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PubMed1991

Evidence for intraluminal Ca++ regulatory site defect in sarcoplasmic reticulum from malignant hyperthermia pig muscle.

Malignant hyperthermia (MH) is a pharmacogenetic disease of humans and various animal species that predisposes to a life-threatening, anesthetic agent-induced syndrome. MH is thought to be a consequence of abnormal, sustained increases in myoplasmic Ca++ and sarcoplasmic reticulum (SR) membranes from MH muscle have been shown to have a Ca++ release channel defect. In the present study we have tested a hypothesis that the abnormal Ca++ release mechanism in MH can be expressed when Ca++ is loaded in the presence of pyrophosphate. SR membrane vesicles isolated from normal and MH pig muscle were loaded with Ca++ in the presence and absence of pyrophosphate until Ca(++)-induced Ca++ release occurred. Under both circumstances the threshold amount of Ca++ loaded until Ca++ release occurred was lower in the SR from MH pig skeletal muscle. This difference in amount of Ca++ preload is not explained by results obtained comparing rates of Ca++ uptake, number of ryanodine binding sites or the amounts of calsequestrin among SR vesicles from MH and normal muscle. We conclude from this study that use of pyrophosphate for Ca++ loading does not ablate the abnormal Ca++ release in SR from MH muscle, suggesting the study can be done on small amounts of SR from biopsied human muscle. The data also suggest that abnormality in an intraluminal, low affinity Ca++ binding site regulating Ca++ release occurs in the SR membrane of MH pig muscle.

Animals↗
PubMed1991

The effect of ryanodine on oxygen free radical-induced dysfunction of cardiac sarcoplasmic reticulum.

The effect of the plant alkaloid ryanodine on the cardiac sarcoplasmic reticulum (SR) function, which plays a major role in the regulation of intracellular calcium and thereby in the generation of force, was studied by determining oxalate-supported calcium uptake, steady-state calcium load, calcium permeability, intravesicular-free calcium and Ca,Mg-adenosine triphosphatase (ATPase) activity of "heavy" vesicles in the presence or absence of the oxygen-free radical-generating system. In vitro generation of oxygen-free radicals by xanthine oxidase (0.09 u/ml), acting on xanthine (25 microM) as a substrate, increased the permeability of the vesicles to calcium, determined by measuring net efflux of calcium after stopping pump-mediated fluxes, and decreased oxalate-supported calcium uptake and steady-state calcium load with no effect on Ca,Mg-ATPase activity. This effect of oxygen-free radicals was inhibited completely by superoxide dismutase, which eliminated completely superoxide anion radical production and caused an anticipated increase in hydrogen peroxide from the xanthine-xanthine oxidase reaction in our system. The xanthine-xanthine oxidase reaction decreased intravesicular-free calcium. The diminished level of intravesicular-free calcium, which was reflected by the decreased steady-state calcium load induced by oxygen-free radicals, was prevented by specific closure of the SR calcium release channel by ryanodine under established optimal conditions; under the same conditions, ryanodine also prevented superoxide dismutase-inhibitable reduction of calcium uptake induced by oxygen-free radicals in the presence or absence of oxalate. Ryanodine was without effect on Ca,Mg-ATPase activity by itself and had no effect on any of the changes in calcium permeability mediated by the generation of oxygen-free radicals under the experimental conditions used.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Comparison of ATP binding in the active sites of (Na+ + K(+)-ATPase, Mg(2+)-ATPase and Ca(2+)-ATPase with low affinity to calcium from cardiac sarcolemma.

