Characterization of the phosphorylated intermediate of the K+-translocating Kdp-ATPase from Escherichia coli

During ATP hydrolysis the K+-translocating Kdp-ATPase from Escherichia coli forms a phosphorylated intermediate as part of the catalytic cycle. The influence of effectors (K+, Na+, Mg2+, ATP, ADP) and inhibitors (vanadate, N-ethylmaleimide, bafilomycin A1) on the phosphointermediate level and on the ATPase activity was analyzed in purified wild-type enzyme (apparent Km = 10 microM) and a KdpA mutant ATPase exhibiting a lower affinity for K+ (Km = 6 mM). Based on these data we propose a minimum reaction scheme consisting of (i) a Mg2+-dependent protein kinase, (ii) a Mg2+-dependent and K+-stimulated phosphoprotein phosphatase, and (iii) a K+-independent basal phosphoprotein phosphatase. The findings of a K+-uncoupled basal activity, inhibition by high K+ concentrations, lower ATP saturation values for the phosphorylation than for the overall ATPase reaction, and presumed reversibility of the phosphoprotein formation by excess ADP indicated similarities in fundamental principles of the reaction cycle between the Kdp-ATPase and eukaryotic E1E2-ATPases. The phosphoprotein was tentatively characterized as an acylphosphate on the basis of its alkali-lability and its sensitivity to hydroxylamine. The KdpB polypeptide was identified as the phosphorylated subunit after electrophoretic separation at pH 2.4, 4 degrees C of cytoplasmic membranes or of purified ATPase labeled with [gamma-32P]ATP.     

Characterization of a High-Affinity Mg2+-Independent Ca2+-ATPase from Rat Brain Synaptosomal Membranes

A high-affinity Mg2+-independent Ca2+-ATPase (Ca2+-ATPase) has been differentiated from the Mg2+-dependent, Ca2+-stimulated ATPase (Ca2+,Mg2+-ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+-ATPase was found to be 1.33 microM with a Km for ATP of 19 microM and a Vmax of 33 nmol/mg/min. Using Ca-ATP as a substrate, the Km for Ca-ATP was found to be 0.22 microM. Unlike Ca2+,Mg2+-ATPase, Ca2+-ATPase was not inhibited by N-ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. Unlike Ca2+, Mg2+-ATPase activity, ATP-dependent Ca2+ uptake was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+-ATPase described. Ca2+-ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+-ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+-ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high-affinity Mg2+-independent Ca2+-ATPase may be a separate form from Ca2+,Mg2+-ATPase. The capacity of Mg2+-independent Ca2+-ATPase to hydrolyze GTP suggests this protein may be involved in GTP-dependent activities within the cell.     

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.     

The relative conformational stability of the alcohol dehydrogenase alleloenzymes of the fruitfly Drosophila melanogaster.

Intact spermatozoa from rat cauda epididymides possess an ecto-(cyclic AMP-dependent protein kinase) activity that causes the transfer of the terminal phosphate group of ATP to the serine residues of all the histone fractions. The enzyme showed a high degree of substrate specificity for the phosphorylation of histones rather than protamine, casein and phosvitin. The cell-external-surface protein kinase requires Mg2+ for activity, and other bivalent cations such as Mn2+ and Co2+ can substitute partially for Mg2+, whereas Ca2+ and Zn2+ are potent inhibitors of the enzyme. The enzyme has markedly higher affinity for cyclic AMP than for other cyclic nucleotides for its activation, with an apparent Km value for cyclic AmP of 80 nM. Spermatozoal ecto-kinase activity is not due to contamination of broken cells or any possible cell damage during incubation and isolation of spermatozoa. There was no loss of kinase activity from the cells when washed with 2 mM-EDTA, and the histones phosphorylated by intact spermatozoa were located outside the cells. Protein kinase activity of intact cells was strongly inhibited (approx. 90%) by p-chloromercuribenzenesulphonic acid (10 microM), which is believed not to enter the cells. These data provide further support for the localization of a protein kinase on the external surface of spermatozoa.     

A plasma-membrane associated ATPase from the acidophilic bacterium Acidiphilium cryptum

A membrane-bound ATPase of Acidiphilium cryptum, an acidophilic bacterium of mine origin, has been studied. The enzyme has a pH optimum of 8.4 Mg2+ is required for its activity and could be replaced by Mn2+, but not by Ca2+. The enzyme shows a strong preference for ATP as substrate, with the apparent Km of about 0.2 mM. Sulphite ion significantly stimulated the enzyme activity. N,N'-Dicyclohexylcarbodiimide, oligomycin, and azide strongly inhibited the enzyme, whereas vanadate was without effect, suggesting that the A. cryptum ATPase might be of F0F1 type.     

