Ca2+ uptake by corpus-luteum plasma membranes. Evidence for the presence of both a Ca2+-pumping ATPase and a Ca2+-dependent nucleoside triphosphatase.

Plasma-membrane vesicles from rat corpus luteum showed an ATP-dependent uptake of Ca2+. Ca2+ was accumulated with a K1/2 (concn. giving half-maximal activity) of 0.2 microM and was released by the bivalent-cation ionophore A23187. A Ca2+-dependent phosphorylated intermediate (Mr 100,000) was detected which showed a low decomposition rate, consistent with it being the phosphorylated intermediate of the transport ATPase responsible for Ca2+ uptake. The Ca2+ uptake and the phosphorylated intermediate (E approximately P) displayed several properties that were different from those of the high-affinity Ca2+-ATPase previously observed in these membranes. Both Ca2+ uptake and E approximately P discriminated against ribonucleoside triphosphates other than ATP, whereas the ATPase split all the ribonucleoside triphosphates equally. Both Ca2+ uptake and E approximately P were sensitive to three different Hg-containing inhibitors, whereas the ATPase was inhibited much less. Ca2+ uptake required added Mg2+ (Km = 2.2 mM), whereas the ATPase required no added Mg2+. The maximum rate of Ca2+ uptake was about 400-fold less than that of ATP splitting; under different conditions, the decomposition rate of E approximately P was 1,000 times too slow to account for the ATPase activity observed. All of these features suggested that Ca2+ uptake was due to an enzyme of low activity, whose ATPase activity was not detected in the presence of the higher-specific-activity Ca2+-dependent ATPase.     

F-actin-like ATPase activity in a polymerization-defective mutant yeast actin (V266G/L267G)

Polymerization increases a low level G-actin ATPase activity yielding ADP-P(i) F-actin and then ADP F-actin following release of P(i). By monitoring P(i) release, we explored the relationship between the ATPase activity and polymerization characteristics of a mutant yeast actin, GG. In this mutant, two hydrophobic residues at the tip of a proposed hydrophobic plug between actin subdomains 3 and 4, Val(266) and Leu(267), were mutated to Gly. Although GG-actin does not polymerize by itself in vitro, GG cells are viable. We show that GG-actin ATPase activity increases under normal polymerization conditions, although stable filaments do not form. A plot of P(i) release rate versus actin concentration yields an apparent critical concentration, like that seen for actin polymerization, of approximately 8 microm for Mg(2+) GG-actin and 11 microm for Ca(2+) GG-actin. In contrast to WT-actin, P(i) release from GG-actin is cold-sensitive, reflecting the temperature sensitivity associated with mutations that decrease hydrophobicity in this region. Thus, under polymerization conditions, GG-actin exhibits a continuous F-actin-like ATPase activity resulting from the temperature-sensitive formation of unstable cycling F-actin oligomers. Tropomyosin limits the extent and rate of this activity and restores polymerization by capturing and stabilizing these oligomers rather than enhancing filament nucleation.     

Kinetic characterization of the normal and procaine-perturbed reaction cycles of the sarcoplasmic reticulum calcium pump.

We investigated the effect of the local anesthetic procaine on the activity of the calcium pump protein of sarcoplasmic reticulum (SR) vesicles. Procaine slowed down the rate of calcium uptake by SR vesicles without enhancing the vesicles' passive permeability. This slowing of the unidirectional pumping rate was reflected by the inhibition of the maximal rate of the transport-coupled Ca(2+)-ATPase activity. The inhibition was dependent on Mg2+ concentration; at optimal (i.e. low) concentrations of magnesium, half-maximal inhibition occurred with procaine concentrations close to 15-20 mM. Inhibition of ATPase was not mediated by a change in the properties of the bulk lipid phase. Procaine moderately reduced the true affinity of ATPase for ATP, whereas equilibrium binding of calcium to ATPase in the absence of ATP was virtually not modified by procaine. In fast-kinetics studies, we explored the various intermediate steps in the ATPase catalytic cycle, in order to determine which of them were targets for inhibition by procaine. We found that procaine slowed down ATPase dephosphorylation, an effect which is at least partly responsible for the observed inhibition of overall ATPase activity. In contrast, procaine accelerated the calcium-induced transconformation of unphosphorylated ATPase in the absence of ATP, and altered neither the rate of the Ca(2+)-dependent phosphorylation of ATPase, nor the rate of the dissociation of Ca2+ from phosphorylated ATPase towards the SR lumen, a critical step, the rate of which was measured by a novel fast-filtration method. These results are discussed with respect to the possible site(s) of binding of this amphiphile on the ATPase, and in relation to the contribution of individual steps in the catalytic cycle to the rate limitation of unperturbed SR ATPase activity.     

