Studies on (Na+ + K+)-activated ATPase. XXXVIII. A 100 000 molecular weight protein as the low-energy phosphorylated intermediate of the enzyme.

Phosphorylation of NaI-treated bovine brain cortex microsomes by inorganic phosphate in the presence of Mg2+ and ouabain has been studied at 0 degrees C (pH 7.4) and 20 degrees C (pH 7.0). Nearly maximal (90%) and half-maximal phosphorylation are achieved at 20 degrees C within 2 min with 50--155 and 5.6--17 muM 32Pi, respectively, and at 0 degrees C within 75 s with 300--600 and 33--66 muM 32Pi, respectively. Maximal phosphorylation yields 146 pmol 32P - mg-1 protein. Without ouabain (20 degrees C, pH 7.0) less than 25% of the incorporation observed in the presence of ouabain is reached. Preincubation of the native microsomes with Mg2+ and K+, in order to decompose possibly present high-energy phosphoryl-bonds prior to ouabain treatment, does not affect the maximal phosphate incorporation. This indicates that the inorganic phosphate incorporation is not due to an exchange with high-energy phosphoryl-bonds, which might have been preserved in the microsomal preparations. Phosphorylation of the native microsomes by ATP in the presence of Mg2+ and Na+ reaches 90 and 50% maximal levels within 15--30 s at 0 degrees C and pH 7.4 at concentrations of [gamma-32P]ATP of 5--32 and 0.5--3.5 muM, respectively. The maximal phosphorylation level is 149 pmol 32P-mg-1 protein, equal to that of ouabain-treated microsomes phosphorylated by inorganic phosphate. Both inorganic phosphate and ATP phosphorylate on site per active enzyme subunit of 135 000 molecular weight. From the equilibrium constants for the phosphorylation of ouabain-treated microsomes by inorganic phosphate at 0 degrees C and 20 degrees C standard free-energy changes of --5.4 and --6.8 kcal/mol, respectively, are calculated. These values yield a standard enthalpy change of 14 kcal/mol and an entropy change of 70 cal/mol - degree K. This characterizes the reaction as a process driven by an entropy change. The intermediate formed by phosphorylation with Pi has maximal stability at acidic pH, as is the case for the intermediate formed with ATP. Solubilization in sodium dodecyl sulfate stabilizes the phosphoryl-bond in the pH range of 4--7. The non-solubilized preparation has optimal stability at pH 2--4, the level of which is equal to that of detergent-solubilized intermediate. Sodium dodecyl sulfate gel electrophoresis of the microsomes at pH 3, following incorporation of 32Pi yields 11 protein bands, only one of which (mol. wt 100 000--106 000) carries the radioactive label. This protein has the same molecular weight as the protein, which is phosphorylated by ATP in the presence of Mg2+ and Na+.     

Difference in kinetic properties of phosphorylated intermediates formed in the forward and backward reactions of solubilized Ca2+-ATPase of sarcoplasmic reticulum

Solubilized Ca2+-ATPase (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The kinetic properties of the phosphorylated intermediate (EP) formed by the reaction of SSR with ATP were compared with those of EP formed by the reaction with Pi. The time course of decay of E32P formed with 4 microM AT32P in the presence of 19 mM CaCl2 and 10 mM MgCl2 (forward reaction) was measured by adding 0.4 mM unlabeled ATP and 10 mM Pi at pH 6.0 and 30 degrees C. The rate of E32P decay was accelerated by 0.4 mM ADP. On the other hand, when the time course of decay of E32P formed with 10 mM 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2 (backward reaction) was measured by adding 0.4 mM unlabeled ATP and 15 mM CaCl2, the rate of E32P decay was unaffected by 0.4 mM ADP. AT32P was produced on adding ADP to E32P formed with AT32P in the presence of 10 mM CaCl2 and 10 mM MgCl2, while no AT32P was produced on adding ADP to E32P formed with 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2, even when 15 mM CaCl2 was added simultaneously with ADP.     

