Effect of eosin Y on Ca2+ -independent, Mg2+ -dependent ATPase activity of smooth muscle cell plasma membranes

The effect of eosin Y (2',4',5',7'-tetrabromofluorescin) on basic kinetic parameters of the reaction of Mg2+ -dependent hydrolysis of ATP catalysed "basal" Mg2+ -ATPase myometrial cells plasma membrane has been studied. The eosin Y (10-100 microM) inhibited initial maximal velocity of the "basal" Mg2+ -ATPase of plasma membrane assayed for Mg2+ and ATP. At the same time the given inhibitor reduces the affinity of Mg2+ -ATPase for ATP. However, the difficult effect of the inhibitor action is observed for Mg ions: eosin Y in concentration of 10-50 microM increases the enzyme affinity for the ion-activator, while in concentration of 100 microM the affinity of Mg2+ -ATPase for Mg2+ is reduced. An analysis of eosin Y effect on catalytic efficiency of "basal" Mg2+ -ATPase of plasma membrane has shown, that at saturating concentrations of ATP (1 mM) the enzyme activity is less sensitive to the action of inhibitor. On this basis the conclusion is made that ATP in high concentrations can compete with eosin Y for active centre of Mg2+ -ATPase of smooth muscle cells plasma membrane.     

Phosphofructokinase from muscle of the marine giant barnacle Austromegabalanus psittacus: kinetic characterization and effect of in vitro phosphorylation

The kinetic properties of phosphofructokinase from muscle of the giant cirripede Austromegabalanus psittacus were characterized, after partial purification by ion exchange chromatography on DEAE-cellulose. This enzyme showed differences regarding PFKs from other marine invertebrates: the affinity for fructose 6-phosphate (Fru 6-P) was very low, with an S(0.5) of 22.6+/-1.4 mM (mean+/-S.D., n=3), and a high cooperativity (n(H) of 2.90+/-0.21; mean+/-S.D., n=3). The barnacle PFK showed hyperbolic saturation kinetics for ATP (apparent K(m ATP)=70 microM, at 5 mM Fru 6-P, in the presence of 2 mM ammonium sulfate). ATP concentrations higher than 1 mM inhibited the enzyme. Ammonium sulfate activated the PFK several folds, increasing the affinity of the enzyme for Fru 6-P and V(max). 5'-AMP (0.2 mM) increased the affinity for Fru 6-P (S(0.5) of 6.2 mM). Fructose 2,6-bisphosphate activated the PFK, with a maximal activation at concentrations higher than 2 microM. Citrate reverted the activation of PFK produced by 0.2 mM 5'-AMP (IC(50 citrate)=2.0 mM), producing a higher inhibition than that exerted on other invertebrate PFKs. Barnacle muscular PFK was activated in vitro after exposure to exogenous cyclic-AMP (0.1 mM) as well as by phosphatidylserine (50 microg/ml), indicating a possible control by protein kinase A and a phospholipid dependent protein kinase (PKC). The results suggest a highly regulated enzyme in vivo, by allosteric mechanisms and also by protein phosphorylation.     

Ca2+-dependent inhibition of smooth muscle adenylate cyclase activity

We have examined the inhibitory regulation by Ca2+ of the adenylate cyclase activity associated with microsomes isolated from bovine aorta smooth muscle. In the presence of 2 mM MgCl2, Ca2+ (0.8-100 microM) inhibited in a noncompetitive manner activation of the enzyme by GTP, Gpp[NH]p, or forskolin. In all instances the value for half-maximal inhibition was between 2 and 3 microM. In contrast, Ca2+ inhibited the activation by MgCl2 (2-50 mM), alone or in the presence of GTP, in a competitive manner. The inhibition of adenylate cyclase by 10 microM Ca2+ was reversed in the presence of either 5 or 25 microM calmodulin or troponin C. These data show that (i) Ca2+, at concentrations similar to those which activate smooth muscle contraction, inhibits the stimulation of adenylate cyclase by several activators; (ii) Ca2+ and Mg2+ compete for a common site on the smooth muscle adenylate cyclase complex; and (iii) the reversal of Ca2+-dependent inhibition by Ca2+-binding proteins may be produced by chelation of the metal by these proteins.     