Regulation of the apolipoprotein B in heterozygous hypobeta-lipoproteinemic knock-out mice expressing truncated apoB,B81. Low production and enhanced clearance of apoB cause low levels of apoB

Meat quality of pigs is dependent on biochemical and biophysical processes in the time course post mortem (p.m.) and is associated with the intracellular Ca2+ homeostasis. However, there is little known about changes in the Ca2+ transporting proteins controlling the Ca2+ uptake of sarcoplasmic reticulum (SR) in the time course p.m. In this study changes in the Ca2+ transporting proteins were investigated in homogenates of longissimus muscles of 4 malignant hyperthermia susceptible (MHS) and 6 malignant hyperthermia resistant (MHR) Pietrain pigs. Muscle samples were obtained at different time intervals: biopsy 2 h prior slaughtering and from the carcass immediately after exsanguination (0 h), 45 min, 4 h, and 22 h p.m. The SR Ca2+ uptake rate was measured immediately after homogenization with closed calcium release channel (CRC), with opened CRC and without manipulation of CRC. Additionally the SR Ca2+ ATPase activity was determined.The results show: (i) The ability of SR to sequester Ca2+ declined to about 60% in the first 45 min p.m. in MHS samples irrespective of CRC state, whereas in MHR samples this decline was about 5%; (ii) Ca2+ uptake and Ca2+ ATPase activity were not different between the biopsy and 0 h samples, i.e. the stress of slaughter was of no immediate influence; (iii) The Ca2+ ATPase activity of the SR declined at about the same rate as the Ca2+ uptake in both MHS and MHR pig samples in the course of time p.m.; (iv) In samples, taken immediately after exsanguination, the Ca2+ ATPase activity of MHS pigs was higher than that of MHR pigs. However, in samples taken 4 h p.m. Ca2+ ATPase activity of MHS pigs has declined to about 30% of the value at 0 h; (v) The CRC can be closed and opened in all samples up to 22 h p.m. and seems to be fully functional at all sampling times; (vi) The CRC of MHS pigs is almost fully open, whereas the CRC of MHR pigs is only partially open at all sampling times; (vii) The permeability of the SR membrane to Ca2+ (determined as the ratio of SR Ca2+ ATPase with and without ionophore A23187) is the same in both MHS and MHR and did not change with ongoing time; (viii) No uncoupling of uptake from ATP hydrolysis occurred up to 4 h p.m., but the coupling differed between MHS and MHR for all time intervals with lower values for MHS pigs. The results suggest that the decreasing Ca2+ uptake rate of homogenates, sampled at different times p.m., is essentially caused by changes in the Ca2+ pump and not by changes in the CRC or an increased phospholipid membrane permeability to Ca2+.     

Effects of exercise of varying duration on sarcoplasmic reticulum function

Sarcoplasmic reticulum (SR) Ca2+ uptake and Ca2+-Mg2+-ATPase activity were examined in muscle homogenates and the purified SR fraction of the superficial and deep fibers of the gastrocnemius and vastus muscles of the rat after treadmill runs of 20 or 45 min or to exhaustion (avg time to exhaustion 140 min). Vesicle intactness and cross-contamination of isolated SR were estimated using a calcium ionophore and mitochondrial and sarcolemmal marker enzymes, respectively. Present findings confirm previously reported fiber-type specific depression in the initial rate and maximum capacity of Ca2+ uptake and altered ATPase activity after exercise. Depression of the Ca2+-stimulated ATPase activity of the enzyme was evident after greater than or equal to 20 min of exercise in SR isolated from the deep fibers of these muscles. The lowered ATPase activity was followed by a depression in the initial rate of Ca2+ uptake in both muscle homogenates and isolated SR fractions after greater than or equal to 45 min of exercise. Maximum Ca2+ uptake capacity was lower in isolated SR only after exhaustive exercise. Ca2+ uptake and Ca2+-sensitive ATPase activity were not affected at any duration of exercise in SR isolated from superficial fibers of these muscles; however, the Mg2+-dependent ATPase activity was increased after 45 min and exhaustive exercise bouts. The alterations in SR function could not be attributed to disrupted vesicles or differential contamination in the SR from exercise groups and were reinforced by similar changes in Ca2+ uptake in crude muscle homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the (Ca2+-Mg2+)ATPase purified by calmodulin-affinity chromatography from bovine aortic smooth muscle

