The effect of taurine on cardiac sarcoplasmic reticulum

A recent communication reported that the rate of calcium uptake by sarcoplasmic reticulum (SR) isolated from rat skeletal muscle could be increased by the isolation of the SR in 15 mM taurine, and that exposure of the SR to taurine throughout the isolation procedure resulted in an increased yield of SR. Because of these results in rat skeletal muscle SR studies were carried out on dog myocardial SR.Sarcoplasmic reticulum isolated from adult dog cardiac muscle was not affected by taurine in concentrations as high as 15 millimolar. The addition of taurine to isolation media did not affect calcium transport, ATPase, binding, or release. Sarcoplasmic reticulum fragments were stored and re-examined over a period of a week without appreciable difference in stability of activity between those isolated in the presence of taurine and the control group. This lack of effect suggests that the role of taurine in cardiac muscle metabolism is not likely to be found in regulation of the sarcoplasmic reticulum.     

Effects of polymyxin B on the sarcoplasmic reticulum membrane

Polymyxin B, a cyclic peptide antibiotic, inhibits Ca²⁺-ATPase, p-nitrophenyl phosphatase and phosphorylase kinase activities associated with rabbit skeletal muscle sarcoplasmic reticulum membranes; 50% inhibition is induced by 100 M, 130M and 550 M of polymyxin respectively. The fluorescence intensity of fluorescein isothiocyanate-labeled Ca²⁺-ATPase, decreases in the presence of polymyxin (50% of the total decrease at 70 M polymyxin). On the other hand, the polypeptide inhibits calmodulin-dependent endogenous phosphorylation of 60 kDa, 20 kDa and 14 kDa membrane proteins, while an increase of calmodulin-dependent phosphorylation is observed in 132 kDa and 86 kDa proteins.     

The effects of sphingosine on sarcoplasmic reticulum membrane calcium release.

In this study, we report that sphingosine is a potent inhibitor of sarcoplasmic reticulum (SR) calcium release. Evidence is presented demonstrating a direct effect of sphingosine on the SR ryanodine receptor. Calcium release from "skinned" rabbit skeletal muscle fibers and isolated junctional SR derived from the terminal cisternae (TC) was measured in response to caffeine, doxorubicin, 5'-adenylyl-beta,gamma-imidodiphosphate or calcium. Sphingosine inhibited caffeine-induced release in a dose-dependent manner with an IC50 of 0.1 microM for the single muscle fibers and 0.5 microM for the isolated TC vesicles. Near complete blockage of TC calcium release rate was observed with 3 microM sphingosine. Neither sphingomyelin nor sphingosylphosphorylcholine had any effect at the 3 microM level, suggesting that the sphingosine effect was specific. Doxorubicin-induced calcium release and spontaneous calcium release were also blocked by sphingosine. Sphingosine was also capable of stimulating calcium transport in the isolated TC vesicles without an effect on Ca-ATPase activity. Ruthenium red was not capable of substantial additional stimulation of calcium transport nor inhibition of calcium release beyond the action of sphingosine. Sphingosine's blockage of calcium release was not reversed by the protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2- methylpiperazine dihydrochloride, suggesting that the action of sphingosine on calcium release was not dependent on ryanodine receptor phosphorylation. Sphingosine significantly increased (8-fold) the Kd for specific [3H]ryanodine binding to TC membranes and decreased the Bmax with a dose dependence similar to the inhibition of calcium release, but sphingosine did not affect the pCa tension relationship of skinned skeletal muscle fibers. These data are consistent with a direct effect of submicromolar sphingosine on the ryanodine receptor. Substantially higher concentrations of sphingosine (30-50 microM) or sphingosylphosphorylcholine (10-20 microM) were capable of inducing calcium release by themselves. Preliminary data indicate that the transverse tubule and not the SR contain substantial sphingomyelinase activity consistent with a transverse tubule source of sphingosine production. Considering that sphingosine is found in micromolar concentrations in some cells, our data indicate that sphingosine generated by the transverse tubule membranes may be a physiologically relevant mechanism for modulating SR calcium release.     

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.     

