Calmodulin-dependent elevation of calcium transport associated with calmodulin-dependent phosphorylation in cardiac sarcoplasmic reticulum

The rate of calcium transport by sarcoplasmic reticulum vesicles from dog heart assayed at 25 degrees C, pH 7.0, in the presence of oxalate and a low free Ca2+ concentration (approx. 0.5 microM) was increased from 0.091 to 0.162 mumol . mg-1 . min-1 with 100 nM calmodulin, when the calcium-, calmodulin-dependent phosphorylation was carried out prior to the determination of calcium uptake in the presence of a higher concentration of free Ca2+ (preincubation with magnesium, ATP and 100 microM CaCl2; approx. 75 microM free Ca2+). Half-maximal activation of calcium uptake occurs under these conditions at 10-20 nM calmodulin. The rate of calcium-activated ATP hydrolysis by the Ca2+-, Mg2+-dependent transport ATPase of sarcoplasmic reticulum was increased by 100 nM calmodulin in parallel with the increase in calcium transport; calcium-independent ATP splitting was unaffected. The calcium-, calmodulin-dependent phosphorylation of sarcoplasmic reticulum, preincubated with approx. 75 microM Ca2+ and assayed at approx. 10 microM Ca2+ approaches maximally 3 nmol/mg protein, with a half-maximal activation at about 8 nM calmodulin; it is abolished by 0.5 mM trifluperazine. More than 90% of the incorporated [32P]phosphate is confined to a 9-11 kDa protein, which is also phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and most probably represents a subunit of phospholamban. The stimulatory effect of 100 nM calmodulin on the rate of calcium uptake assayed at 0.5 microM Ca2+ was smaller following preincubation of sarcoplasmic reticulum vesicles with calmodulin in the presence of approx. 75 microM Ca2+, but in the absence of ATP, and was associated with a significant degree of calmodulin-dependent phosphorylation. However, the stimulatory effect on calcium uptake and that on calmodulin-dependent phosphorylation were both absent after preincubation with calmodulin, without calcium and ATP, suggestive of a causal relationship between these processes.     

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.     

Ca2+, calmodulin-dependent phosphorylation, and inactivation of glycogen synthase by a brain protein kinase

Glycogen synthase from skeletal muscle was phosphorylated by a Ca2+, calmodulin-dependent protein kinase from brain, with concomitant inactivation. About 0.7 mol phosphate/mol subunit was sufficient for a maximal inactivation of glycogen synthase. Further phosphorylation of the enzyme had no effect on the activity. The concentrations required to give half-maximal phosphorylation and inactivation of glycogen synthase were 1.1 and 0.5 microM for Ca2+, and 22 and 11 nM for calmodulin, respectively. The molar ratio of the subunit of the protein kinase to calmodulin was 2-3:1 for half-maximal phosphorylation and inactivation of glycogen synthase. The Km values for glycogen synthase and ATP were 3.6 and 114 microM, respectively, for phosphorylation. Phosphate was incorporated into sites Ia, Ib, and 2 on glycogen synthase, and site 2 was the most rapidly phosphorylated. These results indicate that the brain Ca2+, calmodulin-dependent protein kinase is probably involved in glycogen metabolism in the brain as a glycogen synthase kinase.     

A new activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca2+-, calmodulin-dependent protein kinase. Purification and characterization

Rat brain tryptophan 5-monooxygenase was activated by incubation with ATP, Mg2+, calmodulin, and micromolar concentrations of Ca2+. The activating activity was resolved into two distinct peaks upon gel filtration on Sepharose CL-6B: one, Ca2+-, calmodulin-dependent protein kinase, and the other, a heat-labile activator protein. The activator protein was purified to apparent homogeneity from rat brain by a procedure involving calmodulin-Sepharose 4B, Sephadex G-150, and phenyl-Sepharose CL-4B column chromatography. The molecular weight of the activator protein was determined to be 70,000 by sedimentation equilibrium and by gel filtration on Sephadex G-150. The protein gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of which was estimated to be 35,000, indicating that the protein might be composed of two identical subunits. Analysis of cross-linked activator protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also suggested that the protein might be a dimer of identical subunits. Some other molecular properties of the activator protein were: sedimentation coefficient, 4.3 S; Stokes radius, 3.6 nm; diffusion coefficient, 6.0 x 10(-7) cm2/s; frictional ratio, 1.32; and partial specific volume, 0.73 cm3/g. The activator protein activated tyrosine 5-monooxygenase as well as tryptophan 5-monooxygenase in the presence of ATP, Mg2+, Ca2+, calmodulin, and Ca2+-, calmodulin-dependent protein kinase.     

