Phosphorylation of purified bovine cardiac sarcolemma and potassium-stimulated calcium uptake

Sarcolemmal vesicles were prepared from bovine cardiac muscle by differential and discontinuous sucrose density gradient centrifugation. Na+/K+-ATPase was purified 33-fold to a specific activity of 53 +/- 0.5 (12) mumol Pi X mg-1 X h-1, binding sites for strophantin 20-fold to a density of 56.3 +/- 5.3 (14) pmol/mg and that for the calcium antagonist nitrendipine 5.5-fold to a density of 0.72 +/- 0.07 (6) pmol/mg. The specific activity of the Na+/Ca2+ exchanger was 61.1 +/- 3.7 (6) nmol/mg. The vesicles had an intravesicular volume of 20 +/- 4 (4) microliter/mg and 56.9 +/- 6 (4)% of the vesicles were right-side-out oriented. Several peptides of the purified membranes were phosphorylated in the presence of Mg . ATP and EGTA. Most of the radioactive phosphate was incorporated into a peptide with an apparent molecular mass of 22 kDa. Denaturation of the membranes at 100 degrees C changed the mobility of this peptide to 15 kDa and 11 kDa. This peptide could not be distinguished from a sarcoplasmic reticulum peptide of similar molecular mass. The phosphorylation of the sarcolemmal peptide was stimulated by Ca2+/calmodulin, cAMP and the catalytic subunit of cAMP-dependent protein kinase. A comparison of the phosphorylation of sarcolemmal membranes with that of sarcoplasmic reticulum showed that Ca2+/calmodulin stimulated in each membrane, the phosphorylation of the 22-kDa peptide and a 44-kDa peptide, and in the sarcoplasmic reticulum the phosphorylation of an additional peptide of 55-kDa. Ca2+/calmodulin-dependent phosphorylation of a 55-kDa peptide could not be demonstrated in sarcolemma, regardless if sarcolemmal membranes were incubated together with sarcoplasmic reticulum or if the phosphorylation was carried out in the presence of purified cardiac myosin light chain kinase or phosphorylase kinase. 'Depolarization' induced Ca2+ uptake which was measured according to Bartschat, D.K., Cyr, D.L. and Lindenmayer, G.E. [(1980) J. Biol. Chem. 255, 10044-10047] was 5 nmol/mg protein. This uptake was not enhanced after preincubation of the vesicles with Mg . ATP or Mg . ATP and cAMP-dependent protein kinase. The value of 5 nmol/mg protein is in agreement with the theoretical amount of Ca2+ which can be accumulated by the bovine cardiac sarcolemma in the absence of a driving force other than the Ca2+ gradient. The potassium-stimulated Ca2+ uptake was not blocked by the organic Ca2+ channel blockers. Prolonged incubation of Mg . ATP with sarcolemmal vesicles in the presence of various ATPase inhibitors led to the hydrolysis of ATP. The liberated phosphate precipitated with Ca2+ in the presence of LaCl3. These precipitates amounted to an apparent Ca2+ uptake ranging from 50 to over 1000 nmol/mg. The results suggest that potassium-stimulated Ca2+ uptake of bovine cardiac sarcolemmal vesicles is not enhanced in the presence of ATP or by phosphorylation of a 22-kDa peptide.     

Ionized and bound calcium inside isolated sarcoplasmic reticulum of skeletal muscle and its significance in phosphorylation of adenosine triphosphatase by orthophosphate.

