A Mg2+-independent high-affinity Ca2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg2+

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca2+-stimulated ATPase activity in the absence of Mg2+. The Ca2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity (Km = 0.4 microM) and a low affinity (Km = 0.6 mM) for Ca2+. Distribution of these high-affinity and low-affinity Ca2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg2+ also stimulates the ATPase in the absence of Ca2+. Unlike the Mg2+-ATPase and low-affinity Ca2+-ATPase, the plasmalemmal high-affinity Ca2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca2+-ATPase is noncompetitively inhibited by Mg2+ with respect to Ca2+ stimulation. Such an inhibitory effect of Mg2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg2+-independent, high-affinity Ca2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg2+-dependent, high-affinity Ca2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca2+-pump.     

Mg(Ca)-ATPase of arterial muscle cells]

We could show an ATPase in mitochondrial and microsomal fractions of sheep arteria carotis communis and arteria coronaria of cattle which can be stimulated by Ca2+ of Mg2+, respectively. The enzyme has a higher affinity for Ca2+ than for Mg2+. The maximum activity of the Mg(Ca)-ATPase was found at 2-4 mM Ca2+ or Mg2+, respectively. Higher concentrations of these ions inhibit the enzyme. Mn2+, Sr2+ and Co2+ can substitute Ca2+ in splitting of ATP by the ATPase of both fractions of ateria coronaria of cattle. The ions K+ and Na+, variation of temperature and pH and a variety of pharmacological active compounds has the same effect on the ATPase stimulated by Ca2+ or Mg2+. These findings prove that Ca2+ and Mg2+ act at the same site of the ATPase of the mitochondrial and microsomal fraction of vascular smooth muscle.     

Mechanism of eosin Y action of activity of solubilized Ca2+, Mg2+- ATPase from smooth muscle sarcolemma

The eosin Y inhibitory effect on the activity of smooth muscle plasma membrane Ca(2+)-transporting ATPase was studied: effect of this inhibitor on the maximal initial rate of ATP-hydrolase reaction, catalyzed by Ca2+, Mg(2+)-ATPase, on the affinity of enzyme for the reaction reagents (Ca2+, Mg2+, ATP). Dependence of eosin Y inhibitory effect on some physicochemical factors of incubation medium was studied too. It was determined that eosin Y inhibited reversibly and with high specificity purified Ca2+, Mg(2+)-ATPase solubilized from myometrial cell plasma membrane (Ki--0.8 microM), decreased the turnover rate of this enzyme determined both by Mg2+, ATP and Ca2+. This inhibitor had no effect on the enzyme affinity for Ca2+, increased affinity for Mg2+ and decreased affinity for ATP. It was determined that inhibition of Ca2+, Mg(2+)-ATPase by eosin Y depended on pH and dielectric permeability of the incubation medium: increasing of pH from 6.5 to 8.0 reduced the apparent Ki, decreasing of dielectric permeability from 74.07 to 71.19 increased the apparent Ki.     

Characteristics of Ca2+- and Mg2+-induced tension development in chemically skinned smooth muscle fibers

Chemically skinned fibers from guinea pig taenia caecum were prepared by saponin treatment to study the smooth muscle contractile system in a state as close to the living state as posible. The skinned fibers showed tension development with an increase of Ca2+ in the solution, the threshold tension occurring as 5 X 10(-7) M Ca2+. The maximal tension induced with 10(-4) M Ca2+ was as large and rapid as the potassium-induced contracture in the intact fibers. The slope of the pCa tension curve was less steep than that of skeletal muscle fibers and shifted in the direction of lower pCa with an increase of MgATP. The presence of greater than 1 mM Mg2+ was required for Ca2+-induced contraction in the skinned fibers as well as for the activation of ATPase and superprecipitation in smooth muscle myosin B. Mg2+ above 2 mM caused a slow tension development by itself in the absence of Ca2+. Such a Mg2+-induced tension showed a linear relation to concentrations up to 8 mM in the presence of MgATP. Increase of MgATP concentration revealed a monophasic response without inhibition of Ca2+-induced tension development, unlike the biphasic response in striated muscle. When MgATP was removed from the relaxing solution, the tension developed slowly and slightly, even though the Mg2+ concentrations was fixed at 2 mM. These results suggest a substantial difference in the mode of actin-myosin interaction between smooth and skeletal muscle.     

