Molecular cloning and characterization of the Plasmodium falciparum cytidine triphosphate synthetase gene

6-Anilino-5,8-quinolinedione (LY83583) has been widely used as an agent to reduce levels of nitric oxide (NO)-dependent cGMP in tissues. We report here that suppression of NO formation and production of superoxide during enzymatic reduction of LY83583 by neuronal NO synthase appeared to be potentially involved in the pharmacological action caused by LY83583. LY83583 suppressed neuronal NO synthase activity of 20,000 x g rat cerebellar supernatant preparation in a concentration-dependent manner (IC50 value = 12.9 microM). A kinetic study revealed that LY83583 is a competitive inhibitor with respect to NADPH, with a Ki value of 2.57 microM. With purified neuronal NO synthase it was found that LY83583 was a potent inhibitor of NO formation by the enzyme and served as efficient substrate for reduction with a specific activity of 173 nmol of NADPH oxidized per mg of protein per minute. The reductase activity was stimulated about 19.8-fold by addition of CaCl2/calmodulin, indicating that the presence of CaCl2/calmodulin is essential to express maximal activity of LY83583 reduction. Although LY83583 was a good substrate for both NADPH-cytochrome P450 reductase (P450 reductase) and DT-diaphorase, these flavin enzymes-catalyzed reductions of LY83583 were less than the neuronal NO synthase-mediated reduction in the presence of CaCl2/calmodulin. Enzymatic generation of superoxide during reduction of LY83583 by neuronal NO synthase, P450 reductase or DT-diaphorase was confirmed by electron spin resonance (ESR) experiments. Thus the present results indicate that a benzoquinone derivative LY83583 appears to interact with the P450 reductase domain on neuronal NO synthase, resulting in inhibition of NO formation and superoxide generation, which is involved in suppression of intracellular cGMP content.     

Weaning-induced development of delta-opioid receptors in rat brain: differential effects of guanine nucleotides and sodium upon ligand-receptor recognition

Cibacron Blue F3GA (CB) inhibited the activities of wheat leaves NADH:nitrate reductase and NADH:cytochrome-c reductase in a time-independent and concentration dependent manner. The methyl viologen:nitrate reductase activity of the enzyme was unaffected by various CB concentrations used in the experiment. Inhibition of NADH:nitrate reductase was of mixed type (partial competitive and pure noncompetitive) with respect to NADH and noncompetitive with respect to nitrate. The estimated inhibition constant (Ki) values were 1 microM for NADH and 8.4 microM for nitrate. The secondary plots of inhibition with respect to NADH, indicated a dissociation constant (KI) of 8.8 microM for the enzyme-NADH-CB complex. This KI being greater than the Ki suggested that the noncompetitive inhibition is predominant over the competitive inhibition at the NADH binding site.     

Major changes in the kinetic mechanism of AMP inhibition and AMP cooperativity attend the mutation of Arg49 in fructose-1,6-bisphosphatase

The significance of subunit interface residues Arg49 and Lys50 in the function of porcine liver fructose-1,6-bisphosphatase was explored by site-directed mutagenesis, initial rate kinetics, and circular dichroism spectroscopy. The Lys50 --> Met mutant had kinetic properties similar to the wild-type enzyme but was more thermostable. Mutants Arg49 --> Leu, Arg49 --> Asp, Arg49 --> Cys were less thermostable than the wild-type enzyme yet exhibited wild-type values for kcat and Km. The Ki for the competitive inhibitor fructose 2,6-bisphosphate increased 3- and 5-fold in Arg49 --> Leu and Arg49 --> Asp, respectively. The Ka for Mg2+ increased 4-8-fold for the Arg49 mutants, with no alteration in the cooperativity of Mg2+ binding. Position 49 mutants had 4-10-fold lower AMP affinity. Most significantly, the mechanism of AMP inhibition with respect to fructose 1,6-bisphosphate changed from noncompetitive (wild-type enzyme) to competitive (Arg49 --> Leu and Arg49 --> Asp mutants) and to uncompetitive (Arg49 --> Cys mutant). In addition, AMP cooperativity was absent in the Arg49 mutants. The R and T-state circular dichroism spectra of the position 49 mutants were identical and superimposable on only the R-state spectrum of the wild-type enzyme. Changes from noncompetitive to competitive inhibition by AMP can be accommodated within the framework of a steady-state Random Bi Bi mechanism. The appearance of uncompetitive inhibition, however, suggests that a more complex mechanism may be necessary to account for the kinetic properties of the enzyme.     

