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Plant and essential oil extracts have been used for some time as antimicrobials and antioxidants, although little is known about the interactions between the main components of these plant materials. This knowledge could help to design more potent antimicrobial and antioxidant mixtures. Carvacrol and thymol, the main components of the essential oils of the Lamiaceae family of plants, were assessed in combination to evaluate their antioxidant activity and antimicrobial effect against 19 strains of Staphylococcus aureus ( S. aureus ) of different origins (clinical, meat, milk, and other) and mostly (12) enterotoxin producers. The microdilution test assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the two phenolics alone and in combination. Based on the fractional inhibitory concentration index (FICI), no antimicrobial interaction (0.5 < FICI <4) between carvacrol and thymol was observed against 42% of the S. aureus strains and an antagonistic interaction (FICI >4) was observed in the rest, which indicates different behavior among strains in relation to this antimicrobial combination. Particularly, an antagonistic effect was observed in 29% of the meat origin strains and 57% of the dairy origin strains. Combinations of carvacrol and thymol were bactericidal (differences in MIC and MBC values not more than twofold) for 60% of the tested strains. At low concentrations of both components, the antioxidant effect is additive. However, at high concentrations (2.50 or 2.66 mM) of at least one of the components of the combination, it is antagonistic. The different types of interactions of the components in the combination can depend on many factors (ratio, structural characteristics, and the establishment of intermolecular complexes). The results could be used as reference to apply this combination in foods to control S. aureus , to maintain the organoleptic properties and to extend the shelf-life of them.

The objective of this study was to analyze the response of Phycomyces blakesleeanus to glucose starvation and acetate growth stress. At the onset of the exponential growth phase, the fungus shows a high tolerance to both stresses, being higher for the glucose starvation. In both stresses we have found higher activities of catalase and glutathione peroxidase, and a decrease of the pools of D-erythroascorbate (D-erythroascorbate+D-erythroascorbate monoglucoside) and glutathione (GSH+GSSG), while the intracellular GSH/GSSG redox balance becomes more reducing. Gallic acid was not detected under both stresses. Glycogen breakdown and the high levels of trehalose seem to be part of the stress response. Both stress, under the conditions of this study, seem to lead to a qualitatively similar response in P. blakesleeanus, with regard to the behavior of antioxidant system, the content of secondary metabolites and the role of the reserve carbohydrates., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

An analysis of the components of the antioxidant defence system in exponential and stationary growth phases of filamentous fungus Phycomyces blakesleeanus and the response to the oxidative stress hydrogen peroxide were performed. There is a strong positive correlation between mycelial antioxidant capacity and the contents of gallic acid, d-erythroascorbate (d-EAA) or d-erythroascorbate monoglucoside (d-EAAG). These secondary metabolites are specifically synthesized by this fungus and reach maximal values in the stationary growth phase, suggesting that they can play some role in the antioxidant defence system of this fungus. There is a differential expression of the two more notable antioxidant activities, catalase (CAT) and superoxide dismutase (SOD), depending of the growth stage of P. blakesleeanus, CAT being expressed in the exponential and SOD in the stationary phase. Phycomyces blakesleeanus showed a high resistance to the oxidative stress caused by H2O2 (50 and 200 mM) which was higher in exponential phase. This higher resistance can be explained by the presence of CAT, glutathione peroxidase (GPx), and the probable contribution of glutathione-S-transferase (GST) and high levels of reduced form of glutathione (GSH). The transition to stationary phase was accompanied with a higher physiological oxidative damage illustrated by the higher protein carbonylation. In this growth stage the resistance of the fungus to the oxidative stress caused by H2O2 could be explained by the presence of SOD, GPx, and the probable contribution of GST as well as of secondary metabolites, mainly d-EAA and d-EAAG. These results highlight a specific response to oxidative stress by H2O2 depending on the growth phase of P. blakesleeanus., (Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Six pure phenolic compounds (hydroquinone, thymol, carvacrol, butylated hydroxyanisole, gallic acid, and octyl gallate) were tested for their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against several strains of Staphylococcus aureus isolated from dairy and meat products. In addition, S. aureus reference strains (American Type Culture Collection) for antimicrobial studies and/or isolated from human infections and outbreaks of food poisoning were included in the study. Of the compounds tested, octyl gallate and hydroquinone were the most effective against S. aureus (mean MIC values of 20.89 and 103.05 μg/mL, respectively) and carvacrol and thymol the least (mean MIC values of about 413 μg/mL). The mean MBC values were 40.84, 194.37, 417.46, and 581.90 μg/mL for octyl gallate, hydroquinone, carvacrol, and thymol, respectively. Meat isolates were more resistant than those of dairy origin to hydroquinone, gallic acid, and octyl gallate, as well as to penicillin G (used as a control of the methodology used); gallic acid and penicillin G showed the highest differences in MIC values between the groups of strains (about 10 and 200 times, respectively). On the other hand, when we tested the isolates included in each group of strains (dairy, meat, and other/mixed sources) we only detected significant differences (p < 0.05) among dairy and isolates from other/mixed sources for hydroquinone and thymol, respectively. However, strains of meat origin exhibited significant differences among each other (p < 0.05) to most of the phenolic compounds tested (hydroquinone, carvacrol, gallic acid, and octyl gallate). The relationship between MICs and MBCs for each of the phenolic compounds tested suggested a bactericidal mechanism of action against S. aureus. Gallic acid and octyl gallate exhibited the highest antioxidant capacity and thymol and carvacrol the lowest. So, octyl gallate is an agent with both antimicrobial and antioxidant properties, which would be of interest to use in the food industry.