Sarcolemmal (Ca2(+)+Mg2+)-ATPase of vascular smooth muscle and the effects of protein kinases thereupon

To elucidate the regulation mechanisms for sarcolemmal Ca2(+)-pumping ATPase of vascular smooth muscle, the preparation of the membrane fraction of porcine aorta with which the enzyme activity could be analyzed was attempted. A Ca2(+)-activated, Mg2(+)-dependent ATPase [Ca2(+)+Mg2+)-ATPase) activity with high affinity for Ca2+ (Km = 79 +/- 18 nM) was found in a sarcolemma-enriched fraction obtained from digitonin-treated microsomes that possessed the essential properties of plasma membrane (PM) Ca2(+)-pumping ATPases, as determined for the erythrocyte and cardiac muscle enzymes. The activity was stimulated by calmodulin and inhibited by low concentrations of vanadate. Saponin had a stimulatory effect on it. The existence of the PM enzyme in the membrane fraction was substantiated by the Ca2(+)-dependent, hydroxylamine sensitive phosphorylation of a 130K protein, which could be selectively enhanced by LaCl3. The enzyme activity was potentiated by either cGMP or a purified G-kinase. Purified protein kinase C potentiated the enzyme activity. However, none of these agents stimulated the activity of the enzyme purified from microsomes by calmodulin affinity chromatography. The results suggest that the sarcolemmal Ca2(+)-pumping ATPase of vascular smooth muscle is regulated by these protein kinases not through phosphorylation of the enzyme itself but through phosphorylation of membrane components(s) other than the enzyme. Phosphatidylinositol phosphate was found to stimulate the enzyme, suggesting its role in mediation of the stimulatory effects of the protein kinases.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle: effects of Sr2+ and Cd2+ on Ca2+ uptake and formation of the phosphorylated intermediate of the Ca2+,Mg2+-ATPase

The effects of various divalent cations on the Ca2+ uptake by microsomes from bovine aortic smooth muscle were studied. High concentrations (1 mM) of Co2+, Zn2+, Mn2+, Fe2+, and Ni2+ inhibited neither the Ca2+ uptake by the microsomes nor the formation of the phosphorylated intermediate (E approximately P) of the Ca2+,Mg2+-ATPase of the microsomes. The cadmium ion, however, inhibited both the Ca2+ uptake and the E approximately P formation by the microsomes. Dixon plot analysis indicated Cd2+ inhibited (Ki = 135 microM) the Ca2+ dependent E approximately P formation in a non-competitive manner. The inhibitory effect of Cd2+ was lessened by cysteine or dithiothreitol. The strontium ion inhibited the Ca2+ uptake competitively, while the E approximately P formation increased on the addition of Sr2+ at low Ca2+ concentrations. At a low Ca2+ concentration (1 microM), Sr2+ was taken up by the aortic microsomes in the presence of 1 mM ATP. It is thus suggested that Sr2+ replaces Ca2+ at the Ca2+ binding site on the ATPase.     

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.     

Characterization of the Ca2+- and Mg2+-Dependent ATPases in Electrophorus Electroplax Microsomes