Effect of heparin and dextran sulfate on the activity of glutathione reductase from yeast

Glutathione reductase (EC 1.6.4.2) is a pivotal enzyme of the glutathione antioxidant system in a cell. The kinetic studies of the interaction of glutathione reductase with unfractionated and low molecular weight heparin and dextran sulfate can contribute to explanation of polyanions effect on the conformation changes of glutathione reductase. The tested polyanions inhibit this enzyme and the inhibition effect depends on the ionic strengths and pH value. The most potent inhibitor is dextran sulfate (ID50 is 4.1 micrograms/ml, pH = 6.8, without NaCl). The ionic strength (> 100 mM) allows the reactivating of GR if the concentration of DS is not higher than 80 micrograms/ml. The inhibition effect of tested polyanions is caused by electrostatic interactions with enzyme; the kinetic analyses indicate that it is a mixed inhibition with respect to oxidized glutathione or NADPH.     

Antidepressants and suicide mortality

The aim of this study was to determine the effect of the soluble guanylyl cyclase inhibitors methylene blue and LY83583 (6-anilino-5,8-quinolinedione) on relaxation and increases in intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration ([cGMP]i) induced by sodium nitroprusside, 3-morpholinosydnonimine (SIN-1) and diethylamine-nitric oxide (NO) in porcine tracheal smooth muscle in vitro. We measured (1) the effect of NO donors on isometric force and [cGMP]i and (2) the ability of methylene blue and LY83583 to antagonize these effects. In muscle strips contracted with carbachol (0.1-0.3 microM), both sodium nitroprusside and diethylamine-NO caused relaxation and an increase in [cGMP]i. By contrast, SIN-1 caused a relaxation which was not associated with a concomitant increase in [cGMP]i. Methylene blue (10 microM) and LY83583 (10 microM) completely blocked the increase in [cGMP]i induced by sodium nitroprusside and diethylamine-NO; however substantial relaxation remained. It is concluded that in porcine airway smooth muscle, (1) relaxation induced by some NO donors may occur without a concomitant increase in [cGMP]i; and (2) whereas relaxation induced by some NO donors may be associated with increases in [cGMP]i, the relaxation is not completely dependent upon it.     

Leukotriene C4 is a tight-binding inhibitor of microsomal glutathione transferase-1. Effects of leukotriene pathway modifiers

Microsomal glutathione transferase-1 (MGST-1) is an abundant protein that catalyzes the conjugation of electrophilic compounds with glutathione, as well as the reduction of lipid hydroperoxides. Here we report that leukotriene C4 is a potent inhibitor of MGST-1. Leukotriene C4 was found to be a tight-binding inhibitor, with a Ki of 5.4 nM for the unactivated enzyme, and 9.2 nM for the N-ethylmaleimide activated enzyme. This is the first tight-binding inhibitor characterized for this enzyme. Leukotriene C4 was competitive with respect to glutathione and non-competitive toward the second substrate, CDNB. Analysis of stoichiometry supports binding of one molecule of inhibitor per homotrimer. Leukotrienes A4, D4, and E4 were much weaker inhibitors of the purified enzyme (by at least 3 orders of magnitude). Leukotriene C4 analogues, which have been developed as antagonists of leukotriene receptors, were found to display varying degrees of inhibition of MGST-1. In particular, the cysteinyl-leukotriene analogues SKF 104,353, ONO-1078, and BAYu9773 were strong inhibitors (IC50 values: 0.13, 3. 7, and 7.6 microM, respectively). In view of the partial structural similarity between MGST-1, leukotriene C4 synthase, and 5-lipoxygenase activating protein (FLAP), it was of interest that leukotriene C4 synthesis inhibitors (which antagonize FLAP) also displayed significant inhibition (e.g. IC50 for BAYx1005 was 58 microM). In contrast, selective 5-lipoxygenase inhibitors such as zileuton only marginally inhibited activity at high concentrations (500 microM). Our discovery that leukotriene C4 and drugs developed based on its structure are potent inhibitors of MGST-1 raises the possibility that MGST-1 influences the cellular processing of leukotrienes. These findings may also have implications for the effects and side-effects of drugs developed to manipulate leukotrienes.     