D-erythroascorbate (D-EAA), a five-carbon analogue of L-ascorbate (L-AA), and D-erythroascorbate monoglucoside (D-EAAG) are accumulated in Phycomyces blakesleeanus grown on glucose (99.5 and 1084 μg/g mycelial dry weight, respectively) and also excreted into the culture medium. Both compounds showed UV spectral properties and ionization constants similar to those of L-AA. D-EAAG was much more stable to aerobic oxidation than D-EAA and L-AA at acidic pH. D-EAAG is synthesized from D-erythroascorbate by a mycelial glucosyltransferase activity that uses UDP-glucose as glucose substrate donor with K(m) = 2.5 mM and 41.3 μM for D-EAA. This glucosyltransferase activity was maximal in the stationary growth phase in parallel with maximal production of D-EAAG. The presence of D-arabinose or D-arabinono-1,4-lactone in the culture medium produces the maximal accumulation of D-EAA and D-EAAG (about 30- and 4-fold with respect to that obtained in glucose culture). Both compounds showed greater antioxidant activity than L-AA and other standard antioxidants, with a capacity similar to that of L-AA to inhibit the growth of Escherichia coli.

Seventeen phenolic compounds that are allowed to be used in the European food industry as aromatizants or antioxidants or that are naturally present in plants were tested for their ability to inhibit 19 strains of Staphylococcus aureus by using a standardized paper disc assay. Most of the strains assayed were foodborne (dairy and meat products). Human isolates and/or strains recommended for testing antimicrobial agents were also included in the study, and some of the test strains were enterotoxin producers. When the content was 200 microg/disc, various phenolic compounds had shown antimicrobial activity against all (hydroquinone, thymol, carvacrol, butylated hydroxyanisole, octyl gallate, and tannic acid) or most (gallic acid, propyl gallate, and ellagic acid) of the S. aureus strains tested. Significant differences in the inhibition zones (p < 0.05) among strains of the same, or similar, origin and among the different origins were observed for most of the phenolic compounds that showed antimicrobial activity for all or most of the strains tested.