Pre-steady-state studies of the adenosine triphosphatase activity of coupled submitochondrial particles. Regulation by ADP

ATPase activities were measured in 10 mM MgCl2, 5 mM ATP, 1 mM ADP, and 1 microM FCCP with submitochondrial particles from bovine heart that had been stimulated by delta mu H+-forming substrates and with particles whose natural inhibitor protein was partially removed by heating. The activities were not linear with time. With both particles, the rate of ATP hydrolysis in the 7-fold greater than that in the steady state. Pre-steady-state and steady-state kinetic studies showed that the decrease of ATPase activity was due to the binding of ADP in a high-affinity site of the enzyme (K0.5 of 10 microM). Inhibition of ATP hydrolysis was accompanied by the binding of approximately 1 mol of ADP/mol of particulate F1; 10 microM ADP gave half-maximal binding. ADP could be replaced by IDP, but with an affinity 50-fold lower (K0.5 of 0.5 mM). Maximal inhibition by ADP and IDP was achieved in less than 5 s. Inhibition was enhanced by uncouplers. Even in the presence of pyruvate kinase and phosphoenolpyruvate, the rates of hydrolysis were about 2.5-fold higher in the first seconds of reaction than in the steady state. This decrease of ATPase activity also correlated with the binding of nearly 1 mol of ADP/mol of F1. This inhibitory ADP remained bound to the enzyme after several thousand turnovers. Apparently, it is possible to observe maximal rates of hydrolysis only in the first few catalytic cycles of the enzyme.     

Kinetic cooperativity change after H2O2 modification of (Na,K)-ATPase

The kinetics of hydrolysis of ATP and p-nitrophenylphosphate and the action of the allosteric effectors, Na+ and K+, upon the hydrolysis of these substrates were used to study the H2O2-modified, uncoupled (Na,K)-ATPase isolated from cultured bovine lenses ( Garner , W. H., Garner , M. H., and Spector , A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2044-2048). Pure bovine renal (Na,K)-ATPase was modified by H2O2 in 150 mM KCl and 20 mM MgCl2 to yield an enzyme with kinetic properties similar to the enzyme isolated from the H2O2-treated, cultured bovine lens. H2O2 modification changes the interaction of the ATP hydrolysis site from negative to positive kinetic cooperativity. H2O2 modification dramatically alters Na+ stimulation of ATP hydrolysis and Na+ inhibition of p-nitrophenylphosphate hydrolysis while having little effect upon K+ control of the hydrolysis of these two substrates.     

A high affinity calcium-stimulated magnesium-dependent ATPase in rat liver plasma membranes. Dependence of an endogenous protein activator distinct from calmodulin

A Mg-dependent adenosine triphosphatase (ATPase) activated by submicromolar free Ca2+ was identified in detergent-dispersed rat liver plasma membranes after fractionation by concanavalin A-Ultrogel chromatography. Further resolution by DE-52 chromatography resulted in the separation of an activator from the enzyme. The activator, although sensitive to trypsin hydrolysis, was distinct from calmodulin for it was degraded by boiling for 2 min, and its action was not sensitive to trifluoperazine; in addition, calmodulin at concentrations ranging from 0.25 ng-25 micrograms/assay had no effect on enzyme activity. Ca2+ activation followed a cooperative mechanism (nH = 1.4), half-maximal activation occurring at 13 +/- 5 nM free Ca2+. ATP, ITP, GTP, CTP, UPT, and ADP displayed similar affinities for the enzyme; K0.5 for ATP was 21+/- 9 microM. However, the highest hydrolysis rate (20 mumol of Pi/mg of protein/10 min) was observed at 0.25 mM ATP. For all the substrates tested kinetic studies indicated that two interacting catalytic sites were involved. Half-maximal activity of the enzyme required less than 12 microM total Mg2+. This low requirement for Mg2+ of the high affinity (Ca2+-Mg2+)ATPase was probably the major kinetic difference between this activity and the nonspecific (Ca2+ or Mg2+)ATPase. In fact, definition of new assay conditions, i.e. a low ATP concentration (0.25 mM) and the absence of added Mg2+, allowed us to reveal the (Ca2+-Mg2+)ATPase activity in native rat liver plasma membranes. This enzyme belongs to the class of plasma membrane (Ca2+-Mg2+)ATPases dependent on submicromolar free Ca2+ probably responsible for extrusion of intracellular Ca2+.     