Characterization of the independent and combined effects of two inhibitors on oxidative drug metabolism in rat liver microsomes

To evaluate how two inhibitors influence oxidative drug metabolism, this study investigated the inhibitory effects of mexiletine with cimetidine and mexiletine with lidocaine, both individually and in combination, on the oxidative metabolism of two probe substrates, aminopyrine and aniline in rat liver microsomes. Mexiletine was a competitive inhibitor of aminopyrine N-demethylation, whereas cimetidine was a mixed type of inhibitor (Ki = 2.00 +/- 0.04 and 0.20 +/- 0.02 mM, respectively). For aniline hydroxylation, mexiletine exhibited a mixed type of inhibition, whereas lidocaine was a noncompetitive inhibitor (Ki = 0.60 +/- 0.07 and 8.50 +/- 0.12 mM, respectively). The combined inhibition of either mexiletine with cimetidine or mexiletine with lidocaine on aminopyrine and aniline metabolism was close to the fully additive effects of the individual compounds when their individual concentrations were below a 2-fold Ki concentration, regardless of the apparent kinetic inhibition type. The combined inhibition was less than fully additive when the individual concentrations were twice the Ki or above. These results demonstrate that, when two inhibitors of oxidative drug metabolism are combined, both the Ki values and the concentrations of inhibitors play important roles in determining the extent of additive inhibition of enzyme activity.     

Kinetic study of an enzyme-catalysed reaction in the presence of novel irreversible-type inhibitors that react with the product of enzymatic catalysis

In the present paper a kinetic study is made of the behaviour of a Michaelis-Menten enzyme-catalysed reaction in the presence of irreversible inhibitors rendered unstable in the medium by their reaction with the product of enzymatic catalysis. A general mechanism involving competitive, non-competitive, uncompetitive and mixed irreversible inhibition with one or two steps has been analysed. The differential equation that describes the kinetics of the reaction is non-linear and computer simulations of its dynamic behaviour are presented. The results obtained show that the systems studied here present kinetic co-operativity for a target enzyme that follows the simple Michaelis-Menten mechanism in its action on the substrate, except in the case of an uncompetitive-type inhibitor.     

Guanosine monophosphate reductase from Artemia salina: Inhibition by xanthosine monophosphate and activation by diguanosine tetraphosphate

In the course of studies on the metabolic role of diguanosine tetraphosphate during development of Artemia salina, a guanosine monophosphate (GMP) reductase has been found in partially purified from the 150 000g Artemia cysts supernatant. From Lineweaver-Burk plots, two apparent Km values of 5 and 50 muM were obtained for GMP. Xanthosine monophosphate (XMP) is a very strong inhibitor of the reaction. In the presence of 1.5 muM XMP hyperbolic kinetics are found. Diguanosine tetraphosphate counteracts very effectively the inhibition of the activity by XMP, concomitantly changing to hyperbolic the kinetics of the enzyme, with a unique Km value of about 5 muM. The complex kinetic and the existence of allosteric e-fectors at physiological concentrations, together with our lack of success in resolving two isoenzymes, makes it very likely that GMP reductase presents negative cooperativity towards its substrate. The effect of diguanosine tetraphosphate on the enzyme is very specific; other structural analogues, diadenosine tetraphosphate and diguanosine triphosphate, tested a micromolar concentrations had no detectable effect on the enzyme. Guanosine triphosphate (GTP) (mM) was also able to counteract the inhibition of guanosine monophosphate (GMP) reductase by XMP. The properties of the Artemia GMP reductase are here compared with those of the similar enzyme from calf thymus and Escherchia coli. As a consequence, the regulation of eukaryotic GMP reductase is resulting to be quite different from that of the reductase from prokaryotes.     

Nicotinamide inhibits inducible nitric oxide synthase enzyme activity in macrophages by allowing nitric oxide to inhibit its own formation

Nitric oxide (NO) production by macrophages is mainly regulated by induction of nitric oxide synthase (iNOS) by cytokines and microbial products. Nicotinamide (NIC) inhibits NO production by activated macrophages in a dose dependent manner. NIC also inhibits NOS enzyme activity in extracts from activated macrophages. The inhibition was noncompetitive with L-arginine (Ki 13.37 +/- 4.40 mM, n=3), uncompetitive versus NADPH (Ki 3.06 +/- 0.17 mM, n=3) and tetrahydrobiopterin. Finally, the inhibition by nicotinamide was fully reversed by scavenging NO with hemoglobin. We suggest that NIC acts by allowing NO to inhibit its own formation.     