Trehalose and glycogen are reserve carbohydrates that were shown to accumulate in mycelia of the filamentous fungus Phycomyces blakesleeanus. Both carbohydrates were mobilized under glucose starvation or in the presence of acetate. Glycogen was mobilized faster than trehalose in the presence of acetate. In all cases, glycogen and trehalose mobilization followed single exponential decay. There was a direct relationship between glycogen mobilization and the concentration of the dissociated form of external acetic acid. The half-life of glycogen mobilization increased as the concentration of the external acetate anion decreased, so the dissociated form of acetate was the stressor causing glycogen mobilization. Mobilization was not due to transfer to poor carbon sources, as the dissociated form of other weak acids (butyrate, lactate, pyruvate and propionate) also produced glycogen mobilization. Previous exposure of the mycelia to a lower acetate concentration decreased glycogen mobilization by subsequent exposure to a high acetate concentration. Glycogen mobilization by acetate may be involved in production of ATP necessary for acetate uptake as well as for maintenance of the internal pH homeostasis.

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine in the presence of l-carnitine, thus facilitating the entry of fatty acids to mitochondria, in a process that is physiologically inhibited by malonyl-CoA. To examine the mechanism of CPT1 liver isoform (CPT1A) inhibition by malonyl-CoA, we constructed an in silico model of both its NH2- and COOH-terminal domains. Two malonyl-CoA binding sites were found. One of these, the "CoA site" or "A site," is involved in the interactions between NH2- and COOH-terminal domains and shares the acyl-CoA hemitunnel. The other, the "opposite-to-CoA site" or "O site," is on the opposite side of the enzyme, in the catalytic channel. The two sites share the carnitine-binding locus. To prevent the interaction between NH2- and COOH-terminal regions, we produced CPT1A E26K and K561E mutants. A double mutant E26K/K561E (swap), which was expected to conserve the interaction, was also produced. Inhibition assays showed a 12-fold decrease in the sensitivity (IC50) toward malonyl-CoA for CPT1A E26K and K561E single mutants, whereas swap mutant reverts to wild-type IC50 value. We conclude that structural interaction between both domains is critical for enzyme sensitivity to malonyl-CoA inhibition at the "A site." The location of the "O site" for malonyl-CoA binding was supported by inhibition assays of expressed R243T mutant. The model is also sustained by kinetic experiments that indicated linear mixed type malonyl-CoA inhibition for carnitine. Malonyl-CoA alters the affinity of carnitine, and there appears to be an exponential inverse relation between carnitine Km and malonyl-CoA IC50.

Two forms of acetyl-CoA synthetase (ACS1 and ACS2) have been detected in Phycomyces blakesleeanus. ACS1, encoded by the gene facA, was induced by acetate and repressed by glucose at the transcriptional level. ACS2, not encoded by the gene facA, was detected as a response to carbon starvation both in the wild type and in an facA(-) mutant. Both enzymes were purified and characterized. They can use acetate and propionate as substrates. ACS2 is a much more stable enzyme than ACS1. After 60 min incubation at 55 degrees C, ACS2 retained 50% of its activity whereas ACS1 only retained 3%. The optimum temperature was 50 degrees C for ACS2 and 30 degrees C for ACS1.

D-Erythroascorbate and D-erythroascorbate glucoside have been identified in the Zygomycete fungus Phycomyces blakesleeanus. Ascomycete and Basidiomycete fungi also synthesise D-erythroascorbate instead of l-ascorbate, suggesting that D-erythroascorbate synthesis evolved in the common ancestor of the fungi. Both compounds accumulate in P. blakesleeanus at higher levels than observed in other fungal species. D-Erythroascorbate glucoside reduced dichlorophenolindophenol as effectively as L-ascorbate, but was more stable to autoxidation. D-Erythroascorbate glucoside predominated in spores and stationary phase mycelium. Free D-erythroascorbate accumulated during the exponential phase of mycelial growth and decreased to very low levels in the stationary phase. This suggests an association between growth and free D-erythroascorbate. P. blakesleeanus converted exogenous D-arabinose to D-erythroascorbate and its glucoside. A monomeric NAD-dependent D-arabinose dehydrogenase of 41 kDa was purified to near homogeneity. The enzyme oxidised D-arabinose, L-galactose, and L-fucose. Correspondingly, mycelium converted exogenous L-galactose and L-fucose to L-ascorbate and 6-deoxyascorbate, respectively. The antioxidant role of D-erythroascorbate and its glucoside is discussed.

