Properties of the cyanobacterial coupling factor ATPase from Spirulina platensis. II. Activity of the purified and membrane-bound enzymes

Cyanobacterial (Spirulina platensis) photosynthetic membranes and isolated F1 ATPase were characterized with respect to ATP activity. The following results indicate that the regulation of expression of ATPase activity in Spirulina platensis is similar to that found in chloroplasts: the ATPase activity of Spirulina membranes and isolated F1 ATPase is mostly latent, a characteristic of chloroplast ATPase activity; treatments that elicit ATPase activity in higher plant chloroplast thylakoids and isolated chloroplast coupling factor (CF1) greatly stimulate the activity of Spirulina membranes and F1, and the cation specificity of chloroplast ATPase activity, e. g., light-induced membrane activity that is magnesium dependent and trypsin-activated CF1 activity that is calcium dependent, is also observed in Spirulina. Thus, an 8- to 15-fold increase in specific activity (to 13-15 mumol Pi min-1 mg chl-1) is obtained when Spirulina membranes are treated with trypsin (CaATPase) or with methanol (MgATPase): a light-induced, dithiothreitol-dependent MgATPase activity is also found in the membranes. Purified Spirulina F1 is a CaATPase when activated with trypsin (endogenous activity increases from 4 to 27-37 mumol Pi min-1 mg protein-1) or with dithiothreitol (5.6 mumol Pi min-1 mg-1), but a MgATPase when assayed with methanol (18-20 mumol Pi min-1 mg-1). The effects of varying calcium and ATP concentrations on the kinetics of trypsin-induced CaATPase activity of Spirulina F1 were examined. When the calcium concentration is varied at constant ATP concentration, the velocity plot shows a marked sigmoidicity. By varying Ca-ATP metal-nucleotide complex concentration at constant concentrations of free calcium or ATP, it is shown that the sigmoidicity is due to the effect of free ATP, which changes the Hill constant to 1.6 from 1.0 observed when the free calcium concentration is kept constant at 5 mM. Therefore not only is ATP an inhibitor but it is also an allosteric effector of Spirulina F1 ATPase activity. At 5 mM free calcium, the Km for teh Ca-ATP metal-nucleotide complex is 0.42 mM.     

Calcium binding in cardiac sarcolemma.

The relationship between calcium binding and ATPase activity has been investigated for guinea pig cardiac sarcolemma. The concentrations of calcium ions and of ATP were the main factors in determining the amount of calcium bound. With a saturating concentration of ATP, at low calcium concentrations (0.1 mM) more than 50% of the total calcium bound was ATP dependent while at high concentrations of calcium (10 mM) only 20% of the calcium bound was ATP dependent. The ATP-dependent calcium binding process involves one class of calcium binding sites while the non-ATP-dependent calcium binding process involves two classes of binding sites. Inhibitor studies of Ca2+-stimulated MgATPase, MgATPase, and CaATPase activities suggest Ca2+ and Mg2+ are either interacting with different sites on the same enzyme or with different enzymes.     

Comparison of ATPase activity of cardiac sarcoplasmic reticulum fraction from rat and dog

The purpose of the present study was to compare the ATPase activities of cardiac SR in two species in which the different intrinsic myocardial contractility can only partially be explained by the different properties of cardiac myosins. In cardiac SR isolated from rat heart, the total ATPase activity was 1512.5 +/- 23.3 nmol Pi/mg protein/min, nearly four times as high as in dog cardiac SR (408.8 +/- 28.9 nmol Pi/mg protein/min). The Ca2+-activated ATPase in rat cardiac SR represented only 23.8% of the total ATPase activity, while in dog cardiac SR it was approximately 50% of the total. Thus, the specific Ca2+-activated ATPase was nearly two times higher in the cardiac SR of the rat than in that of the dog. This higher rate of ATP hydrolysis in rat cardiac SR may be, at least in part, responsible for the increased intensity and shorter duration of the active state in the rat myocardium. Polyacrylamide gel electrophoresis of SR showed that the relative amount of Ca2+-pump protein was two times higher in dog heart, similar to the percentage of Ca2+-activated ATPase activity. At the same time, the specific Ca2+-activated ATPase activity and the relative amount of Ca2+ pump protein in both the rat and dog cardiac SR were inversely related.     