Synthesis and differential properties of creatine analogues as inhibitors for human creatine kinase isoenzymes

Fourteen new creatine analogues, all with a guanidine function and either a polar or an apolar group instead of the creatine carboxylic function, were tested as potential inhibitors for human creatine kinase by kinetic analysis of their effects on the reaction rate. Only compounds bearing an apolar aromatic moiety, which was spaced from the guanidine function by at least two bonds, proved to have a significant inhibitory activity and showed a mixed-type inhibition similar to that of creatine. Among these compounds 2,6-dichlorobenzylguanidine (Ki = 5.6 mM and 39.8 mM for muscle-type and brain-type creatine kinases, respectively) and 3-(2,6-dichlorophenyl)propylguanidine (Ki = 15 mM and 4.5 mM) were the more potent inhibitors and showed a significant isoenzyme selectivity between muscle- and brain-type creatine kinases. Our results are in agreement with recent data that suggest the location of a hydrophobic pocket near the guanidine-binding domain of the enzyme. The observed selectivity in isoenzyme inhibition may be useful to study structural differences in catalytic centers.     

Characterization of the gene for human EMMPRIN, a tumor cell surface inducer of matrix metalloproteinases

Taurine is known to increase ATP-dependent calcium ion (Ca2+) uptake in retinal membrane preparations and in isolated rod outer segments (ROS) under low calcium conditions (10 microM) (Pasantes-Morales and Ordó?ez, 1982; Lombardini, 1991). In this report, ATP-dependent Ca2+ uptake in retinal membrane preparations was found to be inhibited by 5 microM cadmium (Cd2+), suggesting the involvement of cation channel activation. The activation of cGMP-gated cation channels, which are found in the ROS, is a crucial step in the phototransduction process. An inhibitor of cGMP-gated channels, LY83583, was found to inhibit taurine-stimulated ATP-dependent Ca2+ uptake but had no effect on ATP-dependent Ca2+ uptake in the absence of taurine, indicating that taurine may be increasing ATP-dependent Ca2+ uptake through a mechanism of action involving the opening of cGMP-gated channels. The activation of cGMP-gated channels with dibutyryl-cGMP and with phosphodiesterase inhibition using zaprinast caused an increase in ATP-dependent Ca2+ uptake in isolated ROS, but not in taurine-stimulated ATP-dependent Ca2+ uptake. LY83583 had the same effects in isolated ROS as in retinal membrane preparations. Another inhibitor of cGMP-gated channels, Rp-8-Br-PET-cGMPS, produced the same pattern of inhibition in isolated ROS as LY83583. Thus, there appears to be a causal link between taurine and the activation of the cGMP-gated channels in the ROS under conditions of low calcium concentration, a connection that suggests an important role for taurine in the visual signalling function of the retina.     

Incidence of spontaneous subarachnoid hemorrhage among Hispanics and non-Hispanic whites in New Mexico

Tri(gamma-glutamylcysteinylglycinyl)trithioarsenite (AsIII(GS)3) is formed in cells and is a more potent mixed-type inhibitor of the reduction of glutathione disulfide (GSSG) by yeast glutathione (GSH) reductase than either arsenite (AsIII) or GSH. The present work examines the effects of valence and complexation of arsenicals with GSH or L-cysteine (Cys) upon potency as competitive inhibitors of the reduction of GSH disulfide (GSSG) by yeast GSH reductase. Trivalent arsenicals were more potent inhibitors than their pentavalent analogs, and methylated trivalent arsenicals were more potent inhibitors than was inorganic trivalent As. Complexation of either inorganic trivalent As or methylarsonous diiodide (CH3As(III)I2) with Cys or GSH produced inhibitors of GSH reductase that were severalfold more potent than the parent arsenicals. In contrast, dimethylarsinous iodide ((CH3)2As(III)I) was a more potent inhibitor than its complexes with either GSH or Cys. Complexes of CH3AsIII with GSH (CH3-AsIII(GS)2) or with Cys (CH3AsIII(Cys)2) were the most potent inhibitors, with Ki's of 0.009 and 0.018 mM, respectively. Inhibition of GSH reductase by arsenicals or arsenothiols was prevented by addition of meso-2,3-dimercaptosuccinic acid (DMSA) to a mixture of enzyme, GSSG, and inhibitor before addition of NADPH. DMSA added to the reaction mixture after NADPH reversed inhibition by (CH3)2As(III)I but had little effect on inhibition by CH3As(III)I2, Ch3AsIII(GS)2, CH3AsIII(Cys)2, or AsIII(GS)3. Partial redox inactivation of the enzyme with NADPH increased the inhibitory potency of CH3As(III)I2 and (CH3)2As(III)I and changed the mode of inhibition for CH3As(III)I2 from competitive to noncompetitive. The greater potency of methylated trivalent arsenicals and arsenothiols than of inorganic trivalent As suggests that biomethylation of As could yield species that inhibit reduction of GSSG and alter the redox status of cells.     

