Biomedical subjects

D De Arriaga

Publications and source records attributed to D De Arriaga.

11 recordsLinked to original sources

PubMed2001

A normalized plot as a novel and time-saving tool in complex enzyme kinetic analysis.

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.

Enzymes↗

PubMed1999

Chemical modification of NADP-isocitrate dehydrogenase from Cephalosporium acremonium evidence of essential histidine and lysine groups at the active site.

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.

Acremonium↗

PubMed1996

Purification and characterization of an extracellular aspartate protease from Phycomyces blakesleeanus.

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.

Amino Acid Sequence↗

PubMed1991

Hysteretic behaviour and GSSG substrate inhibition shown by glutathione reductase from Phycomyces blakesleeanus.

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.

Glutathione↗

PubMed1990

ATP inhibition of Phycomyces pyruvate kinase: a kinetic study of the inhibitory effects on the allosteric kinetics shown by the enzyme.

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.

Adenosine Triphosphate↗

PubMed1989

Effect of glucose on isocitrate lyase in Phycomyces blakesleeanus.

Repression of the synthesis of isocitrate lyase by glucose and/or induction of the synthesis of isocitrate lyase by acetate in Phycomyces blakesleeanus were demonstrated. Both glycerol and ethanol failed to induce isocitrate lyase activity. Furthermore, glucose appeared to cause an in vivo catabolite inactivation of the derepressed enzyme. Isocitrate lyase was inactivated both reversibly and irreversibly by glucose.

Acetates↗

PubMed1988

Carbamoyl-phosphate synthase in Phycomyces blakesleeanus.

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.

Carbamoyl-Phosphate Synthase (Ammonia)↗

PubMed1988

Partial characterization of intracellular protease activity which participates in the inactivation of carbamyl phosphate synthase in Phycomyces blakesleeanus.

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.

Carbamoyl-Phosphate Synthase (Ammonia)↗

PubMed1985

Cytoplasmic malate dehydrogenase from Phycomyces blakesleeanus: kinetics and mechanism.

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.

Cytoplasm↗

PubMed1982

Lactate dehydrogenase in Phycomyces blakesleeanus.

1. An NAD-specific L(+)-lactate dehydrogenase (EC 1.1.1.27) from the mycelium of Phycomyces blakesleeanus N.R.R.L. 1555 (-) was purified approximately 700-fold. The enzyme has a molecular weight of 135,000-140,000. The purified enzyme gave a single, catalytically active, protein band after polyacrylamide-gel electrophoresis. It shows optimum activity between pH 6.7 and 7.5. 2. The Phycomyces blakesleeanus lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate, and its coenzyme, NADH, at pH 7.5, indicating the existence of multiple binding sites in the enzyme for these ligands. 3. At pH 6.0, the enzyme shows high substrate inhibition by pyruvate. 3-hydroxypyruvate and 2-oxovalerate exhibit an analogous effect, whereas glyoxylate does not, when tested as substrates at the same pH. 4. At pH 7.5, ATP, which inhibits the enzyme, acts competitively with NADH and pyruvate, whereas at pH 6.0 and low concentrations of ATP it behaves in a allosteric manner as inhibitor with respect to NADH, GTP, however, has no effect under the same experimental conditions. 5. Partially purified enzyme from sporangiophores behaves in entirely similar kinetic manner as the one exhibited by the enzyme from mycelium.

Adenosine Triphosphate↗

PubMed1982

Influence of pH on the allosteric properties of lactate dehydrogenase activity of Phycomyces blakesleeanus.

1. Lactate dehydrogenase from mycelium of Phycomyces blakesleeanus showed positive homotropic interactions with NADH at all pH values studied (pH 5.0-7.7). The calculated values for the first and last intrinsic association constants remained unaltered with pH, in contrast with the Hill coefficient value, which varied significantly, reaching its maximum values at pH 6.0 and 7.7. This suggests the hypothesis that pH regulates these homotropic effects by changes in the value of the intermediate intrinsic association constants. 2. From pH 7.2 to 7.7 lactate dehydrogenase exhibited, likewise, positive homotropic interactions with pyruvate. There were practically no changes in the first and last intrinsic association constants and in Hill coefficient values with pH. At pH values below 7.2 (pH 5.0-6.8) the enzyme showed high substrate inhibition, which was highly dependent on pH, NADH concentration and temperature. By way of substrate inhibition pH regulates, primarily, lactate dehydrogenase activity towards pyruvate, since the homotropic effects appear not to be dependent on pH. 3. Fructose 1,6-bisphosphate is a true allosteric effector of lactate dehydrogenase of Phycomyces blakesleeanus. it decreases positive co-operativity with NADH, and on the other hand pyruvate co-operativity turns into mixed co-operativity. In addition, the effector decreases the inhibitory effect caused by pyruvate.

Allosteric Site↗