Ca2+– and Calmodulin-Dependent Phosphorylation of Microtubule-Associated Protein 2 and t Factor, and Inhibition of Microtubule Assembly

Microtubule-associated proteins (MAPs) were phosphorylated by a Ca2+- and calmodulin-dependent protein kinase from rat brain cytosol. The maximal amount of phosphate incorporated into MAPs was 25 nmol of phosphate/mg protein. A Ka value of the enzyme for calmodulin was 57.0 nM, with MAPs as substrates. Among MAPs, MAP2 and tau factor were phosphorylated in a Ca2+- and calmodulin-dependent manner. The phosphorylation of MAPs led to an inhibition of microtubule assembly in accordance with its degree. This reaction was dependent on addition of the enzyme, Ca2+, and calmodulin, and had a greater effect on the initial rate of microtubule assembly rather than on the final extent. The critical tubulin concentration for microtubule assembly was unchanged by the MAPs phosphorylation. Therefore assembly and disassembly of brain microtubule are regulated by the Ca2+- and calmodulin-dependent protein kinase that requires only a nanomolar concentration of calmodulin for activation.     

Role of calmodulin in the activation of tryptophan hydroxylase

Tryptophan hydroxylase can be activated 2.0- to 2.5-fold in vitro by ATPa dn Mg2+. This apparent phosphorylation effect is not dependent on cyclic nucleotides but is dependent on the presence of calcium. The activation of tryptophan hydroxylase by ATP-Mg2+ reduces the apparent Km of the enzyme for its cofactor, 6-methyltetrahydropterin, from 0.21 to 0.09 mM. The addition of certain antipsychotic drugs known to bind to calmodulin in a phosphorylation reaction mixture prevents the activation to tryptophan hydroxylase by ATP-Mg2+ in the concentration-dependent fashion. External addition of purified calmodulin protects the enzyme from the drug-induced effects. Preparation of calmodulin-free tryptophan hydroxylase by affinity chromatography on fluphenazine-Sepharose 4B yields an enzyme that is no longer activated by ATP-Mg2+, whereas the readdition of calmodulin to a calmodulin-free enzyme restores the responsiveness of tryptophan hydroxylase to ATP-Mg2+. This restoration is dependent on Ca2+. Taken together, these results indicate that the activation of tryptophan hydroxylase by phosphorylating conditions is dependent on both calcium and calmodulin.     

Regulation of Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II

The 63-kDa subunit, but not the 60-kDa subunit, of brain calmodulin-dependent cyclic nucleotide phosphodiesterase was phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II. When calmodulin was bound to the phosphodiesterase, 1.33 +/- 0.20 mol of phosphate was incorporated per mol of the 63-kDa subunit within 5 min with no significant effect on enzyme activity. Phosphorylation in the presence of low concentrations of calmodulin resulted in a phosphorylation stoichiometry of 2.11 +/- 0.21 and increased about 6-fold the concentration of calmodulin necessary for half-maximal activation of the phosphodiesterase. Peptide mapping analyses of complete tryptic digests of the 63-kDa subunit revealed two major (P1, P4) and two minor (P2, P3) 32P-peptides. Calmodulin-binding to the phosphodiesterase almost completely inhibited phosphorylation of P1 and P2 with reduced phosphorylation rates of P3 and P4, suggesting the affinity change of the enzyme for calmodulin may be caused by phosphorylation of P1 and/or P2. When Ca2+/calmodulin-dependent protein kinase II was added without prior autophosphorylation, there was no phosphorylation of the 63-kDa phosphodiesterase subunit or of the kinase itself in the presence of a low concentration of calmodulin, and with excess calmodulin the phosphodiesterase subunit was phosphorylated only at P3 and P4. Thus the 63-kDa subunit of phosphodiesterase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II and blocked by Ca2+/calmodulin binding to the subunit.     

Effect of calmodulin on myometrial cell membrane Ca2+,Mg2+-ATPase activity

Myometrium cell plasma membrane Ca2+, Mg(2+)-ATPase purified by an affinity chromatography on calmodulin-sepharose 4B is calmodulin-dependent enzyme. Concentration of calmodulin required for half-maximal activation of enzyme was about 26 nM. By unlike to the enzymes originated from other tissues sensitivity to the calmodulin of the myometrial sarcolemma Ca(2+)-transporting ATPase was lower: calmodulin increased Vmax of ATPase about 1.25-fold, the apparent constant of the activation of enzyme by Ca2+ failed to alter independently on the phospholipid presenting at the enzyme isolation.     