Evidence that the Loss of the Voltage-Dependent Mg2+ Block at the N-Methyl-D-Aspartate Receptor Underlies Receptor Activation During Inhibition of Neuronal Metabolism

Both phosphointermediate- and vacuolar-type (P- and V-type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P-type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P-type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 microM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110-kDa subunits. The V-type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 microM. The V-type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V-type ATPase revealed two Michaelis constants for MgATP of approximately 26 microM and 2.0 mM. The V-type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V-type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+,K(+)-ATPase in the synaptic vesicle preparation is demonstrated.     

Studies of gastric Ca2+-stimulated adenosine triphosphatase. I. characterization and general properties

Gastric microsomes do not contain any significant Ca2+-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in significant (2-3 fold) increase in the basal (with Mg2+ as the only cation) ATPase activity, with virtual elimination of the K+-stimulated component. Such treatment causes unmasking of latent Mg2+-dependent Ca2+-stimulation ATPase. Other divalent cations such as Sr2+, Ba2+, Zn2+, and Mn2+ were found ineffective as a substitute for Ca2+. Moreover, those divalent cations acted as inhibitors of the Ca2+-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a Km of 70 microM for ATP and the Ka values for Mg2+ and Ca2+ are about 4 x 10(-4) and 10(-7) M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H+ transport have been discussed.     

Purification and characterization of phosphatidylinositol kinase from porcine liver microsomes

Phosphatidylinositol kinase was solubilized and purified from porcine liver microsomes to apparent homogeneity. The purification procedure includes: solubilization of microsomes by 2% Triton X-100, ammonium sulfate precipitation (20-35% saturation), Reactive blue agarose chromatography, DEAE-Sephacel chromatography and two consecutive hydroxyapatite chromatographies. A total of 4900-fold purification with 8% recovery of enzyme activity was achieved. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 55000. The enzyme is stimulated in a decreasing order by Mg2+, Fe2+, Mn2+, Fe3+ and Co2+. Ca2+ inhibited Mg2+-stimulated activity with an I50 of 0.4 mM. Apparent Km values for phosphatidylinositol and ATP are 120 and 60 microM, respectively. The enzyme is inhibited by adenosine (I50 = 70 microM), ADP (I50 = 120 microM) and quercetin (I50 = 100 microM). The enzyme is also sensitive to sulfhydryl inhibitors. Using the purified enzyme as an immunogen, we have successfully prepared antibodies for phosphatidylinositol kinase in rabbits. The antibodies appear to recognize an antigen of Mr 55000 on SDS-polyacrylamide gel electrophoresis from various porcine tissues in Western blot analysis.     

Protease Ti, a new ATP-dependent protease in Escherichia coli, contains protein-activated ATPase and proteolytic functions in distinct subunits

In addition to protease La (the lon gene product), Escherichia coli contains another ATP-dependent protease, Ti. This enzyme (approximately 340 kDa) is composed of two components, both of which are required for proteolysis. Both have been purified to homogeneity by conventional procedures using [3H]casein as the substrate. The ATP-stabilized component, A, has a subunit molecular weight of 80,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate, but it behaves as a dimer (140 kDa) upon gel filtration. Component P, which is relatively heat stable, is inactivated by diisopropyl fluorophosphate and can be labeled with [3H] diisopropyl fluorophosphate. It has a subunit size of 23 kDa, but the isolated component behaves as a complex (260 kDa) of 10-12 subunits. The isoelectric point of component A is 7.0 and that of P is 8.2, and their amino acid compositions differ considerably. The purified enzyme has an ATPase activity that is stimulated 2-4-fold by casein and other protein substrates but not by nonhydrolyzed proteins. Component A also shows ATPase activity which can be stimulated by casein. Addition of component P (which lacks ATPase activity) inhibits basal ATP hydrolysis by A and makes this ATPase more responsive to casein. Although component P contains the serine active site for proteolysis, it shows no proteolytic activity in the absence of component A, Mg2+, and ATP or dATP. Other nucleoside triphosphates are not hydrolyzed and do not support proteolysis. Protease Ti has a Km for ATP of 210 microM for hydrolysis of both casein and ATP. Casein increases the Vmax for ATP without affecting the Km. A Mg2+ concentration of 5 mM is necessary for half-maximal rates of ATP and casein hydrolysis. Ca2+ and Mn2+ partially support these activities. Thus, protease Ti shares many unusual properties with protease La (e.g. coupled ATP and protein hydrolysis and protein-activated ATPase), but these functions in protease Ti are associated with distinct subunits that modify each other's activities.     