Post-mortem electrical stimulation of muscle and its effects on sarcoplasmic reticulum adenosine triphosphatase.

Sarcoplasmic reticulum (SR) was isolated from control muscles and from muscles which had been subjected to short-term post-mortem electrical stimulation. Both preparations had similar protein compositions but the SR from electrically stimulated muscle had a lower 'extra' ATPase activity. The ability of the SR preparations from electrically stimulated muscles to accumulate Ca2+ was about the same as the controls. There was, therefore, an apparently greater efficiency of Ca2+ transport in the isolated vesicles, the reason for which is not known, but an alteration in the 'leakiness' of the membrane may be involved. Purified ATPase isolated from control and stimulated SR contained, in addition to the ATPase protein, a polypeptide of molecular weight about 30 000. The purified ATPase vesicles from electrically stimulated muscle had a reduced activity as measured by ATP splitting activity, phosphoenzyme formation from either inorganic orthophosphate (Pi) or ATP, or by an ATP in equilibrium Pi exchange reaction. These reduced activities probably result from an alteration in the binding affinities of the ATPase for ATP and Pi. The low affinity site for calcium binding was not affected by electrical stimulation. Purified ATPase vesicles from stimulated muscle were more susceptible to proteolytic attack, suggesting that the conformation of the protein or its association with the membrane lipids had been altered.     

Assembled F1-(alpha beta ) and Hybrid F1-alpha 3beta 3gamma -ATPases from Rhodospirillum rubrum alpha, wild type or mutant beta, and chloroplast gamma subunits. Demonstration of Mg2+versus Ca2+-induced differences in catalytic site structure and function

Refolding together the expressed alpha and beta subunits of the Rhodospirillum rubrum F(1)(RF(1))-ATPase led to assembly of only alpha(1)beta(1) dimers, showing a stable low MgATPase activity. When incubated in the presence of AlCl(3), NaF and either MgAD(T)P or CaAD(T)P, all dimers associated into closed alpha(3)beta(3) hexamers, which also gained a low CaATPase activity. Both hexamer ATPase activities exhibited identical rates and properties to the open dimer MgATPase. These results indicate that: a) the hexamer, as the dimer, has no catalytic cooperativity; b) aluminium fluoride does not inhibit their MgATPase activity; and c) it does enable the assembly of RrF(1)-alpha(3)beta(3) hexamers by stabilizing their noncatalytic alpha/beta interfaces. Refolding of the RrF(1)-alpha and beta subunits together with the spinach chloroplast F(1) (CF(1))-gamma enabled a simple one-step assembly of two different hybrid RrF(1)-alpha(3)beta(3)/CF(1)gamma complexes, containing either wild type RrF(1)-beta or the catalytic site mutant RrF(1)beta-T159S. They exhibited over 100-fold higher CaATPase and MgATPase activities than the stabilized hexamers and showed very different catalytic properties. The hybrid wild type MgATPase activity was, as that of RrF(1) and CF(1) and unlike its higher CaATPase activity, regulated by excess free Mg(2+) ions, stimulated by sulfite, and inhibited by azide. The hybrid mutant had on the other hand a low CaATPase but an exceptionally high MgATPase activity, which was much less sensitive to the specific MgATPase effectors. All these very different ATPase activities were regulated by thiol modulation of the hybrid unique CF(1)-gamma disulfide bond. These hybrid complexes can provide information on the as yet unknown factors that couple ATP binding and hydrolysis to both thiol modulation and rotational motion of their CF(1)-gamma subunit.     