Electrophorus electroplax microsomes were examined for Ca2+- and Mg2+-dependent ATPase activity. In addition to the previously reported low-affinity ATPase, a high-affinity (Ca2+,Mg2+)-ATPase was found. At low ATP and Mg2+ concentrations (200 microM or less), the high-affinity (Ca2+,Mg2+)-ATPase exhibits an activity of 18 nmol Pi mg-1 min-1 with 0.58 microM Ca2+. At higher ATP concentrations (3 mM), the low-affinity Ca2+-ATPase predominates, with an activity of 28 nmol Pi mg-1 min-1 with 1 mM Ca2+. In addition, Mg2+ can also activate the low-affinity ATPase (18 nmol Pi mg-1 min-1). The high-affinity ATPase hydrolyzes ATP at a greater rate than it does GTP, ITP, or UTP and is insensitive to ouabain, oligomycin, or dicyclohexylcarbodiimide inhibition. The high-affinity enzyme is inhibited by vanadate, trifluoperazine, and N-ethylmaleimide. Added calmodulin does not significantly stimulate enzyme activity; rinsing the microsomes with EGTA does not confer calmodulin sensitivity. Thus the high-affinity ATPase from electroplax microsomes is similar to the (Ca2+,Mg2+)-ATPase reported to be associated with Ca2+ transport, based on its affinity for calcium and its response to inhibitors. The low-affinity enzyme hydrolyzes all tested nucleoside triphosphates, as well as diphosphates, but not AMP. Vanadate and N-ethylmaleimide do not inhibit the low-affinity enzymes. The low-affinity enzyme reflects a nonspecific nucleoside triphosphatase, probably an ectoenzyme.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

Sarcoplasmic reticulum Ca(2+)-ATPase of sea cucumber smooth muscle: regulation by K(+) and ATP

Although several Ca(2+)-ATPase isoforms have been described in vertebrates, little is known about Ca(2+)-transport in the muscle of invertebrates. In the microsomal fraction obtained from the sea cucumber (Ludwigothurea grisea) longitudinal body wall smooth muscle, we identified a Ca(2+)-transport ATPase that is able to transport Ca(2+) at the expense of ATP hydrolysis. This enzyme has a high affinity for both Ca(2+) and ATP, an optimum pH around 7.0, and - different from the vertebrate sarcoplasmic reticulum Ca(2+)-ATPases isoforms so far described - is activated 3- to 5-fold by K(+) but not by Li(+), at all temperatures, Ca(2+) and ATP concentrations tested. Calcium accumulation by the sea cucumber microsomes is inhibited by Mg/ATP concentrations >1 mM and the accumulated Ca(2+) is released to the medium when the ATP concentration is raised from 0.1 to 4.0 mM.     

Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle.

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.     

Local anesthetics induce fast Ca2+ efflux through a nonenergized state of the sarcoplasmic reticulum Ca(2+)-ATPase.

The effect of the local anesthetics SKF 525-A, dibucaine, tetracaine, procaine, and benzocaine on sarcoplasmic reticulum vesicles was studied. All the anesthetics tested inhibited the phosphorylation of the Ca(2+)-ATPase by Pi in a competitive manner. Tertiary amine and positively charged anesthetics, in addition to competing with Pi, also decreased the apparent affinity of the ATPase for Mg2+. There was a good correlation between the octanol/water partition coefficients and the inhibitory activity of the different anesthetics. All the anesthetics tested induced a 5- to 10-fold increase in the rate of Ca2+ efflux. This was promoted by the same drug concentration that inhibited the phosphorylation of the ATPase by Pi. The effect on Ca2+ efflux was antagonized by the ligands of the ATPase (Mg2+, K+, Ca2+, MgATP, and ADP) and by the organic polyamines ruthenium red, spermine, spermidine, and putrescine. The natural anion heparin was found to potentiate the effect of the positively charged anesthetics on the rate of Ca2+ efflux. It is concluded that the local anesthetics increase the Ca2+ efflux through a nonenergized state of the Ca(2+)-ATPase, rather than promoting a nonspecific Ca2+ leakage through the membrane.     