Evidence for two α-amanitin-resistant RNA polymerases in vegetative amoebae of Dictyostelium discoideum

Three DNA-dependent RNA polymerases (EC 2.7.7.6), P-I, P-II and P-III, have been isolated from the sonicated nuclear extract of vegetative amoebae of $\text{Dictyostelium discoideum}$ by phosphocellulose chromatography. P-I was inhibited by α-amanitin, while P-II and P-III were not. Rifampicin did not prevent all the polymerase activities. These polymerases were more active in the presence of Mg²⁺ than Mn²⁺. P-III was reduced in the enzyme activity by being passed through DEAE-Sephadex column and not obtained from the nuclear extract of amoebae at the culmination stage during morphogenetic development.     

Ca2+ translocation and catalytic activity of the sarcoplasmic reticulum ATPase. Modulation by ATP, Ca2+, and Pi

The ratio between Ca2+ uptake and Ca(2+)-dependent ATP hydrolysis measured in sarcoplasmic reticulum vesicles of rabbit skeletal muscle was found to vary greatly depending on the concentrations of oxalate or Pi used. In the presence of 5 mM oxalate, 20 mM Pi, and 1 mM Pi, the ratios found were in the range of 1.4-2.3, 0.6-0.8, and 0.01-0.10, respectively. The rates of Ca2+ exchange and ATP synthesis were measured at the steady state by adding trace amounts of 45Ca and 32Pi, after the vesicles had been loaded with Ca2+. In the presence of 1 mM Pi, 10 mM MgCl2, and 0.2 mM CaCl2, the ratio between Ca2+ exchange and ATP synthesis varied from 9 to 14. This ratio approached two when Ca2+ in the medium was reduced to a very low level, or when in the presence of Ca2+, dimethyl sulfoxide was added to the assay medium, or when the Pi concentration was raised from 1 to 20 mM. A ratio of two was also measured when the steady state was attained using ITP instead of ATP. In all the conditions that led to a ratio close to two, there was an increase in the fraction of enzyme phosphorylated by Pi. It is proposed that the coupling between Ca2+ translocation and ATP hydrolysis or synthesis is modulated by the phosphorylation of the ATPase by Pi.     

ATP reversible Pi exchange and membrane phosphorylation in sarcoplasmic reticulum vesicles: activation by silver in the absence of a Ca2+ concentration gradient.

The activation of ATP reversible Pi exchange, normally associated with a Ca2+ concentration gradient in sarcoplasmic reticulum vesicles, can be obtained in "leaky" vesicles in 4-10 mM CaCl2. In the micromolar range, Ag+ activates the ATP reversible Pi exchange two- to fourfold. Similar concentrations of Ag+ promote a parallel inhibition of Ca2+- activated ATP hydrolysis and Ca2+ uptake in intact vesicles. Maximal inhibition of these activities by Ag+ leaves the Mg2+-dependent ATPase unaffected. No net synthesis of ATP was demonstrated in leaky vesicles. The effects of Ag+ depends on the protein concentration and persist after removal of Ag+ from the medium. Membrane phosphorylation from Pi or from ATP is respectively activated or inhibited by Ag+ in reciprocal fashion.     

Modulation of 32Pi incorporation into phospholipids of polymorphonuclear leukocytes by ionophore A23187.