Characterization of the antiproliferative signal mediated by the somatostatin receptor subtype sst5

We investigated cell proliferation modulated by cholecystokinin (CCK) and somatostatin analogue RC-160 in CHO cells bearing endogenous CCKA receptors and stably transfected by human subtype sst5 somatostatin receptor. CCK stimulated cell proliferation of CHO cells. This effect was suppressed by inhibitor of the soluble guanylate cyclase, LY 83583, the inhibitor of the cGMP dependent kinases, KT 5823, and the inhibitor of mitogen-activated protein (MAP) kinase kinase, PD 98059. CCK treatment induced an increase of intracellular cGMP concentrations, but concomitant addition of LY 83583 virtually suppressed this increase. CCK also activated both phosphorylation and activity of p42-MAP kinase; these effects were inhibited by KT 5823. All the effects of CCK depended on a pertussis toxin-dependent G protein. Somatostatin analogue RC-160 inhibited CCK-induced stimulation of cell proliferation but it did not potentiate the suppressive effect of the inhibitors LY 83583 and KT 5823. RC-160 inhibited both CCK-induced intracellular cGMP formation as well as activation of p42-MAP kinase phosphorylation and activity. This inhibitory effect was observed at doses of RC-160 similar to those necessary to occupy the sst5 recombinant receptor and to inhibit CCK-induced cell proliferation. We conclude that, in CHO cells, the proliferation and the MAP kinase signaling cascade depend on a cGMP-dependent pathway. These effects are positively regulated by CCK and negatively influenced by RC-160, interacting through CCKA and sst5 receptors, respectively. These studies provide a characterization of the antiproliferative signal mediated by sst5 receptor.     

Depletion of brain glutathione results in a decrease of glutathione reductase activity; an enzyme susceptible to oxidative damage

Loss of the intracellular antioxidant glutathione (GSH) from the substantia nigra is considered to be an early event in the pathogenesis of Parkinson's disease (PD). While the cause of the loss is unclear, an imbalance in the enzymes associated with the synthesis, utilisation, degradation and translocation of GSH has been implicated. The enzyme glutathione reductase is also important in GSH homeostasis: it regenerates GSH from the oxidised from (GSSG). However, to date the activity and regulation of glutathione reductase in conditions such as PD have not been explored. In view of this we have measured the effects of GSH depletion on glutathione reductase activity of the rat brain. Other glutathione related enzymes were also measured. Using pre-weanling rats, brain GSH was depleted by up to 60% by subcutaneous administration of L-buthionine sulfoximine. The only enzyme affected by GSH depletion was glutathione reductase; its activity being reduced by approximately 40%. As GSH inactivates a number of oxidising species including peroxynitrite (ONOO-), we additionally investigated the susceptibility of glutathione reductase to ONOO- in vitro, using purified enzyme. ONOO- decreased glutathione reductase activity in a concentration dependent manner with an apparent 50% inhibition occurring at an initial concentration of 0.09 mM. These data suggest that GSH is important in the maintenance glutathione reductase activity. This may arise in part from its ability to inactivate oxidising agents such as ONOO-.     

2,4,6-Trinitrotoluene Transformation Using Spinacia Oleracea: Saturation Kinetics of the Nitrate Reductase Enzyme

A series of aqueous phase and soil-slurry phase microcosm studies were conducted on 2,4,6-trinitrotoluene (TNT) to obtain kinetic data for optimizing a treatment protocol using an enzyme extract from spinach (Spinacia oleracea). Crude extract was obtained by homogenization of fresh leaves with a buffered protease inhibitor, and employed as phytoremediation agent. Aqueous phase microcosms containing 20?mg/L TNT and soil–slurry microcosms containing 1 g of a characterized sandy loam soil contaminated with 2,500?mg/kg TNT and 1,000?mg/kg hexahydro-1,3,5-trinitro-1,3,5-triazine were dosed with fixed aliquots of extract and analyzed for TNT transformation over time. The TNT concentration was monitored using a colorimetric method for nitroaromatic compounds based on EPA Method 8515. Nitrate reductase activity of the applied crude extract was simultaneously quantified. The transformation of TNT was described by a pseudofirst-order reaction. Coupling kinetic rate information with enzyme activity allowed for estimation of a second-order rate constant with respect to activity. A rectangular hyperbola function normalized for enzyme activity described observed kinetic data based on enzyme saturation, similar to a Michaelis–Menten relationship. Pseudofirst-order rate constants for the aqueous phase and soil–slurry phase experiments were fit to this function. The maximum rate of reaction (kmax) for TNT transformation was 0.50 and 0.04?h?1 for aqueous phase and soil–slurry phase experiments, respectively, while respective half-saturation constants (Ksat*) were comparable in value at 0.63 and 0.28?U/μmol–NO2, respectively. A Hanes–Woolf plot of reaction velocity versus TNT concentration with and without soil suggests an uncompetitive inhibition mechanism may be affecting overall nitrate reductase efficacy. Temperature effects for both aqueous phase and soil–slurry phase microcosms followed the Arrhenius relationship with estimated activation energies of 54.7 and 26.1?kJ/mol, respectively.     

Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase

The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase were investigated via the stopped-flow technique using the fluorescent labels RH421 and BIPM. After the enzyme is mixed with MgATP, both labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau1 approximately 180 s-1 (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3 --> E2P(Na+)3 + ADP). The rate of the phosphorylation reaction measured by the acid quenched-flow technique was 190 s-1 at 100 microM ATP, suggesting that phosphorylation controls the kinetics of the RH421 signal and that the conformational change is very fast (>/=600 s-1). The rate of the RH421 signal was optimal at pH 7.5. The Na+ concentration dependence of 1/tau1 showed half-saturation at a Na+ concentration of 8-10 mM with positive cooperativity involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high affinity ATP binding site determined from the ATP concentration dependence of 1/tau1 was 7.0 (+/-0.6) microM, while the apparent Kd for the low affinity site and the rate constant for the E2 to E1 conformational change evaluated in the absence of Mg2+ were 143 (+/-17) microM and 相似文献   

Effect of preincubation on smooth muscle myosin light chain kinase activity

Phosphorylation of myosin regulatory light chain (RLC) catalysed by myosin light chain kinase (MLCK) is a key reaction in the regulation of actin-myosin interaction in smooth muscle. The activation of MLCK by calmodulin (CaM) and Ca2+ was investigated over a wide range of the enzyme concentrations using myosin or its RLC with Mw = 20 kDa as substrates. Kinase activation by CaM (at saturating Ca2+ concentrations) was characterized by positive cooperativity even though noncooperative activation would be expected from the established 1:1 binding stoichiometry between MLCK and CaM. The activation of the kinase by Ca2+ was also cooperative but only at relatively low CaM levels. This cooperativity was shown to result from time dependent changes in MLCK that take place during its incubation with Ca2+ and CaM before substrate addition in phosphorylation assays. As a result the kinase activity as a function of its concentration at constant CaM level was biphasic: there was the activity optimum at 1:1 ratio of CaM to MLCK and almost complete inhibition at 3 to 7 molar excess of kinase over CaM. Such changes that take place during 10 to 15 min preincubation with Ca2+ and CaM may involve the kinase supramolecular structure formation or/and its conformational rearrangements.     

Coenzyme A disulfide reductase, the primary low molecular weight disulfide reductase from Staphylococcus aureus. Purification and characterization of the native enzyme

The human pathogen Staphylococcus aureus does not utilize the glutathione thiol/disulfide redox system employed by eukaryotes and many bacteria. Instead, this organism produces CoA as its major low molecular weight thiol. We report the identification and purification of the disulfide reductase component of this thiol/disulfide redox system. Coenzyme A disulfide reductase (CoADR) catalyzes the specific reduction of CoA disulfide by NADPH. CoADR has a pH optimum of 7.5-8.0 and is a dimer of identical subunits of Mr 49,000 each. The visible absorbance spectrum is indicative of a flavoprotein with a lambdamax = 452 nm. The liberated flavin from thermally denatured enzyme was identified as flavin adenine dinucleotide. Steady-state kinetic analysis revealed that CoADR catalyzes the reduction of CoA disulfide by NADPH at pH 7.8 with a Km for NADPH of 2 muM and for CoA disulfide of 11 muM. In addition to CoA disulfide CoADR reduces 4,4'-diphosphopantethine but has no measurable ability to reduce oxidized glutathione, cystine, pantethine, or H2O2. CoADR demonstrates a sequential kinetic mechanism and employs a single active site cysteine residue that forms a stable mixed disulfide with CoA during catalysis. These data suggest that S. aureus employs a thiol/disulfide redox system based on CoA/CoA-disulfide and CoADR, an unorthodox new member of the pyridine nucleotide-disulfide reductase superfamily.