Inhibition by excess of free ATP, and free Mg2+ ions of the mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus.

High concentrations of either Mg-ATP complex, free ATP, or free Mg2+ ions were inhibitors of the mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus. Free Mg2+ acts as a linear competitive inhibitor with regard to Mg-ATP hydrolysis with a Ki value of 2.8 mM. The inhibition by free ATP was markedly biphasic and thus simple competitive inhibition alone is not sufficient to explain the inhibitory effect. From these results conclusions were drawn about the binding of the substrate, Mg-ATP complex, to the enzyme.     

Partial purification and some kinetic properties of glucose-6-phosphate dehydrogenase from Phycomyces blakesleeanus

Glucose-6-phosphate dehydrogenase from sporangiophores of Phycomyces blakesleeanus NRRL 1555 (-) was partially purified. The enzyme showed a molecular weight of 85 700 as determined by gel-filtration. NADP+ protected the enzyme from inactivation. Magnesium ions did not affect the enzyme activity. Glucose-6-phosphate dehydrogenase was specific for NADP+ as coenzyme. The reaction rates were hyperbolic functions of substrate and coenzyme concentrations. The Km values for NADP+ and glucose 6-phosphate were 39.8 and 154.4 microM, respectively. The kinetic patterns, with respect to coenzyme and substrate, indicated a sequential mechanism. NADPH was a competitive inhibitor with respect to NADP+ (Ki = 45.5 microM) and a non-competitive inhibitor with respect to glucose 6-phosphate. ATP inhibited the activity of glucose-6-phosphate dehydrogenase. The inhibition was of the linear-mixed type with respect to NADP+, the dissociation constant of the enzyme-ATP complex being 2.6 mM, and the enzyme-NADP+-ATP dissociation constant 12.8 mM.     

Some kinetic properties of pyruvate kinase from Phycomyces blakesleeanus

• 1.1. Pyruvate kinase from mycelium of Phycomyces blakesleeanus NRRL 1555(−) has been partially purified and some kinetic properties has been investigated at pH 7.5.
• 2.2. Positive homotropic interactions were observed with phosphoenolpyruvate and Mg²⁺, showing Hill coefficient values of 2.8 and 2.5, respectively, whereas hyperbolic kinetics are found when ADP was the variable substrate.
• 3.3. Fructose 1,6-bisphosphate acts as a heterotropic allosteric activator, markedly decreasing the S_0.5 value for phosphoenolpyruvate saturation curve from a sigmoidal to a hyperbolic form.
• 4.4. ATP inhibits pyruvate kinase from mycelium of Phycomyces blakesleeanus. ATP appears to be a non-competitive inhibitor with respect PEP and competitive inhibitor with respect ADP.

     

Isolation and characterization of Phycomyces blakesleeanus ferritin.

Ferritin was isolated from the fungus Phycomyces blakesleeanus and compared biochemically and immunologically with horse spleen ferritin. Phycomyces and horse spleen ferritins were shown to exhibit similar electrophoretic patterns on polyacrylamide gels. Both preparations yielded an identical single band on sodium dodecyl sulfate-containing polyacrylamide gels. Tryptic digests of Phycomyces ferritin yielded 17 ninhydrin-positive spots as compared to 26 for horse spleen ferritin tryptic digests. Phycomyces ferritin was immunologically unrelated to horse spleen ferritin.     

The F1-ATPase from Streptococcus cremoris: isolation, purification and partial characterization

1. The F1-ATPase from the plasma membrane of Streptococcus cremoris HA was released by low ionic shock wash and purified by gel filtration and ion exchange chromatography. 2. The specific activity of the purified F1-ATPase was 25.8 mumol Pi/mg protein/min. 3. Km for ATP was 0.80 mM, and Ki for ADP as a competetive inhibitor 0.40 mM. 4. The purified F1-ATPase consisted of five subunits, alpha, beta, gamma, delta and epsilon, with molecular masses of 47.0, 45.0, 29.5, 22.0 and 13.0 kDa, respectively. 5. The isoelectric point of the enzyme complex was found to be 4.4.     

