum: evidence for sulfhydryl groups in the active site of ATPase and for conformational changes induced by adenosine tri- and diphosphate.

The time course of binding of N-ethylmaleimide (NEM) to the SR was measured at pH 7.5 in the presence or absence of ATP or ADP. The following results were obtained. 1. Both in the presence and absence of nucleotide, the ATPase [EC 3.6.1.3] activity decreased linearly with increase in the amount of NEM bound to the fragmented sarcoplasmic reticulum (SR), and was inhibited almost completely by the binding of 2 moles of NEM per 10(5) g of the SR protein. 2. The amount of NEM incorporated into the ATPase (M.W.=105,000) was measured by SDS disc-gel electrophoresis. It was shown that the ATPase activity was inhibited almost completely by the binding of 2 moles of NEM per mole of ATPase. 3. The rate of binding of NEM to SR decreased by 30-40% in the presence of either ATP or ADP. The concentrations of both ATP and ADP for half-saturation were 0.1-0.2mM. 4. The effect of nucleotide on the rate of binding of NEM was not changed by the presence of Ca2+ and Mg2+ ions. Similar effects were also observed even when the SR membranes were solubilized with Triton X-100. It is suggested from these results that one or two SH groups are located in the active site of the SR ATPase, and that conformational changes are induced by the addition of ATP and ADP.

Adenosine Diphosphate↗
PubMed1978

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.

Adenosine Triphosphatases↗
PubMed1976

Isolation and characterization of tryptic fragments of the adenosine triphosphatase of sarcoplasmic reticulum.

Exposure of sarcoplasmic reticulum to trypsin in the presence of 1 M sucrose results in degradation of the Mr = 102,000 ATPase enzyme to two fragments of Mr = 55,000 and 45,000 with subsequent appearance of fragments of Mr = 30,000 and 20,000. These fragments were purified by column chromatography in sodium dodecyl sulfate. Antibodies were raised against the ATPase and the Mr = 55,000, 45,000, and 20,000 fragments. There was no antigenic cross-reactivity between the Mr = 55,000 and 45,000 fragments, indicating that they were derived from a single linear cleavage of the larger enzyme. There was antigenic cross-reactivity between the Mr = 20,000 and 55,000 fragments, indicating an origin of the Mr = 20,000 fragment in the Mr = 55,000 fragment. None of the antibodies inhibited (Ca2+ + Mg2+)-dependent ATPase or Ca2+ transport. The Mr = 20,000 fragment and the Mr = 55,000 fragment were active in Ca2+ ionophore assays. The active site of ATP hydrolysis was labeled with [gamma-32P]ATP and the site of ATP binding was labeled with tritiated N-ethylmaleimide. In both cases radioactivity was found in the intact ATPase and in the Mr = 55,000 and 30,000 fragments, indicating that the Mr = 30,000 fragment was also derived from the Mr = 55,000 fragment. Amino acid composition data showed that the Mr = 45,000 fragment contained about 60% nonpolar and 40% polar amino acids, while the Mr = 55,000 fragment and the Mr = 20,0000 fragment contained about equal amounts of polar and nonpolar amino acids. Studies of the reaction of various antibodies at the external surface of sarcoplasmic reticulum vesicles showed that the ATPase was exposed, whereas calsequestrin and the high affinity Ca2+-binding protein were not. The use of antibodies against the various fragments indicated that the Mr = 55,000 fragment was in large part exposed, whereas the Mr = 20,000 and the 45,000 fragments were only poorly exposed. It is probable that the site of ATP hydrolysis in the Mr = 55,000 fragment is external, whereas the ionophore site is only partially exposed and the Mr = 45,000 fragment is largely buried within the membrane.

Adenosine Triphosphatases↗
PubMed1976

Effects of exercise training and exhaustion on 45Ca uptake by rat skeletal muscle mitochondria and sarcoplasmic reticulum.

Mitochondrial and sarcoplasmic reticular 45Ca2+ uptake and Ca2+-ATPase activity were determined in skeletal muscle from exercise trained and non-trained rats at rest or following short-term exhaustive exercise. In trained rats exercised to exhaustion, mitochondrial 45Ca2+ uptake was significantly depressed when compared to non-trained rats at rest. Ca2+-ATPase activity of sarcoplasmic reticulum from trained rats exercised to exhaustion was significantly increased as compared to trained rats at rest. These data suggest that the disruptive influence of Ca2+ accumulation in mitochondria isolated following exhaustive exercise may be diminished as a result of training.

Adenosine Triphosphatases↗
PubMed1978

[Several properties of the Ca-pump of rabbit skeletal muscle sarcoplasmic reticulum in hypercholesteremia].