Characterisation of an ATP diphosphohydrolase (Apyrase, EC 3.6.1.5) activity in Trichomonas vaginalis

In the present report the enzymatic properties of an ATP diphosphohydrolase (apyrase, EC 3.6.1.5) in Trichomonas vaginalis were determined. The enzyme hydrolyses purine and pyrimidine nucleoside 5'-di- and 5'-triphosphates in an optimum pH range of 6.0--8.0. It is Ca(2+)-dependent and is insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (5 mM). A significant inhibition of ADP hydrolysis (37%) was observed in the presence of 20 mM sodium azide, an inhibitor of ATP diphosphohydrolase. Levamisole, a specific inhibitor of alkaline phosphatase, and P(1), P(5)-di (adenosine 5'-) pentaphosphate, a specific inhibitor of adenylate kinase, did not inhibit the enzyme activity. The enzyme has apparent K(m) (Michaelis Constant) values of 49.2+/-2.8 and 49.9+/-10.4 microM and V(max) (maximum velocity) values of 49.4+/-7.1 and 48.3+/-6.9 nmol of inorganic phosphate x min(-1) x mg of protein(-1) for ATP and ADP, respectively. The parallel behaviour of ATPase and ADPase activities and the competition plot suggest that ATP and ADP hydrolysis occur at the same active site. The presence of an ATP diphosphohydrolase activity in T. vaginalis may be important for the modulation of nucleotide concentration in the extracellular space, protecting the parasite from the cytolytic effects of the nucleotides, mainly ATP.     

The locus of nucleotide specificity in the reaction mechanism of (Na+ + K+)-ATPase determined with ATP and GTP as substrates

ATP and GTP have been compared as substrates for (Na+ + K+)-ATPase in Na+-activated hydrolysis, Na+-activated phosphorylation, and the E2K----E1K transition. Without added K+ the optimal Na+-activated hydrolysis rates in imidazole-HCl (pH 7.2) are equal, but are reached at different Na+ concentrations: 80 mM Na+ for GTP, 300 mM Na+ for ATP. The affinities of the substrates for the enzyme are widely different: Km for ATP 0.6 microM, for GTP 147 microM. The Mg-complexed nucleotides antagonize activation as well as inhibition by Na+, depending on the affinity and concentration of the substrate. The optimal 3-s phosphorylation levels in imidazole-HCl (pH 7.0) are equally high for the two substrates (3.6 nmol/mg protein). The Km value for ATP is 0.1-0.2 microM and for GTP it ranges from 50 to 170 microM, depending on the Na+ concentration. The affinity of Na+ for the enzyme in phosphorylation is lower with the lower affinity substrate: Km (Na+) is 1.1 mM with ATP and 3.6 mM with GTP. The GTP-phosphorylated intermediate exists, like the ATP-phosphorylated intermediate, in the E2P conformation. Addition of K+ increases the optimal hydrolytic activity 30-fold for ATP (at 100 mM Na+ + 10 mM K+) and 2-fold for GTP (at 100 mM Na+ + 0.16 mM K+). K+ greatly increases the Km values for both substrates (to 430 microM for ATP and 320 microM for GTP). Above 0.16 mM K+ inhibits GTP hydrolysis. GTP does not reverse the quenching effect of K+ on the fluorescence of the 5-iodoacetamidofluorescein-labeled enzyme. ATP fully reverses this effect, which represents the transition from E1K to E2K. Hence GTP is unable to drive the E2K----E1K transition.     