The kinetic mechanism of pyruvate reduction by lactate dehydrogenase from Phycomyces blakesleeanus

The kinetics of pyruvate reduction by lactate dehydrogenase from Phycomyces blakesleeanus NRRL 1555 (-) have been determined at pH 6.0. Initial rate studies performed in the pyruvate reduction direction suggest that a sequential mechanism is operating. Product inhibition studies with NAD+ and L(+)-lactate are consistent with an ordered sequential mechanism if we considered that NAD+ mimics the NADH that binds cooperatively on the enzyme and also the existence of dead-end complex responsible for substrate inhibition by pyruvate at this pH value.     

Large-scale purification and characterization of the five subunits of F1-ATPase from pig heart mitochondria.

A large-scale purification procedure was developed to isolate the five subunits of F1-ATPase from pig heart mitochondria. The previously described procedure (Williams, N. and Pedersen, P.L. (1986) Methods Enzymol. 126, 484-489) to dissociate the rat liver F1-ATPase by cold treatment followed by warming at 37 degrees C has been adapted for the pig heart enzyme. Removal of endogenous nucleotides from that enzyme before dissociation led to the efficient separation of the alpha and gamma subunits from beta, delta and epsilon subunits. The beta subunit was purified in the hundred-milligram range by anion-exchange chromatography in the absence of any denaturing agent. This subunit was free from any bound nucleotide and almost no ATPase and adenylate kinase-like activities were detected. The delta and epsilon subunits were purified by reversed-phase chromatography (RP-HPLC) in the milligram range. As recently reported (Penin, F., Deléage, G., Gagliardi, D., Roux, B. and Gautheron, D.C. (1990) Biochemistry 29, 9358-9364), these purified subunits kept biophysical features of folded proteins and their ability to reconstitute the tight delta epsilon complex. The alpha and gamma subunits remained poorly soluble and required dissociation by 8 M guanidinium chloride prior to their purification by RP-HPLC. In addition, characterizations of the five subunits by IEF and SDS-polyacrylamide gel electrophoresis are reported, as well as ultraviolet spectra and solubility properties of the beta, delta and epsilon subunits.     

A study of the kinetic mechanism followed by glutathione reductase from mycelium of Phycomyces blakesleeanus

An investigation of the reaction mechanism of glutathione reductase isolated from the mycelium of Phycomyces blakesleeanus NRRL 1555(-) was conducted. The enzyme showed GSSG concentration-dependent substrate inhibition by NADPH and pH-dependent substrate inhibition by GSSG. At pH 7.5, the kinetic data were consistent with a basic scheme corresponding to the branching mechanism, involving a ping-pong with formation of a dead-end F.NADPH complex and an ordered sequential mechanism. Both pathways have in common the step in which NADPH binds to the free oxidized form (E) of the glutathione reductase. At low concentrations of GSSG the ping-pong mechanism prevails, whereas at high concentrations the ordered mechanism appears to dominate. The data were analyzed on the basis of the limiting ping-pong mechanism with F.NADPH complex formation and of the hybrid mechanism, and the kinetic constants of the model were calculated. The data obtained at acidic pH values do not rule out the possibility that the kinetic model may be more complicated than the basic scheme studied.     

Purification and characterization of the F1-ATPase from Clostridium thermoaceticum.

The F1 portion of the H+-ATPase from Clostridium thermoaceticum was purified to homogeneity by solubilization at low ionic strength, ion-exchange chromatography, and gel filtration. The last indicated the Mr to be 370,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the pure enzyme revealed four bands with Mr corresponding to 60,000, 55,000, 37,000, and 17,000 in an apparent molar ratio of 3:3:1:1. The purified enzyme would bind to stripped membranes to reconstitute dicyclohexylcarbodiimide-sensitive ATPase activity. Phosphohydrolase activity, measured at 58 degrees C, was optimal at pH 8.5. In the presence of a 1 mM excess of Mg2+ over the concentration of ATP, the Km for ATP was 0.4 mM, and the Vmax was 6.7 mumol min-1 mg-1. Unlike the membrane-bound F1F0 complex, the F1-ATPase was relatively insensitive to the inhibitors dicyclohexylcarbodiimide and tributyltin chloride. Both the complex and the F1-ATPase were inhibited by quercetin, azide, 7-chloro-4-nitro-benz-2-oxa-1,3-diazole, and free magnesium, and both were stimulated by primary alcohols and sulfite. In whole cells, the F1F0-ATPase catalyzed the synthesis of ATP in response to a pH gradient.     