Intact Phycomyces blaskesleeanus mycelia are capable of reducing extracellular ferricyanide and this transmembrane reduction is an enzymatic process, which is enhanced by the presence of 10 mM lactate. It is modulated in response to intracellular iron levels and negatively regulated by iron and copper. It is inhibited by NEM, p CMB, iodoacetate, Zn2+, Cd2+, dicumarol, and capsaicine analog, but not by cloroquine, and activated by Ca2+, Mg2+, Na+, and K+. Ferricyanide reduction was concomitant with proton release into the extracellular medium, both processes being greatly promoted by vitamin K3 following hyperbolic saturation kinetics with regard to ferricyanide concentration. No stoichiometric proton release was observed with regard to ferricyanide reduction in the absence or the presence of vitamin K3. Proton release coupled with ferricyanide reductase activity does not appear to be due to H+-ATPase. The relevance of these findings to the relationship between the two processes is discussed.

Normalisation of kinetic data is a useful tool in the study of complex enzyme systems. In the present paper, we have applied the premises of the normalised plot to the description of uni-uni enzyme inhibition. Guidelines to the design of the experiments and to data managing using the freeware program SIMFIT (http:\\www.simfit.man.ac.uk) are offered. The treatment has a lessened demand in experimental data while ensuring biological consistence of the results. Moreover, the results are obtained without resorting to secondary plots, and the election between rival mechanisms is statistically granted. Hyperbolic mixed-type inhibition is studied as a general model for enzyme-inhibitor/activator interaction, and equations describing classical cases of linear inhibition are also considered.

Phycomyces blakesleeanus isocitrate lyase (EC 4.1.3.1) is in vivo reversibly inactivated by hydrogen peroxide. The purified enzyme showed reversible inactivation by an ascorbate plus Fe(2+) system under aerobic conditions. Inactivation requires hydrogen peroxide; was prevented by catalase, EDTA, Mg(2+), isocitrate, GSH, DTT, or cysteine; and was reversed by thiols. The ascorbate served as a source of hydrogen peroxide and also reduced the Fe(3+) ions produced in a "site-specific" Fenton reaction. Two redox-active cysteine residues per enzyme subunit are targets of oxidative modification; one of them is located at the catalytic site and the other at the metal regulatory site. The oxidized enzyme showed covalent and conformational changes that led to inactivation, decreased thermal stability, and also increased inactivation by trypsin. These results represent an example of redox regulation of an enzymatic activity, which may play a role as a sensor of redox cellular status.

A new data treatment is described for designing kinetic experiments and analysing kinetic results for multi-substrate enzymes. Normalized velocities are plotted against normalized substrate concentrations. Data are grouped into n + 1 families across the range of substrate or product tested, n being the number of substrates plus products assayed. It has the following advantages over traditional methods: (1) it reduces to less than a half the amount of data necessary for a proper description of the system; (2) it introduces a self-consistency checking parameter that ensures the 'scientific reliability' of the mathematical output; (3) it eliminates the need for a prior knowledge of Vmax; (4) the normalization of data allows the use of robust and fuzzy methods suitable for managing really 'noisy' data; (5) it is appropriate for analysing complex systems, as the complete general equation is used, and the actual influence of effectors can be typified; (6) it is amenable to being implemented as a software that incorporates testing and electing among rival kinetic models.

NADP-isocitrate dehydrogenase from Cephalosporium acremonium CW-19 has been inactivated by diethyl pyrocarbonate following a first-order process giving a second-order rate constant of 3.0 m-1. s-1 at pH 6.5 and 25 degrees C. The pH-inactivation rate data indicated the participation of a group with a pK value of 6.9. Quantifying the increase in absorbance at 240 nm showed that six histidine residues per subunit were modified during total inactivation, only one of which was essential for catalysis, and substrate protection analysis would seem to indicate its location at the substrate binding site. The enzyme was not inactivated by 5, 5'-dithiobis(2-nitrobenzoate), N-ethylmaleimide or iodoacetate, which would point to the absence of an essential reactive cysteine residue at the active site. Pyridoxal 5'-phosphate reversibly inactivated the enzyme at pH 7.7 and 5 degrees C, with enzyme activity declining to an equilibrium value within 15 min. The remaining activity depended on the modifier concentration up to about 2 mm. The kinetic analysis of inactivation and reactivation rate data is consistent with a reversible two-step inactivation mechanism with formation of a noncovalent enzyme-pyridoxal 5'-phosphate complex prior to Schiff base formation with a probable lysyl residue of the enzyme. The analysis of substrate protection shows the essential residue(s) to be at the active site of the enzyme and probably to be involved in catalysis.