白细胞介素-2对大鼠心肌Ca2+ATPase和Na+ /K+ATPase的影响

为了探讨IL－2对心肌细胞内钙影响的可能机制,用光学法检测心肌肌浆网Ca^2 ATPase的活性,以及细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性。结果：（1）用IL－2（10、40、200、800U/ml）灌流心脏后,其肌浆网Ca^2 ATPase的活性随IL－2浓度的升高而增强;（2）在ATP浓度为0.1-4mmol/L时,Ca^2 ATPase的活性随ATP浓度的升庙则增强,由IL－2（200U／ml）灌流后的心脏获得肌浆网（SR）,其Ca^2 ATPase的活性对ATP的反应强于对照组;（3）在[Ca^2 ]为1－40μmol/L时,心脏SR Ca^2 ATPase的活性随[Ca^2 ]增加而增强,而IL－2灌流心脏后分离的SR,其Ca^2 ATPase活性在[Ca^2 ]升高时没有明显改变;（4）用nor-BNI(10nmol/L）预处理5min后,IL－2（200U／ml）灌流后不再使SR Ca^2 ATPase的活性增强;（5）用PTX（5mg/L）预处理后,IL－2对SR Ca^2 ATPase的影响减弱;（6）用磷脂酶C（PLC）抑制剂U73122（5μmol/L）处理后,IL－2不再使SR Ca^2 ATPase活性增高;（7）用IL－2直接处理从正常大鼠分离的SR后,对SR Ca^2 ATPase活性无明显影响;（8）IL－2灌流后,对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase活性没有显著。上述结果表明,IL－2灌流心脏后使心肌肌浆网Ca^2 ATPase的活性增加,心肌细胞膜上的κ－阿片受体及其下游的G蛋白和PLC介导了IL－2的作用。尽管IL－2提高SR Ca^2 ATPase对ATP的反应性,但却抑制SR Ca^2 ATPase对钙离子的敏感性。IL－2对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性无明显影响。     

Measurement of sarcoplasmic reticulum Ca2+-ATPase activity and E-type Mg2+-ATPase activity in rat heart homogenates

The presence of a high and nonlinear Ca2+-independent (or basal) ATPase activity in rat heart preparations makes difficult the reliable measurement of sarcoplasmic reticulum (SR) Ca2+-ATPase activity by usual methods. A spectrophotometric assay for the accurate determination of SR Ca2+-ATPase activity in unfractionated homogenates from rat heart is described. The procedure is based on that reported by Simonides and van Hardeveld (1990, Anal. Biochem. 191, 321-331) for skeletal muscle homogenates. To avoid overestimation of the Ca2+-ATPase activity of cardiac homogenates that occurs when sequential measurements of total and basal ATPase activities are performed, two parallel and independent assays are required: one with low (micromolar) and other high (millimolar) calcium concentration. Addition of thapsigargin (0.2 microM) blocked totally the activity considered as Ca2+-ATPase activity. Using this method, the rat heart homogenate Ca2+-ATPase activity was 10.5 +/- 2.0 micromol. min-1 x g-1 tissue wet weight (n = 8). Likewise, a spectrophotometric assay for measuring E-type Mg2+-ATPase activity in cardiac total homogenates has been developed, comparing the following characteristics of the enzymatic activity in homogenate and a membrane-enriched fraction: first-order rate constant for ATP-dependent inactivation, Km for ATP, and effects of concanavalin A, Triton X-100, and specific inhibitors.     

Unsaturated aminophospholipids are preferentially retained by the fast skeletal muscle CaATPase during detergent solubilization. Evidence for a specific association between aminophospholipids and the calcium pump protein.

When fast twitch skeletal muscle vesicles (SR) and purified calcium pump protein are stripped with the nonionic detergent C12E8 (octaethylene glycol dodecyl ether), not all the membrane phospholipids are removed from the calcium pump protein. Maximal extraction produces a remnant of 6-8 mol of phospholipid/mole of calcium ATPase (CaATPase). In contrast to native SR and the prestripped purified CaATPase, the remaining phospholipid is markedly enriched in phosphatidylethanolamine (PE) and phosphatidylserine (PS) in both preparations; the remaining lipid is also enriched in phospholipid that is predominantly unsaturated. In addition, virtually all of the associated PE is plasmalogenic (96% as opposed to 63% in the native SR). The amino-specific cross-linking reagent DFDNB (1,5-difluoro-2,4-dinitrobenzene sulfonic acid) and the amino binding reagent TNBS (2,4,6-trinitrobenzene sulfonic acid) were utilized to identify the monolayer of the native preparation where these phospholipids reside, and to determine which phospholipids are closely associated with the calcium pump protein following detergent treatment. These studies demonstrate that PE and PS are closely associated with the pump protein, PE residing almost exclusively in the outer monolayer of SR, while PS resides in the inner monolayer. Nonspecific phospholipid exchange protein was shown to be capable of exchanging phospholipids from donor vesicles into those phospholipids associated with the CaATPase; stripping of lipid-exchanged vesicles with C12E8 exhibited the same specificity with regard to head-group species (i.e., PE is markedly enriched in the extracted protein associated fraction). The results suggest that specific protein-lipid interactions exist, favoring the association of plasmalogenic aminophospholipids with the calcium pump protein.     