The chemical composition of the active sites of cardiac sarcolemmal (Na+ + K(+)-ATPase, Mg(2+)-ATPase and Ca(2+)-ATPase has not been determined definitely. The present study deals with investigation of the role of OH group in position two on the ribose moiety of the ATP molecule in its interaction with the specific ATP binding sites on the above ATPases. Experiments with application of ATP and deoxyATP (the OH group in position 2 on the ribose absent revealed that neither Ca(2+)-ATPase nor Mg(2+)-ATPase is able to distinguish between ATP and deoxyATP as substrates). This indicates that the OH group investigated may play a negligible role only in ATP binding and splitting by the latter ATPases. On the contrary, kinetic studies of Na+ + K(+)-ATPase activation by deoxyATP revealed that the latter compound is a considerably less suitable substrate for the enzyme than ATP. Consequently the OH group in position 2 on the ribose moiety proved to be important both for ATP binding in the active site and for proper substrate turnover by (Na+ + K(+)-ATPase interaction of the ATP binding site of heart sarcolemmal ATPases. Results of the experiments showed that Ca(2+)-ATPase and Mg(2+)-ATPase cannot distinguish between ATP and deoxyATP as substrates. Kinetic studies of (Na+ + K+)-ATPase activation by deoxyATP revealed that the latter is a considerably less good substrate for the enzyme than ATP. It means that the OH group in position two on the ribose moiety proved to be important for both binding of ATP in the active site and for proper substrate turnover by (Na+ + K+)-ATPase.

Adenosine Triphosphatases↗
PubMedDate not supplied

Influence of calcium antagonists on heart sarcolemmal (Na+ + K(+)-ATPase.

The effect of calcium entry blocking agents nitrendipine and flunarizine on Mg(2+)-ATPase, (Mg2+ + Na(+)-ATPase, (Na+ + K(+)-ATPase and (Mg2+ + Ca(2+)-ATPase activities was studied. Nitrendipine (1 mumol/l-1) exerted a stimulatory effect on (Mg2+ + Na(+)-ATPase activity. Kinetic analysis of this effect revealed a two-fold rise in Vmax value and lowered Km value for activation of the enzyme by Na+ ions. In concentrations 10(-7) and 10(-5) mol.l-1 flunarizine behaved as a non-specific inhibitor of all sarcolemmal ATPases investigated. Nevertheless, in concentration of 10(-6) mol.l-1 flunarizine inhibited selectively the (Mg2+ + Na(+)-ATPase and (Na+ + K(+)-ATPase activities of myocardial sarcolemma. These observations provided evidence that both flunarizine and nitrendipine, in the concentration 10(-6) mol.l-1, modulate the (Mg2+ + Na(+)-ATPase and (Na+ + K(+)-ATPase activities in cardiac sarcolemma. These side effects of the above drugs particularly that of nitrendipine might have potential physiological relevance.

Adenosine Triphosphatases↗
PubMed1990

[Characteristics of adenosine triphosphatase activity in carp erythrocytes treated with saponin].

The activities of Ca-ATPase and Na,K-ATPase in saponin-treated erythrocytes of man, rat and carp were compared. It was shown that at free calcium concentrations lower than 1 microM the activity of Ca-ATPase in carp erythrocytes was by one order of magnitude lower than in rat erythrocytes and 3-4 times lower than in human red blood cells. At [Ca2+] = 0.4 microM the activities of Na,K-ATPase in all species under study were essentially the same. The increase in Ca2+ concentration up to 1 microM resulted in a 2-5-fold activation of Na,K-ATPase in rat and carp erythrocytes, respectively. In all cases studied a further elevation of free calcium concentration was accompanied by a decline of the Na,K-ATPase activity. It was shown that the Pi content in carp erythrocytes is 5-6 times as high as that in mammalian cells. This circumstance is a considerable obstacle to a detailed analysis of mechanisms of ATPase activity regulation in carp erythrocytes by methods used for determination of inorganic phosphate production.

Animals↗
PubMedDate not supplied

[Indices of structure and function of erythrocyte membranes in rats with vitamin E deficiency and their correction with antioxidant drugs].