Calcium loading of skeletal muscle sarcoplasmic reticulum performed passively by incubation with high calcium concentrations (0.5--15 mM) on ice gives calcium loads of 50--60 nmol/mg sarcoplasmic reticulum protein. This accumulated calcium is not released by EGTA [ethyleneglycol bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid], but almost completely released by ionophore X-537A plus EGTA or phospholipase A plus EGTA treatment and is therefore assumed to be inside the sarcoplasmic reticulum. This calcium is distributed in one saturable and one non-saturable calcium compartment, as derived from the dependence of the calcium load on the calcium concentration in the medium. These compartments are assigned to bound and ionized calcium inside the sarcoplasmic reticulum, respectively. Maximum calcium binding under these conditions was 33 nmol/mg protein with an apparent half-saturation constant of 5,8 nmol/mg free calcium inside, or between 1.2 and 0.6 mM free calcium inside, assuming an average vesicular water space of 5 or 10 microliter/mg protein, respectively. Calcium-dependent phosphorylation of sarcoplasmic reticulum calcium-transport ATPase from orthophosphate depends on the square of free calcium inside, whilst inhibition of phosphorylation depends on the square of free calcium in the medium. Calcium-dependent phosphorylation appears to be determined by the free calcium concentrations inside or outside allowing calcium binding to the ATPase according to the two classes of calcium binding constants for low affinity calcium binding or high affinity calcium binding, respectively. It is further suggested that the saturation of the low-affinity calcium-binding sites of the ATPase facing the inside of the sarcoplasmic reticulum membrane is responsible for the greater apparent orthophosphate and magnesium affinity in calcium-dependent phosphorylation than in calcium-independent phosphorylation from orthophosphate. Maximum calcium-dependent phosphoprotein formation at 20 degrees C and pH 7.0 is about 4 nmol/mg sarcoplasmic reticulum protein.     

Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum

The mycotoxin, cyclopiazonic acid (CPA), inhibits the Ca2+-stimulated ATPase (EC 3.6.1.38) and Ca2+ transport activity of sarcoplasmic reticulum (Goeger, D. E., Riley, R. T., Dorner, J. W., and Cole, R. J. (1988) Biochem. Pharmacol. 37, 978-981). We found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase. Cyclopiazonic acid caused similar inhibition of the Ca2+-stimulated ATP hydrolysis in intact sarcoplasmic reticulum and in a purified preparation of Ca2+-ATPase. Cyclopiazonic acid also inhibited the Ca2+-dependent acetylphosphate, p-nitrophenylphosphate and carbamylphosphate hydrolysis by sarcoplasmic reticulum. ATP protected the enzyme in a competitive manner against inhibition by CPA, while a 10(5)-fold change in free Ca2+ concentration had only moderate effect on the extent of inhibition. CPA did not influence the crystallization of Ca2+-ATPase by vanadate or the reaction of fluorescein-5'-isothiocyanate with the Ca2+-ATPase, but it completely blocked at concentrations as low as 1-2 mol of CPA/mol of ATPase the fluorescence changes induced by Ca2+ and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in FITC-labeled sarcoplasmic reticulum and inhibited the cleavage of Ca2+-ATPase by trypsin at the T2 cleavage site in the presence of EGTA. These observations suggest that CPA interferes with the ATP-induced conformational changes related to Ca2+ transport. The effect of CPA on the sarcoplasmic reticulum Ca2+-ATPase appears to be fairly specific, since the kidney and brain Na+,K+-ATPase (EC 3.6.1.37), the gastric H+,K+-ATPase (EC 3.6.1.36), the mitochondrial F1-ATPase (EC 3.6.1.34), the Ca2+-ATPase of erythrocytes, and the Mg2+-activated ATPase of T-tubules and surface membranes of rat skeletal muscle were not inhibited by CPA, even at concentrations as high as 1000 nmol/mg protein.     

GM1对肌质网Ca~(2+)-ATPase活性及膜流动性的影响

外源性GM1对肌质网Ca2+-ATPase的水解及转运活性都有明显的抑制作用.在GM1浓度为0～8nmol/mg蛋白质范围内抑制作用具有浓度依赖性.当GM1浓度达到8nmol/mg蛋白质时,酶活性受到最大抑制,此时水解活性降低51%,转运活性降低49%.荧光偏振测定结果表明:GM1参入后,肌质网膜流动性降低.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca²⁺ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37°C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 μM CaCl₂, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca²⁺ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca²⁺ uptake, a second phase of Ca²⁺ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca²⁺ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca²⁺ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca²⁺ influx of sarcoplasmic reticulum near steady state of Ca²⁺ uptake was measured by pulse labeling with ⁴⁵Ca²⁺. The Ca²⁺ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca²⁺ uptake in normal medium, Ca²⁺ influx was balanced by Ca²⁺ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca²⁺ exchange rate at the first plateau of Ca²⁺ uptake was about half of that in normal medium. When the second phase of Ca²⁺ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca²⁺ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 μg/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca²⁺ uptake was also observed. These data suggest that the Ca²⁺ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca²⁺ efflux, which subsequently stimulates Ca²⁺ exchange.     