A calmodulin inhibitor, trifluoperazine, suppresses ATP-dependent Ca2+ uptake into microsomes prepared from bovine aortic smooth muscle. From this microsomal preparation which we expected to contain calmodulin-dependent Ca2+-transport ATPase [EC 3.6.1.3], we purified (Ca2+-Mg2+)ATPase by calmodulin affinity chromatography. The protein peak eluted by EDTA had calmodulin-dependent (Ca2+-Mg2+)ATPase activity. The major band (135,000 daltons) obtained after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) accounted for about 80% of the total protein eluted. This major band was phosphorylated by [gamma-32P]ATP in a Ca2+-dependent manner. All the 32P incorporated into the major band was released by hydroxylaminolysis. The ATPase reconstituted in soybean phospholipid liposomes showed ATP, calmodulin-dependent Ca2+ uptake. The affinity of the ATPase for Ca2+, Km, was 7 microM and the maximum ATPase activity was 1.4 mumol/mg/min. These values were changed to 0.17 microM and 3.5 mumol/mg/min, respectively by the addition of calmodulin. The activity of the purified (Ca2+-Mg2+)ATPase was inhibited by orthovanadate, and the concentration required for half-maximal inhibition was about 1.8 microM which is close to that of plasma membrane ATPases. Judging from the effect of orthovanadate and the molecular weight, the purified (Ca2+-Mg2+)ATPase was considered to have originated from the plasma membrane not from the sarcoplasmic reticulum.     

BDM compared with P(i) and low Ca2+ in the cross-bridge reaction initiated by flash photolysis of caged ATP.

Using flash photolysis of caged ATP in skinned muscle fibers from rat psoas, we examined the inhibitory effects of 2,3-butanedione monoxime (BDM) on the contraction kinetics and the rate of ATP hydrolysis of the cross-bridges at approximately 10 degrees C. The hydrolysis rate was estimated from the stiffness records. The effects of BDM were compared with those of orthophosphate (P(i)) and of reduction in [Ca2+] (low Ca2+), and it was found that i) BDM and low Ca2+ inhibited ATPase activity to the same extent as they inhibited the steady tension, whereas P(i) inhibited ATPase activity much less than tension; ii) BDM and P(i) decreased tension per stiffness during the steady contraction more than did low Ca2+; iii) neither BDM nor low Ca2+ affected the initial relaxation of the fiber on release of ATP, but P(i) slightly slowed it; and iv) BDM hardly influenced the rate of contraction development after relaxation, although P(i) and low Ca2+ accelerated it. We concluded that BDM inhibits the Ca(2+)-regulated attachment of the cross-bridges and force-generation of the attached cross-bridges.     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.     

Correlation between steady-state ATP hydrolysis and vanadate-induced ADP trapping in Human P-glycoprotein. Evidence for ADP release as the rate-limiting step in the catalytic cycle and its modulation by substrates

