Ca<Superscript>2+</Superscript>, Mg<Superscript>2+</Superscript>-dependent DNase Involvement in Apoptotic Effects in Spermatozoa of Sea Urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> Induced by Two-Headed Sphingolipid Rhizochalin

Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca²⁺, Mg²⁺-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn²⁺ blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.     

Calcium uptake and Ca2+-ATPase activity in goat spermatozoa membrane vesicles do not require Mg2+

Microsomal membrane vesicles isolated from goat spermatozoa contain Ca²⁺-ATPase, and exhibit Ca²⁺ transport activities that do not require exogenous Mg²⁺ .The enzyme activity is inhibited by calcium-channel inhibitors,e.g. verapamil and diltiazem, like the well known Ca²⁺ , Mg²⁺-ATPase. The uptake of calcium is ATP (energy)-dependent and the accumulated Ca²⁺ can be completely released by the Ca²⁺ ionophore A23187, suggesting that a significant fraction of the vesicles are oriented inside out     

A comparative study of the rat heart sarcolemmal Ca2+-dependent ATPase and myosin ATPase

In order to gain some information regarding Ca²⁺-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca²⁺-ATPase activity of myosin purified from rat heart. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by Ca²⁺ but the maximal activation of Ca²⁺-dependent ATPase required 4 mM Ca²⁺ whereas that of myosin ATPase required 10 mM Ca²⁺. These ATPases were also activated by other divalent cations in the order of Ca²⁺ > Mn²⁺ > Sr²⁺ > Br²⁺ > Mg²⁺; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca²⁺-dependent ATPase was not activated by actin. The pH optima of Ca²⁺-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na⁺ markedly inhibited Ca²⁺-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca²⁺-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca²⁺-dependent ATPase. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH₄-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca²⁺-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca²⁺ and 3 to 4 mM ATP like sarcolemmal Ca²⁺-dependent ATPase. K⁺ stimulated both ATPase activities in the absence of Ca²⁺ and inhibited in the presence of Ca²⁺. Both enzymes were inhibited by Na⁺, Mg²⁺, La³⁺, and azide similarly. However, Ca²⁺ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca²⁺ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca²⁺-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca²⁺-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca²⁺-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca²⁺-ATPase. These observations suggest that Ca²⁺-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils.     

Disulfiram lowers Ca2+, Mg2+-ATPase activity of rat brain synaptosomes

The chronic administration of disulfiram (DS) to rats resulted in significant decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase activity. In vitro studies indicated that DS (ID₅₀=20 M) produced a dose-dependent inhibition of Ca²⁺, Mg²⁺-ATPase. However, diethyldithio-carbamate, a metabolite of DS, failed to modify Ca²⁺, Mg²⁺-ATPase activity, implying that the decrease in ATPase activity in DS administered rats was due to the effect of parent compound. The DS-mediated inhibition (48%) of ATPase activity was comparable with a similar degree of inhibition (49%) achieved by treating the synaptosomal membranes with N-ethylmaleimide (ID₅₀=20 M) in vitro. Furthermore, the inhibition by DS was neither altered by washing the membranes with EGTA nor reversed by treatment with sulfhydryl reagents such as GSH or dithiothreitol. About 74% and 68% decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase specific activity was observed when treated with DS (30 M) and EGTA (100 M) respectively. The remaining 25–30% of total activity is suggested to be of Mg²⁺-dependent ATPase activity. This indicates that both these drugs may act on a common target, calmodulin component that represents 70–75% of total Ca²⁺, Mg²⁺-ATPase activity. Therefore, DS-mediated modulation of synaptosomal Ca²⁺, Mg²⁺-ATPase activity could affect its function of maintaining intracellular Ca²⁺ concentration. This could contribute to the deleterious effects on CNS.     

Relationship between Ca2+-transport and ATP hydrolytic activities in guinea-pig pancreatic acinar plasma membranes

Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca²⁺-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca²⁺-transporter showed high affinity for Ca²⁺ (K_Ca ²⁺ = 0.04 ± 0.01 M), an apparent requirement for Mg²⁺ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca²⁺ or Mg²⁺ but showed a low affinity for these cations. At low concentrations, Mg²⁺ appeared to inhibit the Ca²⁺-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg²⁺ concentration (0.5–1 mM), a high affinity Ca²⁺-dependent ATPase activity was observed (K_Ca ²⁺ = 0.08 ± 0.02 M). The hydrolytic activity showed little specificity towards ATP. Neither the Ca²⁺-transport nor high affinity Ca²⁺-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca²⁺ (or Mg⁺)-ATPase activity, the presence of both a high affinity Ca²⁺-pump and high affinity Ca²⁺-dependent ATPase. However, the high affinity Ca²⁺-ATPase activity does not appear to be the biochemical expression of the Ca²⁺-pump.Abbreviations Ca²⁺-ATPase calcium-activated, magnesium-dependent adenosine triphosphatase - CaM calmodulin - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate - EDTA ethylene-diaminetetraacetate - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate - NADPH reduced form of nicotinamide adenine dinucleotide phosphate     

Purification and characterization of a Ca2+/Mg2+ ecto-ATPase from rat heart sarcolemma

The Ca²⁺/Mg²⁺ ATPase of the rat heart sarcolemmal particles was solublized with Triton X-100 after treating the membranes with trypsin and purified by high speed centrifugation, ammonium sulfate fractionation, hydrophobic chromatography and gel filtration. The purified enzyme was seen as a single protein band in nondenaturing polyacrylamide gel electrophoresis and its molecular weight by gel filtration was found to be about 240000. The enzyme utilized Ca-ATP or Mg-ATP as a substrate with high affinity sites (Km = 0.12 – 0.16 mM) and low affinity sites (Km = 1 mM). The enzyme also utilized CTP, GTP, ITP, UTP and ADP as substrates but at a lower rate in comparison to ATP. The enzyme was activated by Ca²⁺ (Ka = 0.4 mM) and Mg²⁺ (Ka = 0.2 mM) as well as by other cations in the order Ca^2– > Mg²⁺ > Mn²⁺ > Sr²⁺ > Ba²⁺ > Ni²⁺ > Cu²⁺. The ATPase activity in the presence of Ca²⁺ was markedly inhibited by Mg²⁺, Mn²⁺, Ni²⁺ and Cu²⁺ whereas the monovalent cations such as Na⁺ and K⁺ were without effect. The enzyme did not exhibit Ca²⁺ stimulated Mg²⁺ dependent ATPase activity and was insensitive to calmodulin, ouabain, verapamil, D-600, oligomycin, azide and vanadate. Optimum pH for Ca²⁺ or Mg²⁺ ATPase activity was 8.5 – 9.0. In view of the possible ectoenzyme nature of the ATPase, its role in adenine nucleotide and Ca²⁺ metabolism in the myocardium is discussed.     

Monoclonal antibodies to the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum identify polymorphic forms of the enzyme and indicate the presence in the enzyme of a classical high-affinity Ca2+ binding site

In order to determine whether polymorphic forms of the Ca²⁺ + Mg²⁺-dependent ATPase exist, we have examined the cross-reactivity of five monoclonal antibodies prepared against the rabbit skeletal muscle sarcoplasmic reticulum enzyme with proteins from microsomal fractions isolated from a variety of muscle and nonmuscle tissues. All of the monoclonal antibodies cross-reacted in immunoblots against rat skeletal muscle Ca²⁺ + Mg²⁺-dependent ATPase but they cross-reacted differentially with the enzyme from chicken skeletal muscle. No cross-reactivity was observed with the Ca²⁺ + Mg²⁺-dependent ATPase of lobster skeletal muscle. The pattern of antibody cross-reactivity with a 100,000 dalton protein from sarcoplasmic reticulum and microsomes isolated from various muscle and nonmuscle tissues of rabbit demonstrated the presence of common epitopes in multiple polymorphic forms of the Ca²⁺ + Mg²⁺-dependent ATPase. One of the monoclonal antibodies prepared against the purified Ca²⁺ + Mg²⁺-dependent ATPase of rabbit skeletal muscle sarcoplasmic reticulum was found to cross-react with calsequestrin and with a series of other Ca²⁺-binding proteins and their proteolytic fragments. Its cross-reactivity was enhanced in the presence of EGTA and diminished in the presence of Ca²⁺. Its lack of cross-reactivity with proteins that do not bind Ca²⁺ suggests that it has specificity for antigenic determinants that make up the Ca²⁺-binding sites in several Ca²⁺-binding proteins including the Ca²⁺ + Mg²⁺-dependent ATPase.This paper is dedicated to the memory of Dr. David E. Green.     

