The correction of reaction rates in continuous fluorometric assays of enzymes.

The kinetic data obtained from the action of a cathepsin D-like enzyme from Biomphalaria glabrata hepatopancreas (digestive gland) on MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNp)-D-Arg-NH(2), was studied as a data prototype, generated by means of a fluorogenic substrate. An initial fluorescence, due to incomplete energy transfer, of about 8% of the values attained after complete substrate hydrolysis; a non-linear standard curve even at microM concentrations and an exponential decay of the steady state fluorescence of reaction product of the order of 10(-4) x s(-1) were the main analytical problems encountered. The standard curves for fluorescence of the substrate reaction product after 48 h of hydrolysis, and the reference compound MOCAc-Pro-Leu-Gly-NH(2), were fitted by polynomial approximation and the point derivates used as calibration factors. Time dependence of the calibration factor for the reaction product was -2.96 x 10(-4) a.u microM(-1) x s(-1) that is, in the same order of observed enzymic reaction rates. A mathematical treatment was devised for obtaining rates corrected for errors derived from the three analytical problems indicated. The method is of general application in continuous fluorometric assays, irrespective of the particular enzyme used, but of special value for substrates that present significant initial fluorescence. The reaction rates were 11% higher; as calculated by means of the calibration factor [substrate]/(final-initial fluorescence intensities), which is the prevalent procedure in the literature; leading to underestimation of K(m) and overestimation of V(max).

Mechanism of action and determination of the best substrate for a thrombin-like enzyme from Lachesis muta muta venom by regression analysis of the kinetic parameters determined with peptidyl p-nitroanilide substrates.

The kinetic behavior of a thrombin-like enzyme from Lachesis muta muta venom has been studied with 13 tripeptidyl p-nitroanilide substrates. Eight substrates were unprotected at the N terminus and were used for the regression analysis of the experimentally determined kinetic parameters 1/Km, kcat and kcat/Km. The individual contribution of each amino acid side chain to the kinetic parameters was calculated. The amino acid sequence of the ideal substrate (D-Pro-Leu-Arg-pNA) was determined from a regression analysis for each kinetic parameter. This result was confirmed experimentally. The structural analysis of the tripeptides showed that the binding to the S3 sub-site had a small effect on Km. The binding of L-Leu to the S2 sub-site increased kcat without changing the value of Km. The analysis of the kinetic parameters revealed that, in the binding of L-Leu to the S2 sub-site, the enzyme bound the transition state configuration of the substrate/product transformation more tightly than that of the substrate.

Inhibition of the effects of different agonists on the rat uterus and guinea-pig ileum by Mandevilla velutina extract

O extrato de Mandevilla velutina inibe as contraçöes induzidas pela bradicinina e cininogenases (calicreína, tripsina e tonina) no útero de rata. As contraçöes por angiotensina II e mesmo por cloreto de bário também foram inibidas, quando o íleo de cobaia foi usado, a contraçäo evocada pela histamina foi inibida. Esses experimentos sugerem que a inibiçäo causada pelo extrato de Mandevilla velutina näo é seletiva para bradicinina