The effect of electromagnetic radiation on the membranes of the sarcoplasmic reticulum

On the sarcoplasmic reticulum membranes has been shown that at temperature of Ca2(+)-ATPase activity change of dependence in the Arrhenius plot the microwaves (2450 MHz, specific absorption rate 12 w/kg) inhibit the ATP-hydrolase and Ca2(+)-transporting activity of Ca2(+)-ATPase. The effect of radiation exhibits within the narrow temperature range (approximately 1 degree C) and quantitatively corresponds to the decrease of Ca2(+)-ATPase activity caused by the decrease of temperature by 1.6 degrees C from 18 degrees C. The fluorescence intensity of naphthalene sulfonic probes reduces under the influence of microwaves at 18 degrees C.     

Calcium fluxes across the membrane of sarcoplasmic reticulum vesicles

The relationship between calcium exchange across the membrane of sarcoplasmic reticulum vesicles and phosphoenzyme (EP) was examined in calcium transport reactions using a limited amount of ATP as substrate. Rapid calcium influx and efflux (approximately 385 nmol.(mg.min)-1), measured in reactions in which ATP concentration fell from 20 microM, was accompanied by a shift in the equilibrium between an ADP-sensitive EP and an ADP-insensitive EP toward the former. Rapid exchange between ATP and ADP (approximately 1500 nmol.(mg.min)-1) was also observed under conditions where no significant incorporation of Pi into ATP took place, suggesting that ATP in equilibrium ADP exchange can occur without Cao in equilibrium Cai exchange. Ca2+ permeability during the calcium transport reaction was estimated in reactions carried out with acetylphosphate, which produces a hydrolytic product that does not participate in the backward reaction of the calcium pump. Under conditions where the calcium content exceeded 43 nmol.mg-1, a level that may reflect the binding of calcium ions to sites inside the sarcoplasmic reticulum, the rate constant for Ca2+ efflux was 0.33 min-1. These data allow the rate of passive Ca2+ efflux to be estimated as approximately 17 nmol.(mg.min)-1 at the time when calcium content was maximal and a rapid Cao in equilibrium Cai was observed. It is concluded that the majority of the rapid Ca2+ efflux is mediated by partial backward reactions of the calcium pump ATPase.     

Ca2+-ATPase membrane crystals in sarcoplasmic reticulum. The effect of trypsin digestion

Vanadate induces the formation of two-dimensional crystalline arrays of Ca2+-ATPase molecules in sarcoplasmic reticulum. The Ca2+-ATPase membrane crystals are evenly distributed among the terminal cisternae and longitudinal tubules of sarcoplasmic reticulum, but very few crystals were observed in the T tubules. Tryptic cleavage of the Ca2+ transport ATPase into two major fragments (A and B) did not interfere with the vanadate-induced formation of membrane crystals. The ability of Ca2+-ATPase to crystallize was lost after further cleavage of the A fragment into the A1 and A2 subfragments that is known to be accompanied by loss of Ca2+ uptake. Vanadate (0.1-5 mM) inhibited the secondary cleavage of Ca2+-ATPase by trypsin suggesting that the susceptibility of the tryptic cleavage sites is influenced either by the conformation of the enzyme or by the formation of ATPase crystals.     

Permeability of sarcoplasmic reticulum membrane. The effect of changed ionic environments on Ca2+ release

Summary Permeability properties and the effects of a changed membrane potential on Ca²⁺ release of sarcoplasmic reticulum vesicles of rabbit skeletal muscle were investigated by Millipore filtration. The relative permeability of sarcoplasmic reticulum to solutes determined under conditions of isotope exchange at equilibrium and/or under conditions of net flow of solute and water into the vesicles was as follows: sucrose, Ca²⁺, Mn²⁺–, choline⁺, Tris⁺–+, Na⁺, Li⁺, Cl^–. Transient membrane potentials were induced by rapidly changing the ionic environment of the vesicles. Knowledge of the relative permeation rates of the above ions allowed prediction of the direction and extent of membrane polarization. Osmotic effects in the polarization measurements due to the rapid influx of solute and water into the vesicles were minimized by using media containing a fast (K⁺ or Cl^–) and a relatively slow (gluconate^– or choline⁺) penetrating ion.⁴⁵Ca²⁺ efflux from vesicles derived from different parts of the sarcoplasmic reticulum structure was not appreciably changed when vesicles were made more positive inside (choline chloride potassium gluconate) or more negative inside (potassium gluconate choline chloride). These studies suggest that part or all of the ion-induced changes in sarcoplasmic reticulum membrane permeability, previously interpreted to indicate depolarization-induced Ca²⁺ release, may be due to osmotic effects.     