It is found that the amount of saturated fatty acids grows, while that of unsaturated--falls in the erythrocyte membranes of rats maintained on a diet without vitamin E. In this case transmembrane calcium transport catalyzed by Ca2(+)-ATPase (EC 3.6.1.3) is not broken and activity of antioxidant enzyme superoxide dismutase (EC 1.15.1.11) in lysate of erythrocytes falls. The found shifts are corrected with administration of antioxidant preparations.

Animals↗
PubMed1990

Enzymatic properties, metal composition and SH-group reactivity of fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle.

Comparison of properties of fragmented sarcoplasmic reticulum samples isolated by several methods is reported. It was found that the protein composition does not differ significantly in samples which were or were not washed with 0.6 M KCl when isolated. In the case of samples washed with KCl solution the Zn concentration, the Ca/Mg ratio (determined from experimental data), acetylcholinesterase and superoxide dismutase activities were higher whereas Ca+Mg-activated ATPase and p-nitrophenylphosphatase activities were lower than those of samples which were not washed with 0.6 M KCl. In the latter samples the amount of SH-groups and the reactivity of fast SH-s are higher in Ca+EGTA containing media than in media containing only EGTA. In contrast in the case of samples washed with KCl solution the results are the opposite. In conclusion, washing of FSR with 0.6 M KCl alters the metal composition, enzymatic properties, SH-group reactivity and as a result of these probably the conformation of the protein samples, as well.

4-Nitrophenylphosphatase↗
PubMed1990

Cardioprotection by beta-blockers: molecular and structural aspects in experimental hypertension.

This paper deals with some changes at the cardiac and aortic levels observed in normotensive rats and in hypertensive rats and turkeys by using two different beta-blockers, namely propranolol and oxprenolol. Chronic treatment with propranolol induced in the heart of normotensive rats a shift in the ventricular myosin pattern toward the "slow" V2 and V3 isoforms which are characterized by a reduced oxygen consumption. Oxprenolol treatment did not modify the blood pressure levels in the renal hypertensive rats nor in the spontaneously hypertensive turkeys. Nevertheless, in both experimental models a substantial modification of the media and intima, respectively, took place. In untreated hypertensive and normal rats the thickness of the aortic media was significantly higher than that of the treated ones, therefore suggesting a direct effect of oxprenolol on the smooth muscle cells of the aortic media. In the spontaneously hypertensive turkeys the atherosclerotic plaques appeared to be more frequent and thicker than those found in the oxprenolol-treated animals. These two experiments demonstrate that beta-blockers can prevent the development of hypertrophy of the media and decrease both the incidence and severity of intimal proliferations independently of blood pressure control. It therefore appears that the well-known myocardial protective effect played by beta-blockers, which mainly consists of a reduced myocardial oxygen consumption, is certainly obtained by reducing blood pressure and heart rate but also by changing the contractile protein pattern. In addition, an indirect myocardial protective effect could be exerted by beta-blockers at the vascular level by preventing medial hypertrophy and the development of atherosclerosis.

Adrenergic beta-Antagonists↗
PubMed1977

Ca2+-activated ATPase of rat liver plasma membrane.

Rat liver plasma membranes hydrolyze ATP in the presence of Ca2+. The rate of hydrolysis is different when Mg2+ions are present in the incubation system. Several parameters differentiate Ca2+-ATPase from Mg2+-ATPase: a) the Km of ATP hydrolysis for Ca2+ (2.25 x 10(-4) M) is lower than for Mg2+ (2.14 x 10(-3) M); b) the shape of the activation curve is hyperbolic in the presence of Ca2+ and sigmoid in the presence of Mg2+; c) Mg2+-ATPase shows two different values of activation energy while Ca2+-ATPase presents only a single value; d) Ca2+-ATPase is inhibited, while Mg2+-ATPase is unaffected by cyclic AMP. Ca2+-ATPase is localized on the plasma membrane and is not inhibited by cysteine. It does not hydrolyze substrates different from nucleotides triphosphate, such as glucose-1-phosphate or alpha-glycero-phosphate. The enzyme is probably related to a mechanism of calcium transport.