Identification and characterization of a Mg+2-dependent and an independent Ca+2-ATPase in microsomal membranes of rat testis

Rat testicular microsomal membrane fraction contains both Mg⁺²-dependent and Mg⁺²-independent Ca⁺²-ATPase activity. The latter activity is about two times higher than the former. Calcium ion required for maximum activation of Mg⁺²-independent Ca⁺²-ATPase in 3.0 mM, whereas for the dependent one it is 2.5 mM. Both the enzymes are resistant to cold shock upto seven days. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities, respectively. The pH optima for dependent one is 7.5, whereas for the independent one it is 8.5. Temperature optima for the former is 37°C and for latter one it is 40°C. Among all the nuclestides tested, ATP is found to be the best substrate for both the enzymes. The optimum concentration of ATP for dependent and independent enzyme activities are 3.0 mM and 1.5 mM respectively. Divalent metal ions like Zn⁺², Ba⁺² and Mn⁺² have been found to inhibit Mg⁺²-dependent Ca⁺²-ATPase activity whereas Mg⁺²-independent Ca⁺²-ATPase activity is inhibited by the divalent ions except zinc which is found to stimulate the enzyme activity. Both the enzymes are inhibited by vanadata, EDTA and EGTA. I₅₀, for vanadate is 0.05 and 0.125 mM for dependent and independent activities, respectively. Sulfhydral groups modifying agents e.g., NEM, DTNB and chlorpromazine are found to affect the enzyme activities in different ways. Thus NEM and chlorpromazine are found to inhibit and DTNB stimulate the enzyme activities in both the cases.     

Opiates inhibit calmodulin activation of a high-affinity Ca2+-stimulated Mg2+-dependent ATPase in synaptic membranes

A high affinity Ca²⁺/Mg²⁺ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca²⁺ (1 M) believed to approximate the range of Ca²⁺ in the synaptosomal cytosol (0.1 to 5.0 M). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca²⁺-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca²⁺-stimulated ATP hydrolysis was characterized by a reduction inV _max for Ca²⁺. Levorphanol pretreatment reduced the Hill coefficient (H_N) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca²⁺ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca²⁺/Mg²⁺ ATPase in synaptic membranes. Levorphanol (10 M), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca²⁺-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca²⁺-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inV _max for both Ca²⁺ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca²⁺-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca²⁺/Mg²⁺ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca²⁺ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca²⁺ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca²⁺ following inhibition of Ca²⁺/Mg²⁺ ATPase may underlie some of the pharmacological effects of opiate drugs.     

Phosphorylation and dephosphorylation of Mg2+-independent Ca2+-ATPase from goat spermatozoa

We reported previously that a Ca²⁺-ATPase in rat testes and goat spermatozoa could be activated by Ca²⁺ alone without Mg²⁺, though it has a lot of similarities with the well known Ca²⁺, Mg²⁺-ATPase. Recently, we were successful in isolating the phosphorylated intermediate of the former enzyme under control conditions i.e., in the presence of low concentration of Ca²⁺ and at low temperature. Increase of the concentration of Ca²⁺ and/or temperature lead to dephosphorylation. Based on our observations, we proposed a reaction scheme comparable to that of Ca²⁺, Mg²⁺-ATPase. The findings strengthened our previous report that Mg²⁺-independent Ca²⁺-ATPase is involved in Ca²⁺ transport and Ca²⁺ uptake like Ca²⁺, Mg²⁺-ATPase.     

Biophysical characterization of rat cardiac Ca2+/Mg2+ ecto-ATPase (Myoglein)

Sarcolemmal Ca²⁺/Mg²⁺ ecto-ATPase (Myoglein; MW 180 kD) is a membrane bound enzyme which requires a millimolar concentration of either Ca²⁺ or Mg²⁺ for maximal hydrolysis of ATP. The isoelectric point (pI) of the cardiac ecto-ATPase was 5.7. The purified Ca²⁺/Mg²⁺ ecto-ATPase from the rat heart sarcolemmal appeared as a single band with MW 90 kD in the SDS-PAGE. In order to understand the nature of this enzyme, the 90 kD band in the SDS-PAGE was electroeluted; the analysis of the eluate showed 2 prominent bands with MW 90 and 85 kD. The presence of 2 bands was further confirmed by gradient gel (10-20%) electrophoresis in 0.375 M Tris-HCl buffer, pH 8.8. Analysis of the purified Ca²⁺/Mg²⁺ ecto-ATPase as well as the electroeluted protein in a non-equilibrium linear two dimensional electrophoresis (Ampholyte pI 3.0-10.0) also showed two distinct bands. Mass spectroscopic analysis of the enzyme using different matrix combinations revealed the presence of multi-components indicating microheterogeneity in the protein structure. Treatment of the ecto-ATPase with DL-dithiothreitol did not alter the pattern of mass spectroscopic analysis and this indicated that the microheterogeneity may be due to some posttranslational modifications. It is concluded that rat cardiac Ca²⁺/Mg²⁺ ecto-ATPase is an acidic protein having two subunits. Furthermore, the enzyme shows microheterogeneity in its molecular structure.     

