Interaction of spin-labelled phosphatidyl choline with Ca-dependent ATPase from sarcoplasmic reticulum

The interaction between 3-acyl-2-(6-doxylpalmitoyl) phosphatidyl choline used as a hydrophobic spin probe and Ca2+-dependent ATPase from sarcoplasmic reticulum membranes of rabbit and carp white skeletal muscles was studied. The spin label incorporation into ATPase preparations was performed at initial steps of ATPase isolation by incubating reticulum membranes with the spin probe in the presence of cholic acid. A comparison of the molecular mobility of the probe incorporated into ATPase preparations and into liposomes formed from ATPase phospholipids demonstrated that the presence of the protein in the membrane produces the same effect on the probe mobility as does the decrease of temperature by 10-15 degrees C. The molecular mobility of the probe in the ATPase preparation is increased during protein thermal denaturation. The breaks on the Arrhenius plots for the probe molecular mobility are revealed at the same temperatures (25 degrees for rabbit reticulum and 16 degrees for carp reticulum) as those for the ATPase activity.     

Intracellular localization of the caffeine-sensitive form of Ca-dependent ATPase in the sarcoplasmic reticulum

A Ca-selective electrode was used to study the effect of caffeine on different fractions of sarcoplasmic reticulum membranes of rabbit skeletal muscles. Caffeine was found to uncouple Ca2+ transport and ATP hydrolysis in a fraction, which is enriched with fragments of terminal cisterns according to the electron microscopy data. Caffeine does not produce any effect on the light fraction containing no fragments of terminal cisterns. It is concluded that caffeine-sensitive Ca2+-dependent ATPase is localized in terminal cisterns of the sarcoplasmic reticulum.     

Dinitrophenylation of rabbit skeletal sarcoplasmic reticulum ATPase protein

The ATPase (ATP phosphohydrolase (EC 3.6.1.3)) protein of rabbit skeletal sarcoplasmic reticulum rapidly incorporated three mol of 1-fluoro-2,4-dinitrobenzene per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-dependent ATPase activity was inhibited. The dinitrophenyl group was located mainly in the ATPase protein and a small amount of the label was found in the proteolipid component of the ATPase preparation as judged by dissociation experiments in sodium dodecyl sulfate. Cysteine and tyrosine residues were dinitrophenylated in the modified ATPase protein. Thiolysis of the dinitrophenylated ATPase protein with 2-mercaptoethanol under various conditions did not restore the Ca2+-dependent ATPase activity. Solubilization of the ATPase protein with sodium deoxycholate increased the reactivity of the reagent and the Ca2+-dependent ATPase activity was inhibited to a greater extent. Dinitrophenylation of the ATPase protein was Ca2+-dependent; in the presence of high Ca2+ the incorporation increased by 50% and a large decrease in the Ca2+-ATPase activity was noted. The half-maximal changes for the incorporation of the reagent and the inhibition of the Ca2+-ATPase activity occurred at 3--4 microgram Ca2+-concentration, consistent with the binding of Ca2+ to high affinity sites on the ATPase protein. These results indicate that the ATPase protein as a Ca2+-free and a Ca2+-bound conformation. The reagent, 1-fluoro-2,4-dinitrobenzene reacts differentially and thus characterizes these two conformations.     

Calcium release from vesicles of heavy sarcoplasmic reticulum of rabbit skeletal muscles

The release of Ca2+ from vesicles of heavy sarcoplasmic reticulum after its accumulation due to hydrolysis of ATP, GTP, CTP, UTP or ITP has been studied using Antipyrylazo III, a metal-chromic Ca-indicator. All the studied substrates of the Ca-pump provide Ca2+ accumulation inside the heavy sarcoplasmic reticulum vesicles, the spontaneous Ca2+ outflux rate being different for different nucleoside triphosphates. It is only ATP that provides Ca-(caffeine)-induced Ca2+ release, however AMP, ADP, beta, gamma-methylene-ATP induce Ca2+ ejection in the presence of nonadenylic nucleotides. The ruthenium red (10(-7M) inhibits the induced ejection of Ca2+ from vesicles of the heavy sarcoplasmic reticulum, but does not prevent the spontaneous release of Ca2+ in the same concentrations. A conclusion is drawn that besides Ca-channels sensitive to Ca2+ and caffeine in the presence of ATP (or to AMP, ADP, beta, gamma-methylene-ATP in the presence of nonadenylic nucleotides) and possessing high sensitivity to the ruthenium red there is another pathway for Ca2+ in the heavy reticulum membranes along which its spontaneous release occurs after the substrate exhaustion. It is supposed that this release is provided by the presence of the Ca-ATPase protein.     