Purification and characterization of a myosin heavy chain kinase from Dictyostelium discoideum

A Dictyostelium discoideum myosin heavy chain kinase has been purified 14,000-fold to near homogeneity. The enzyme has a Mr = 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and greater than 700,000 as determined by gel filtration on Bio-Gel A-1.5m. The enzyme has a specific activity of 1 mumol/min X mg when assayed at a Dictyostelium myosin concentration of 0.3 mg/ml. A maximum of 2 mol of phosphate/mol of myosin is incorporated by the kinase, and the phosphorylated amino acid is threonine. Phosphate is incorporated only into the myosin heavy chains, not into the light chains. The actin-activated Mg2+-ATPase of Dictyostelium myosin is inhibited 70-80% following maximal phosphorylation with the kinase. The myosin heavy chain kinase requires 1-2 mM Mg2+ for activity and is most active at pH 7.0-7.5. The activity of the enzyme is not significantly altered by the presence of Ca2+, Ca2+ and calmodulin, EGTA, cAMP, or cGMP. When incubated with Mg2+ and ATP, phosphate is incorporated into the myosin heavy chain kinase, perhaps by autophosphorylation.     

Purification and characterization of a Ca2+/calmodulin-dependent protein kinase from rat brain

A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.     

Partial purification and characterization of a soluble protein kinase from Leishmania donovani promastigotes

A soluble protein kinase from the promastigote form of the parasitic protozoon Leishmania donovani was partially purified using DEAE-cellulose, Sephadex G-200 and phosphocellulose columns. The enzyme preferentially utilized protamine as exogenous phosphate acceptor. The native molecular mass of the enzyme was about 85 kDa. Mg2+ ions were essential for enzyme activity; other metal ions, e.g. Ca2+, Co2+, Zn2+ and Mn2+, could not substitute for Mg2+. cAMP, cGMP, Ca2+/calmodulin and Ca2+/phospholipid did not stimulate enzyme activity. The pH optimum of the enzyme was 7.0-7.5, and the temperature optimum 37 degrees C. The apparent Km for ATP was 60 microM. Phosphoamino acid analysis revealed that the protein kinase transferred the gamma-phosphate of ATP to serine residues in protamine. The thiol reagents p-hydroxymercuribenzoic acid, 5-5'-dithio-bis(2-nitrobenzoic acid) and N-ethylmaleimide inhibited enzyme activity; the inhibition by p-hydroxymercuribenzoic acid and 5-5'-dithio-bis(2-nitrobenzoic acid) was reversed by dithiothreitol.     

A brain-specific Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) is regulated by autophosphorylation. Relevance to neuronal Ca2+ signaling.

A neuronal Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) undergoes autophosphorylation on a serine residue(s) in response to Ca2+ and calmodulin. Phosphate incorporation leads to the formation of a Ca(2+)-independent (autonomous) activity state, as well as potentiation of the Ca2+/calmodulin-dependent response. The autonomous enzyme activity of the phosphorylated enzyme approximately equals the Ca2+/calmodulin-stimulated activity of the unphosphorylated enzyme, but displays diminished affinity toward ATP and the synthetic substrate, syntide-2. The Km(app) for ATP and syntide-2 increased 4.3- and 1.7-fold, respectively. Further activation of the autonomous enzyme by Ca2+/calmodulin yields a marked increase in the affinity for ATP and peptide substrate such that the Km(app) for ATP and syntide-2 decreased by 14- and 8-fold, respectively. Both autophosphorylation and the addition of Ca2+/calmodulin are required to produce the maximum level of enzyme activation and to increase substrate affinity. Unlike Ca2+/calmodulin-dependent protein kinase type II that is dephosphorylated by the Mg(2+)-independent phosphoprotein phosphatases 1 and 2A, CaM kinase-Gr is dephosphorylated by a Mg(2+)-dependent phosphoprotein phosphatase that may be related to the type 2C enzyme. Dephosphorylation of CaM kinase-Gr reverses the effects of autophosphorylation on enzyme activity. A comparison between the autophosphorylation and dephosphorylation reactions of CaM kinase-Gr and Ca2+/calmodulin-dependent protein kinase type II provides useful insights into the operation of Ca(2+)-sensitive molecular switches.     