Purification, kinetic behavior, and regulation of NAD(P)+ malic enzyme of tumor mitochondria

The purification and kinetic characterization of an NAD(P)+-malic enzyme from 22aH mouse hepatoma mitochondria are described. The enzyme was purified 328-fold with a final yield of 51% and specific activity of 38.1 units/mg of protein by employing DEAE-cellulose chromatography and an ATP affinity column. Sephadex G-200 chromatography yielded a native Mr = 240,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major subunit with Mr = 61,000, suggesting a tetrameric structure, and also showed that the preparation contained less than 10% polypeptide impurities. Use of the ATP affinity column required the presence of MnCl2 and fumarate (an allosteric activator) in the elution buffers. In the absence of fumarate, the Michaelis constants for malate, NAD+, and NADP+ were 3.6 mM, 55 microM, and 72 microM, respectively; in the presence of fumarate (2 mM), the constants were 0.34 mM, 9 microM, and 13 microM, respectively. ATP was shown to be an allosteric inhibitor, competitive with malate. However, the inhibition by ATP displayed hyperbolic competitive kinetics with a KI (ATP) of 80 microM (minus fumarate) and 0.5 mM (plus 2 mM fumarate). The allosteric properties of the enzyme are integrated into a rationale for its specific role in the pathways of malate and glutamate oxidation in tumor mitochondria.     

Identification of P1 and P2 purinoceptors in the aorta of the lizard (Agama sp.)

In the isolated Agama lizard aorta, acetylcholine (ACh; 3 nM-100 microM), noradrenaline (NA; 30 nM-0.3 mM), adrenaline (Adr; 30 nM-300 microM), adenosine 5'-triphosphate (ATP; 30 nM-1 mM), alpha,beta-methylene ATP (alpha,beta-meATP; 10 nM-10 microM), beta,gamma-methylene ATP (beta,gamma-meATP; 0.1-300 microM), 2-methylthio ATP (2-meSATP; 30 nM-30 microM) and high concentrations of uridine triphosphate (UTP; 1 microM-1 mM), all produced constriction. The P2 receptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 30 microM), suramin (0.1 mM) and Reactive blue 2 (30 microM) all raised vascular tone and could not be utilized and the antagonist 2'-O-(trinitrophenyl) ATP (TNP-ATP; 0.1 microM) had no effect on responses to the ATP analogues. alpha,beta-MeATP (3 microMx3) desensitised responses to alpha,beta-meATP (10 microM) and beta,gamma-meATP (0.3 mM), but not to ATP (0.3 mM) or 2-meSATP (30 microM). On pre-constricted aorta (EC50 concentration of either ACh or Adr), adenosine (1 microM-1 mM), the A1-selective agonist N6-cyclopentyl adenosine (CPA; 1-300 microM) [but not the A2- and A3-selective agonists CGS 21680 and IB-MECA respectively (both up to 30 microM)] and sodium nitroprusside (10 nM-100 microM) produced vasodilatation. Adenosine vasodilatation was antagonised by 8-p-sulfophenyl-theophylline (8-pSPT; 30 microM) but not by N(omega)-nitro-L-arginine methyl ester (L-NAME; 0.1 mM). ATP (up to 0.3 mM), 2-meSATP (up to 10 microM) and UTP (up to 1 mM) were not vasodilators. In summary, A1 receptors mediating relaxation and excitatory P2X1 receptors were identified in the smooth muscle of the lizard aorta. However, in contrast to mammalian aorta, P2Y receptors on endothelial cells mediating vasodilatation via nitric oxide do not appear to be present.     

Specific enhancement of the cardiac myofibrillar ATPase by bound creatine kinase.

The kinetic influence of bound creatine kinase (CK) on the Ca(2+)-activated myosin ATPase was evaluated. ATPase rates were measured from 0.8 microM to 3.2 mM MgATP. Under control conditions, the apparent KmATP was 79.9 +/- 13.3 microM. In contrast, the addition of 12.2 mM phosphocreatine (PCr) decreased the apparent KmATP to a value of 13.6 +/- 1.4 microM. To determine if this reduction was merely the result of an ATP maintenance system, ATP was regenerated using either phosphoenolpyruvate and pyruvate kinase (PEP-PK), or PCr and soluble bovine cardiac CK. Data obtained with PEP + PK indicated an apparent KmATP of 65.5 +/- 7.3 microM. To study the effects of exogenous CK, the endogenous CK was irreversibly inhibited with 1 mM iodoacetamide. The kinetics of the ATPase were then examined by adding soluble CK to the incubation medium. Under these conditions, the KmATP was 56.4 +/- 0.86 microM. Therefore, these two ATP regeneration systems could not duplicate the effects of endogenous CK. The reduction of the apparent KmATP by endogenous CK was not the result of an altered inhibition by MgADP. MgADP inhibition was determined to be non-competitive, with a Ki of 5.0 +/- 0.1 mM. These data suggest that the observed kinetic effects reflect the proximity of the enzymes in the myofibrillar bundle, thus emphasizing the importance of bound CK for the localized regeneration of MgATP utilized by the myosin ATPase.     