A saturable and accumulative transport system for pyruvate has been detected in Phycomyces blakesleeanus NRRL 1555(-) mycelium. It was strongly inhibited by alpha-cyano-4-hydroxycinnamate. l-Lactate and acetate were competitive inhibitors of pyruvate transport. The initial pyruvate uptake velocity and accumulation ratio was dependent on the external pH. The Vmax of transport greatly decreased with increasing pH, whereas the affinity of the carrier for pyruvate was not affected. The pyruvate transport system mediated its homologous exchange, which was essentially pH independent, and efflux, which increased with increasing external pH. The uptake of pyruvate was energy dependent and was strongly inhibited by inhibitors of oxidative phosphorylation and of the formation of proton gradients. Glucose counteracted the inhibitory effect of the pyruvate transport produced by inhibitors of mitochondrial ATP synthesis. Our results are consistent with a pyruvate/proton cotransport in P. blakesleeanus probably driven by an electrochemical gradient of H+ generated by a plasma membrane H+-ATPase., (Copyright 1998 Academic Press.)

An acid protease has been found in the culture broth of Phycomyces blakesleeanus growing under standard conditions. It has been induced up to 70-fold with several complex growth media and the enzyme has been purified to homogeneity and characterized. The molecular mass of the native enzyme was estimated by gel filtration to be 40 kDa. The acid protease of Phycomyces migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to a molecular mass of 35 kDa. The glycoprotein nature for the acid protease was deduced from its binding to a concanavalin A-Sepharose 4B column. The carbohydrate moiety is composed of mannose and rhamnose. Its amino acid composition was determined, and its isoelectric point was estimated to be 4.2, the optimum pH was 2.5 to 3, and the optimum temperature was 70 degrees C, using hemoglobin as a substrate. The enzyme showed thermal stability between 37 and 50 degrees C. The thermodynamic parameters for hemoglobin hydrolysis and thermal inactivation were calculated. With Lys-Pro-Ile-Glu-Phe-Phe(4-N02)-Arg-Leu as the substrate, the Km, kcat, and Vmax values were 8.78 microM, 1.25 s(-1), and 2.12 mumol min(-1) mg(-1), respectively. The protease was insensitive to phenylmethylsulfonyl fluoride, O-phenanthroline, N-ethylmaleimide, iodoacetamide, ethylenediaminetetraacetate, [ethyl-enebis(oxyethylenenitrilo)]tetraacetic acid, and trypsin inhibitor. However, pepstatin A established a strong competitive inhibition against it, with a K(i) value of 1.33 nM. The data suggest that this protease has properties of an aspartate-type proteinase.

Two forms of NAD+-isocitrate dehydrogenase, named ICDH-1 and ICDH-2, have been identified and purified in Phycomyces blakesleeanus NRRL-1555(-). These enzymes forms may be separated by chromatography on DEAE-Sephacel. ICDH-2 induction was a response to the adaptation of Phycomyes growth on acetate as the carbon source. Both enzyme forms were octamers of 388 + or - 30 kDa with apparently identical subunits of 40.5 +/- 5 kDa, but they were distinguishable by their electrophoretic mobilities on polyacrylamide gel electrophoresis. Isoelectric pH values were 5.28 and 4.96 for ICDH-1 and ICDH-2, respectively. ICDH-2 was more stable to urea denaturation than ICDH-1. At pH 7.6, ICDH-1 showed a markedly sigmoidal kinetic behavior with respect to isocitrate. However, ICDH-1 and ICDH-2 showed hyperbolic kinetics with respect to NAD+. THe tribasic form of isocitrate (I3-) and its magnesium complex (MI-) are the true substrates for both enzyme forms. Kinetic data obtained with Mg2+ as a divalent cation for both enzyme forms are compatible with the kinetic mechanism proposed by Cohen and Colman (1974) [Eur. J. Biochem. 47, 35-45] but assuming some degree of interaction between binding sites for the active form of isocitrate. This report describes for the first time the existence of two forms of NAD+-isocitrate dehydrogenase in filamentous fungi. From the changes in activity levels for each form, during adaptation of Phycomyces to growth on acetate and taking into account the kinetic and regulatory properties of both enzyme forms, we discuss the role of ICDH-1 and ICDH-2 in the control of isocitrate flux in Phycomyces.