Nd3+ and Co2+ binding to sarcoplasmic reticulum CaATPase. An estimation of the distance from the ATP binding site to the high-affinity calcium binding sites

Nd3+ binding to sarcoplasmic reticulum (SR) was detected by inhibition of ATPase activity and directly by a fluorimetric assay. Both methods indicated that Nd3+ inhibited the ATPase activity by binding in the high-affinity Ca2+ binding sites. The stoichiometry of binding was about 11 nmol of Nd3+ bound per mg of SR proteins at pNd = 6.5. At higher [Nd3+], substantial nonspecific binding occurred. The association constant for Nd3+ binding to the high-affinity Ca2+ binding sites was estimated to be near 2 X 10(9) M-1. When the CaATPase was inactivated with fluorescein isothiocyanate (FITC), 5.3 nmol were bound per mg of SR protein. This fluorescent probe is known to bind in the ATP binding site. The stoichiometry of Nd3+ binding to FITC-labeled CaATPase was the same, within experimental error, as to the unlabeled CaATPase. Fluorescence energy transfer between FITC in the ATP site and Nd3+ in the Ca2+ sites was found to be very small. This donor-acceptor pair has a critical distance of 0.93 nm and the distance between the ATP site and the closest Ca2+ was estimated to be greater than 2.1 nm. Parallel measurements with FITC-labeled SR and Co2+, an acceptor with a critical distance 1.2 nm, suggested the ATP and Ca2+ binding sites are greater than 2.6 nm apart.     

Inhibition of skeletal muscle sarcoplasmic reticulum CaATPase activity by calmidazolium

Calmidazolium, a lipophilic cation and putative calmodulin-specific antagonist, inhibited potently the calcium ATPase of sarcoplasmic reticulum (SR) vesicles isolated from skeletal muscle. Based on steady-state measurements of catalytic activity over a range of MgATP, calmidazolium, and SR protein concentrations, the calculated values of the inhibition constant (KI) and binding stoichiometry were 0.06 microM and 770 nmol/mg protein, respectively. SR CaATPase inhibition apparently is not a general property of lipophilic cations since the hydrophobic anion tetraphenylboron inhibited catalysis, whereas its cationic analog, tetraphenylarsonium, did not. Enzyme inhibition by calmidazolium was noncompetitive with respect to the substrates Ca2+ and MgATP. In the presence of other SR CaATPase inhibitors, calmidazolium was competitive with respect to quercetin and noncompetitive with respect to trifluoperazine and propranolol. While calmidazolium inhibited enzyme phosphorylation by MgATP, catalysis was more sensitive to the inhibitor. Binding of calmidazolium to SR membranes produced morphological changes seen by electron microscopy as membrane thickening and loss of resolution of surface detail. Our results show that calmidazolium is a high-affinity, noncompetitive inhibitor of skeletal SR CaATPase activity, and they suggest that this inhibition is based on binding to the membrane phospholipids rather than specific antagonism of enzyme activation by calmodulin.     

Ca2+-overload inhibits the cardiac SR Ca2+-calmodulin protein kinase activity

There is increasing evidence to suggest that Ca2+-calmodulin dependent protein kinase (CaMK) regulates the sarcoplasmic reticulum (SR) function and thus plays an important role in modulating the cardiac performance. Because intracellular Ca2+-overload is an important factor underlying cardiac dysfunction in a heart disease, its effect on SR CaMK was examined in the isolated rat heart preparations. Ca2+-depletion for 5 min followed by Ca2+-repletion for 30 min, which is known to produce intracellular Ca2+-overload, was observed to attenuate cardiac function as well as SR Ca2+-uptake and Ca2+-release activities. Attenuated SR function in the heart was associated with reduced CaMK phosphorylation of the SR Ca2+-cycling proteins such as Ca2+-release channel, Ca2+-pump ATPase, and phospholamban, decreased CaMK activity, and depressed levels of SR Ca2+-cycling proteins. These results indicate that alterations in cardiac performance and SR function following the occurrence of intracellular Ca2+-overload may partly be due to changes in the SR CaMK activity.     