Structural and functional properties of a Ca2+-ATPase from human platelets

An antibody prepared against highly purified rabbit muscle Ca2+-ATPase from sarcoplasmic reticulum has been observed to cross-react with proteins in human platelet membrane vesicles. The antibody specifically precipitated Ca2+-ATPase activity from solubilized human platelet membranes and recognized two platelet polypeptides denatured in sodium dodecyl sulfate with Mr = 107,000 and 101,000. Ca2+-ATPase activity from Brij 78-solubilized platelet membranes was purified up to 10-fold. The purified preparation consisted mainly of two polypeptides with Mr approximately 100,000, and 40,000. The lower molecular weight protein appeared unrelated to Ca2+-ATPase activity. The Ca2+-ATPase in human platelet membrane vesicles exhibited "negative cooperativity" with respect to the kinetics of ATP hydrolysis. The apparent Km for Ca2+ activation of ATPase activity was 0.1 microM. Ca2+-dependent phosphorylation of platelet vesicles by [gamma-32P]ATP at 0 degrees C yielded a maximum of 0.2-0.4 nmol of PO4/mg of protein that was labile at pH 7.0 and 20 degrees C. This result suggests that only about 2-4% of the total protein in platelet membrane vesicles is the Ca2+-ATPase, which agrees with an estimate based on the specific activity of the Ca2+-ATPase in platelet membranes (20-50 nmol of ATP hydrolyzed/min/mg of protein at 30 degrees C). Calmodulin resulted in only a 1.6-fold stimulation of Ca2+-ATPase activity even after extensive washing of membranes with a calcium chelator or chlorpromazine. It is concluded that human platelets contain a Ca2+-ATPase immunochemically related to the Ca2+ pump from rabbit sarcoplasmic reticulum and that the enzymatic characteristics and molecular weight of the platelet ATPase are quite similar to those of the muscle ATPase.     

Stoichiometric and electrostatic characterization of calcium binding to native and lipid-substituted adenosinetriphosphatase of sarcoplasmic reticulum

The stoichiometry of calcium binding to specific sites (i.e., those producing enzyme activation) was found to be 8-10 nmol/mg protein in native sarcoplasmic reticulum vesicles, and 13.9-15.4 nmol/mg of ATPase purified by non-ionic detergent solubilization and anion exchange chromatography. Parallel measurements of phosphoenzyme yielded levels of 4.0-4.9 and 6.0-7.7 nmol/mg of protein in the two preparations, respectively, demonstrating that each 115 kDa ATPase chain includes one catalytic site and two calcium binding sites. The apparent association constant, K = (6 +/- 2) X 10(5) M-1, and the binding cooperativity, nH = 1.9, were unchanged when measurements were carried out with native sarcoplasmic reticulum vesicles and when the membrane surface charge was altered by lipid substitution with phosphatidylcholine or phosphatidylserine, at neutral pH in the presence of 10 mM MgCl2 and 80 mM KCl. On the other hand, the apparent association constant was increased in the absence of Mg2+ or, to a lesser extent, in the absence of monovalent cations. It was also observed that the cooperative character of the calcium binding isotherms was reduced in low ionic-strength media. Analysis of the electrostatic effects indicates that the calcium-binding domain is shielded from the membrane phospholipid surface charge by virtue of its location within the ATPase protein. The effects of various electrolytes are attributed to monovalent-cation binding in the calcium-binding domain. The apparent loss of cooperativity of the calcium binding isotherms at low ionic strength is attributed to a progressive displacement of the titration curve which is minimal at low degrees of saturation and becomes larger at higher degrees of saturation. This behavior is described quantitatively by the progressive effect of calcium binding on an electrostatic potential generated by localized protein charge densities within, or near, the calcium-binding domain.     

Isolation of calcium pump system and purification of calcium ion-dependent ATPase from heart muscle.