Adenosine Triphosphate↗
PubMed1977

Studies on adenosine triphosphatase activity of rat cardiac myosin in isoproterenol-induced cardiac hypertrophy.

Ca2+- and K+ -activated ATPase activity of cardiac myosin from normal and hypertrophied rat hearts was investigated. Cardiac hypertrophy was induced by isoproterenol treatment. A nearly 40% increase in heart mass was seen after seven consecutive days of isoproterenol injection (5 mg/kg) as determined by either heart weight expressed as per cent of body weight or by dry heart weight and total protein content. The measurement of ATP hydrolysis revealed that cardiac myosin from isoproterenol-treated rats had a significant decrease (P less than 0.01) in Ca2+-activated ATPase activity at low ionic strength (0.05 M KCl) in the presence of 5 and 10 mM Ca2+. In contrast, in a high ionic strength medium (0.50 M KCl) the K+- and Ca2+-activated ATPase activity of myosin prepared from hypertrophied myocardium remained unchanged. Comparative analysis of protein present in the light chains of myosin showed no alteration in the proportion of LC1 to LC2 in the myosin from hypertrophied hearts, however, a decrease in the absorption of myosin in the u.v. region was observed. On the basis of our results one can hypothesize that there may be some conformational change in the myosin molecule from hypertrophied myocardium, thereby modifying both Ca2+-sensitivity and ATPase activity at a low KCl concentration.

Animals↗
PubMed1990

Ca2(+)-induced conformational changes and location of Ca2+ transport sites in sarcoplasmic reticulum Ca2(+)-ATPase as detected by the use of proteolytic enzyme (V8).

Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureus produced appreciable amounts of a Ca2(+)-ATPase fragment (p85) in the presence of Ca2+ (E1 conformation of the enzyme), along with many other peptide fragments that were also formed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (E2 conformation). p85 was formed as a carboxyl-terminal cleavage product of Ca2(+)-ATPase by a split of the peptide bond between Glu-231 and Ile-232. Other conformation-dependent V8 splits were localized to the "hinge" region, involved in ATP binding, between the middle and COOH-terminal one-third of the Ca2(+)-ATPase polypeptide chain. Representative split products in this region (p48,p31) were identified as NH2-terminal and COOH-terminal cleavage products of p85. In the membrane p85 probably remains associated with its complementary NH2-terminal fragment(s) and retains the capacity to bind Ca2+ as evidenced by resistance to V8 degradation in Ca2+ and ability to become phosphorylated by ATP. However, the hydrolysis rate of the phosphorylated enzyme is reduced, indicating that peptide cleavage at Glu-231 interferes with Ca2+ transport steps after phosphorylation. Binding of Ca2+ to V8 and tryptic fragments of Ca2(+)-ATPase was studied on the basis of Ca2(+)-induced changes in electrophoretic mobility and 45Ca2+ autoradiography after transfer of peptides to Immobilon membranes. These data indicate binding by the NH2-terminal 1-198 amino acid residues (corresponding to the tryptic A2 fragment) and the COOH-terminal 715-1001 amino acid residues (corresponding to p31). By contrast the central portion of Ca2(+)-ATPase, including the NH2-terminal portion of p85, is devoid of Ca2+ binding. These results question an earlier proposition that Ca2(+)-binding is located to the "stalk" region of Ca2(+)-ATPase (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H.) (1986) Cell 44, 597-607) but are in agreement with recent data obtained by oligonucleotide-directed mutagenesis of Ca2(+)-ATPase (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). These different studies suggest that Ca2+ translocation sites may have an intramembranous location and are formed predominantly by the carboxyl-terminal part of the Ca2(+)-ATPase polypeptide chain.

Animals↗
PubMed1990

Functional reconstitution of the cardiac sarcoplasmic reticulum Ca2(+)-ATPase with phospholamban in phospholipid vesicles.