The effects of ionophore A23187 on the incorporation of 32Pi into phospholipids and on 45Ca2+ uptake and release by polymorphonuclear leukocytes were examined. A23187 increased 32Pi incorporation into phosphatidic acid, phosphatidylglycerol, phosphatidylserine, and the phosphoinositides. It also promoted a rapid burst uptake and release of 45Ca2+ by leukocytes. External Ca2+, but not Mg2+, was required for full stimulation of 32Pi incorporation into phosphatidic acid and the phosphoinositides. In the absence of external Ca2+, the increased radiophosphorus activity of phosphatidic acid, phosphatidylserine and the phosphoinositides was grossly reduced but not eliminated, and the decreased radiophosphorus activity of phosphatidylcholine became pronounced. In addition, the ionophore effect on 32Pi incorporation into leukocyte phospholipids was not abolished by ethyleneglycol bis(beta-amino-ethylether)-N,N'-tetraacetic acid. ATP radiophosphorus activity was also enhanced by the presence of A23187, but the enhancement was much less than that of the acidic phospholipids. Based on these findings, it is suggested that the increased 32Pi incorporation into the acidic phospholipids of leukocytes induced by A23187 was not solely derived from the higher radioactivity of ATP, increased Ca2+ fluxes and perturbation of cellular Ca2+ distribution of leukocytes exposed to A 23187 may trigger part of the altered 32Pi incorporation into phospholipids.     

Isolation of Alkaline and Neutral Proteases from Aspergillus flavus var. columnaris, a Soy Sauce Koji Mold

Two different extracellular proteases, protease I (P-I), an alkaline protease, and protease II (P-II) a neutral protease, from Aspergillus flavus var. columnaris were partially purified by using (NH(4))(2)SO(4) precipitation, diethylaminoethyl-Sephadex A-50 chromatography, carboxymethylcellulose CM-52 chromatography, and Sephadex G-100 gel filtration. The degree of purity was followed using polyacrylamide gel electrophoresis. The activity of P-I was completely inhibited by 0.1 mM phenylmethylsulfonyl fluoride, and that of P-II was completely inhibited by 1 mM ethylenediaminetetraacetate. By using these inhibitors with extracts of wheat bran koji, the proportions of total activity that could be assigned to P-I and P-II were 80 and 20%, respectively. This compared favorably with activities estimated by using polyacrylamide gel electrophoresis slices (82 and 18%, respectively). Extracts from factory-run soybean koji gave comparable results. Both enzymes demonstrated maximum activity at 50 to 55 degrees C and only small changes in activity between pH 6 and 11. For P-I, activity was somewhat higher from pH 8.0 to 11.0, whereas for P-II it was somewhat higher from pH 6 to 9. In the presence of 18% NaCl, the activities of both P-I and P-II dropped by approximately 90 and 85%, respectively. P-I was inferred to possess aminopeptidase activity since it could hydrolyze l-leucyl-p-nitroanilide hydrochloride. P-II was devoid of such activity. The ramifications of the results for factory-produced soy sauce koji are discussed.     

Inhibitory effect of glucose on the maturation of rat liver mitochondria at birth. Phospholipid and oxidative metabolism

(1) The rate of ATP synthesis coupled with succinate oxidation in rat liver mitochondria is low at birth and increases rapidly during the first postnatal hours (Nakazawa, T., Asami, K., Suzuki, H. and Yakawa, O. (1973) J. Biochem. 73, 397-406). A glucose injection given to newborn rats immediately after birth seemed to delay this maturation process. (2) Glucose administration specifically diminished the rate of 32Pi incorporation into phosphatidylcholine both in microsomes and in mitochondria while other phospholipids remained unaffected. (3) In newborn rat liver, 32Pi incorporation into phospholipids can be explained by de novo synthesis of phospholipids in microsomes followed by transfer to mitochondria with two exceptions phosphatidylserine and sphingomyelin. Indeed, after a 20-min incorporation of 32Pi into phospholipids, the specific radioactivity of phosphatidylserine and sphingomyelin was higher in mitochondria than in microsomes. (4) As far as phospholipid synthesis is concerned, no precursor-product relationship could be observed between light and heavy mitochondria.     

Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes.