The procedure for the isolation of the highly active fraction of sarcoplasmic reticulum from pigeon and dog hearts is described. The method is based on the partial loading of heart microsomes with calcium and oxalate ions and the precipitation of loaded vesicles in sucrose and potassium chloride concentration gradients. Preparations obtained possess high activity of Ca2+-dependent ATPase and are also able to accumulate up to 10 mumol Ca2+ per mg protein. Purification of sarcoplasmic reticulum membranes is accompanied by a decrease in concentration of cytochrome a+a3 and an increase in the content of [32P]phosphoenzyme. The basic components in "calcium-oxalate preparation" from hearts are proteins with molecular weights of about 100000 (Ca2+-dependent ATPase) and 55000 Calcium-oxalate preparation from pigeon hearts was used for subsequent purification of Ca2+-dependent ATPase. Specific activity of purified enzyme from pigeon hearts is 12-16 mumol Pi/min per mg protein. Enzyme activity of purified Ca2+-dependent ATPase is inhibited by EGTA and is not sensitive to azide, 2,4-dinitrophenol and ouabain. The data obtained demonstrate the similarity of calcium pump systems and Ca2+-dependent ATPases isolated from heart and skeletal muscles.     

Interaction of fluorescein isothiocyanate with the (H+ + K+)-ATPase

Fluorescein isothiocyanate was used to covalently label the gastric (H⁺ + K⁺)-ATPase. FITC treatment of the enzyme inhibited the ATPase activity while largely sparing partial reactions such as the associated $\text{p-}\text{nitrophenylphosphatase}$ activity. ATP protected against inhibition suggesting the ligand binds at or near an ATP binding site. At 100% inhibition the stoichiometry of binding was 1.5 nmol FITC per mg Lowry protein a value corresponding to maximal phosphoenzyme formation. Binding occurred largely to a peptide of 6.2 isoelectric point, although minor labelling of a peptide of $\text{p}\text{I}$ 5.6 was also noted. Fluorescence was quenched by K⁺, Rb⁺ and Tl⁺ in a dose-dependent manner, and the $\text{K}{}_{0.5}$ values of 0.28, 0.83 and 0.025 mM correspond rather well to the values required for dephosphorylation at a luminal site. Vanadate, a known inhibitor of the gastric ATPase produced a slow Mg²⁺-dependent fluorescent quench. Ca²⁺ reversed the K⁺-dependent loss of fluorescence and inhibited it when added prior to K⁺. This may relate to the slow phosphorylation in the presence of ATP found when Ca²⁺ was substituted for Mg²⁺ and the absence of K⁺-dependent dephosphorylation. The results with FITC-modified gastric ATPase provide evidence for a conformational change with K⁺ binding to the enzyme.     

Stoichiometric photolabeling of two distinct low and high affinity nucleotide sites in sarcoplasmic reticulum ATPase

Highly purified 3'-arylazido-ATP (aATP) was obtained by high performance liquid chromatography. In the dark, this photoactivatable ATP analog was a competitive inhibitor of ATP hydrolysis catalyzed by purified sarcoplasmic reticulum (SR) ATPase with a Ki of 10 microM. The analog itself was hydrolyzed by the enzyme in the dark. A biphasic curve of velocity of hydrolysis of the analog versus aATP concentration was obtained, indicating the presence of high and low affinity sites with K0.5 of approximately 10 microM and 300 microM, respectively. Upon irradiation with visible light, a biphasic curve was obtained for the level of covalent photolabeling of the enzyme versus [beta-32P]aATP concentrations. Levels of 6.5-9 nmol of analog/mg of protein and 20-22 nmol of analog/mg of protein were obtained when labeling with 20-30 or with 400 microM aATP, respectively, showing the existence of 1 mol of high affinity sites/mol of ATPase and 1-1.5 mol of low affinity sites/mol of enzyme. The rate of light-dependent incorporation of [beta-32P]aATP was decreased by the presence of ATP, Pi, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene-ATP, or Ca2+ in the illumination media. Photolabeling of the high affinity sites had little effect on the velocity of ATP hydrolysis but significantly inhibited the splitting of additional aATP added in the dark. Photolabeling the low affinity sites caused irreversible inhibition of the ATPase activity. The inhibition was prevented by having ATP in the illumination medium, which protected it from labeling. Gel filtration chromatography in the presence of detergent showed that radioactive photolabel was incorporated in the SR ATPase protein. The results indicate that aATP is a useful tool for stoichiometrically labeling and probing the nucleotide binding domains of the SR ATPase.     