Damage to the sarcoplasmic reticulum of skeletal muscles in leukemia: role of lipid peroxidation]

Ischemia development was accompanied by inhibition of the enzymatic transport system (ETS) of Ca2+ (reduction of the Ca2+/ATP value and of the Ca2+-dependent ATPase activity), this correlating with the accumulation of primary and secondary molecular products of lipid peroxidation (LPO) in the sarcoplasmic reticulum membranes of the skeletal muscles, in vivo. Administration of antioxidants (2,6-ditretbutyl-4-methylphenol, alpha-tocopherol) prevented the LPO activation in the ischemic muscle and partially protected the ETS of Ca2+ from damage. The blood supply restoration after prolonged ischemia led to further ETS of Ca2+ inhibition against the background of unchanges LPO products level.     

Regulation of Ca2+ release from sarcoplasmic reticulum in skeletal muscles

Ca²⁺ release from skeletal sarcoplasmic reticulum (SR) could be regulated by at least three mechanisms: 1) Ca²⁺, 2) calmodulin, and 3) Ca²⁺/calmodulin-dependent phosphorylation. Bell-shaped Ca²⁺-dependence, of Ca²⁺ release from both actively- and passively-loaded SR vesicles suggest that opening and closing of the Ca²⁺ release channel could be regulated by [Ca²⁺ _o] . The time- and concentration-dependent inhibition of Ca ²⁺ release from skeletal SR by calmodulin was also studied using passively-Ca²⁺ loaded SR vesicles. Up to 50% of Ca ²⁺ release was inhibited by calmodulin (0.01–0.5 µM); this inhibition required 5–15 min preincubation time. The hypothesis that Ca²⁺/calmodulin-dependent phosphorylation of a 60 kDa protein regulates Ca²⁺ release from skeletal SR was tested by stopped-flow fluorometry using passively-Ca²⁺-loaded SR vesicles. Approximately 80% of the initial rates of Ca²⁺-induced Ca²⁺ release was inhibited by the phosphorylation within 2 min of incubation of the SR with Mg·ATP and calmodulin. We identified two types of 60 kDa phosphoproteins in the rabbit skeletal SR, which was distinguished by solubility of the protein in CHAPS. The CHAPS-soluble 60 kDa phosphoprotein was purified by column chromatography on DEAE-Sephacel, heparin-agarose, and hydroxylapatite. Analyses of the purified protein indicate that the CHAPS-soluble 60 kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding isoforms of PGM were cloned and sequenced using synthetic oligonucleotides. Two types of PGM isoforms (Type I and Type 11) were identified. The translated amino acid sequences show that Type II isoform is SR-form. Our results are significant in terms of understanding evidence of an association of glycolytic and glycogenolytic enzymes with SR and a role in the regulation of SR functions. (Mol Cell Biochem 114: 105-108, 1992)     

Enzymatically active Ca2+ ATPase from sarcoplasmic reticulum membranes, solubilized by nonionic detergents. Role of lipid for aggregation of the protein.

The present study provides data on the properties of Ca2+-dependent Atpase of sarcoplasmic reticulum in states intermediary between the fully detergent-solubilized and vesicular form. After solubilization of ATPase vesicles by dodecyloctaoxyethylene glycol monoether (C12E8), the protein is mainly present as a monomer exhibiting enzymatic activity. Gel chromatography in presence or absence of Tween 80 gives rise to formation of oligomers of various size and smaller amounts of monomeric ATPase. Only the oligomeric species retain enzymatic activity (half-life, 3 to 4 days), while the gel chromatographic monomer is enzymatically inactive. Teteramers or trimers of ATPase, containing approximately 22 mol of phospholipid/mol of ATPase, are the smallest enzymatically active units after gel chromatography. Formation of larger sized particles and vesicles of ATPase appears to depend on the presence of sufficient lipid to make a cohesion between the tetrameric or trimeric units. The protein appears to be partially deaggregated by a relatively high Tween 80 concentration in the eluant (0.5 mg/ml) and under these conditions, phospholipid binding is reduced to a low level (approximately 11 mol/mol of protein). The data indicate that any bonds between ATPase polypeptide chains are easily disrupted by detergent and that lipid also may play a role in mediating contact between individual polypeptide chains in the tetrameric or trimeric units. Phospholipid analysis and exchange experiments indicate that the phospholipid left on ATPase after solubilization has a similar composition to that of the whole membrane. The binding of Tween 80 by soluble ATPase above the critical micellar concentration is 0.23 to 0.29 g/g of protein. The inactive monomer of ATPase binds phospholipid and Tween 80 to about the same extent, but has a slightly different circular dichroism spectrum, than oligomeric ATPase.     