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)     

Interaction of protein kinase C with membranes is regulated by Ca2+, phorbol esters, and ATP

Physiologic regulation of protein kinase C activity requires its interaction with cellular membranes. We have recently shown that binding of the enzyme to plasma membranes is controlled by Ca2+, whereas enzyme activators, like phorbol esters, regulate both membrane binding and enzyme activity. Here we describe the factors which control the dissociation of protein kinase C from the plasma membrane. In the absence of phorbol esters, the dissociation reaction is rapid and is determined by varying the Ca2+ concentration between 0.1 and 1 microM. However, the presence of 4-beta-phorbol 12,13-dibutyrate greatly reduces enzyme release in response to Ca2+ depletion; removal of the phorbol ester itself permits efficient membrane-enzyme dissociation. The stabilization of the membrane-protein kinase C complex by phorbol esters can be reversed by ATP with an apparent Km for the nucleotide of 6.5 microM. The ATP effect requires MgCl2 and cannot be reproduced by other nucleotides or by a nonhydrolyzable analogue, suggesting that an ATP-dependent phosphorylation reaction may be involved. 4-beta-Phorbol 12,13-dibutyrate appears to stabilize membrane-enzyme association by reducing the apparent Km for Ca2+ to about 15 nM, whereas ATP reverses the phorbol ester effect by increasing the Km for Ca2+ to about 760 nM. Furthermore, the strong degree of negative cooperativity displayed by the Ca2+-dependent enzyme-membrane dissociation is consistent with the presence of multiple interacting Ca2+-binding sites on protein kinase C.     

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.     

Enzymic characteristics of ecto-adenosine triphosphatase in rat epididymal intact spermatozoa.

Ecto-ATPase in rat cauda-epididymal intact spermatozoa has a high degree of substrate specificity for the hydrolysis of ATP and dATP rather than of ADP, AMP, GTP, dGTP, CTP, dCTP, TTP and UTP. The enzyme is activated by bivalent metal ions in the order Mg2+ greater than Mn2+ greater than Co2+ greater than Ca2+. The apparent Km values of the enzyme for Mg2+, Mn2+, Co2+ and Ca2+ are approx. 80, 100, 100 and 150 microM respectively. Addition of Ca2+ (0.1 or 1 mM) gives no further stimulation of the Mg2+-activated ecto-ATPase activity. The apparent Km value of the enzyme for ATP is 95 microM. Pi (16 mM) inhibits the enzymic activity (by 25%), whereas Na+ (50 mM) or K+ (10 mM) alone or in combination, polyamines (spermine and spermidine; 1--12.5mM) and nucleic acids (yeast RNA and calf thymus DNA; 0.12 or 0.62 mg/ml) had no significant effect on the activity of the enzyme. Orthovanadate at a relatively low concentration (20 microM) strongly inhibits (approx. 50%) the ecto-ATPase activity. Vanadate inhibition can be reversed by noradrenaline (2.5 mM). The vanadate-sensitivity of the enzyme increases markedly during spermatozoal maturation in the epididymis. However, the activity of the spermatozoal ecto-ATPase decreases progressively during the epididymal transit of the testicular spermatozoa.     

Kinetic studies on the membrane-bound and the purified coupling factor-ATPase from Rhodopseudomonas sphaeroides

The activity of membrane-bound and purified ATPase (EC 3.6.1.3) was potentiated by several divalent cations. Highest rates of ATP hydrolysis were obtained when the activity was measured with the (cation-ATP)2- complex. Free ATP and free divalent cations in excess were found to be competitive inhibitors to the complex. The apparent Km (complex) values were lower than the Ki values for free ATP indicating that the (cation-ATP)2- complex is bound more tightly to the enzyme than the free ATP. Based on these results, a binding of the complex to the active site at two points is suggested, namely through the ATP and through the cation. Removal of the coupling factor from the membrane apparently caused conformational changes which resulted in a pronounced alteration of the kinetic parameters of ATPase activity. Whereas highest values in chromatophore-bound ATPase activity were observed in the presence of Mg2+, the purified enzyme became even more active in the presence of Ca2+. The Ki values for free ATP decreased upon solubilization of the enzyme. Free Mg2+ in excess was more inhibitory on the purified ATPase than Ca2+, while free Ca2+ in excess was more inhibitory on the membrane-bound enzyme if compared to Mg2+. Ki values for product inhibition by ADP and Pi were determined. Kinetic analyses of photophosphorylation activity revealed that the (cation-ADP)- complex is the functional substrate. The apparent Km values for the complex and for Pi were estimated. Excess of free cations and ADP inhibited competitively the phosphorylation. Ki(ADP), Ki(Ca2+), and Ki(Mg2+) were calculated by Dixon analyses.     