The Ca2(+)-ATPase in cardiac sarcoplasmic reticulum (SR) is under regulation by phospholamban, an oligomeric proteolipid. To determine the molecular mechanism by which phospholamban regulates the Ca2(+)-ATPase, a reconstitution system was developed, using a freeze-thaw sonication procedure. The best rates of Ca2+ uptake (700 nmol/min/mg reconstituted vesicles compared with 800 nmol/min/mg SR vesicles) were observed when cholate and phosphatidylcholine were used at a ratio of cholate/phosphatidylcholine/Ca2(+)-ATPase of 2:80:1. The EC50 values for Ca2+ were 0.05 microM for both Ca2+ uptake and Ca2(+)-ATPase activity in the reconstituted vesicles compared with 0.63 microM Ca2+ in native SR vesicles. Inclusion of phospholamban in the reconstituted vesicles was associated with a significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0. However, phosphorylation of phospholamban by the catalytic subunit of the cAMP-dependent protein kinase reversed the inhibitory effect on the Ca2+ pump. Similar findings were observed when a peptide, corresponding to amino acids 1-25 of phospholamban, was used. These findings indicate that phospholamban is an inhibitor of the Ca2(+)-ATPase in cardiac SR and phosphorylation of phospholamban relieves this inhibition. The mechanism by which phospholamban inhibits the Ca2+ pump is unknown, but our findings with the synthetic peptide suggest that a direct interaction between the Ca2(+)-ATPase and the hydrophilic portion of phospholamban may be one of the mechanisms for regulation.

Animals↗
PubMed1990

The E2 in equilibrium E1 transition of the Ca2(+)-ATPase from plasma membranes studied by phosphorylation.

The relative abundance of the two conformers (E1 and E2) of the Ca2(+)-ATPase of plasma membranes and the rates of their interconversion were estimated measuring the initial velocity of phosphorylation of the Ca2(+)-ATPase in pig red cell membranes at 37 degrees C. This was based on the hypothesis that only E1 catalyzes phosphorylation from ATP. In the absence of ligands near 90% of the Ca2(+)-ATPase was in the E2 conformation. Ca2+ shifted the equilibrium toward E1. The K0.5 of Ca2+ for this effect was 15 microM, suggesting that it acted at the transport site. The conversion of E2 into E1 was slow (t1/2 = 911 s) while the conversion of E1 into E2 was faster (t1/2 less than or equal to 60 s). Mg2+ accelerated the E2----E1 reaction lowering its t1/2 to 0.25 s. In the presence of 4 mM Ca2+ t1/2 was 7.8 s, as if at this concentration to some extent Ca2+ replaced Mg2+ in accelerating the E2----E1 reaction. Results suggest that in intact membranes at 37 degrees C Ca2+ stabilized E1 and that the Ca2(+)-induced E2----E1 transition was strongly accelerated by Mg2+. Both cations were effective at near physiological concentrations and in the absence of other ligands like ATP or calmodulin that could also modify the reaction. After partial proteolysis with trypsin the Ca2(+)-ATPase behaved during phosphorylation as if it were E1.

Animals↗
PubMed1990

Functional consequences of alterations to polar amino acids located in the transmembrane domain of the Ca2(+)-ATPase of sarcoplasmic reticulum.