Effect of the lipid environment on protein motion and enzymatic activity of sarcoplasmic reticulum calcium ATPase.

In order to investigate the roles of the physical states of phospholipid and protein in the enzymatic behavior of the Ca2+ -ATPase from sarcoplasmic reticulum, we have modified the lipid phase of the enzyme, observed the effects on the enzymatic activity at low temperatures, and correlated these effects with spectroscopic measurements of the rotational motions of both the lipid and protein components. Replacement of the native lipids with dipalmitoyl phosphatidylcholine inhibits ATPase activity and decreases both lipid fluidity, as monitored by EPR spectroscopy on a stearic acid spin label, and protein rotational mobility, as monitored by saturation transfer EPR spectroscopy on the covalently spin-labeled enzyme. Solubilization of the lipid-replaced enzyme with Triton X-100 reverses all three of these effects. Ten millimolar CaCl2 added either to the enzyme associated with the endogenous lipids or to the Triton X-100 soulbilized enzyme inhibits both ATPase activity and protein rotational mobility but has no detectable effect on the lipid mobility. These results are consistent with the proposal that both lipid fluidity and protein rotational mobility are essential for enzymatic activity.     

The influence of phenothiazines on the sarcoplasmic reticulum Ca-ATPase from skeletal and cardiac muscles

Phenothiazines--trifluoperazine, chloropromazine and ethmozine-- inhibit the sarcoplasmic reticulum Ca-ATPase from skeletal and cardiac muscles of the rabbit. The inhibition constants for both preparations are of the same order of magnitude. The experimental data suggest that the effect of phenothiazine on the sarcoplasmic reticulum Ca-ATPase is not mediated by CaM, but is directed toward the enzyme molecule.     

Endogenous phosphorylation of sarcoplasmic reticulum fragments of rabbit fast skeletal muscles

The purified membrane fragments of sarcoplasmic reticulum (SR) of rabbit fast skeletal muscles were found to incorporate 32P from[gamma-32P]ATP in endogenous membrane substrates and in histone H1. The existence of membrane-bound protein kinase of SR was demonstrated by steady state binding of [3H]-cAMP to the SR membranes. The constant of [3H]cAMP binding to the membranes is 2.5 +/- 0.003 x 10(6) M-1, the number of binding sites is 6.1 +/- 0.8 pmol per 1 mg of protein. The endogenous phosphorylation of SR components was inhibited by cAMP and cGMP at concentrations of 10(-7)-10(-6) and depended on Mg2+ and Ca2+. The thermostable protein inhibitor of cAMP-dependent protein kinase inhibited the endogenous phosphorylation of SR membranes by 30-40%. The protein phosphoproduct of SR membranes revealed the properties of a phosphoester. The membrane-bound protein kinase was active towards the exogenous substrate--histone H1. Phosphorylation in the presence of histones was independent of cyclic nucleotides, Mg2+ and Ca2+. Fractionation of 32P-labelled solubilized membranes in polyacrylamide gel in the presence of Na-SDS showed that the radioactivity is bound to protein zones with molecular weights of 95 000 and 6000.     

Non-proteolytic functions of calpain-3 in sarcoplasmic reticulum in skeletal muscles