Adenosine triphosphatases associated with bovine brain microtubules. II. Properties of ATPases I and II

The catalytic properties of two ATPases which had been purified from bovine brain microtubules (Tominaga, S. & Kaziro, Y. (1983) J. Biochem. 93, 1085-1092) were studied. ATPase I, which had a molecular weight of 33,000, required the presence of 1.0 microM tubulin, 0.2 mM Mg2+, and 10 mM Ca2+ for maximal activity. The activation of ATPase I by tubulin was specific to the native form of tubulin, which could not be replaced by F-actin or tubulin denatured either by heat or more mildly by dialysis in the absence of glycerol. ATPase I was not specific to ATP, and GTP, and to a lesser extent, UTP and CTP were also hydrolyzed. Km for ATP of ATPase I was about 0.04 mM. ATPase I was inhibited by 5 mM Mg2+, 0.04 M K+, 10(-3) M vanadate, 10 mM N-ethylmaleimide, or 20% (v/v) glycerol. ATPase II, which was associated with membrane vesicles, required the presence of 0.2-2.0 mM Mg2+ and 20 mM KCl for activity. Tubulin stimulated the reaction of ATPase II only partially, and the addition of Ca2+ was rather inhibitory. ATPase II was specific to ATP with a Km value of 0.14 mM. It was inhibited by 1.6 mM N-ethylmaleimide and 20% (v/v) glycerol, but was not very sensitive to vanadate. Instead, ATPase II was inhibited by trifluoperazine, chlorpromazine, and nicardipin at 10(-3) M.     

Purification and some properties of ATP-sulfurylase from developing sea urchin embryos

ATP-sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4), purified about 200-fold from sea urchin embryos, was free of ATPase and inorganic pyrophosphatase. The molecular weight of the enzyme was approx. 280 000 measured by gel filtration. The enzyme was activated by Mg2+, Ca2+ or Zn2+; EDTA and p-chloromercuriphenylsulfonate inhibited the enzyme activity. The inhibition was reversed by addition of Mg2+ and dithiothreitol, respectively. The enzyme activity increased continuously as the pH was raised from 5.6 to 10.6. The Km values for the enzyme were calculated to be 13 microM for adenosine 5'-phosphosulfate and 23 microM for pyrophosphate.     

Two Ca2+-requiring p-nitrophenylphosphatase activities of the highly purified Ca2+-pumping adenosinetriphosphatase of human erythrocyte membranes, one requiring calmodulin and the other ATP

The highly purified Ca2+-pumping ATPase from human erythrocyte membranes displays two p-nitrophenylphosphatase (NPPase) activities: one of these requires calmodulin and low concentrations of Ca2+, while the other requires ATP and higher Ca2+ concentrations. The free Ca2+ concentrations required for the expression of the two NPPase activities differed very substantially. Both activities required high free Mg2+ concentrations and displayed simple hyperbolic kinetics toward p-nitrophenyl phosphate (NPP) with a Km in the range of 5-20 mM. Study of the dependence of the calmodulin-stimulated NPPase on Mg2+ and NPP indicated that the Mg-NPP complex is not the substrate of the enzyme. Under conditions optimal for ATP-requiring NPPase (1 mM free Ca2+), the Ca2+-ATPase displayed simple hyperbolic kinetics with a low Km for ATP. NPP competitively inhibited this activity, and the apparent Ki for NPP was less than 1 mM, much lower than the Km for NPP as a substrate. If NPP were inhibiting the ATPase by binding at the same site at which NPP is hydrolyzed, the apparent Ki for NPP as inhibitor would be the same as the Km for NPP as substrate. (Under these circumstances, the apparent Ki and the Km can be directly compared, since NPP was being hydrolyzed under both circumstances.) Since Ki was much lower than Km, NPP must have been inhibiting at another site; thus, these data show the existence of two types of NPP sites on the enzyme, one at which NPP is hydrolyzed and the other at which it inhibits ATP hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+/Ca2+ exchange in coated microvesicles.