P-glycoprotein (Pgp) is a transmembrane protein conferring multidrug resistance to cells by extruding a variety of amphipathic cytotoxic agents using energy from ATP hydrolysis. The objective of this study was to understand how substrates affect the catalytic cycle of ATP hydrolysis by Pgp. The ATPase activity of purified and reconstituted recombinant human Pgp was measured using a continuous cycling assay. Pgp hydrolyzes ATP in the absence of drug at a basal rate of 0.5 micromol x min x mg(-1) with a K(m) for ATP of 0.33 mm. This basal rate can be either increased or decreased depending on the Pgp substrate used, without an effect on the K(m) for ATP or 8-azidoATP and K(i) for ADP, suggesting that substrates do not affect nucleotide binding to Pgp. Although inhibitors of Pgp activity, cyclosporin A, its analog PSC833, and rapamycin decrease the rate of ATP hydrolysis with respect to the basal rate, they do not completely inhibit the activity. Therefore, these drugs can be classified as substrates. Vanadate (Vi)-induced trapping of [alpha-(32)P]8-azidoADP was used to probe the effect of substrates on the transition state of the ATP hydrolysis reaction. The K(m) for [alpha-(32)P]8-azidoATP (20 microm) is decreased in the presence of Vi; however, it is not changed by drugs such as verapamil or cyclosporin A. Strikingly, the extent of Vi-induced [alpha-(32)P]8-azidoADP trapping correlates directly with the fold stimulation of ATPase activity at steady state. Furthermore, P(i) exhibits very low affinity for Pgp (K(i) approximately 30 mm for Vi-induced 8-azidoADP trapping). In aggregate, these data demonstrate that the release of Vi trapped [alpha-(32)P]8-azidoADP from Pgp is the rate-limiting step in the steady-state reaction. We suggest that substrates modulate the rate of ATPase activity of Pgp by controlling the rate of dissociation of ADP following ATP hydrolysis and that ADP release is the rate-limiting step in the normal catalytic cycle of Pgp.     

The metal-binding sites of the zinc-transporting P-type ATPase of Escherichia coli. Lys693 and Asp714 in the seventh and eighth transmembrane segments of ZntA contribute to the coupling of metal binding and ATPase activity

ZntA is a P-type ATPase which transports Zn(2+), Pb(2+) and Cd(2+) out of the cell. Two cysteine-containing motifs, CAAC near the N-terminus and CPC in transmembrane helix 6, are involved in binding of the translocated metal. We have studied these motifs by mutating the cysteines to serines. The roles of two other possible metal-binding residues, K(693) and D(714), in transmembrane helices 7 and 8, were also addressed. The mutation CAAC-->SAAS reduces the ATPase activity by 50%. The SAAS mutant is phosphorylated with ATP almost as efficiently as the wild type. However, its phosphorylation with P(i) is poorer than that of the wild type and its dephosphorylation rate is faster than that of the wild type ATPase. The CPC-->SPS mutant is inactive but residual phosphorylation with ATP could still be observed. The most important findings of this work deal with the prospective metal-binding residues K(693) and D(714): the substitution K693N eliminates the Zn(2+)-stimulated ATPase activity completely, although significant Zn(2+)-dependent phosphorylation by ATP remains. The K693N ATPase is hyperphosphorylated by P(i). ZntA carrying the change D714M has strong metal-independent ATPase activity and is very weakly phosphorylated both by ATP and P(i). In conclusion, K(693) and D(714) are functionally essential and appear to contribute to the metal specificity of ZntA, most probably by being parts of the metal-binding site made up by the CPC motif.     

Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromysin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ

Transient kinetic studies of Mg(2+)-dependent heavy-meromyosin ATPase (adenosine triphosphatase) were done by monitoring the release of both ADP and P(i) into the reaction medium by using linked assay systems. The release of P(i) was monitored by its quantitative transfer to ADP, with concomitant reduction of NAD(+) in the presence of d-glyceraldehyde 3-phosphate, d-glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase. The dissociation rates of the products, ADP and P(i), from heavy meromyosin were shown to be faster than the rate-controlling process, which occurs after the initial bond cleavage of ATP. The chromophoric ATP analogue, 6-mercapto-9-beta-d-ribofuranosylpurine 5'-triphosphate (thioATP) was used as a substrate and spectral changes associated with a single turnover of heavy meromyosin could be assigned to elementary processes of the mechanism. It was shown that the dissociation rate of thioADP was not the rate-controlling process of the thioATPase, whose catalytic-centre activity was 7.6 times that of the ATPase at pH8. The dissociation rate of ADP from heavy meromyosin was measured by using thioATP as displacing agent and was found to be 2.3s(-1), which is about 50 times the catalytic-centre activity of the ATPase at pH8. Transient kinetic studies with chromophoric adenosine phosphate analogues have general application for kinases and ATPases both in characterizing the chemical states of the intermediates and in delineating the elementary processes of the enzyme mechanism.     