Myocardial Ca-sequestration failure and compensatory increase in Ca-ATPase with congestive cardiomyopathy: kinetic characterization by a homogenate microassay using real-time ratiometric indo-1 spectrofluorometry

A novel, simple, rapid and reproducible microassay is used for kinetic analysis of Ca-sequestration by homogenates of myocardium of turkeys with furazolidone-induced congestive cardiomyopathy. The assay monitors Ca in real-time using dual-emission ratiometric spectrofluorometry and the Ca-indicator dye indo-1. Using this assay and isolated SR studies we make several novel findings regarding the mechanism of SR failure in furazolidone cardiomyopathy.Qualitative differences in Ca-sequestration were not detected between groups. However, compared to controls the furazolidone treatment resulted in: 1) 50% depression in maximal activities (1.54 ± 0.36 vs 0.73 ± 0.12 µM/sec); 2) 2-fold increases in post-sequestration concentrations of ionized Ca(79 ± 23 vs 141 ±13 nmol Ca/L homogenate); 3) 2-fold increases in Ca half-life (415 vs 790 msec); and 4) 25% increased passive Ca-binding capacity of homogenates. The Ca-ATPase specific activity of isolated sarcoplasmic reticulum was 60% increased in congestive cardiomyopathy (543 ± 140 vs 873 ± 108 nmol ATP hydrolyzed/min/mg membrane protein) although membrane yield was 20% decreased (0.79 ± 0.09 vs 0.63 ± 0.03 mg/g heart). The increased ATPase and decreased Ca-uptake activities in combination with the occurrence of 36% cardiac hypertrophy and 19% decreased body weights resulted in estimates of the relative energy cost to the animal for myocardial Ca transport being 5.5-fold increased with cardiomyopathy (20.5 vs 111 nmol ATP hydrolyzed per µM decrease of sarcoplasmic free Ca/kg body weight).These data indicate that congestive cardiomyopathy is associated with markedly increased permeability of sarcoplasmic reticulum to Ca and compensatorily increased Ca-ATPase activity. Accelerated energy consumption due to the increased energy cost of Ca transport and increased time of myocyte activation are predicted to predispose the myocardium to fatigue and irreversible failure.     

Highly purified sarcoplasmic reticulum vesicles are devoid of Ca2+-independent (‘basal’) ATPase activity

On solubilization with Triton X-100 of sarcoplasmic reticulum vesicles isolated by differential centrifugation, the Ca²⁺-ATPase is selectively extracted while approximately half of the initial Mg²⁺-, or ‘basal’, ATPase remains in the Triton X-100 insoluble residue. The insoluble fraction, which does not contain the 100 000 dalton polypeptide of the Ca²⁺-ATPase, contains high levels of cytochrome c oxidase. Furthermore, its Mg²⁺-ATPase activity is inhibited by specific inhibitors of mitochondrial ATPase, indicating that the ‘basal’ ATPase separated from the Ca²⁺-ATPase by detergent extraction originates from mitochondrial contaminants.To minimize mitochondrial contamination, sarcoplasmic reticulum vesicles were fractionated by sedimentation in discontinuous sucrose density gradients into four fractions: heavy, intermediate and light, comprising among them 90–95% of the initial sarcoplasmic reticulum protein, and a very light fraction, which contains high levels of Mg²⁺-ATPase. Only the heavy, intermediate and light fractions originate from sarcoplasmic reticulum; the very light fraction is of surface membrane origin. Each fraction of sarcoplasmic reticulum origin was incubated with calcium phosphate in the presence of ATP and the loaded fractions were separated from the unloaded fractions by sedimentation in discontinuous sucrose density gradients. It was found that vesicles from the intermediate fraction had, after loading, minimal amounts of mitochondrial and surface membrane contamination, and displayed little or no Ca²⁺-independent basal ATPase activity. This shows conclusively that the basal ATPase is not an intrinsic enzymatic activity of the sarcoplasmic reticulum membrane, but probably originates from variable amounts of mitochondrial and surface membrane contamination in sarcoplasmic reticulum preparations isolated by conventional procedures.     