The effect of deuterium ion concentration on the properties of sarcoplasmic reticulum

Summary 99.8% Deuterium oxide, as obtained commerically, has been shown to contain a contaminant which strongly inhibits calcium transport and binding by sarcoplasmic reticulum (S.R.) and the associated ATPase activity. The contaminant is removed by distillation of deuterium oxide. Calcium binding by S.R. is maximal at pH 6.5 whereas calcium transport (in the presence of oxalate) is maximal at a pH of 7.2 to 7.5. In the presence of deuterium oxide, these maxima are shifted to a pD of 7.2 and a pD of 7.5 to 8.0, respectively. The maximum binding and transport rates are not affected by the change from aqueous to deuterium oxide medium. The same phenomena are observed with the ATPase activity. In the presence of oxalate, calcium;magnesium ATPase is maximal at pH 7.2 and pD 8.0. The maximum rate is unchanged, however,At pH 7.2 or higher, the amount of calcium which may be bound by S.R. remains constant with time. At lower pH, calcium initially bound is slowly displaced from the membrane with time. It has been reported that deuterium oxide inhibits excitation-contraction coupling. The results presented here indicate that S.R. is probably not the site of deuterium oxide inhibition, and raise the possibility that the measured inhibition is due to an impurity in the deuterium oxide.     

The effect of calcium load on the calcium permeability of sarcoplasmic reticulum

The effect of intravesicular and extravesicular calcium concentration on the passive efflux from sarcoplasmic reticulum (SR) vesicles isolated from cardiac and skeletal muscle was determined by measuring net efflux of calcium after stopping pump-mediated fluxes. The apparent permeability, calculated as the passive efflux divided by the total intravesicular calcium, depended on calcium load. This dependence of the apparent permeability on calcium load could be explained by the presence of intravesicular calcium-binding sites with a dissociation constant less than 10(-3) M. When the intravesicular bound calcium was taken into account, passive calcium efflux was found to be linearly related to the difference in calcium concentration across the SR membrane. Thus the permeability of the SR membrane is independent of intravesicular and extravesicular calcium concentration in the ranges investigated. The average first order rate constant for passive calcium efflux for six preparations was 0.8 +/- 0.2 min-1 for skeletal and 0.7 +/- 0.1 min-1 for cardiac SR. The amount of intravesicular bound calcium for the same preparations was 33 +/- 6 nmol mg-1 for skeletal and 13 +/- 2 nmol mg-1 for cardiac SR. The first order rate constants were unaffected by Mg concentration between 0.1 +/- 15.1 mM and by the presence of an ATP-regenerating system. The results suggest that some minimal calcium load may be required in order to observe a substantial passive calcium efflux, the passive calcium efflux is not carrier mediated, and passive calcium efflux is not a likely route of calcium release during excitation-contraction coupling.     

Dolichol kinase in rat sarcoplasmic reticulum membrane preparations

A dolichol kinase (EC 2.7.1.108) was found in sarcoplasmic reticulum membrane fractions from rat leg muscle. This enzyme specifically required CTP as a phosphoryl donor and relatively little activity was found in the absence of exogenous detergent-suspended dolichol. Unlike other reported dolichol kinases, the kinase from skeletal muscle was activated almost equally well by Ca2+, Zn2+, or Mg2+, but not Mn2+. No effect of calmodulin was seen. The kinase exhibited a single pH optimum at pH 7-8 in contrast to kinases from certain other tissues. Despite the low level of dolichol present in skeletal muscle, the kinase in the sarcoplasmic reticulum fraction had an activity comparable to that of microsomal preparations from tissues such as brain and liver, which may indicate that skeletal muscle has a high capacity for dolichol phosphorylation and protein glycosylation.     

The effect of hypothyroidism on sarcoplasmic reticulum in fast-twitch muscle of the rat