We determined the variation with pH of the kinetic parameters for the isocitrate cleavage reaction catalyzed by Phycomyces isocitrate lyase, with the aim of elucidating the role played by ionising amino acid residues in binding and catalysis. The log VmaxpH profile shows that the enzyme possesses two ionising groups with pK values of 6.1 and 8.3. The first group is also observed in the VmaxpH/KmpH and pKmpH profiles, so this group is involved in catalysis. The last two profiles exhibit a similar pK value of 16 on the basic side, which represents the sum of the pK values for two ionising groups with pK values that differ by less than two pH units. Diethyl pyrocarbonate inactivated isocitrate lyase from Phycomyces with a second-order rate constant of 18.58 M-1 s-1 (at pH 6.0 and 20 degrees C). The difference spectra of the modified enzyme revealed an absorption maximum at 242 nm, characteristic of N-carbethoxyhistidine isocitrate lyase. No trough at around 280 nm due to O-carbethoxytyrosine is observed. Quantification of the increase in absorbance to 242 nm due to N-carbethoxyhistidine showed that ten histidine residues/active site were modified during total inactivation. However, only one of them was essential for catalysis. Treatment of the partially inactivated enzyme with hydroxylamine led to recovery of a substantial part of the original activity. The reactivity of isocitrate lyase towards diethyl pyrocarbonate declined with pH, following a titration curve for a group of pK 6.1. The presence of substrate decreased the rate of inactivation. Data-protection analyses indicate that the reactive histidine residues are within the active site of the enzyme.

Isocitrate lyase was purified from Cephalosporium acremonium CW-19 from cultures growing with poly(oxyethylene)sorbitan monopalmitate as the carbon source. Its subunit M(r) and native M(r) were 63,000 +/- 2000 and 250,000 +/- 5000, respectively. We found the Mg(2+)-isocitrate complex to be the true substrate and that Mg2+ ions act as a nonessential activator, according to the model reported by Giachetti et al. (1988) [Giachetti, E., Pinzauti, G., Bonaccorsi, R., & Vanni, P. (1988) Eur. J. Biochem. 172, 85-91], from which the kinetic parameters were calculated. The kinetic study is consistent with an ordered Uni-Bi mechanism, and the kinetic and rate constants of the model were calculated. pH dependence of the cleavage reaction indicated that the catalysis was dependent on two dissociable groups on the enzyme-substrate complex. The enzyme was inactivated by diethyl pyrocarbonate following first-order kinetics at all reagent concentrations used. The pseudo-first-order rate constant of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.0. Hydroxylamine added to the inactivated enzyme quickly restored the incremental absorption at 240 nm and most of the activity. Data analyses indicated that diethyl pyrocarbonate inactivation is a consequence of modification of 11 histidine residues per enzyme subunit, and from statistical analysis, we concluded that one is catalytically important. Mg(2+)-isocitrate protects the enzyme against diethyl pyrocarbonate inactivation with a Ks value of 26.8 +/- 2.1 microM, close to the Km value. Isocitrate protects the enzyme but a high concentration, suggesting its binding to the catalytic site of the nonactivated enzyme. Mg2+ ions also produced total competitive protection.