Calcium gradient-dependent and calcium gradient-independent phosphorylation of sarcoplasmic reticulum by orthophosphate. The role of magnesium.

Phosphorylation of the calcium-transport ATPase of skeletal muscle sarcoplasmic reticulum by inorganic phosphate was investigated in the presence or absence of a calcium gradient. The maximum phosphoprotein formation in the presence of a calcium gradient at 20 degrees C and pH 7.0 is approximately 4 nmol/mg sarcoplasmic reticulum protein, but only between 2.4 and 2.8 nmol/mg protein in the absence of a calcium gradient, using Ionophore X-537 A or phospholipase-A-treated sarcoplasmic reticulum vesicles. Maximum phosphoprotein formation independent of calcium gradient at 20 degrees C and pH 6.2 is in the range of 3.6--4 nmol/mg protein. Half-maximum phosphoprotein formation dependent on calcium gradient was achieved with 0.1--0.2 mM free orthophosphate at 10 mM free magnesium or at 0.1--0.2 mM free magnesium at 10 mM free orthophosphate. Phosphoprotein formation independent of calcium gradient is in accordance with a model which assumes, firstly, the formation of a ternary complex of the ATPase protein with orthophosphate and magnesium (E . Pi . Mg) in equilibrium with the phosphoprotein (E-Pi . Mg) and, secondly, an interdependence of both ions in the formation of the ternary complex. The apparent equilibrium constant was 0.6 and the apparent dissociation constants KMg, KMg', KPi and KPi' were 8.8, 1.9, 7.2 and 1.5 mM respectively, assuming a total concentration of the phosphorylation site per enzyme of 7 nmol/mg protein.     

Stoichiometry and mapping of the nucleotide sites in sarcoplasmic reticulum ATPase with the use of UTP

Purified sarcoplasmic reticulum ATPase was phosphorylated by either ATP or UTP under otherwise identical conditions. Calcium, pH, and nucleotide concentrations were adjusted to permit maximal steady-state accumulation of phosphoenzyme (EP). Either 4 or 8.5 nmol of EP/mg of protein were obtained with ATP or UTP, respectively. Tryptic digestion of phosphorylated ATPase followed by acid gel electrophoresis showed that EP from UTP was on fragment A1, similar to the report in the literature for EP from ATP. Phosphorylation with Pi in the absence of calcium gave EP levels similar to those obtained from UTP. Thus, comparison of EP levels from different substrates measured in parallel in the same preparation reveal that with ATP half of the sites are phosphorylated. Illumination of the ATPase with UV light in the presence of [3H]UTP caused photolabeling of the ATPase at a maximal level of 1 nmol of [3H]UTP incorporated/mg of ATPase. The UTP concentration dependence for photolabeling was the same as that for promoting catalysis. ATP when present in the illumination protected with a competitive pattern against photolabeling with UTP. Tryptic digestion and autoradiography of photolabeled ATPase revealed that UTP was covalently attached to tryptic fragment A2. The data indicate that a peptide sequence of fragment A2 is involved in the binding of the nucleoside moiety of UTP and possibly belongs to the nucleotide domain of the ATPase in addition to the sequence of fragment A1 which contains the phosphorylation residue.     

Transmembranous organization of (Ca2(+)-Mg2+)-ATPase from sarcoplasmic reticulum. Evidence for lumenal location of residues 877-888

An antipeptide antibody was produced against a peptide corresponding to residues 877-888 of fast twitch rabbit sarcoplasmic reticulum ATPase. This antipeptide antibody bound strongly to the ATPase in sarcoplasmic reticulum vesicles only after the vesicles had been solubilized with the detergent C12E8 indicating that its epitope was located in the lumen of the sarcoplasmic reticulum. Digestion of sarcoplasmic reticulum or purified (Ca2(+)-MG2+)-ATPase by proteinase K for up to 1 h resulted in a stable ATPase fragment of 30 kDa containing the epitope for the above antibody and the epitope for an antibody directed against the C terminus. Further proteolysis revealed smaller fragments (Mr 19,000 and 13,000) containing both epitopes. By contrast, small fragments of the ATPase (less than 29 kDa) containing the N-terminal epitope were not observed even after short exposures to proteinase K. These data support the view that the (Ca2(+)-MG2+)-ATPase has 10 transmembranous helices.     