Activation of brain calcineurin phosphatase towards nonprotein phosphoesters by Ca2+, calmodulin, and Mg2+

Calcineurin purified from bovine brain was found to be active towards beta-naphthyl phosphate greater than p-nitrophenyl phosphate greater than alpha-naphthyl phosphate much greater than phosphotyrosine. In its native state, calcineurin shows little activity. It requires the synergistic action of Ca2+, calmodulin, and Mg2+ for maximum activation. Ca2+ and Ca2+ X calmodulin exert their activating effects by transforming the enzyme into a potentially active form which requires Mg2+ to express the full activity. Ni2+, Mn2+, and Co2+, but not Ca2+ or Zn2+, can substitute for Mg2+. The pH optimum, and the Vm and Km values of the phosphatase reaction are characteristics of the divalent cation cofactor. Ca2+ plus calmodulin increases the Vm in the presence of a given divalent cation, but has little effect on the Km for p-nitrophenyl phosphate. The activating effects of Mg2+ are different from those of the transition metal ions in terms of effects on Km, Vm, pH optimum of the phosphatase reaction and their affinity for calcineurin. Based on the Vm values determined in their respective optimum conditions, the order of effectiveness is: Mg2+ greater than or equal to Ni2+ greater than Mn2+ much greater than Co2+. The catalytic properties of calcineurin are markedly similar to those of p-nitrophenyl phosphatase activity associated with protein phosphatase 3C and with its catalytic subunit of Mr = 35,000, suggesting that there are common features in the catalytic sites of these two different classes of phosphatase.     

Purification and characterization of a calcium-calmodulin-dependent phospholamban kinase from canine myocardium

A Ca2+-calmodulin-dependent protein kinase was purified to apparent homogeneity from the cytosolic fraction of canine myocardium, with phospholamban as substrate. Purification involved sequential chromatography on DEAE-cellulose, calmodulin-agarose, DEAE-Bio-Gel A, and phosphocellulose. This procedure resulted in a 987-fold purification with a 5.4% yield. The purified enzyme migrated as a single band on native polyacrylamide gels, and it exhibited an apparent molecular weight of 550,000 upon gel filtration. Gel electrophoresis under denaturing conditions revealed a single protein band with Mr 55,000. The purified kinase could be autophosphorylated in a Ca2+-calmodulin-dependent manner, and under optimal conditions, 6 mol of Pi was incorporated per mole of 55,000-dalton subunit. The activity of the enzyme was dependent on Ca2+, calmodulin, and ATP.Mg2+. Other ions which could partially substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations were Sr2+ greater than Mn2+ greater than Zn2+ greater than Fe2+. The substrate specificity of the purified Ca2+-calmodulin-dependent protein kinase for cardiac proteins was determined by using phospholamban, troponin I, sarcoplasmic reticulum membranes, myofibrils, highly enriched sarcolemma, and mitochondria. The protein kinase could only phosphorylate phospholamban and troponin I either in their purified forms or in sarcoplasmic reticulum membranes and myofibrils, respectively. Exogenous proteins which could also be phosphorylated by the purified protein kinase were skeletal muscle glycogen synthase greater than gizzard myosin light chain greater than brain myelin basic protein greater than casein. However, phospholamban appeared to be phosphorylated with a higher rate as well as affinity than glycogen synthase.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro activation of rat liver phenylalanine hydroxylase by phosphorylation.

Essentially pure phenylalanine hydroxylase from rat liver can be activated between 2.5- and 3.0-fold by treatment with Mg2+, ATP, protein kinase, and cyclic AMP. The activation is seen when the hydroxylase is assayed in the presence of tetrahydrobiopterin, but not in the presence of 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine. In the presence of [gamma-32P]ATP, activation is accompanied by incorporation of 32P into the protein to the extent of 0.7 mol/mol of hydroxylase subunit (Mr = 50,000). Cehmical analysis of the untreated enzyme shows that it already contains about 0.3 mol of Pi/mol of hydroxylase. These results suggest that the activity of the hydroxylase may be regulated by phosphorylation.     