Exposure of lactate dehydrogenase from rabbit muscle to the Fe(III)/EDTA/ascorbate oxidation system leads to a time-dependent enzymatic inactivation (rate of inactivation of 7.35 x 10(-3) min-1), as well as to a spontaneous fragmentation of the protein. Fe(III) is the most important compound in this system, having the highest inactivating effects at concentrations above 10 microM. The substrate pyruvate and the products of the enzymatic reaction, when added at high concentration to the full mixture of the system, have a partial protective effect on the catalytic activity.

Mitochondrial F1-ATPase was purified from the mycelium of Phycomyces blakesleeanus NRRL 1555(-) and its kinetic characteristics were studied. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme reveals five bands (alpha, beta, gamma, delta, and epsilon) characteristic of the F1 portion with apparent molecular weights of 60,000, 53,000, 31,000, 25,000, and 21,000, respectively. The molecular weight of the native F1-ATPase from Phycomyces blakesleeanus was in agreement with the stoichiometry alpha 3 beta 3 gamma delta epsilon. The MgATP complex is the true substrate for ATPase activity which has a Km value of 0.15 mM. High concentrations of free ATP or free Mg2+ ions inhibit the ATPase activity. ADP appears to act as a negative allosteric effector with regard to MgATP hydrolysis, with the apparent Vmax remaining unchanged.

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.

Phycomyces blakesleeanus glutathione reductase shows hysteretic behaviour under experimental conditions, when GSSG substrate inhibition is observed. The progress curves for the reaction show an acceleration phase. The degree of hysteresis varied inversely as the enzyme concentration. It increased when GSSG or NADPH concentration increased, whereas the addition of GSH or NADP+ to the initial reaction mixture prevented it from occurring. In addition, hysteresis was dependent on pH, ionic strength and temperature, decreasing as any of these parameters increased. The parallel effects of pH and ionic strength on the GSSG substrate inhibition and hysteretic behaviour suggest a relationship between these two mechanisms. From the overall results reported in this paper, we propose that the hysteretic behaviour shown by Phycomyces glutathione reductase could be due to a process of time-dependent accumulation of reaction products rather than to a slow conformational change.

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.

Studies on ATP effects on the allosteric kinetics shown by pyruvate kinase from Phycomyces blakesleeanus NRRL 1555 (-) are reported. Phosphoenolpyruvate showed an allosteric ATP-dependent substrate inhibition. The results supported the existence of spatially distinct catalytic binding sites and the inhibitory binding sites for phosphoenolpyruvate, and ATP showed opposite heterotropic effects with respect to these two types of binding site. With respect to Mg2+ ions, ATP caused a negative heterotropic effect. The global inhibitory effect of ATP was in agreement with the predictions postulated by the two-state concerted-symmetry model of Monod, Wyman and Changeux.

A carbamoyl-phosphate synthase has been purified from mycelia of Phycomyces blakesleeanus NRRL 1555 (-). The molecular weight of the enzyme was estimated to be 188,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consists of two unequal subunits with molecular weights of 130,000 and 55,000. The purified enzyme has been shown to be highly unstable. The carbamoyl-phosphate synthase from Phycomyces uses ammonia and not L-glutamine as a primary N donor and does not require activation by N-acetyl-L-glutamate, but it does require free Mg2+ for maximal activity. Kinetic studies showed a hyperbolic behavior with respect to ammonia (Km 6.34 mM), bicarbonate (Km 10.5 mM) and ATP.2 Mg2+ (Km 0.93 mM). The optimum pH of the enzyme activity was 7.4-7.8. The Phycomyces carbamoyl-phosphate synthase showed a transition temperature at 38.5 degrees C. It was completely indifferent to ornithine, cysteine, glycine, IMP, dithiothreitol, glycerol, UMP, UDP and UTP. The enzyme was inhibited by reaction with 5 mM N-ethylmaleimide.

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.

1. The inhibition of ATP, ADP and AMP on lactate dehydrogenase activity from Phycomyces blakesleeanus was investigated kinetically at pH 6.0 and 7.5. 2. At pH 6.0 ATP was more effective as inhibitor than ADP and AMP. 3. All three nucleotides bind cooperatively to the enzyme, ATP and ADP decrease the positive homotropic interactions of NADH at pH 6.0. 4. The results obtained may contribute to explain the regulation of lactate dehydrogenase from Phycomyces blakesleeanus at acid pH values.