Endogenous,Ca2+-dependent cysteine-protease cleaves specifically the ryanodine receptor/Ca2+ release channel in skeletal muscle

The association of an endogenous, Ca²⁺-dependent cysteine-protease with the junctional sarcoplasmic reticulum (SR) is demonstrated. The activity of this protease is strongly stimulated by dithiothreitol (DTT), cysteine and β-mercaptoethanol, and is inhibited by iodoacetamide, mercuric chloride and leupeptin, but not by PMSF. The activity of this thiol-protease is dependent on Ca²⁺ with half-maximal activity obtained at 0.1 μm and maximal activity at 10 μm. Mg²⁺ is also an activator of this enzyme (CI₅₀=22 μm). These observations, together with the neutral pH optima and inhibition by the calpain I inhibitor, suggest that this enzyme is of calpain I type. This protease specifically cleaves the ryanodine receptor monomer (510 kD) at one site to produce two fragments with apparent molecular masses of 375 and 150 kD. The proteolytic fragments remain associated as shown by purification of the cleaved ryanodine receptor. The calpain binding site is identified as a PEST (proline, glutamic acid, serine, threonine-rich) region in the amino acid sequence GTPGGTPQPGVE, at positions 1356–1367 of the RyR and the cleavage site, the calmodulin binding site, at residues 1383–1400. The RyR cleavage by the Ca²⁺-dependent thiol-protease is prevented in the presence of ATP (1–5 mm) and by high NaCl concentrations. This cleavage of the RyR has no effect on ryanodine binding activity but stimulates Ca²⁺ efflux. A possible involvement of this specific cleavage of the RyR/Ca²⁺ release channel in the control of calpain activity is discussed.     

Sarcolemmal Na+-Ca2+ exchange and Ca2+-pump activities in cardiomyopathies due to intracellular Ca2+-overload

In order to identify defects in Na⁺-Ca²⁺ exchange and Ca²⁺-pump systems in cardiomyopathic hearts, the activities of sarcolemmal Na⁺-dependent Ca²⁺ uptake, Na⁺-induced Ca²⁺ release, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase were examined by employing cardiomyopathic hamsters (UM-X7.1) and catecholamine-induced cardiomyopathy produced by injecting isoproterenol into rats. The rates of Na⁺-dependent Ca²⁺ uptake, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities of sarcolemmal vesicles from genetically-linked cardiomyopathic as well as catecholamine-induced cardiomyopathic hearts were decreased without any changes in Na⁺-induced Ca²⁺-release. Similar results were obtained in Ca²⁺-paradox when isolated rat hearts were perfused for 5 min with a medium containing 1.25 mM Ca²⁺ following a 5 min perfusion with Ca²⁺-free medium. Although a 2 min reperfusion of the Ca²⁺-free perfused hearts depressed sarcolemmal Ca²⁺-pump activities without any changes in Na⁺-induced Ca²⁺-release, Na⁺-dependent Ca²⁺ uptake was increased. These results indicate that alterations in the sarcolemmal Ca²⁺-efflux mechanisms may play an important role in cardiomyopathies associated with the development of intracellular Ca²⁺ overload.     

Asymmetric effects of divalent cations and protons on active Ca2+ efflux and Ca2+-ATPase in intact red blood cells

Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca²⁺ transport have been examined in intact human red blood cells. Active Ca²⁺ efflux was determined from the initial rate of⁴⁵Ca²⁺ loss after CoCl₂ was added to block Ca²⁺ loading via the ionophore A23187. Ca²⁺-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca²⁺ in the presence of A23187. The apparent Ca affinity of active Ca²⁺ efflux (K _0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca²⁺-ATPase assay (K _0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca²⁺ efflux and Ca²⁺-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg²⁺-depleted cells, and completely restored by reintroduction of intracellular Mg²⁺. Active Ca²⁺ efflux was inhibited almost completely by raising external CaCl₂ (but not MgCl₂) to 20mm, probably by interaction of Ca²⁺ at the externally oriented E₂P conformation of the pump. Cd²⁺ was more potent than Ca²⁺ in this inhibition, while Mn²⁺ was less potent and 10mm Ba²⁺ was without effect. A Ca²⁺: proton exchange mechanism for active Ca²⁺ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca²⁺ efflux at least twofold above the efflux rate at pH 7.8 Ca²⁺ transport was not affected by decreasing the membrane potential across the red cell.     