Coated microvesicles isolated from bovine neurohypophyses could be loaded with Ca2+ in two different ways, either by incubation in the presence of ATP or by imposition of an outwardly directed Na+ gradient. Na+, but not K+, was able to release Ca2+ accumulated by the coated microvesicles. These results suggest the existence of an ATP-dependent Ca2+-transport system as well as of a Na+/Ca2+ carrier in the membrane of coated microvesicles similar to that present in the membranes of secretory vesicles from the neurohypophysis. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ of the ATP-dependent uptake was 0.8 microM. The average Vmax. was 2 nmol of Ca2+/5 min per mg of protein. The total capacity of microvesicles for Ca2+ uptake was 3.7 nmol/mg of protein. Both nifedipine (10 microM) and NH4Cl (50 mM) inhibited Ca2+ uptake. The ATPase activity in purified coated-microvesicles fractions from brain and neurohypophysis was characterized. Micromolar concentrations of Ca2+ in the presence of millimolar concentrations of Mg2+ did not change enzyme activity. Ionophores increasing the proton permeability across membranes activated the ATPase activity in preparations of coated microvesicles from brain as well as from the neurohypophysis. Thus the enzyme exhibits properties of a proton-transporting ATPase. This enzyme seems to be linked to the ion accumulation by coated microvesicles, although the precise coupling of the proton transport to Ca2+ and Na+ fluxes remains to be determined.     

Guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Subunit structure and characterization of the purified enzyme.

cGMP-dependent protein kinase from bovine lung has been purified to homogeneity using 8-(2-aminoethyl)-amino adenosine 3':5'-monophosphate/Sepharose. Conditions for adsorption of holoenzyme to the affinity chromatography media followed by competitive ligand elution with cGMP have been determined. The holoenzyme of 150,000 molecular weight is composed of two 74,000 molecular weight subunits which are linked in part by disulfide bridges. Two moles of cGMP are bound per mol of holoenzyme compatible with 1 mol of cGMP/monomer. Dissociation of subunits does not occur upon cGMP binding and protein kinase activation. cGMP-dependent protein kinase has an isoelectric point of 5.4 and a Stokes radius of 50 A. The enzyme is asymmetric with an f/f0 of 1.42 and an axial ratio of 7.4. Determination of enzyme activity at varying concentrations of ATP revealed that cGMP increased the Vmax for ATP without significant effect on the Km. The purified enzyme was maximally active at 5 mM Mg2+; other divalent cations could not substitute for Mg2+. In the presence of Mg2+, strong inhibitory effects of other cations were observed with Mn2+, greater than Zn2+, greater than Co2+ greater than Ca2+. Although maximal cGMP-dependence was observed at pH 5.7 to 7.0, basal activity rose at higher pH values to approach activity observed with cGMP. A molecular model comparing cGMP-dependent protein kinase with cAMP-dependnet protein kinase is presented.     

Some properties of the H-ATPase activity present in root plasmalemma of Avena sativa L. Two different enzymes or one enzyme with two ATP sites?

The effects of Mg2+, K+ and ATP on a H-ATPase activity from a native plasmalemma fraction of oat roots were explored at 20 degrees C and pH 6.5. In the presence of 3 mM ATP and no K+, H-ATPase activity vs. [Mg2+] approached a monotonic activation but it became biphasic, with a decline above 3 mM Mg2+, in the presence of 20 mM K+. Mg2+ inhibition occurred also in K-free solutions when [ATP] was lowered to 0.05 mM. Also, an apparent monotonic H-ATPase activation by [K+] at 3.0 mM ATP was transformed in biphasic (inhibition by high [K+]) when [ATP] was reduced to 0.05 mM. The best fits of the ATP stimulation curves of hydrolysis satisfied the sum of two Michaelian functions where that with higher affinity had lower Vmx. Taking into consideration all conditions of activity assay, the high-affinity component (1) had a Km about 11-16 microM and a Vmx around 0.14-0.28 mumol Pi/mg per min whereas that with lower affinity (2) had a Km of 220-540 microM and a Vmx of 0.5-1.0 mumol Pi/mg per min. Km2 was markedly affected by the [K+] and [Mg2+]; at optimal concentrations of these cations (1 mM Mg2+ and 10 mM K+) it had a value of 235 +/- 24 microM which was increased to 540 +/- 35 microM at 20 mM [Mg2+] and 60 mM [K+]. In addition, Vmx1 was reduced to about a half when the concentrations of Mg2+ and K+ were increased to inhibitory levels. These results could be explained by the existence of two different enzymes or one enzyme with two ATP sites. In the second case, we could not tell at this stage if both are catalytic or one is regulatory.