The kinetics and reaction mechanism of cytoplasmic malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) from mycelium of Phycomyces blakesleeanus NRRL 1555 (-) in 0.1 M potassium phosphate buffer (pH 7.5) at 30 degrees C have been investigated. The initial rate and product inhibition studies were consistent with an ordered bi-bi mechanism that involved more than one kinetically significant ternary complex and also with the coenzyme binding first. The dissociation of the coenzyme from the enzyme-coenzyme complex appeared to be the slowest step in either direction of the reaction. The kinetic and rate constants for the individual steps of the reaction were determined.

Some kinetic properties of the D(-)-lactate dehydrogenase (EC 1.1.1.28) of Escherichia coli have been investigated. There were marked differences between the kinetic properties of the enzyme studied in situ compared with the in vitro D(-)-lactate dehydrogenase. D(-)-Lactate dehydrogenase in situ showed high substrate inhibition with pyruvate over the pH range 6.0-7.0, whereas the enzyme in vitro did not. The pH optimum for pyruvate reduction by the in situ D(-)-lactate dehydrogenase ranged between pH 7.5 and 7.8, whereas the in vitro enzyme showed its pH optimum between pH 6.8 and 7.0. The pK values of the prototropic groups that controlled the enzymatic activity shift to the acidic region for the in vitro enzyme with respect to the in situ enzyme. In vitro D(-)-lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate and its coenzyme, NADH, at pH values ranging between pH 6.0 and 8.5, but the in situ enzyme showed homotropic interactions neither with pyruvate nor with NADH at all pH values studied.

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.

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.

The influence of fructose 1,6-bisphosphate and L-alanine on the kinetics of pyruvate kinase (ATP:pyruvate O2-phosphotransferase, EC 2.7.1.40) from Phycomyces blakesleeanus NRRL 1555 (-) was studied at pH 7.5. By addition of fructose 1,6-bisphosphate the sigmoid kinetics with respect to phosphoenol pyruvate and Mg2+ were abolished and the velocity curves became hyperbolic. In the presence of L-alanine the positive homotropic cooperativity with respect to phosphoenol pyruvate increased with Hill coefficient values close to 4, while the sigmoid kinetics with respect to Mg2+ became hyperbolic. Fructose 1,6-bisphosphate overcomes the inhibition produced by L-alanine, the antagonism between phosphoenol pyruvate and L-alanine also being evident. Inhibition has been found at high Mg2+ concentrations, compatible with the binding of the magnesium ions to an inactive conformational state of the enzyme. The data were analysed on the basis of the two-states concerted-symmetry model of Monod, Wyman and Changeux, and the parameters of the model were calculated. Phosphoenol pyruvate and fructose 1,6-bisphosphate appeared to show exclusive binding to the active conformational state (R), whereas magnesium ions bind preferentially, by a factor of 45, to the R state. L-Alanine binds more readily to the inactive T state of the enzyme.

The inductive effect of different sugars on beta-galactosidase synthesis in Phycomyces blakesleeanus has been studied. The enzyme was inducible by galactose and fructose. When grown on these sugars the enzyme level was 10-20 times greater than when grown on glucose. We have detected both intra- and extracellular beta-galactosidase activity when Phycomyces blakesleeanus was grown on galactose, but only extracellular beta-galactosidase activity when grown on fructose plus lactose.

We have partially characterized an intracellular fraction from Phycomyces blakesleeanus which shows proteolytic activity. The apparent thermal inactivation constant (Kd) was 0.12 min-1 at 50 degrees C. This proteolytic fraction was split into two active fractions by ultrafiltration using a membrane with an exclusion size of 30,000. Both fractions were inhibited by phenyl methyl sulphonyl fluoride. The Ki value for the fraction with molecular weight greater than 30,000 was 0.075 mM. The fraction with molecular weight less than 30,000 inactivated the Phycomyces CPS.