Calcium binding to the H+,K(+)-ATPase. Evidence for a divalent cation site that is occupied during the catalytic cycle

The binding and conformational properties of the divalent cation site required for H+,K(+)-ATPase catalysis have been explored by using Ca2+ as a substitute for Mg2+. 45Ca2+ binding was measured with either a filtration assay or by passage over Dowex cation exchange columns on ice. In the absence of ATP, Ca2+ was bound in a saturating fashion with a stoichiometry of 0.9 mol of Ca2+ per active site and an apparent Kd for free Ca2+ of 332 +/- 39 microM. At ATP concentrations sufficient for maximal phosphorylation (10 microM), 1.2 mol of Ca2+ was bound per active site with an apparent Kd for free Ca2+ of 110 +/- 22 microM. At ATP concentrations greater than or equal to 100 microM, 2.2 mol of Ca2+ were bound per active site, suggesting that an additional mole of Ca2+ bound in association with low affinity nucleotide binding. At concentrations sufficient for maximal phosphorylation by ATP (less than or equal to 10 microM), APD, ADP + Pi, beta,gamma-methylene-ATP, CTP, and GTP were unable to substitute for ATP. Active site ligands such as acetyl phosphate, phosphate, and p-nitrophenyl phosphate were also ineffective at increasing the Ca2+ affinity. However, vanadate, a transition state analog of the phosphoenzyme, gave a binding capacity of 1.0 mol/active site and the apparent Kd for free Ca2+ was less than or equal to 18 microM. Mg2+ displaced bound Ca2+ in the absence and presence of ATP but Ca2+ was bound about 10-20 times more tightly than Mg2+. The free Mg2+ affinity, like Ca2+, increased in the presence of ATP. Monovalent cations had no effect on Ca2+ binding in the absence of ATP but dit reduce Ca2+ binding in the presence of ATP (K+ = Rb+ = NH4 + greater than Na+ greater than Li+ greater than Cs+ greater than TMA+, where TMA is tetramethylammonium chloride) by reducing phosphorylation. These results indicate that the Ca2+ and Mg2+ bound more tightly to the phosphoenzyme conformation. Eosin fluorescence changes showed that both Ca2+ and Mg2+ stabilized E1 conformations (i.e. cytosolic conformations of the monovalent cation site(s)) (Ca.E1 and Mg.E1). Addition of the substrate acetyl phosphate to either Ca.E1 or Mg.E1 produced identical eosin fluorescence showing that Ca2+ and Mg2+ gave similar E2 (extracytosolic) conformations at the eosin (nucleotide) site. In the presence of acetyl phosphate and K+, the conformations with Ca2+ or Mg2+ were also similar. Comparison of the kinetics of the phosphoenzyme and Ca2+ binding showed that Ca2+ bound prior to phosphorylation and dissociated after dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

ATP in equilibrium with 32Pi exchange catalyzed by plasma membrane Ca(2+)-ATPase from kidney proximal tubules.

The Ca(2+)-stimulated adenosine 5'-triphosphate-orthophosphate (ATP in equilibrium with 32Pi) exchange reaction was studied using a vesicular preparation derived from plasma membrane of kidney proximal tubules. With native inside-out vesicles, ATP in equilibrium with 32Pi was stimulated by micromolar Ca2+ concentrations. Treatment of the vesicles with the Ca2+ ionophore A23187 that abolished Ca2+ accumulation, strongly inhibited ATP in equilibrium with 32Pi. When Ca(2+)-ATPase was solubilized with the nonionic detergent octaethylene glycol mono n-dodecyl ether, maximal activation of ATP in equilibrium with 32Pi required millimolar Ca2+ concentrations. These Ca2+ concentrations inhibited ATP hydrolysis. ATP in equilibrium with 32Pi exhibited a Michaelian dependence on Pi and Mg2+, was stimulated by ATP, and depended on the ATP/ADP ratio. ATP in equilibrium with 32Pi was modified by the osmolytes urea, trimethylamine-N-oxide, and sucrose, which are representative of the methylamines and polyols that normally accumulate in renal tissue. These compounds did not modify the apparent affinity for Pi; they affected the response to ADP in the same fashion as the overall rate of ATP in equilibrium 32Pi, and their effects depended on medium pH. These data show that the Ca(2+)-ATPase from plasma membrane kidney proximal tubules can operate simultaneously in forward and backward directions. They also show that ATP in equilibrium with 32Pi is modulated by the ligands Ca2+, ATP, ADP, Pi, Mg2+, and H+, and by organic solutes found in renal tissue.     