Chemical modification of an arginine residue in the ATP-binding site of Ca2+-transporting ATPase of sarcoplasmic reticulum by phenylglyoxal

Phenylglyoxal (PGO) was used as a reagent for chemical modification of the ATP-binding site of Ca2+-transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum (SR-ATPase). When 1 mM PGO was reacted with SR-ATPase at 30°C at pH 8.5, PGO was bound to the ATPase molecule in two-to-one stoichiometry with concomitant loss of activity of the ATPase to form the phosphorylated intermediate (E-P). ATP and ADP prevented the binding of PGO and thereby protected the enzyme from inactivation. The SR membranes were labeled with [14C]PGO and then digested with pepsin to identify the attachment site of PGO. A 14C-labeled peptide (402lle-Arg*-Ser-Gly-Gln406) was purified to homogeneity by C18-reversed phase HPLC (Arg* denotes the binding site of [14C]PGO). These results indicate that Arg403 is located in the ATP binding site of the SR-ATPase.     

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。     

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.     

Fourier transform infrared spectroscopic studies of lipid-protein interaction in native and reconstituted sarcoplasmic reticulum

Fourier transform infrared spectroscopy has been used to monitor lipid-protein interaction and protein secondary structure in native and reconstituted sarcoplasmic reticulum vesicles. Studies of the temperature dependence of the CH₂ symmetric stretching frequency reveal no cooperative phase transitions in purified sarcoplasmic reticulum or in vesicles reconstituted with dioleoylphosphatidylcholine, although a continuous introduction of disorder into the lipid acyl chains is observed as the temperature is raised. In addition, temperature-dependent changes are observed in the Amide I and Amide II vibrations arising from protein peptide bonds. A comparison of lipid order in native sarcoplasmic reticulum and its lipid extract showed that the introduction of protein is accompanied by a slight increase in lipid order. Reconstitution of Ca²⁺-ATPase from sarcoplasmic reticulum with dipalmitoylphosphatidylcholine (lipid/protein ratio 30:1), reveals a perturbed lipid melting event broadened and reduced in midpoint temperature from multilamellar lipid vesicles. The onset of melting (27–28°C) correlates well with the onset of ATPase activity and confirms a suggestion (Hesketh, T.R., Smith G.A., Houslay M.D., McGill, K.A., Birdsall, N.J.M., Metcalfe, J.C. and Warren, G.B. (1976) Biochemistry 15, 4145–4151) that a liquid crystalline environment is a requirement for optimal protein function. Finally, Ca²⁺-ATPase has been reconstituted into binary lipid mixtures of DOPC and acyl-chain perdeuterated DPPC. The effect of protein on the structure and melting behavior of each lipid component was monitored. The protein appears to preferentially interact with the DOPC component.     

Isolation of calcium pump system and purification of calcium ion-dependent ATPase from heart muscle

The procedure for the isolation of the highly active fraction of sarcoplasmic reticulum from pigeon and dog hearts is described. The method is based on the partial loading of heart microsomes with calcium and oxalate ions and the precipitation of loaded vesicles in sucrose and potassium chloride concentration gradients. Preparations obtained possess high activity of Ca²⁺-dependent ATPase and are also able to accumulate up to 10 μmol Ca²⁺ per mg protein. Purification of sarcoplasmic reticulum membranes is accompanied by a decrease in concentration of cytochrome a+a₃ and an increase in the content of [³²P]phosphoenzyme. The basic components in “calcium-oxalate preparation” from hearts are proteins with molecular weights of about 100 000 (Ca²⁺-dependent ATPase) and 55 000 Calcium-oxalate preparation from pigeon hearts was used for subsequent purification of Ca²⁺-dependent ATPase. Specific activity of purified enzyme from pigeon hearts is 12–16 μmol P_i/min per mg protein. Enzyme activity of purified Ca²⁺-dependent ATPase is inhibited by EGTA and is not sensitive to azide, 2,4-dinitrophenol and ouabain. The data obtained demonstrate the similarity of calcium pump systems and Ca²⁺-dependent ATPases isolated from heart and skeletal muscles.