Agents known to inphorylation of specific endogenous proteins in intact synaptosomes from rat brain. Synaptosome preparations, preincubated in vitro with 32Pi, incorporated 32P into a variety of specific proteins. Veratridine and high (60 mM) K+, which increase Ca2+ transport across membranes, through a mechanism involving membrane depolarization, as well as the calcium ionophore A23187, each markedly stimulated the incorporation of 32P into two specific proteins (80,000 and 86,000 daltons) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. All three agents failed to stimulate protein phosphorylation in calcium-free medium containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Moreover, the Ca2+-dependent protein phosphorylation could be reversed by the addition of sufficient EGTA to chelate all free extracellular Ca2+. Veratridine, high K+, and A23187 also stimulated 45Ca2+ accumulation by synaptosomes. Tetrodotoxin blocked the stimulation both of protein phosphorylation and of 45Ca2+ accumulation by veratridine but not by high K+ or A23187. Cyclic nucleotides and several putative neurotransmitters were without effect on protein phosphorylation in these intact synaptosome preparations. The absence of any endogenous protein phosphorylation in osmotically shocked synaptosome preparations incubated with 32Pi, and the inability of added [gamma-32P]ATP to serve as a substrate for veratridine-stimulated protein phosphorylation in intact preparations, indicated that the Ca2+-dependent protein phosphorylation occurred within intact subcellular organelles. Fractionation of a crude synaptosome preparation on a discontinuous Ficoll/sucrose flotation gradient indicated that these organelles were synaptosomes rather than mitochondria. The data suggest that conditions which cause an accumulation of calcium by synaptosomes lead to a calcium-dependent increase in phosphorylation of specific endogenous proteins. These phosphoproteins may be involved in the regulation of certain calcium-dependent nerve terminal functions such as neurotransmitter synthesis and release.     

Observations on the kinetics, subunit composition, and sulfhydryl reactivity of myosin from Physarum polycephalum.

A highly purified preparation of myosin from Physarum polycephalum has been shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis to contain heavy chains and only one molecular weight class of light chains, of approx. 15 000 daltons. Kinetic investigations of the Ca2+-ATPase and Mg2+-ATPase (ATP phosphohydrolases, EC 3.6.1.3) at pH 8.0 gave Km and V values of 17.3 muM and 1.25 mumol Pi/min per mg, and 2.4 muM and 0.12 mumol Pi/min per mg, respectively. Adenylyl imidodiphosphate, a beta-gamma-imido ATP analog, inhibited the ATPase activity of Physarum myosin competitively with Ki values equal to 350 and 12 muM in the presence of Ca2+ and Mg2+, respectively. The ATPase activity of Physarum myosin was inhibited at a very low rate (t1/2 = 24 h) by the ATP analog, 6,6'-dithiobis(inosinyl imidodiphosphate), with concentrations of inhibitor previously shown to inactivate (t1/2 approximately 10 min) skeletal and cardiac myosins rapidly by reacting with key cysteines.     

Biochemical Characterization of Secreted Proteases During Growth in Tetrahymena pyriformis WH-14: Comparison of Extracellular with Intracellular Proteases

Tetrahymena pyriformis strain WH-14 secreted large quantities of intracellular proteases into its culture medium during growth. Extracellular enzymes were purified to homogeneity from cell-free medium by ammonium sulfate precipitation, CM-Sephadex column chromatography, gel filtration, and DEAE-cellulose column chromatography. The DEAE-cellulose eluates were separated into four peaks (P-I, P-II, P-III, and P-IV), each of which exhibited a different specific activity toward azocasein and α-N-benzoyl-DL-arginine-ρ-nitroanilide (Bz-Arg-Nan). These four forms of the protease showed similarity in amino acid composition, molecular weight (21,000–24,000), and antigenic reactivity. They had pH optima at neutral range. P-I showed the highest specificity to azocasein whereas P-IV was most effective toward the synthetic substrates. The Km values for hydrolysis of Bz-Arg-Nan were 2.4, 1.6, 1.3, and 1.4 mM for P-I, P-II. P-III, and P-IV, respectively, and the corresponding Kcat/Km values were 5.0, 9.4, 28.5, and 114.3 S^-1.M^-1. These properties of secreted proteases were compared with those of intracellular proteases purified by the same procedure except for the initial Triton X-100 extraction. There were similarities in specific activity toward two substrates, molecular weight, Km, pH optima, and antigenic reactivity between the proteases from two sources, providing evidence that the intracellular proteases may be secreted into the extracellular medium without modification.     