Cyclic GMP regulation of the plasma membrane (Ca2+-Mg2+)ATPase in vascular smooth muscle

Plasma membrane (Ca2+-Mg2+)ATPase purified from bovine aortic microsomes by calmodulin affinity chromatography was incorporated into soybean phospholipid liposomes. In the reconstituted proteoliposomes, a protein corresponding to the ATPase was phosphorylated by [gamma-32P]ATP in the presence of cGMP and cGMP-dependent protein kinase. Both the affinity for Ca2+ and the maximum Ca2+ uptake activity by the proteoliposomes were increased by the cGMP-dependent phosphorylation, and there was good parallelism between the Ca2+-uptake rate and the extent of phosphorylation. These results strongly suggest that the Ca2+-transport ATPase of the vascular smooth muscle plasma membrane is regulated through its cGMP-dependent phosphorylation.     

Correlative changes in sucrose uptake, ATPase activity and membrane fluidity in carnation petals during senescence

Apparent sucrose uptake. ATPase activity and membrane fluidity changes were studied during the development and senescence of carnation flowers ( Dianthus caryophyllus L., cv. Cerise Royallette). Typical changes associated with senescence of a cut flower, such as respiration, ethylene production and fresh weight, were measured. Concomitant with a rise in respiration and ethylene production and a decline in fresh weight, a sharp decrease in apparent sucrose uptake was observed. Sucrose uptake was pH dependent (pH optimum, 5.5) and influenced by membrane integrity. Apparently, the activity of ATPase is related to sucrose uptake, because similar changes occurred during flower development. In addition, the activity of ATPase was well correlated with membrane fluidity.
It is suggested that sucrose uptake is controlled by ATPase activity, which in turn is modulated by membrane lipid fluidity. The decline in membrane fluidity associated with senescence leads to a corresponding reduction in ATPase activity and sucrose uptake. Further evidence supporting this view comes from experiments in which senescence was enhanced by 1-aminocyclopropane-l-carboxylic acid. It shortened the time to decline in fresh weight, rise in respiration and ethylene production. In parallel, reduction in membrane fluidity, ATPase activity and sucrose uptake were observed.     

Influence of inorganic phosphate and pH on ATP utilization in fast and slow skeletal muscle fibers.

The influence of P(i) and pH was studied on myofibrillar ATP turnover and force development during maximally activated isometric contractions, in skinned single fibers from rabbit soleus and psoas muscle. ATP hydrolysis was coupled to the breakdown of NADH, which was monitored photometrically at 340 nm. In psoas the depression by phosphate of force is twice that of ATP turnover, but in soleus force and ATP turnover are depressed equally by P(i). Most, but not all, of the ATPase and force values observed for a combination of high P(i) and low pH could be explained by independent effects of P(i) and pH. The effects of P(i) and pH on ATP turnover can be understood by a three-state cross-bridge scheme. Mass action of phosphate on the reaction from the actomyosin(AM).ADP state to the AM.ADP.P(i) state may largely account for the phosphate dependencies of ATPase activity found. Protons affect cross-bridge detachment from the AM.ADP state and the rate of the AM.ADP.P(i)-to-AM.ADP transition. In this scheme, the effects of P(i) and pH on cross-bridge kinetics appeared to be largely independent.     

Inhibition of Na+/Ca2+ exchanger by peroxynitrite in microsomes of pulmonary smooth muscle: role of matrix metalloproteinase-2