Mg2,Ca2+-ATPase activity of sarcolemmas of intestinal smooth muscle cells in the rabbit

Preparations of rabbit small intestine smooth muscle cell sarcolemma are capable of hydrolyzing ATP in the presence of millimolar concentrations of Mg2+ and Ca2+ and possess the activity of Mg2+,Ca2+-ATPase having a high affinity for Ca2+ (Km = 5.8 X 10(-6) M). The optimal conditions for the Mg2+,Ca2+-ATPase reaction were established. It was demonstrated that sarcolemmal preparations hydrolyze ATP, GTP, ITP and UTP almost at the same rates. The enzyme contains SH-groups that are unequally exposed to the water phase and are inhibited by 50% by p-chloromercurybenzoate and by 90% by dithionitrobenzoate. The Mg2+,Ca2+-ATPase activity is highly sensitive to oxytocin: at the concentration of 10(-7) MU/ml, the hormone completely inhibits the enzyme without affecting its Mg2+-, Ca2+- and Na+,K+-ATPase activities.     

AIF4-induced inhibition of the ATPase activity, the Ca2+-transport activity and the phosphoprotein-intermediate formation of plasma-membrane and endo(sarco)plasmic-reticulum Ca2+-transport ATPases in different tissues. Evidence for a tissue-dependent functional difference.

AIF4- inhibits the (Ca2+ + Mg2+)-ATPase activity of the plasma-membrane and the sarcoplasmic-reticulum Ca2+-transport ATPase [Missiaen, Wuytack, De Smedt, Vrolix & Casteels (1988) Biochem. J. 253, 827-833]. The aim of the present work was to investigate this inhibition further. We now report that AIF4- inhibits not only the (Ca2+ + Mg2+)-ATPase activity, but also the ATP-dependent 45Ca2+ transport, and the formation of the phosphoprotein intermediate by these pumps. Mg2+ potentiated the effect of AIF4-, whereas K+ had no such effect. The plasma-membrane Ca2+-transport ATPase from erythrocytes was 20 times less sensitive to inhibition by AIF4- as compared with the Ca2+-transport ATPase from smooth muscle. The endoplasmic-reticulum Ca2+-transport ATPase from smooth muscle was inhibited to a greater extent than the sarcoplasmic-reticulum Ca2+-transport ATPase of slow and fast skeletal muscle.     

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.     

The enhancement of Ca2+ efflux from sarcoplasmic reticulum vesicles by urea.

Urea, in nondenaturing concentrations, inhibited Ca2+ uptake by sarcoplasmic reticulum vesicles with no concomitant effect on ATP hydrolysis. This inhibition was antagonized by 5 mM oxalate and 20 mM orthophosphate. At concentrations of 0.2 to 1.0 M, urea induced an increase in the Ca2+ efflux from preloaded vesicles diluted in a medium at pH 7.0 containing 2 mM ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid, 0.1 mM orthophosphate, and 0.1 mM MgCl2. The urea-induced efflux was arrested by ligands of the (Ca(2+)-Mg2+) ATPase, namely, K+, Mg2+, Ca2+, and ADP, and by ruthenium red and the polyamines spermine, spermidine, and putrescine. In the case of polyamines a dissociation between the effect on the efflux and the net Ca2+ uptake was observed, as only the efflux could be blocked by the drugs. Glycine betaine, trimethylamine-N-oxide, and sucrose antagonized the effects of urea on both the net Ca2+ uptake and the rate of Ca2+ efflux.     

Characterization of the high and low affinity components of the renal Ca2(+)-Mg2+ ATPase

The purpose of this study was to characterize the interrelationship between free calcium (Ca2+) and magnesium (Mg2+) in the Ca2+ ATPase enzyme cycle of kidney membranes. Experiments were performed with basolateral membranes from rat renal cortex and microdissected proximal and distal tubules from mice. Results were similar in the three types of preparations. We first investigated the effect of ATP concentration on Ca2(+)- and Mg2(+)-dependent ATP hydrolysis. With 0.2 microM Ca2+, the enzyme activity, as a function of ATP concentration, showed two saturable components: a high affinity component with a Km of 33 microM ATP and a low affinity component with a Km of 0.63 mM ATP. These components may represent either two distinct sites of ATP binding or two forms of the same site. For the sake of simplicity, it was assumed that the two components correspond to a high affinity and a low affinity substrate site. At the high affinity site (ATP = 50 microM), the Ca2+ dependence of ATP hydrolysis followed a single Michaelis-Menten kinetics with Km for Ca2+ of 0.08 microM. The addition of 1 mM Mg2+ resulted in a relatively constant increase in ATP hydrolysis at all Ca2+ concentrations, indicating that the effects of the two cations were additive. With high ATP concentration (ATP = 3 mM), Ca2+ also induced an ATP hydrolysis according to a saturable process, with a Km for Ca2+ of 0.2 microM. In contrast with what occurred with low concentrations of ATP, addition of millimolar Mg2+ completely curtailed the sensitivity of the enzyme to Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of eosin Y on Mg2+-dependent ATPase activity of the myometrium actomyosin