The effects of hypothyroidism on the Ca2+-transport capabilities of fast-twitch muscle (m. gastrocnemius) of the rat were studied in whole-muscle homogenate and isolated sarcoplasmic reticulum. Hypothyroidism did not affect the percentage recovery and the vesicle composition of the sarcoplasmic reticulum fraction, the total lipid and phospholipid-to-protein ratios and the protein composition (both qualitative and quantitative). Also the Ca2+-loading capacity of purified sarcoplasmic reticulum, in the presence of oxalate, and the Ca2+ and pH dependence of both the uptake reaction and the coupled ATPase activity were unchanged. However, the homogenate Ca2+-loading capacity and the Ca2+-uptake activity were depressed, as was the yield of purified sarcoplasmic reticulum. The results indicate a 31% reduction of the entire sarcoplasmic reticulum membrane system per volume of muscle. Ca2+/ATP coupling ratios, determined in purified sarcoplasmic reticulum vesicles by measurement of initial rates of net Ca2+ uptake and Ca2+-Mg2+-dependent hydrolysis of ATP, were found to be 1.48 +/- 0.06 and 2.08 +/- 0.05 in the euthyroid and hypothyroid groups, respectively. Identical values were obtained with a recently described Ca2+-pulse method (Meltzer, S. and Berman, M.C. (1984) Anal. Biochem. 138, 458-464), i.e., 1.53 +/- 0.06 and 2.01 +/- 0.03 in the euthyroid and hypothyroid groups, respectively. Passive Ca2+ efflux from sarcoplasmic reticulum was the same in both groups (30 nmol/mg per min), as was the fraction of vesicles that did not show net uptake of Ca2+ (less than 10%), which makes it unlikely that these parameters provide an explanation for the differences in the coupling ratio. The energy of activation of the (Ca2+ + Mg2+)-ATPase was increased in hypothyroidism, which may point to changes in the phospholipid environment of the enzyme. Physiological concentrations of T3 and T4 had no effect on the (Ca2+ + Mg2+)-ATPase in vitro, but all observed changes in the hypothyroid state could be reversed within 14 days by administration of T3 to hypothyroid animals. Approximate calculations indicate that the observed changes in the sarcoplasmic reticulum as a result of thyroid-hormone depletion may contribute significantly to the decrease in relaxation rate and the decrease in energy consumption during contraction.     

Interaction of lactate dehydrogenase and the sarcoplasmic reticulum membrane]

The binding of lactate dehydrogenase (LDH) to sarcoplasmic reticulum membranes results in a 60-70% decrease of the enzyme specific activity. This binding occurs both in high (Kd = 1 microM) and low affinity sites. Addition of NADH or NAD+ and a increase of ionic strength lead to the solubilization of the bound enzyme. A similar effect is observed after addition of the fluorescent probes--anilinonaphthalene sulfonate (ANS) and auramine O (A0). The effect of ANS consists predominantly in its binding to the membrane, while that of A0 is due to the probe interaction with the enzyme. At low concentrations of toluidinylnaphthalene sulfonate (TNS) under conditions of predominant binding of the probe to the membrane, the LDH binding to microsomes is enhanced. A rise in the TNS concentration leads to the formation of the probe-LDH complex which interaction with membrane is hampered. The sites of the probes binding to the protein are located outside the enzyme active center but are, nevertheless, sensitive to it states. It is assumed that these sites of the LDH molecule are involved in its interaction with the membrane. The decline of activity of the bound enzyme is interpreted in terms of alterations of the physico-chemical properties of the medium during the enzyme transition from the solution to the perimembrane space.     

Peroxide modification of skeletal muscle sarcoplasmic reticulum in antioxidant deficiency and under the effect of ionol. II. Physico- chemical properties of the sarcoplasmic reticulum membrane

Physico-chemical parameters of membranes of skeletal muscles' sarcoplasmic reticulum in antioxidant insufficiency, which was modelled by excluding alpha-tocopherol from the animals ration, and after treatment with phenol antioxidant ionol were studied. It was shown that activation of lipid peroxidation in vitamin E insufficiency results in a significant lowering of microviscosity of lipid bilayer membranes of sarcoplasmic reticulum. Using polarography significant changes in membrane protein conformation were revealed, which were characterized by lowering of integrity and by disorganization of protein globules. Treatment of animals with antioxidant insufficiency with ionol led to certain normalization of changes of physico-chemical characteristics of the learned membrane structures caused by lipid peroxidation.     

Allosteric effect of substrate on sarcoplasmic reticulum Ca-ATPase]

High concentration of ATP is found to activate Ca-dependent ATPase from sarcoplasmic reticulum both in membrane fraction and in purified enzyme preparation. The treatment of Ca-ATPase preparation with tripsin results in the elimination of the activating effect of ATP, which is accompanied by the disappearance of 100.000 molecular weight protein and by the appearance of fragments with molecular weight of 45.000 and 55.000. Repeated freezing of the enzyme preparation eliminates activating effect of ATP. ATP action is analysed from the viewpoint of allosteric kinetics, which postulates the existence of two Ca-ATPase conformers, their mutual conversion being induced by ATP binding at allosteric center. Kinetic parameters of the conformers studied are calculated.