Treatment of bovine pulmonary artery smooth muscle microsomes with peroxynitrite (ONOO-) (100 microM) markedly stimulated matrix metalloproteinase-2 (MMP-2) activity and also enhanced Ca2+ATPase activity and ATP-dependent Ca2+ uptake. Pretreatment of the microsomes with vitamin E (1 mM) and TIMP-2 (50 microg/ml) preserved the increase in MMP-2 activity, Ca2+ATPase activity and also ATP-dependent Ca2+ uptake in the microsomes. In contrast, Na(+)-dependent Ca2+ uptake in the microsomes was inhibited by ONOO- and this was found to be reversed by vitamin E (1 mM) and TIMP-2 (50 microg/ml). However, changes caused by ONOO- in MMP-2 activity, ATP-dependent Ca2+ uptake and Na(+)-dependent Ca2+ uptake were not reversed upon pretreatment of the microsomes with a low concentration of 5 microg/ml of TIMP-2 which, on the contrary, reversed MMP-2 (1 microg/ml)-mediated alteration on these parameters. The inhibition of Na(+)-dependent Ca2+ uptake by ONOO- and MMP-2 overpowered the stimulation of ATP-dependent Ca2+ uptake in the microsomes. Treatment with ONOO- abolished the inhibitory effect of TIMP-2 (5 microg/ml) on MMP-2 (1 microg/ml) causing 14C-gelatin degradation. Overall, the present study suggests that ONOO- inactivated TIMP-2, the ambient inhibitor of MMP-2, leading to activation of the ambient proteinase, MMP-2, and subsequently stimulated Ca2+ATPase activity and ATP-dependent Ca2+ uptake, but inhibited Na(+)-dependent Ca2+ uptake, resulting in a marked decrease in Ca2+ uptake in microsomes of bovine pulmonary artery smooth muscle.     

Expression of rat, renal NHE2 and NHE3 during postnatal development

In apical membrane vesicles from beef tracheal epithelia expressing up to 30% of the proteins as functional cystic fibrosis transmembrane conductance regulator (CFTR)-- i.e. a voltage-independent and PKA-sensitive 36Cl- flux--an ATPase activity, different from P, F0F1 and V types, was reproducibly detected. Its specific activity averaged 20 micromol Pi h(-1) mg(-1) with an apparent affinity for ATP of 530 +/- 30 microM. Its possible involvement in CFTR functions was supported by (1) the linear relationship between the ATPase activity and the magnitude of 36Cl- fluxes (turnover rate: 3 ATP hydrolyzed per CFTR per second), (2) the same rank of potency of ATP, ITP, GTP, UTP and CTP to be hydrolyzed and to open CFTR chloride channels, (3) the similar and parallel inhibition of the ATPase and CFTR Cl- fluxes by NS004 (IC50: 60 microM) and (4) the potency of anti-R domain antibodies to increase by 18% the ATPase activity.     

Behaviour of cardiac microsomal Ca2+ pump under conditions that may simulate pathological situations

The behaviour of Ca2+ ATPase activity in relation to Ca2+ transport process was studied under different experimental conditions in canine cardiac microsomal fraction predominantly containing sarcoplasmic reticulum. The total Ca2+ concentration required for half maximal activation (Ka) of microsomal Ca2+ ATPase and Ca2+ uptake did not differ significantly, unless 0.1 mmol/l EGTA was present in the incubation media. Pretreatment of cardiac microsomes with membrane disruptive agents like phospholipase A, trypsin as well as deoxycholate strongly increased (2-3 fold) Ca2+ ATPase activity but uptake rate of Ca2+ declined. Only in phospholipase C and beta-glucuronidase pretreatment, a parallel decrease of Ca2+ ATPase and uptake was observed. In presence of excess (free)Ca2+ (greater than 10 mumol/l) both Ca2+ ATPase as well as Ca2+ uptake were inhibited, however, Ca2+ binding process remained unaltered. Likewise, low pH completely altered the relation between Ca2+ binding and ATPase activity; whereas Ca2+ ATPase was inhibited, Ca2+ binding did not change. Our present data provide evidence for some cellular factors that may be involved in producing uncoupling of microsomal Ca2+ ATPase from Ca2+ accumulation process that was previously observed in various pathological situations.     

Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum with 4-(bromomethyl)-6,7-dimethoxycoumarin: detection of conformational changes.