Phosphorylated intermediate of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in endoplasmic reticulum from rat pancreatic acinar cells

Formation and decomposition of the phosphorylated intermediate of endoplasmic reticulum (Ca2+ + Mg2+)-ATPase from pancreatic acinar cells have been studied using lithium dodecyl sulfate- and tetradecyltrimethylammonium bromide-polyacrylamide gel electrophoresis. Incorporation of 32P from [gamma-32P]ATP is Ca2+-dependent (approximate Km for free [Ca2+] = 2-3 X 10(-8) mol/liter). Formation of the 100-kDa phosphoprotein is rapid, reaching maximal 32Pi incorporation within 1 s at room temperature. At 4 degrees C, phosphorylation is slower and dephosphorylation is drastically decreased. For dephosphorylation, Mg2+ and monovalent cations such as K+ or Na+ are necessary. Vanadate inhibits both 32P incorporation and 32P liberation dose dependently (Km = 3 X 10(-6) mol/liter), whereas mitochondrial inhibitors and ouabain have no effect. The phosphoprotein is stable at pH 2 and destabilizes with increasing pH being completely decomposed at pH 9. Reduction of 32P incorporation in the presence of high concentrations of cold ATP and hydroxylamine suggests formation of acylphosphate present in the ATPase intermediate. The characteristics of Ca2+, cation, and pH dependencies of the ATPase activity are similar to those previously described for MgATP-dependent Ca2+ transport into rough endoplasmic reticulum from pancreatic acinar cells (Bayerd?rffer, E., Streb, H., Eckhardt, L., Haase, W., and Schulz, I. (1984) J. Membr. Biol. 81, 69-82). The data suggest that the 100-kDa phosphoprotein as described in this study is the intermediate of this Ca2+ transport ATPase.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle. Identification and characterization of an acid-stable phosphorylated intermediate of the Ca2+,Mg2+-ATPase

An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 microM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 microM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 microM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 microM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15 degrees C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16 degrees C and 14.6 kcal/mol between 16 and 30 degrees C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes.     

Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum.

Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopycnic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg [gamma-32P]ATP significant amounts of 32P were incorporated into all these proteins. Incorporation of 32P into the 15 000 dalton protein was moderately and 32P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by Ca2+ concentrations up to 0.1 mM and by ethyleneglycol-bis-(beta-aminoethyl-ether)-N,N'-tetraacetic acid in the absence of added Ca2+. Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [32P]-phosphate were incorporated into threonine and serine residues of this protein, when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP. The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate.     

Preganglionic stimulation increases the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion by both cAMP-dependent and Ca2+-dependent protein kinases

Electrical stimulation of the preganglionic cervical sympathetic trunk increases the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion of the rat by a nicotinic mechanism and by a noncholinergic mechanism. We have measured the incorporation of [32P]Pi into specific tryptic phosphopeptides in tyrosine hydroxylase in order to identify the protein kinases that phosphorylate this enzyme in electrically stimulated ganglia. 32P-labeled tyrosine hydroxylase was isolated from the ganglion by immunoprecipitation and polyacrylamide gel electrophoresis and was subjected to tryptic hydrolysis. Seven tryptic peptides were resolved from these hydrolysates by two-dimensional thin-layer electrophoresis and chromatography. Preganglionic stimulation (20 Hz, 5 min) increased the incorporation of 32P into four of these peptides. In the presence of cholinergic antagonists, however, electrical stimulation increased the labeling of only one phosphopeptide. From a comparison of the effects of preganglionic stimulation with the effects of agonists that activate specific protein kinases, we conclude that electrical stimulation increases the phosphorylation of tyrosine hydroxylase by both a cAMP-dependent protein kinase and a Ca2+/calmodulin-dependent protein kinase. The nicotinic component of preganglionic stimulation appears to be mediated by a Ca2+/calmodulin-dependent protein kinase, while the noncholinergic component appears to be mediated by cAMP-dependent protein kinase. Although protein kinase C can phosphorylate tyrosine hydroxylase, this kinase does not appear to participate in the stimulation-induced phosphorylation of tyrosine hydroxylase in the superior cervical ganglion.     