Interaction of Sr2+ with Ca2+-induced Ca2+ release in mitochondria

Respiring rat liver mitochondria are known to spontaneously release the Ca²⁺ taken up when they have accumulated Ca²⁺ over a certain threshold, while Sr²⁺ and Mn²⁺ are well tolerated and retained. We have studied the interaction of Sr²⁺ with Ca²⁺ release. When Sr²⁺ was added to respiring mitochondria simultaneously with or soon after the addition of Ca²⁺, the release was potently inhibited or reversed. On the other hand, when Sr²⁺ was added before Ca²⁺, the release was stimulated. Ca²⁺-induced mitochondrial damage and release of accumulated Ca²⁺ is generally believed to be due to activation of mitochondrial phospholipase A (EC 3.1.1.4.) by Ca²⁺. However, isolated mitochondrial phospholipase A activity was little if at all inhibited by Sr²⁺. The Ca²⁺ -release may thus be triggered by some Ca²⁺ -dependent function other than phospholipase.     

Immunolocalization of the sarcolemmal Ca2+/Mg2+ ecto-ATPase (myoglein) in rat myocardium

Cardiac plasma membrane Ca²⁺/Mg²⁺ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca²⁺ or Mg²⁺ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca²⁺/Mg²⁺ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca²⁺/Mg²⁺ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or sarcolemmal Ca²⁺-pump ATPase. On the other hand, the cardiac Ca²⁺/Mg²⁺ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or plasma membrane Ca²⁺-pump ATPase. Furthermore, the immune serum inhibited the Ca²⁺/Mg²⁺ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca²⁺/Mg²⁺ ecto-ATPase antibodies indicated the localization of Ca²⁺/Mg²⁺ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca²⁺/Mg²⁺ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca²⁺/Mg²⁺ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle.     

Characteristics of Ca2+-stimulated ATPase in rat heart sarcolemma in the presence of dithiothreitol and alamethicin

We have studied the activities of Ca²⁺-stimulated ATPase in rat heart sarcolemma upon modulating the redox state of membrane thiol groups with dithiothreitol (DTT). The suitability of alamethicin to unmask the latent activity of this enzyme was also investigated. The Ca²⁺-stimulated ATPase in sarcolemma exhibited two activation sites — one with low affinity (Km = 0.70 ± 0.2 mM; Vmax = 10.0 ± 2.2 mol Pi/mg/h) and the other with high affinity (Km = 0.16 ± 0.7 mM; Vmax = 4.6 ± 0.8 mol Pi/mg/h) for Mg²⁺ATP. Alamethicin at a ratio of 1:1 with the sarcolemmal protein caused a 3-fold activation of Ca²⁺-stimulated ATPase without affecting its sensitivity to Ca²⁺ or Mg²⁺ATP. Treatment of sarcolemma with deoxycholate or sodium dodecyl sulfate resulted in a total loss of the enzyme activity; high concentrations of alamethicin also showed a detergent-like action on the sarcolemmal vesicles. DTT at 5–10 mM concentrations caused a 4–5 fold activation of Ca²⁺-stimulated ATPase in sarcolemma and this effect was observed to be dependent on the concentration of Mg²⁺ATP. DTT increased the affinity of the enzyme to Mg²⁺ATP at the high affinity site and enhanced the Vmax at the low affinity site in addition to increasing the sensitivity of Ca²⁺-stimulated ATPase to Ca²⁺. DTT protected the Ca²⁺-stimulated ATPase against deterioration by detergents and restored the enzyme activity after treatment with N-ethylmaleimide. The mechanism of action of DTT on Ca²⁺-stimulated ATPase may involve the reduction of essential thiols at the active site of the enzyme or its interaction with specific DTT-dependent inhibitor protein. No changes in the sensitivity of sarcolemmal Ca²⁺-stimulated ATPase to orthovanadate was evident in the absence or presence of DTT and alamethicin. The results suggest the use of both DTT and alamethicin for the determination of Ca²⁺-stimulated ATPase activity in sarcolemmal preparations.     