Isolation, purification and kinetic characterization of plasma membrane H(+)-ATPase of Candida albicans.

The plasma membrane ATPase of Candida albicans was solubilized by Tween 40 and purified to homogeneity on glycerol step gradient. The purified protein appeared as a single band of 100 +/- 4 KDa, represented greater than 98% of the total pure protein on densitometer scan. The purified PM-ATPase which was very specific to MgATP, had Km of about 0.77 mM and a sharp pH optimum at 6.6. Orthovanadate was able to inhibit the enzyme in a non-competitive manner, however, at higher concentrations the nature of inhibition changed to uncompetitive type. Based on molecular size, immuno cross-reactivity and sensitivity to different inhibitors, PM-ATPase of C. albicans appears to be similar to other ion pumps.     

A kinetic study of the pH effect on the allosteric properties of pyruvate kinase from Phycomyces blakesleeanus

This paper reports the pH-dependence of the allosteric kinetics of Phycomyces blakeseeanus pyruvate kinase with phosphoenol pyruvate and Mg2+ ions in the presence and in the absence of fructose 1,6-bisphosphate (allosteric activator) and L-alanine (allosteric inhibitor). Hydrogen ions increase the affinity of the inhibitory binding sites for phosphoenol pyruvate and Mg2+ ions. Assuming partial conformational states of high and low affinity for inhibitory binding sites, the data presented are in good agreement with the predictions postulated by the two-state concerted-symmetry model of Monod, Wyman, and Changeux. Fructose-1,6-bisphosphate and L-alanine show opposite effects on the interactions of phosphoenol pyruvate and Mg2+ ions with their respective catalytic and inhibitory binding sites. At pH 6.0, the regulation of the Phycomyces pyruvate kinase activity by the concentrations of phosphoenol pyruvate and Mg2+ ions is controlled mainly by L-alanine.     

In vitro characterization of two different Phycomyces blakesleeanus mutants with impaired phytoene desaturation.

In vitro phytoene desaturation was investigated in two Phycomyces blakesleeanus mutants, C5 and S442, in which phytoene is accumulated instead of beta-carotene. For strain C5 but not strain S442 the phenotypic block of phytoene conversion could be overcome in vitro by the addition of Tween 40. Immunodetection of phytoene desaturase revealed in all cases the presence of a 40-kilodalton protein.     

Mitochondrial ATP synthase: dramatic Mg2+-induced alterations in the structure and function of the F1-ATPase moiety

The ATPase activity of the F1 moiety of rat liver ATP synthase is inactivated when incubated prior to assay at 25 degrees C in the presence of MgCl2. The concentration of MgCl2 (130 microM) required to induce half-maximal inactivation is over 30 times higher than the apparent Km (MgCl2) during catalysis. Moreover, the relative efficacy of divalent cations in inducing inactivation during prior incubation follows an order significantly different from that promoting catalysis. Inactivation of F1-ATPase activity by Mg2+ is accompanied by the dramatic dissociation from the F1 complex of alpha subunits and part of the gamma-subunit population. The latter form a precipitate while the beta, delta, and epsilon subunits, and the remaining part of the gamma-subunit population, remain soluble. Dissociation is not a sudden "all or none" event but parallels loss of ATPase activity until alpha subunits have almost completely dissociated together with about 50% of the gamma-subunit population. Mg2+-induced loss of F1-ATPase activity cannot be prevented by including either the hydrolytic substrates ATP, GTP, or ITP in the incubation medium or the product ADP. Ethylenediaminetetraacetic acid, mercaptoethanol, and dithiothreitol are also ineffective in preventing loss of ATPase activity. Significantly, KPi at high concentration (greater than or equal to 200 mM) is effective in partially protecting F1 against inactivation. However, the most effective means of preventing Mg2+-induced inactivation of F1-ATPase activity is to rebind F1 to its F0 moiety in F1-depleted particles. When bound to F0, F1 is protected completely against divalent cation induced inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

F1F0-ATPase from Escherichia coli with mutant F0 subunits. Partial purification and immunoprecipitation of F1F0 complexes