Kinetic properties of the purified Ca2+-translocating ATPase from human erythrocyte plasma membrane

The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied. A complex interaction between the major ligands (i.e., Ca2+, Mg2+, H+, calmodulin and ATP) and the enzyme was found. The apparent affinity of the enzyme for Ca2+ was inversely proportional to the concentration of free Mg2+ and H+, both in the presence or absence of calmodulin. In addition, the apparent affinity of the enzyme for Ca2+ was significantly increased by the presence of calmodulin at high concentrations of MgCl2 (5 mM), while it was hardly affected at low concentrations of MgCl2 (2 mM or less). In addition, the ATPase activity was inhibited by free Mg2+ in the millimolar concentration range. Evidence for a high degree of positive cooperativity for Ca2+ activation of the enzyme (Hill coefficient near to 4) was found in the presence of calmodulin in the slightly alkaline pH range. The degree of cooperativity induced by Ca2+ in the presence of calmodulin was decreased strongly as the pH decreased to acid values (Hill coefficient below 2). In the absence of calmodulin, the Hill coefficient was 2 or slightly below over the whole pH range tested. Two binding affinities of the enzyme for ATP were found. The apparent affinity of the enzyme for calmodulin was around 6 nM and independent of the Mg2+ concentration. The degree of stimulation of the ATPase activity by calmodulin was dependent on the concentrations of both Ca2+ and Mg2+ in the assay system.     

Phosphorylation and modulation of the enzymic activity of native and protease-cleaved purified hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase by a calcium/calmodulin-dependent protein kinase

A Ca2+/calmodulin-dependent kinase has been purified which catalyzed the phosphorylation and concomitant inactivation of both the microsomal native (100,000 Da) and protease-cleaved purified 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) (53,000 Da) fragments. This low molecular weight brain cytosolic Ca2+/calmodulin-dependent kinase phosphorylates histone H1, synapsin I, and purified HMG-CoA reductase as major substrates. The kinase, purified by sequential chromatography on DEAE-cellulose, calmodulin affinity resin, and high performance liquid chromatography (TSKG 3000 SW) is an electrophoretically homogeneous protein of approximately 110,000 Da. The molecular weight of the holoenzyme, substrate specificity, subunit protein composition, subunit autophosphorylation, subunit isoelectric points, and subunit phosphopeptide analysis suggest that this kinase of Mr 110,000 may be different from other previously reported Ca2+/calmodulin-dependent kinases. Maximal phosphorylation by the low molecular form of Ca2+/calmodulin-dependent kinase of purified HMG-CoA reductase revealed a stoichiometry of approximately 0.5 mol of phosphate/mol of 53,000-Da enzyme. Dephosphorylation of phosphorylated and inactivated native and purified HMG-CoA reductase revealed a time-dependent loss of 32P-bound radioactivity and reactivation of enzyme activity. Based on the results reported here, we propose that HMG-CoA reductase activity may be modulated by yet another kinase system involving covalent phosphorylation. The elucidation of a Ca2+/calmodulin-dependent HMG-CoA reductase kinase-mediated modulation of HMG-CoA reductase activity involving reversible phosphorylation may provide new insights into the molecular mechanisms involved in the regulation of cholesterol biosynthesis.     

Calmodulin-mediated regulation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity in isolated cardiac sarcoplasmic reticulum

A severalfold activation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity by micromolar concentrations of calmodulin was observed in sarcoplasmic reticulum vesicles obtained from canine ventricles. This activation was seen in the presence of 120 mM KCl. The ratio of moles of calcium transported per mol of ATP hydrolyzed remained at about 0.75 when calcium transport and (Ca2+ + Mg2+)-activated ATPase activity were measured in the presence and absence of calmodulin. Thus, the efficiency of the calcium transport process did not change. Stimulation of calcium transport by calmodulin involves the phosphorylation of one or more proteins. The major 32P-labeled protein, as determined by sodium dodecyl sulfate slab gel electrophoresis, was the 22,000-dalton protein called phospholamban. The Ca2+ concentration dependency of calmodulin-stimulated microsomal phosphorylation corresponded to that of calmodulin-stimulated (Ca2+ + Mg2+)-activated ATPase activity. Proteins of 11,000 and 6,000 daltons and other proteins were labeled to a lesser extent. A similar phosphorylation pattern was obtained when microsomes were incubated with cAMP-dependent protein kinase and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Phosphorylation produced by added cAMP-dependent protein kinase and calmodulin was additive. These studies provided further evidence for Ca2+-dependent regulation of calcium transport by calmodulin in sarcoplasmic reticulum that could play a role in the beat-to-beat regulation of cardiac relaxation in the intact heart.