The effect of the reversible inhibitor of membrane-bound Ca2+ -transporting system in smooth muscle--eosin Y--on apparent kinetic parameters that characterize the sensitivity to Mg2+ of myometrium actomyosine ATPase reaction was investigated. It is shown that eosin Y decreases an affinity of actomyosin for Mg2+ and does not influence the number of turns of the smooth muscle actomyosin ATPase activity that was defined by Mg2+. This suggests possible competition of eosin Y with Mg2+ for the active center of actomyosin ATPase. However, the negatively charged inhibitor cannot be adsorbed on Mg2+-binding site of the active center because of essential differences in size, form and charge between eosin Y and Mg2+. Most likely, eosin Y acts on uterus smooth muscle actomyosin as an allosteric inhibitor. Consequently, the mechanism of eosin Y action on ATPase activity of myometrium contractile proteins is different from the mechanism of its influence on ATP-hydrolase enzyme systems of plasmatic membranes.     

Modulation of glycogen synthase kinase activity of skeletal and smooth muscle casein kinase I by spermine

Casein kinase I (CK-I) from skeletal muscle was stimulated 2-3 fold by 0.25-1 mM spermine. The polyamine also stimulated the phosphorylation of glycogen synthase by another casein kinase purified from aortic smooth muscle [DiSalvo et al. (1986) Biochem. Biophys. Res. Commun. 136, 789-796]. Phosphopeptide maps and phosphoamino acid analysis of [32P]glycogen synthase revealed that smooth muscle casein kinase phosphorylated glycogen synthase in the same sites that undergo phosphorylation by CK-I. The stimulatory effect of spermine on glycogen synthase kinase activity of CK-I was accompanied by increased phosphorylation of all peptide sites of glycogen synthase. Increased phosphorylation was observed in both seryl and threonyl residues. Higher concentrations (4 mM) of spermine inhibited CK-I activity by about 50%. These results indicate that aortic smooth muscle casein kinase is a CK-I enzyme and that skeletal and smooth muscle CK-I can be modulated by spermine.     

Ca2+ gradient and drugs reveal different binding sites for Pi and Mg2+ in phosphorylation of the sarcoplasmic reticulum ATPase.

The first step towards ATP synthesis by the Ca2-ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by Pi. Phosphoenzyme formation requires both Pi and Mg2+. At 35 degrees C, the presence of a Ca2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for Pi more than 10-fold, whereas it had no effect on the apparent affinity for Mg2+. In the absence of a Ca2+ gradient, the phosphorylation reaction is inhibited by both K+ and Na+ at all Mg2+ concentrations used. However, in the presence of 1 mM Mg2+ and of a transmembrane Ca2+ gradient, the reaction is still inhibited by Na+, but the inhibition promoted by K+ is greatly decreased. When the Mg2+ concentration is raised above 2 mM, the enzyme no longer discriminates between K+ and Na+, and the phosphorylation reaction is equally inhibited by the two cations. Trifluoperazine, ruthenium red and spermidine were found to inhibit the phosphorylation reaction by different mechanisms. In the absence of a Ca2+ gradient, trifluoperazine competes with the binding to the enzyme of both Pi and Mg2+, whereas spermidine and ruthenium red were found to compete only with Mg2+. The data presented suggest that the enzyme has different binding sites for Mg2+ and for Pi.