Previously identified mutations in subunits a and b of the F0 sector of the F1F0-ATPase from Escherichia coli are further characterized by isolating detergent-solubilized, partially purified F1F0 complexes from cells bearing these mutations. The composition of the various F1F0 complexes was judged by quantitating the amount of each subunit present in the detergent-solubilized preparations. The composition of the F0 sectors containing altered polypeptides was determined by quantitating the F0 subunits that were immunoprecipitated by antibodies directed against the F1 portion. In this way, the relative amounts of F0 subunits (a, b, c) which survived the isolation procedure bound to F1 were determined for each mutation. This analysis indicates that both missense mutations in subunit a (aser206----leu and ahis245----tyr) resulted in the isolation of F1F0 complexes with normal subunit composition. The nonsense mutation in subunit a (atyr235----end) resulted in isolation of a complex containing the b and c subunits. The bgly131----asp mutation in the b subunit results in an F0 complex which does not assemble or survive the isolation. The isolated F1F0 complex containing the mutation bgly9----asp in the b subunit was defective in two regards: first, a reduction in F1 content relative to F0 and second, the absence of the a subunit. Immunoprecipitations of this preparation demonstrated that F1 interacts with both c and mutant b subunits. A strain carrying the mutation, bgly9----asp, and the compensating suppressor mutation apro240----leu (previously shown to be partially unc+) yielded an F1F0 ++ complex that remained partially defective in F1 binding to F0 but normal in the subunit composition of the F0 sector. The assembly, structure, and function of the F1F0-ATPase is discussed.     

Kinin-converting aminopeptidase from human urine. Further purification and characterization through kinetic and inhibitory studies

An aminopeptidase from human urine (HUA) able to hydrolyze L-aminoacyl-2-naphthylamides, L-Leu-p-nitroanilide and to convert both MLBK and LBK to BK has been further purified and characterized. The preparation now obtained showed a 3-fold higher specific activity than the previously described one and a single active protein band in 7% polyacrylamide gel electrophoresis accounting for 86% of total protein. Kinetic constants for this kinin-converting enzyme were determined using L-aminoacyl-2-naphthylamides, L-Leu-p-nitroanilide and LBK. The Km values for different naphthylamides were in the 10(-5) M range while that for L-Leu-p-nitroanilide was 3.6 X 10(-4) M. With LBK as substrate the aminopeptidase activity showed the highest catalytic efficiency in spite of a Km in the mM range. The enzyme was poorly inhibited by -SH and -S-S- group reagents. Some L-aminoacids, as well as mono- and diamines, indomethacin, puromycin and bestatin were equipotent competitive inhibitors of both arylamidase and aminopeptidase activities. Results obtained in this paper are compatible with our conclusion that human urine, unlike other enzyme sources, contains only one aminopeptidase, and that this enzyme displays both arylamidase and kinin-converting activities. The enzyme's action may be important in the metabolism of kinins, yielding peptides which could interact with both B-1 and B-2 kinin receptors in the kidney.     

Activation of chitin synthetase from Phycomyces blakesleeanus by calcium and calmodulin

Levels of basal chitin synthetase in cell-free extracts from Phycomyces blakesleeanus were reduced by breakage of cells in the presence of EDTA or EGTA. Addition of Ca²⁺ to these extracts activated chitin synthetase. Maximal activation was obtained after 2 h at a Ca²⁺ concentration of 2–5 mM. Activation by calcium was not reduced by any protease inhibitor tested but benzamidine, whereas the weak proteolytic activity of the extracts was inhibited by antipain. Larger levels of chitin synthetase activation were obtained by the simultaneous addition of calcium and calmodulin in most, but not all extracts. This further activation by calmodulin was prevented by TFP. ATP or cAMP did not stimulate activation by calcium or calcium-calmodulin.Abbreviations EGTA ethylene glycol-bis(B-aminoethylether)-N,NN-tetraacetic acid - GlcNAc N-acetyl-d-glucosamine - PMSF phenylmethylsulfonyl fluoride - SBTI soybean trypsin inhibitor - TFP trifluoperazine - TLCK N-p-tosyl-l-lysine choromethyl ketone - UDPGlcNAc uridine diphosphate N-acetyl-d-glucosamine