Intercellular communication: role of gap junctions in establishing the pattern of ATP-elicited Ca2+ oscillations and Ca2+-dependent currents in freshly isolated aortic smooth muscle cells

Cytosolic-free [Ca2+] was evaluated in freshly dissociated smooth muscle cells from mouse thoracic aorta by the ratio of Fura Red and Fluo 4 emitted fluorescence using confocal microscopy. The role of intercellular communication in forming and shaping ATP-elicited responses was demonstrated. Extracellular ATP (250 microM) elicited [Ca2+]i transient responses, sustained [Ca2+]i rise, periodic [Ca2+]i oscillations and aperiodic repetitive [Ca2+]i transients. Quantity of smooth muscle cells in the preparation responding to ATP with periodical [Ca2+]i oscillations depended on the density of isolated cells on the cover slip. ATP-elicited bursts of [Ca2+]i spikes in 66+/-7% of cells in dense and in 33+/-8.5% of cells in non-dense preparations. The number of cells responding to ATP with bursts of [Ca2+]i spikes decreased from 55+/-5% (n=84) to 14+/-3% (n=141) in dense preparations pretreated with carbenoxolone. Simultaneous measurement of [Ca2+]i and ion currents revealed a correlation between [Ca2+]i and current oscillations. ATP-elicited bursts of current spikes in 76% of cells regrouped in small clusters and in 9% of isolated cells. Clustered cells responding to ATP with current oscillations had higher membrane capacity than clustered cells with transient and sustained ATP-elicited responses. Lucifer Yellow (1% in 130 mM KCl) injected into one of clustered cells was transferred to the neighboring cell only when ATP-elicited oscillations. Fast application of carbenoxolone (100 microM) inhibited ATP (250 microM) elicited Ca2+-dependent current oscillations. Taken together these results suggest that the probability of ATP (250 microM) triggered cytosolic [Ca2+]i oscillations accompanied with K+ and Cl- current oscillations increased with the coupling of smooth muscle cells.     

Kaempferol inhibits myosin light chain kinase

Kaempferol, 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, was found to inhibit bovine aorta myosin light chain kinase with a Ki of 0.3-0.5 microM. It was found to be competitive with ATP and non-competitive with isolated myosin light chains. The specificity of this inhibitor was studied relative to protein kinase C and cAMP dependent protein kinase (IC50 = 15 microM and 150 microM, respectively). It appears not to interact strongly with calmodulin binding proteins, such as Ca2+-calmodulin dependent phosphodiesterase (IC50 = 45 microM), and had little effect on actin-activated myosin subfragment-1 ATPase activity (IC50 greater than 100 microM) or smooth muscle phosphatase activities (IC50 greater than 100 microM).     

The phosphoenolpyruvate carboxykinase of Trypanosoma (Schizotrypanum) cruzi epimastigotes: molecular, kinetic, and regulatory properties

The phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) of the epimastigote form of Trypanosoma (Schizotrypanum) cruzi has been purified to homogeneity. The enzyme is composed of two apparently identical 42,000 +/- 500 subunits, is highly specific for adenine nucleotides, and has a strict requirement of Mn2+ ions for activity; the activation of the enzyme by ionic Mn2+ reveals that one Mn2+ ion required for each 42,000 subunit. Hyperbolic kinetics are observed for all substrates in the carboxylation reaction with Km (phosphoenolpyruvate) of 0.36 +/- 0.08 mM, Km (HCO-3) of 3.7 +/- 0.2 mM, and Km (Mg-ADP) of 39 +/- 1 microM. In the decarboxylation reaction the kinetics with respect to oxalacetic acid are also hyperbolic with a Km of 27 +/- 3 microM, but towards Mg-ATP there is a biphasic response: hyperbolic at low (less than 250 microM) concentrations with a Km of 39 +/- 1 microM, but at higher concentrations the nucleotide produces a strong inhibition of the enzyme activity. This inhibition is also observed with Mg-GTP and Mg-ITP which are not substrates of the reaction. The results are consistent with an important regulatory function of the enzyme in the amino-acid catabolism of T. cruzi.     