Inhibition of dicarboxylic anion transport by fluorescein isothiocyanate in skeletal sarcoplasmic reticulum.

It has been demonstrated previously that dicarboxylic anions are cotransported during ATP-dependent Ca2+ transport by skeletal muscle sarcoplasmic reticulum (SR) membranes, and that anion cotransport stimulates Ca2+ transport. In the current study, we present evidence that dicarboxylic anion cotransport and Ca2+ transport are kinetically distinct in SR, but both functions are mediated by the CaATPase protein. Preincubation of SR with 40 microM fluorescein isothiocyanate (FITC) (pH 7.0) inhibited essentially all of the Ca2+ ATPase activity, as well as active oxalate-supported and oxalate-independent 45Ca2+ accumulation. The addition of 1 mM beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) to the preincubation media fully protected the dicarboxylic anion-independent Ca2+ ATPase activity and the oxalate-independent active 45Ca2+ accumulation from the inhibitory effects of FITC; however, the ATP-associated [14C]oxalate accumulation, the oxalate-dependent 45Ca2+ accumulation, and the oxalate- and maleate-dependent stimulation of Ca2+ ATPase activity were not protected by AMP-PCP. Thus, the dicarboxylic anion accumulation and the stimulation of Ca2+ uptake by dicarboxylic anions could be functionally separated from the ATP-dependent, anion-independent Ca2+ translocation. FITC bound exclusively to the 100-kDa (CaATPase) and 92-kDa (phosphorylase) proteins in the SR membranes and to purified CaATPase in sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 1 mM AMP-PCP inhibited 50-55% of the FITC fluorescence on the 100-kDa protein, but did not significantly alter fluorescence on the 92-kDa protein. Two-dimensional gel analysis demonstrated a single 100-kDa protein in longitudinal SR membranes. FITC appears to inhibit ATP-dependent Ca2+ transport, and dicarboxylic anion translocation through interaction at separate domains of the CaATPase protein.     

Decreased expression of cardiac sarcoplasmic reticulum Ca2+-pump ATPase in congestive heart failure due to myocardial infarction

Myocardial infarction in rats induced by occluding the left coronary artery for 4, 8 and 16 weeks has been shown to result in congestive heart failure (CHF) characterized by hypertrophy of the viable ventricular myocardial tissue. We have previously demonstrated a decreased calcium transport activity in the sarcoplasmic reticulum (SR) of post-myocardial infarction failing rat hearts. In this study we have measured the steady state levels of the cardiac SR Ca²⁺-pump ATPase (SERCA2) mRNA using Northern blot and slot blot analyses. The relative amounts of SERCA2 mRNA were decreased with respect to GAPDH mRNA and 28 S rRNA in experimental failing hearts at 4 and 8 weeks post myocardial infarction by about 20% whereas those at 16 weeks declined by about 35% of control values. The results obtained by Western blot analysis, revealed that the immunodetectable levels of SERCA2 protein in 8 and 16 weeks postinfarcted animals were decreased by about 20% and 30%, respectively. The left ventricular SR Ca²⁺-pump ATPase specific activity was depressed in the SR preparations of failing hearts as early as 4 weeks post myocardial infarction and declined by about 65% at 16 weeks compared to control. These results indicate that the depressed SR Ca²⁺-pump ATPase activity in CHF may partly be due to decreased steady state amounts of SERCA2 mRNA and SERCA2 protein in the failing myocardium.     

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.     

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.     