Sarcoplasmic reticulum (SR) fragments from the skeletal muscles of rabbit with marked atherosclerosis possessed decreased Ca2+-accumulating capacity. Lowering of transport efficiency, namely reduction of the Ca/ATP ratio from 1.9--normal value--to 0.9 during the experiment at 26 degrees C was accompanied by activation of Ca-ATPase and simultaneously of the rate of Ca2+ outflux from the SR. Arrhenius plots of Ca-ATPase temperature dependence characterized under normal conditions by a break at 20--21 degrees C was linearized under hypercholesterolemia. At the same time there was a rise (from 0.03 under normal conditions to 0.15 in atherosclerosis) of cholesterol/protein ratio in the SR membrane preparations. Activation energy for Ca-ATPase crude membranes under normal conditions was equal to 15.6 and 28.7 kcal/mol above and below the break point respectively; this value for Ca-ATPase of membranes with increased cholesterol level was 19 kcal/mol for all the temperatures investigated.

Adenosine Triphosphatases↗
PubMedDate not supplied

The effect of ethylene glycol and DMSO on fusion of isolated sarcoplasmic reticulum membranes.

Fragmented sarcoplasmic reticulum (FSR) vesicles from rabbit skeletal muscle were suspended in 5-10% ethylene glycol (EG) or in 5, 10, or 15% dimethylsulfoxide (DMSO) and were pelleted onto flat aluminum foil disks. No vesicle fusion o-curs with either 5 or 10% EG treatment and 2-21/2 hours drying. After 4-5 hours drying, 5% EG-treated vesicles have established more areas of close contact and individual vesicles have begun to flatten when compared with the 10% EG vesicles remained round as in the 2-21/2 hour samples of all treatments. Overnight drying also causes extended sheets of bilayer pairs to form in the 5% DMSO-treated samples but, with 10 and 25% treated vesicles, destroys the double bilayers and only occasional dense regions of membrane whorls remain. Both EG and DMSO promote morerapid fusion of FSR vesicles than does glycerol but overnight drying after treatment with 10 or 25% DMSO DESTROYS THE FUSED MEMBRANE.

Animals↗
PubMedDate not supplied

Fusion of isolated sarcoplasmic reticulum membranes.

Fragmented sarcoplasmic reticulum (FSR) vesicles from rabbit muscle were suspended in 1.5-5% glycerol solutions and were pelleted onto aluminum foil disks in a modified centrifuge tube. Examination of these pellets in the electron microscope after drying for 2--2.5, 4--5.5, and 21 hours revealed a progression of changes. First, distances between individual, round vesicles decreases. Next, somewhat flattened vesicles establish limited areas of contact with adjacent vesicles. Finally, vesicle fusion occurs and extended areas of double bilayers are formed. A water loss-time interaction appears to be needed for the fusion process. A Hg-phenyl azoferritin compound was used as a marker to identify intra- and extra-vesicular space in the fused samples. Quantitative measurements of birefringence during imbibition of pellet slices in a graded series (eta-1.41-1.43) is reached. The plateau seen in this part of the curve is again followed by steadily increasing birefringence at higher glycerol concentrations. This interruption in the birefringence curve is presumably due to a matching of the refractive indices of the glycerol solution and a lipid component in the membranes.

Animals↗
PubMed1979

Post-mortem electrical stimulation of muscle and its effects on sarcoplasmic reticulum adenosine triphosphatase.

Sarcoplasmic reticulum (SR) was isolated from control muscles and from muscles which had been subjected to short-term post-mortem electrical stimulation. Both preparations had similar protein compositions but the SR from electrically stimulated muscle had a lower 'extra' ATPase activity. The ability of the SR preparations from electrically stimulated muscles to accumulate Ca2+ was about the same as the controls. There was, therefore, an apparently greater efficiency of Ca2+ transport in the isolated vesicles, the reason for which is not known, but an alteration in the 'leakiness' of the membrane may be involved. Purified ATPase isolated from control and stimulated SR contained, in addition to the ATPase protein, a polypeptide of molecular weight about 30 000. The purified ATPase vesicles from electrically stimulated muscle had a reduced activity as measured by ATP splitting activity, phosphoenzyme formation from either inorganic orthophosphate (Pi) or ATP, or by an ATP in equilibrium Pi exchange reaction. These reduced activities probably result from an alteration in the binding affinities of the ATPase for ATP and Pi. The low affinity site for calcium binding was not affected by electrical stimulation. Purified ATPase vesicles from stimulated muscle were more susceptible to proteolytic attack, suggesting that the conformation of the protein or its association with the membrane lipids had been altered.

Adenosine Triphosphatases↗