Purification and kinetic characterization of a dopamine-sulfating form of phenol sulfotransferase from human brain

The kinetic and biochemical properties of a purified, monoamine-sulfating form of phenol sulfotransferase (M-PST) from human brain are described. M-PST activity was separated and purified from phenol-sulfating activity by anion-exchange chromatography on DEAE-cellulose and subsequently purified on AffiGel Blue and Sephacryl S-200, routinely giving a final purification of over 20 000-fold, with approximately a 3% yield. The molecular weight of the active species, as estimated by gel filtration chromatography, was 250 000. The purified enzyme was inhibited by NaCl (50% at 325 mM) and showed an optimum for dopamine sulfation at pH 7.0. Of the monoamine substrates examined, 4-methoxytyramine was the most extensively sulfated at 20 microM, while at higher substrate concentrations (200 microM), tyramine was the apparent preferred substrate. Kinetic analysis demonstrated that sulfation by M-PST proceeds via an ordered, bisubstrate reaction mechanism, where 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is the leading substrate. True Km values for dopamine and PAPS were 2.9 and 0.35 microM, respectively. The product inhibitor 3'-phosphoadenosine 5'-phosphate possessed a Ki of 0.07 microM, while the dead-end inhibitor ATP exhibited a Ki of 170 microM.     

Proximate sulfhydryl groups in the acetylglutamate complex of rat carbamylphosphate synthetase I: their reaction with the affinity reagent 5'-p-fluorosulfonylbenzoyladenosine

A preparation of rat carbamylphosphate synthetase I, isolated in the presence of antipain and stable without glycerol, has been used to investigate the effect of the allosteric activator, N-acetyl-L-glutamate (AcGlu), on the sulfhydryl chemistry of the enzyme. The enzyme X AcGlu complex was rapidly inactivated by several sulfhydryl group reagents and the ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSO2BzAdo), with the loss of two sulfhydryl groups per monomer. Inactivation was much slower without AcGlu, and ATP/Mg2+/K+ provided complete protection. Reaction with a 1.1 molar excess of 4,4'-dipyridyldisulfide resulted in an intramonomer disulfide bond between groups that are probably juxtaposed in the activated enzyme, because 1.1 equivalents of the vicinal dithiol reagent, phenylarsine oxide, eliminated the rapid reaction with the disulfide. Evidence is presented that the same disulfide bond was formed in the reactions with 5-thiocyano-2-nitrobenzoic acid and FSO2BzAdo. Inactivation by FSO2BzAdo was a pseudo-first-order reaction. The concentration dependence of the rate is consistent with the reaction proceeding through a noncovalent complex (KI = 67 microM and k2 = 0.23 min-1 at pH 7.0, 30 degrees C). Protection from FSO2BzAdo by ATP required Mg2+ in excess of ATP with KMgATP = 4.5 microM at saturating free Mg2+ (0.1 M K+) and KMg2+ = 6.5 mM. KMgATP is close to Kd for the molecule of ATP that contributes the phosphoryl group of carbamylphosphate (H.B. Britton, V. Rubio, and S. Grisolia, (1979) Eur. J. Biochem. 102, 521-530]; KMg2+ agrees with the minimum value for the steady-state kinetic parameter, Ki,Mg2+, obtained under the same conditions. Dissociation constants for adenosine (320 microM), MgADP (110 microM) at 10 mM Mg2+, and AcGlu (100 microM) were also estimated.     

