William J. Canady

Interaction of aromatic hydrocarbons and drugs with adrenal microsomal cytochrome P-450 in the guinea pig.

Abstract Addition of simple aromatic hydrocarbons (benzene, ethylbenzene, naphthalene) to guinea pig adrenal microsomes produced typical Type I difference spectra (ΔOD 385-420 ). Spectral dissociation constants ( K s ) for each indicated a far higher affinity for adrenal than hepatic cytochrome P-450. Hydrocarbon affinities for adrenal cytochrome P-450 were similar to that for progesterone, an endogenous steroid substrate. Ethylmorphine and aniline produced Type I and Type II spectral changes respectively in adrenal microsomes. The K s , and magnitude of spectrum for each in adrenals was similar to that in livers. Nonetheless, demethylation of ethylmorphine proceeded far more rapidly in adrenal than hepatic tissue. The Michaelis constants ( K m ) for ethylmorphine metabolism in both tissues were similar. Although the aniline-induced difference spectra in adrenal and hepatic microsomes did not differ substantially, aniline hydroxylase activity was far greater in liver. Pretreatment of guinea pigs with phenobarbital or 3-methylcholanthrene increased hepatic but not adrenal ethylmorphine metabolism. Spironolactone pretreatment, in contrast, did not affect hepatic metabolism, but significantly lowered adrenal demethylase activity. The results indicate a relative non-specificity of guinea pig adrenal microsomal cytochrome P-450 and suggest that the adrenal cortex may represent a significant site for the extra-hepatic metabolism of foreign compounds in the guinea pig.

Solvation effects upon the thermodynamic substrate activity; correlation with the kinetics of enzyme catalyzed reactions. I. Effects of added reagents such as methanol upon alpha-chymotrypsin

Solvents, detergents, etc., have often been added to the medium to study the kinetics of enzyme action and for binding studies. They have been employed for diverse reasons such as solubilization of substrates or to stabilize an enzyme that was originally membrane bound. Thermodynamic considerations dictate that any added substance, such as methanol, which is present in significant quantity must affect the thermodynamic activities of the enzyme, enzyme-substrate complex, substrate and any other intermediates although cancellation effects may occur in this regard. The influence upon substrate activities is the only one that is easily experimentally accessible. These effects are shown, from the data of Bernard and Laidler, to be large in the case of the alpha-chymotrypsin catalyzed hydrolysis of methylhydrocinnamate. The variation of the Michaelis-Menten constant is quantitatively explainable in terms of the alteration of the thermodynamic activity of the substrate by methanol.

Solvation effects upon the thermodynamic substrate activity : correlation with the kinetics of enzyme catalyzed reactions. II. More complex interactions of alpha-chymotrypsin with dioxane and acetone which are also competitive inhibitors

It is shown that the effects of the addition of various amounts of dioxane and acetone (solvent modifiers) upon the alpha-chymotrypsin-catalyzed hydrolysis of methylhippurate can be explained in terms of three factors. (A) The effects of the above modifiers on the chemical potential of the substrate. (B) The solvent modifiers dioxane and acetone also act as classical competitive inhibitors. The means of sorting out these contributions is presented

Association of hydrophobic substances with hemin: Characterization of the reverse type i binding spectrum and its relationship to cytochrome P-450

When hydrophobic compounds were added to a solution of protoferriheme, a a reverse type I spectral change was produced when observed by difference spectroscopy. The spectrum had a peak at 422 nm and a trough at 387 nm, and the characteristics were dependent on the pH of the sample. An association constant for the complex could be determined and was also found to be pH sensitive, with the association constant dropping to zero at values below pH 7.0 and above pH 8.5. The determination of the delta Absmax for the ethylbenzene-hemin complex at various hemin concentrations indicates monomeric heme to be the species responsible for binding the hydrocarbon with the concomitant generation of the reverse type I spectral change.

The nature of human Cl-esterase: The hydrophobic nature of its binding site and pH dependence of the kinetic constants

Abstract The active site of human Cl-esterase is composed in part of a highly hydrophobic area that is capable of binding simple hydrocarbons which contain no functional groups. The binding constants (Kivalues) show a size dependence which is consistent with the concept of the enzyme acting as a second ‘organic phase’, the hydrocarbon inhibitors being ‘extracted’ by the enzyme. The ability of the enzyme to bind hydrocarbons increases as the molecular size of the hydrocarbon with which it is presented is increased. The evidence indicates that there is one such hydrophobic site per enzyme molecule. The pH dependence of the kinetic constants for N- acetyl- l -tyrosine ethylester and p-toluene sulfonyl- l -arginine methylester indicates that a terminal amino group may be involved in catalysis.

A new mechanism for the terminal stages of complement hemolysis based on kinetic and thermodynamic rationales

Okada, Boyle and Borsos (1) made a study, reported in this journal, of the temperature dependence of and effects of preincubation on the terminal reactions in complement hemolysis. They suggested that EAC1–9inserted could go on to lysis by either of two reaction paths and also that there is an enzyme catalyzed step. In this paper we show by kinetic and thermodynamic analysis a) that their results are inconsistent with such a double path mechanism and b) that their data are quantitatively compatible with a fairly conventional Michaelis-Menten mechanism. Although we agree with Okada et. al that their data are consistant with the participation of an enzyme in the terminal stages of complement hemolysis, the type of rate laws referred to in this paper could apply to some non-enzymatic reactions.