Ca2+-dependent protein phosphorylation associated with microsomal fraction of rat pancreas

Microsomes isolated from cat pancreas were incubated with [gamma-32P]ATP in the presence or absence of Ca2+. Following fractionation of phosphoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single microsomal protein with an apparent molecular mass of 77,000 dalton (77K) was found to be phosphorylated in a Ca2+-dependent mechanism. Maximal phosphate incorporation into the 77K protein was observed at 10(-6) mol/l [Ca2+] and was 4-fold higher than in the absence of Ca2+. The 77K phosphoprotein showed characteristic of a stable phosphoester rather than an acyl phosphate. Measurable phosphate incorporation into the 77K protein was noted 5 s following addition of [gamma-32P]ATP and reached maximum at 9-10th min. The lack of effect of exogenous cyclic AMP, cyclic AMP-dependent protein kinase, calmodulin, the calmodulin antagonist trifluoperazine, leupeptin and the suppression of phosphorylation by some phospholipid-interacting drugs suggested that the 77K protein is a substrate for cyclic AMP- and calmodulin-independent, Ca2+-activated phospholipid-sensitive kinase activity. Centrifugation of the pancreatic homogenate in a ficoll-sucrose density gradient indicated that both the 77K protein and enzyme were associated in a fraction enriched in rough endoplasmic reticulum.     

On the sidedness of membrane phosphorylation by Pi and ATP synthesis during reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles.

The membrane sidedness of Pi interaction in reactions which characterize reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was investigated. Vesicles previously loaded with calcium [32P]phosphate were incubated with 0.1 mM ADP and different concentrations of nonradioactive Pi. Alternatively, vesicles loaded with nonradioactive calcium phosphate were incubated in a medium containing 32Pi. The rates of Ca2+ efflux and ATP synthesis were siginficantly activated only when Pi was included in the assay medium. Although the Pi contained by the vesicles crosses the membrane at a rate proportional to the Ca2+ efflux, [gamma-32P]ATP was synthesized only when 32Pi interacted with the outer surface of the membrane. Similarly, ATP in equilibrium 32Pi or ITP in equilibrium 32Pi exchange could be measured only when the external pool of Pi was labeled. Both for ATP synthesis and for the ITP in equilibrium Pi exchange reaction, membrane phosphorylation by 32Pi was negligible unless the external pool of Pi was labeled. The ionophore X-537 A increased the rate of Ca2+ efflux but inhibited the synthesis of ATP. During reversal of the Ca2+ pump, Pi apparently interacts with the membrane only at the outer surface, and at a site different from that where Ca2+ crosses the membrane.     

Protein phosphorylation and dephosphorylation in liver plasma membrane. Effect of inorganic phosphate

