Quantitative structure-activity relationship study of the biophysicochemical behavior of nitrosamine.

The partition coefficients of 14 aliphatic nitrosamines were measured in six water-organic solvent systems. Correlations between Hansch's hydrophobic parameter (pi) and Kier and Hall's topological index (1 chi) allowed pi to be substituted by 1 chi in the structure-activity correlations of the carcinogenicity data of the nitrosamines studied. The use of high-performance liquid chromatographic capacity factors of nitrosamines in these correlations is also discussed.

Kinetic study of the enzyme NADPH cytochrome-C (P-450) reductase: non-Michaelian behaviour.

1. Contrary to what has been accepted until now, the enzyme exhibits non-Michaelian kinetics both against NADPH and against cytochrome-c as substrates; deviations were detected that have led to the proposition of a rate equation of minimum degree 2:2. 2. A general mechanism is proposed that includes, apart from the binding of the enzyme to NADPH, the formation of an enzyme-cytochrome-c complex, both routes leading to the formation of a ternary-complex NADPH-enzyme-acceptor. 3. From the latter, a series of intermediate steps finally leads to the release of the enzyme in conditions to start a new catalytic cycle. 4. Application of the King-Altman method to this mechanism yields a kinetic equation of degree 2:2 with respect to the cytochrome-c and NADPH, in accordance with our experimental results.

Kinetic study of the inhibition of the amidolytic activity of thrombin by benzamidine and N-dansyl-(p-guanidino)-phenylalanine-piperidide (I-2581).

1. v([I]) data were obtained for the hydrolysis of the chromogenic substrate H-D-Phe-L-Pip-L-Arg-pNA (S-2238) by native human thrombin in the presence of the synthetic inhibitors Benzamidine and N-dansyl-(p-guanidino)-phenylalanine-piperidide (I-2581). v([S]) data were also obtained in the absence and presence of fixed concentrations of each of the inhibitors. 2. Analysis of the kinetic data was based on the numerical fitting to rate equations of the polynomial quotient type of degree n:m using nonlinear regression methods. The discrimination between rate equations with different degrees was performed by application of the statistical F test. 3. Of eight v([I]) curves fitted, in six cases it was found that degree 1:2 was significantly better than degree 1:1 at a confidence level of 95% or higher; in no case was a significant improvement found with rate equations with a higher number of parameters. For the v([S]) data, of eleven curves fitted it was found that in nine cases degree 2:2 significantly improved degree 1:1 at confidence levels 99% and in one case at a level of 95%; no significant improvement was found with rate equations of higher degree for these data either. 4. Our findings allow us to propose that inhibition of the amidolytic activity of native human thrombin by benzamidine and I-2581 may be accounted for by mechanisms whose v([I]) rate equation will be a minimum of degree 1:2, thus implying a pure inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

A mechanism for fibrin monomer aggregation based on a kinetic study.

A kinetic study was carried out of the aggregation of fibrin monomers under different reaction conditions. At either acid or base pH values, second-order kinetic processes were observed throughout the concentration range studied. At neutral pH (6.8 less than or equal to pH less than or equal to 7.3) the kinetics were of second order at fibrin monomer concentrations of less than 0.3-0.4 mg/ml, while at higher concentrations they changed to first order. Both the second order rate constants and the opacity of the fibrin gels are highly dependent on pH, ionic strength, the concentration of calcium ions and on temperature. A three-step reaction mechanism is proposed for the aggregation of fibrin monomers to explain the kinetic behaviour observed in the different reaction media.

Negative cooperativity in alkaline phosphatase from E. col: new kinetic evidence from a steady-state study.

1. A study has been carried out on the steady-state kinetics followed by the alkaline phosphatase from Escherichia coli at different pH, temperatures, ionic strengths, phosphate concentrations and in the presence of the effectors such as Tris, NH4+--NH3 and CH3OH; p-nitrophenyl phosphate was used as substrate. 2. Contrary to what has generally been accepted, in most cases the enzyme follows non-Michaelian kinetics for a wide substrate concentration range, giving concave-down Lineweaver-Burk plots. Only at high phosphate concentrations (5 . 10(-3) M) and at high ionic strengths (2.0 M) is a linear Lineweaver-Burk plot obtained (Michaelian kinetics). 3. In order to analyse the kind of kinetics obtained, a non-linear regression fitting method was used to obtain rate vs substrate concentration equations as polynomial quotients of minimum degree with positive coefficients. 4. Most of the data obtained follows 2:2 degree type equations. 5. These results tend to suggest an idea of cooperativity rather than one of independence between the sites of the dimeric enzyme. A model is discussed for cooperativity between the sites with a wide concentration range giving concave-down Lineweaver-Burk plots.

Kinetic studies of the transphosphorylation reactions catalyzed by alkaline phosphatase from E. coli: hydrolysis of p-nitrophenyl phosphate and o-carboxyphenyl phosphate in presence of Tris.

1. Transphosphorylation of p-nitrophenyl phosphate and o-carboxyphenyl phosphate to Tris, has been studied at alkaline and acid pH. 2. The rate of release for all reactions products was Tris-dependent for both substrates, with a slight maximum for phenol at alkaline pH. These dependences have been analyzed from a mechanistic standpoint. 3. Individual constants of rate of a simple transphosphorylation mechanism have been determined. 4. At high Tris concentration (greater than 1.0 M) a slight competitive inhibition has been observed. 5. Inhibition in NH4+-NH3Cl buffer has been found at alkaline pH but not at acid pH. It would therefore seem that the non-protonated NH2 group of Tris is responsible for inhibition. 6. The results suggest the formation of complexes between Tris and the enzyme. Other possible alternatives are also analyzed.