Mutations in CAPN3/Capn3, which codes for skeletal muscle-specific calpain-3/p94 protease, are responsible for limb-girdle muscular dystrophy type 2A. Using “knock-in” (referred to as Capn3^CS/CS) mice, in which the endogenous calpain-3 is replaced with a mutant calpain-3:C129S, which is a proteolytically inactive but structurally intact calpain-3, we demonstrated in our previous studies that loss of calpain-3 protease activity causes muscular dystrophy [Ojima, K. et al. (2010) J. Clin. Invest. 120, 2672-2683]. However, compared to Capn3-null (Capn3^−/−) mice, Capn3^CS/CS mice showed less severe dystrophic symptoms. This suggests that calpain-3 also has a non-proteolytic function. This study aimed to elucidate the non-proteolytic functions of calpain-3 through comparison of Capn3^CS/CS mice with Capn3^−/− mice. We found that calpain-3 is a component of the sarcoplasmic reticulum (SR), and that calpain-3 interacts with, but does not proteolyze, typical SR components such as ryanodine receptor and calsequestrin. Furthermore, Capn3^CS/CS mice showed that the nonenzymatic role of calpain-3 is required for proper Ca²⁺ efflux from the SR to cytosol during muscle contraction. These results indicate that calpain-3 functions as a nonenzymatic element for the Ca²⁺ efflux machinery in the SR, rather than as a protease. Thus, defects in the nonenzymatic function of calpain-3 must also be involved in the pathogenesis of limb-girdle muscular dystrophy type 2A.     

Effect of phenothiazines on Ca-ATPase in the sarcoplasmic reticulum of rabbit skeletal muscles

The phenothiazines trifluoroperazine , chlorpromazine and etmozine inhibit Ca-ATPase of the sarcoplasmic reticulum of rabbit skeletal muscles. The inhibitory action decreases in the order of trifluoroperazine greater than chlorpromazine greater than etmozine . The data are provided, indicating that the inhibitory effects of the phenothiazines on Ca-ATPase of the reticulum of the skeletal muscles are not mediated via calmodulin.     

Ca-release by phosphoinositides from sarcoplasmic reticulum of frog skeletal muscle

To clarify the biological role of phosphoinositides including inositol trisphosphate (IP3) in the skeletal muscle, we examined the Ca-releasing action on the heavy fraction of sarcoplasmic reticulum (HFSR) from bullfrog skeletal muscle of IP3, phosphatidylinositol monophosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and glycerophosphoinositol 4,5-bisphosphate (GPIP2). Only PIP2 caused dose-dependent Ca release. IP3 (up to 55 microM), PIP (up to 37 microM), and GPIP2 (up to 33 microM) were ineffective. The PIP2-induced Ca release is due to the direct action of PIP2, but not its metabolite(s). The properties of the PIP2-induced Ca release are unique and cannot be accounted for by the Ca release mechanisms already reported, such as Ca2+-induced, ionic substitution-induced, or IP3-induced Ca release. The rate of the PIP2-induced Ca release, however, is so slow that it may have no physiological relevance unless stimulating factors or agents exist.     

Pathway for ATP synthesis by sarcoplasmic reticulum ATPase

Millisecond mixing and quenching experiments were performed in order to study the rate of phosphorylation by P_i of the Ca²⁺-dependent ATPase of sarcoplasmic reticulum vesicles. A rapid phosphoenzyme formation was observed when the vesicles were preincubated in the absence of Ca²⁺ prior to the addition of P_i and Mg²⁺ to the medium, the half-time being in the range of 6 to 10 ms. A lag phase and a 5- to 10-fold slower rate of phosphoenzyme formation were observed when the enzyme was preincubated with Ca²⁺ prior to the addition to the reaction mixture of P_i, Mg²⁺, and an excess of ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid. The rate of phosphoenzyme hydrolysis was measured either by the addition of Ca²⁺ or, in the absence of Ca²⁺, by tracing the hydrolysis of radioactive phosphoenzyme upon the addition of nonradioactive P_i. In the presence of Ca²⁺, the rate of phosphoenzyme hydrolysis was found to be one order of magnitude slower than the rate of hydrolysis measured in the absence of Ca²⁺. Different rates of phosphoenzyme formation and cleavage were found depending on whether sarcoplasmic reticulum vesicles or purified Ca²⁺-dependent ATPase were used. A transient phosphorylation by P_i was observed when the enzyme was preincubated in the absence of Ca²⁺ and then added to a medium containing P_i, Mg²⁺, and excess of Ca²⁺. The enzyme was phosphorylated during the initial 100 ms, the phosphoenzyme formed being slowly hydrolyzed in the subsequent incubation intervals. In these conditions ATP synthesis was observed if ADP was added to the mixture 100 ms after starting the reaction. No transient phosphorylation by P_i was observed when the enzyme was preincubated with Ca²⁺. Synthesis of a small but significant amount of ATP was observed when the enzyme was preincubated in the absence of Ca²⁺ and then added to a medium containing P_i, ADP, Mg²⁺, and 20 mm CaCl₂. This was not observed when the enzyme was preincubated in the presence of Ca²⁺.