When a plasma membrane preparation isolated from rat liver was incubated with [gamma-32P]ATP and Mg2+, protein-bound 32P increased rapidly, followed by a gradual decrease. The time course suggested the existence of membrane-bound kinase(s) and phosphatase(s) phosphorylating and dephosphorylating endogenous proteins. The extent of phosphorylation was not affected by inclusion of cyclic AMP in the reaction mixture. The extent of the maximum phosphorylation was dependent on membrane concentration, owing to rapid hydrolysis of ATP by the membrane-bound ATPase activity. Thus, phosphorylation proceeded further on repeated addition of ATP. Both phosphorylation and dephosphorylation were stimulated by Mg2+, an effective rate of phosphorylation being obtained at 15 mM. Pi up to 20 mM stimulated phosphorylation with little effect on the rate of dephosphorylation. At higher phosphate concentrations, the maximum 32P-incorporation decreased again, and at 100 mM, dephosphorylation was prevented significantly. Autoradiography after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea revealed six main phosphorylated bands, two of which (Band 3 and 5) were partly extractable with 1 M NaCl. In the presence of 100 mM Pi, very strong phosphorylation of Band 5 (about 23,000 daltons) was noted, and a new strongly labeled band (Band P, about 20,000 daltons) was observed. It was concluded that the phosphoproteins in the membrane may be turned over at different rates and high concentrations of Pi may affect the turnover rate of some phosphoproteins, probably through interference with the phosphatase.     

Proteolytic activation of the canine cardiac sarcoplasmic reticulum calcium pump

Mild trypsin treatment of canine cardiac microsomes consisting largely of sarcoplasmic reticulum vesicles produced a severalfold activation of oxalate-facilitated calcium uptake. The increase in calcium uptake was associated with an increase in ATP hydrolysis. Proteases other than trypsin were also effective although to a lesser degree. Trypsin produced a shift of the Ca2+ concentration dependency curve for calcium uptake toward lower Ca2+ concentrations, which was almost identical with that produced by phosphorylation of microsomes by cyclic AMP dependent protein kinase when the trypsin and the protein kinase were present at maximally activating concentrations. The Hill numbers (+/- SD) of the Ca2+ dependency after treatment of microsomes with trypsin (1.5 +/- 0.1) or protein kinase (1.7 +/- 0.1) were similar and were not significantly different from those for untreated control microsomes (1.6 +/- 0.1 and 1.8 +/- 0.1, respectively). Autoradiograms of sodium dodecyl sulfate-polyacrylamide electrophoretic gels indicate that 32P incorporation into phospholamban (Mr 27.3K) or its presumed monomeric subunit (Mr 5.5K) was markedly reduced when trypsin-treated microsomes were incubated in the presence of cyclic AMP dependent protein kinase and [gamma-32P]ATP compared to control microsomes incubated similarly but pretreated with trypsin inhibitor inactivated trypsin. The activation of calcium uptake by increasing concentrations of trypsin was paralleled by the reduction of phosphorylation of phospholamban. Trypsin treatment of microsomes previously thiophosphorylated in the presence of cyclic AMP dependent protein kinase and [gamma-35S]thio-ATP did not result in a loss of 35S label from phospholamban, which suggests that phosphorylation of phospholamban protects against trypsin attack.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transient state kinetic studies of phosphorylation by ATP and Pi of the calcium-dependent ATPase from sarcoplasmic reticulum.

The ATPase of the sarcoplasmic reticulum is phosphorylated by ATP in the presence of Ca2+. A rapid phosphorylation was observed when the enzyme was preincubated with Ca2+ prior to the addition of 0.1 or 1 mM ATP. The rate of phosphorylation was decreased when Ca2+ was omitted from the preincubation medium and added with ATP when the reaction was started. The rate of phosphorylation by ATP was further decreased when Pi was included in the preincubation medium without Ca2+. In this case, the enzyme was phosphorylated by Pi during the preincubation. When Ca2+ and ATP were added, a burst of phosphorylation by ATP was observed in the initial 16 ms. In the subsequent incubation intervals, the phosphorylation by ATP was synchronous with the hydrolysis of the phosphoenzyme formed by Pi. The rate of hydrolysis of the phosphoenzyme formed by Pi was measured when either the Pi concentration was decreased 10 fold, or when Ca2+, ATP or ATP plus Ca2+ was added to the medium. Upon the single addition of Ca2+, the time for half-maximal decay was in the range 500--1000 ms. In all other conditions it was in the range 70--90 ms.