The (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum was labeled with 4-(bromomethyl)-6,7-dimethoxycoumarin. It was shown that a single cysteine residue (Cys-344) was labeled on the ATPase, with a 25% reduction in steady-state ATPase activity and no reduction in the steady-state rate of hydrolysis of p-nitrophenyl phosphate. The fluorescence intensity of the labeled ATPase was sensitive to pH, consistent with an effect of protonation of a residue of pK 6.8. Fluorescence changes were observed on binding Mg2+, consistent with binding to a single site of Kd 4 mM. Comparable changes in fluorescence intensity were observed on binding ADP in the presence of Ca2+. Binding of AMP-PCP produced larger fluorescence changes, comparable to those observed on phosphorylation with ATP or acetyl phosphate. Phosphorylation with P(i) also resulted in fluorescence changes; the effect of pH on the fluorescence changes was greater than that on the level of phosphorylation measured directly using [32P]P(i). It is suggested that different conformational states of the phosphorylated ATPase are obtained at steady state in the presence of Ca2+ and ATP and at equilibrium in the presence of P(i) and absence of Ca2+.     

Protein kinase C mediated activation and phosphorylation of Ca(2+)-pump in cardiac sarcolemma.

The effects of purified protein kinase C (PKC) on the Ca(2+)-pumping ATPase of cardiac sarcolemma were investigated. The addition of PKC to sarcolemmal vesicles resulted in a significant increase in ATP-dependent Ca2+ uptake, by increasing the calcium affinity by 2.8-fold (Km 0.14 vs. 0.4 microM for control) and by increasing Vmax from 5 to 6.8 nmol.mg protein-1.min-1. The addition of PKC also stimulated Ca2+ ATPase activity in sarcolemmal preparations. This activity was increased further upon the addition of calmodulin. These results suggest that PKC stimulates Ca2+ ATPase through a kinase-directed phosphorylation. The addition of PKC to a purified preparation of Ca2+ ATPase in the presence of [gamma-32P]ATP resulted in a 100% increase in phosphorylation that was dependent on the presence of Ca2+, phosphatidylserine, and phorbol 12,13-dibutyrate. These results demonstrate that the Ca2+ ATPase of canine cardiac muscle can be phosphorylated by PKC in vitro, resulting in increased affinity of the Ca2+ ATPase for Ca2+ and increase in the Ca2+ pump pumping rate. The results suggest that the Ca(2+)-pumping ATPase in heart tissue can be stimulated by PKC, thereby regulating the intracellular Ca2+ levels in whole heart.     

Isolation and partial characterization of magnesium ion-and calcium ion-dependent adenosine triphosphatase activity from bovine brain microsomal fraction

Microsomal fraction was prepared by ultracentrifugation of homogenates of cortical tissue from bovine brains. The preparation displayed ATPase (adenosine triphosphatase) activity in the presence of Mg(2+) (6.4mumol of P(i)/h per mg of protein) and Ca(2+) (3.4mumol of P(i)/h per mg of protein). Kinetic analysis of the activation of the enzyme preparation by Ca(2+) resulted in the demonstration of two apparent K(m) values for Ca(2+) (6.0x10(-8)m and 1.2x10(-6)m). Treatment of the microsomal membranes with Triton X-100 resulted in solubilization of the ATPase, though with some loss of activity. The solubilized microsomal proteins were incorporated into liposomes. By incubation of the liposomes in media containing (45)Ca(2+) an ATP-dependent uptake of Ca(2+) was demonstrated. The solubilized preparation was subjected to preparative isoelectric focusing in granulated gel beds. Two distinct peaks of Mg(2+)- and Ca(2+)-dependent ATPase activity were observed at pH4.8 (peak 4.8) and at pH6.3 (peak 6.3). The material isolated in peaks 4.8 and 6.3 was focused in polyacrylamide gel with pH gradients. The material corresponding to peak 4.8 consisted of a single protein, whereas peak 6.3 contained one major and at least one minor protein. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis confirmed these results and indicated that the major component of peak 4.8 and the protein of peak 6.3 both had a molecular weight of 105000. The material in peaks 4.8 and 6.3 was assayed for ATPase activity in the presence of various concentrations of Ca(2+). Kinetic analysis of the results for peak 4.8 demonstrated an apparent K(m) value for Ca(2+) of 4.1x10(-8)m. The enzyme isolated at pH6.3 had an apparent K(m) value of 3.8x10(-6)m. However, when the material from peak 4.8 was incubated in the presence of 1mm-Mg(2+) the ATPase could not be activated by Ca(2+).