Glu309, Glu771, Asn796, Thr799, Asp800, and Glu908 (ligands 1 to 6, respectively) appear to form the high affinity Ca2(+)-binding sites of the Ca2(+)-ATPase. The plasticity of the Ca2(+)-binding sites was tested by separate replacement of each of the ligands with a structurally similar oxygen-containing residue using site-specific mutagenesis. Mutant cDNAs were transfected into COS-1 cells, and ATP-dependent Ca2+ transport or partial reactions were studied in microsomes containing the expressed Ca2(+)-ATPases. In most cases where amino acid substitutions were carried out, the expressed enzymes lacked Ca2+ transport function and Ca2(+)-dependent phosphorylation by ATP. Furthermore, the mutant enzymes were phosphorylated by inorganic phosphate, even in the presence of Ca2+, which inhibits phosphorylation of the wild-type enzyme possessing intact Ca2(+)-binding sites. On mutant, however, containing an isosteric replacement of Glu by Gln at ligand 6, exhibited wild-type levels of Ca2+ transport activity and Ca2+ affinity. Two mutants exhibited properties consistent with a reduction in Ca2+ affinity. In the mutant in which Thr was replaced by Ser at ligand 4, Ca2+ transport activity was 70% of wild-type, while half-maximal activation by Ca2+ occurred at 0.8 microM as compared to 0.3 microM for the wild-type enzyme. In the mutant Glu309----Asp at ligand 1, Ca2+ transport activity was lost, but Ca2(+)-activated phosphorylation by ATP was retained. The concentration of Ca2+ required to activate phosphorylation was increased about 10-fold, however, compared to wild type. These results support our hypothesis that ligands 1 to 6, believed to reside within the transmembrane domain, interact with Ca2+ ions during the transport process. The roles of 12 other oxygen-containing residues and of His278 located in the transmembrane domain were also examined by mutation. Although the oxygen-containing side chains of these residues are potential Ca2+ ligands, their replacement with nonpolar amino acids did not abolish Ca2+ transport function, leading to the conclusion that they are not essential ligands for high affinity Ca2+ binding by the Ca2+ pump.

Amino Acid Sequence↗
PubMed1990

Numerical and areal density estimates of fibre type composition in a skeletal muscle (rat extensor digitorum longus).

The composition of a mixed fast skeletal muscle (rat extensor digitorum longus) was examined to quantify the difference between the relative number of the three major fibre types in a representative muscle and their relative contribution to muscle cross-section, i.e. numerical (NN) and areal (AA) densities, respectively. These two indices clearly differ in their physiological relevance. While the former may be useful in describing hyperplasia, the latter allows for differences in size among fibre types. When estimated as NN, over 20% of fields contained 5-10% SO fibres and less than 5% had 75-80% FG fibres. In contrast, only 2% of fields had an AA of 5-10% for SO fibres while around 30% contained 75-80% FG fibres. The importance of a direct method for estimating AA is emphasised, as an indirect approach may have an error of 20-30% when used for oxidative fibre types. The use of an unbiased sampling regime to minimise error in determining both numerical and areal densities of different fibre types is illustrated.

Animals↗
PubMed1990

Ca2(+)-dependent binding of tamoxifen to calmodulin isolated from bovine brain.

The interaction of the antiestrogen tamoxifen (Tx) with calmodulin (CaM) was investigated by cross-linking between the protein and [3H] tamoxifen aziridine. We observed that CaM binds Tx in a Ca2(+)-dependent manner and that two components are involved in the binding, with apparent dissociation constants (Kd) of about 6 nM and 9 microM. The high affinity binding site has a maximal capacity of 25 pmol/mg protein, whereas the low affinity binding site has a Bmax value of 120 nmol/mg protein. The stimulatory effect of Ca2+ is maximal at the pCa value of 5, and it is noncompetitively inhibited by Mg2+. In the micromolar range, the cation-dependent interaction of Tx with CaM exhibits positive cooperativity (nH = 1.4) and it is specific in the sense that it is inhibited by unlabeled Tx and by the CaM antagonist trifluoperazine. In contrast, no specificity was observed for the Tx binding, which is cation independent. Tx in the nanomolar range forms complexes with CaM which can be visualized by fluorography after electrophoretic separation in a polyacrylamide gel. Furthermore, CaM antagonism of Tx was observed with respect to inhibition of the CaM effect on the RBC membrane (Ca2(+) + Mg2+)-ATPase. The results indicate that Tx may alter Ca2(+)-dependent processes by interacting directly with CaM.

Animals↗