Role of Ca2+, Mg2+ and K+ ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture

We have addressed the possibility that Ca²⁺, Mg²⁺ and K⁺ ions play a central role in governing the morphological and biochemical changes attributed to apoptotic cell death. By removing Ca²⁺, Mg²⁺ or K⁺ ions from the cell culture medium we were able to assess the contribution of each ion to hybridoma cell growth and viability. The differences were explained in terms of a possible reduction in their respective intracellular levels. From several lines of evidence, the deprivation of K⁺ ions was the most detrimental to cellular growth and viability and induced significant levels of early apoptotic cells. Another effect of this deprivation was to weaken the plasma membranes without causing membrane breakdown; exposure to high agitation rates confirmed fragility of the cell membranes. Removal of Mg²⁺ caused a reduction in the levels of early apoptotic cells and predisposed cells to high levels of primary necrotic death. The lower levels of apoptotic cells failed to demonstrate the classic nuclear morphology associated with apoptosis, while retaining other apoptotic features. These results highlighted the importance of utilizing several assays for the determination of apoptosis. The absence of Ca²⁺ appeared to be the mildest insult, but its deprivation did accelerate a significant decline in culture by increasing apoptotic death. Hybridoma cells overexpressing the apoptotic suppresser gene bcl-2 were protected from the predominantly necrosis inducing effects of Mg²⁺ ion deprivation and apoptosis inducing effects of Ca²⁺ ion deprivation. However, apoptosis was not as effectively suppressed in bcl-2 cells responding to incubation in K⁺ free medium. The inclusion of bcl-2 activity in the mechanisms of Ca²⁺ Mg²⁺ or K⁺ deprivation induced cell death emphasizes a close relationship between ionic dissipation and the apoptotic process.     

Uncoupling of Ca2+ transport from ATP hydrolysis activity of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

Summary In reconstituted rabbit skeletal muscle (Ca²⁺ + Mg²⁺)-ATPase proteoliposomes, Ca²⁺-uptake is decreased by more than 90% with T₂ cleavage (Arg-198). However, no difference in the ATP dependence of hydrolysis activity is seen between SR and trypsin-treated SR. A large decrease in E-P formation and hydrolysis activity of the enzyme appear only at T₃ cleavage, which represents the cleavage of A₁ fragment to A_1a + A_1b forms. The disappearance of hydrolysis activity due to digestion is prior to the disappearance of E-P formation. No significant difference is found in the passive Ca²⁺ efflux between control SR and tryptically digested SR in the absence of Mg⁺ ruthenium red or in the presence of ATP. However, the passive Ca²⁺ efflux rate for tryptically digested SR is much larger than control SR in the presence of Mg²⁺ + ruthenium red. These results show that the Ca²⁺ channel cannot be closed after trypsin digestion of SR membranes by the presence of the Ca²⁺ channel inhibitors, Mg²⁺ and ruthenium red. In the reconstituted ATPase proteoliposomes, the Ca²⁺ efflux rates are the same regardless of digestion (T₂); also, efflux is not affected by the presence or absence of Mg²⁺ + ruthenium red. These results indicate that T₂ cleavage causes uncoupling of the Ca²⁺-pump from ATP hydrolytic activity.A theoretical model is developed in order to fit the extent of tryptic digestion of the A fragment of the (Ca²⁺ + Mg²⁺)-ATPase polypeptide with the loss of Ca²⁺-transport. Fits of the theoretical equations to the data are consistent with that Ca²⁺-transport system appears to require a dimer of the polypeptide (Ca²⁺ + Mg²⁺)-ATPase.     

Mg2+ release coupled to Ca2+ uptake: A novel Ca2+ accumulation mechanism in rat liver

Isolated hepatocytes release 2–3 nmol Mg²⁺/mg protein or ~10% of the total cellular Mg²⁺ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During Mg²⁺ release, a quantitatively similar amount of Ca²⁺ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg²⁺:1Ca²⁺. Calcium induced Mg²⁺ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg²⁺:1Ca²⁺ exchange ratio. The uptake of Ca²⁺ for the release of Mg²⁺ occurs in the absence of significant changes in Δψ as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium (TPP⁺) electrode or ³H-TPP⁺. Collapsing the Δψ by high concentrations of TPP⁺ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca²⁺-induced Mg²⁺ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca²⁺ uptake and Mg²⁺ release. These data demonstrate the operation of an electroneutral Ca²⁺/Mg²⁺ exchanger which represents a novel pathway for Ca²⁺ accumulation in liver cells following adrenergic receptor stimulation. This work was supported by National Institutes of Health Grant HL 18708.