Investigation of Ca2+ -induced changes of membrane potential of smooth muscle mitochondria using flow cytometric analysis

Using flow cytometric analysis and potential-sensitive fluorescent dye TMRM Ca2+ -induced changes of membrane potential of isolated smooth muscle mitochondria were studied. It was shown, that Ca2+ (100 microM) addition to the incubation medium induced mitochondrial membrane depolarization that probably could be explained by Ca2+/H+ -exchanger activation which functioning lead to membrane potential dissipation. In the case of ruthenium red (10 microM) preliminary presence in incubation medium, Ca2+ (100 microM) addition did not lead to membrane potential dissipation. Hence, membrane potential dissipation was caused by an increase of matrix Ca2+ concentration. In the presence of Mg2+ (3 mM) and ATP (3 mM), Ca2+ addition did not cause depolarization. It was supposed that in this case ATP synthase acted in the opposite direction as H+ -pump and prevented from mitochondrial membrane potential dissipation. Thus, the flow cytometry method allows to register membrane potential of isolated smooth muscle mitochondria and also to test the effectors, capable to modulate this parameter.     

Human placental cytoplasmic 5'-nucleotidase. Kinetic properties and inhibition

The kinetic properties of highly purified human placental cytoplasmic 5'-nucleotidase were investigated. Initial velocity studies gave Michaelis constants for AMP, IMP, and CMP of 18, 30, and 2.2 microM, respectively. The enzyme shows the following relative Vmax values: CMP greater than UMP greater than dUMP greater than GMP greater than AMP greater than dCMP greater than IMP. The activity was magnesium-dependent, and this cation binds sequentially with a Km of 14 microM for AMP and an apparent Km of 6 mM for magnesium. A large variety of purine, pyrimidine, and pyridine compounds exert an inhibitory effect on enzyme activity. IMP, GMP, and NADH produce almost 100% inhibition at 1.0 mM. Nucleoside di- and triphosphates are potent inhibitors. ATP and ADP are competitive inhibitors with respect to AMP and IMP as substrates with Ki values of 100 and 15 microM, respectively. Inorganic phosphate is a noncompetitive inhibitor with Ki values of 19 and 43 mM. Nucleosides and other compounds studied produce only a modest decrease of enzyme activity at 1 mM. Our findings suggest that the enzyme is regulated under physiological conditions by the concentrations of magnesium, nucleoside 5'-monophosphates, and nucleoside di- and triphosphates. The nucleotide pool concentration regulates the enzyme possibly by a mechanism of heterogeneous metabolic pool inhibition. These properties of human placental cytoplasmic 5'-nucleotidase may be related to the control of nucleotide degradation in vivo.     

Study of the interaction of Na+,K+-ATPase protomers using the molecular target method

The radiation inactivation analysis of Na+, K+-ATPase, (EC 3.6.1.37) from two different sources was carried out using ATP, CTP, GTP and p-NPP as substrates. In the case of Na+, K+-ATPase from the bovine brain the relation between the logarithm of the residual activity and the radiation dose is strictly linear, which permits calculating 75-90 kDa (for 3 mM GTP and 10 mM p-NPP). Duck salt glands Na+, K+-ATPase reveals larger target sizes: 350 kDa for ATP hydrolysis in saturating concentrations and 145-190 kDa in the case of GTP and p-NPP or low concentration of ATP (30 microM). A conclusion is drawn that while hydrolyzing substrates with complex kinetics (ATP and CTP) the enzyme functions like oligomer. The interaction of nucleotide with substrate-binding site of low affinity induces the aggregation of monomers.     

Purification of a myosin phosphatase from bovine aortic smooth muscle

A myosin phosphatase has been purified to homogeneity from bovine aortic smooth muscle. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme eluted from nondenaturing gels revealed two subunits (Mr = 67,000 and 38,000). Densitometric scans of the subunits indicated a molar ratio of 1:1. Several phosphoproteins were substrates for the phosphatase including histone II-A, isolated 20,000-dalton smooth muscle myosin light chains, phosphorylase a, and smooth muscle myosin. In the presence of 0.25 M NaCl and a substrate concentration of 2 microM, myosin was preferentially dephosphorylated. The specific activity of the phosphatase for myosin at a concentration of 10 microM was found to be 5 mumol/mg/min. The phosphatase required Mn2+ or Co2+ ions for activity. Mg2+, Ca2+, or Mg-ATP would not substitute for Mn2+ or Co2+ at equimolar concentrations. This phosphatase may play an important role in regulating actin-myosin interaction in smooth muscle by serving to dephosphorylate myosin.