The interaction between ruthenium red and the isolated sarcoplasmic reticulum

The multivalent anions, ATP and oxalate, present at 5mM concentrations in incubation mixtures with isolated sarcoplasmic reticulum (SR) preparations, reduce the binding of ruthenium red (RR) to the SR. When oxalate is omitted from the incubation mixture and ATP is used at reduced concentrations, it is possible to observe an inhibitory effect of RR on calcium uptake by the SR and on ATPase activity. However, this inhibition is only partial and it remains clear that calcium transport in the SR is much less susceptible to inhibition by RR than is calcium transport in mitochondria. The effect of multivalent anions in suppressing the effect of RR, a hexavalent cation, is probably due to the formation of soluble complexes with RR.     

Phosphorylation of a 100 000 dalton component and its relationship to calcium transport in sarcoplasmic reticulum from rabbit skeletal muscle

Sarcomplasmic reticulum from rabbit fast skeletal muscle contains intrinsic protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) and a substrate. The protein kinase activity was Mg2+ dependent and could also phosphorylate exogenous protein substrates. Autophosphorylation of sarcoplasmic reticulum vesicles was not stimulated by cyclic AMP, neither was it inhibited by the heat-stable protein kinase inhibitor protein. The phosphorylated membranes had the characteristics of a protein with a phosphoester bond. An average of 73 pmol Pi/mg protein were incorporated in 10 min at 30 degrees C. Addition of exogenous cyclic AMP-dependent protein kinase increased the endogenous level of phosphorylation by 25-100%. Sarcoplasmic reticulum membrane phosphorylation, mediated by either endogenous cyclic AMP-independent or exogenous cyclic AMP-dependent protein kinase, occurred on a 100 000 dalton protein and both enzyme activities resulted in enhanced calcium uptake and Ca2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3), in a manner similar to cardiac microsomal preparations. Regulation of Ca2+ transport in skeletal sarcoplasmic reticulum may be mediated by phosphorylation of a 100 000 dalton component of these membranes.     

Overexpression of SERCA2b in the heart leads to an increase in sarcoplasmic reticulum calcium transport function and increased cardiac contractility

The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca(2+) affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately approximately 20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calcium-dependent calcium uptake measurements showed that the maximal velocity of Ca(2+) uptake was not changed, but the apparent pump affinity for Ca(2+) (K(0.5)) was increased in SERCA2b transgenic mice (0.199 +/- 0.011 micrometer) compared with wild-type control mice (0.269 +/- 0.012 micrometer, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.     

Pharmacological intervention in oxidant-induced calcium pump dysfunction of dog heart

Micromolar concentrations of HOCl, an oxidant produced by activated neutrophils, inhibited Ca2+ uptake and Ca2+ATPase of isolated dog heart sarcoplasmic reticulum (SR). DTT antagonized completely the HOCl effect only when it was given within 5 min after the addition of HOCl. When the pharmacological intervention was delayed, the recovery with DTT was not complete, and administration of DTT 30 min after the start of HOCl's reaction with SR resulted in only a small improvement in SR Ca2+ uptake. Although H2O2 and Fe ion-chelate (a free radical-generating procedure) also inhibited Ca2+ uptake and ATPase, the concentrations required were very large. The response of cardiac sarcolemmal and skeletal muscle SR calcium pumps to oxidants was similar to that of the cardiac SR calcium pump.     

Seasonal variations in the rate and capacity of cardiac SR calcium accumulation in a hibernating species.

The rate of calcium uptake and the level of calcium accumulation was measured in cardiac muscle SR from hibernating and nonhibernating Richardson's ground squirrels. In whole heart homogenates, the rate of calcium uptake was higher (P less than 0.05) in hibernating animals than it was in active animals. Further purification of homogenates into sacroplasmic reticulum (SR) preparations showed that the hibernating animals had the highest rate of calcium uptake and the greatest level of calcium accumulation. These results could not be explained by variations in non-SR membrane contaminants nor by changes in the maximal activity or total amount of a SR marker enzyme, the Ca(2+)-ATPase. The addition of ryanodine to the calcium uptake medium increased the level of calcium accumulation in all groups by a similar amount. It is concluded that the high rate of calcium uptake by isolated cardiac SR vesicles from hibernating ground squirrels reflects the activity of the organelle in vivo, and that the ability of the ryanodine-insensitive population of SR vesicles to accumulate calcium is affected by hibernation.