Dominick L. Cinti

Kinetic parameters of drug-metabolizing enzymes in Ca2+-sedimented microsomes from rat liver☆

Abstract Kinetic parameters of five substrates of the mixed function oxidase system were determined to ensure that the mixed function oxidases were unaltered by the Ca 2+ -sedimented microsomes. Using either of the microsomal preparation procedures, no differences were noted in the V max and K m for aminopyrine, ethylmorphine and p -nitroanisole demethylation, aniline hydroxylation and hexobarbital oxidase. Also, no differences were seen in the NADPH cytochrome P-450 reductase activities. The Ca 2+ -sedimentation procedure was further simplified to allow microsomal preparation within 1 hr.

[6] Preparation of microsomes with calcium

Publisher Summary This chapter focuses on the preparation of microsomes with calcium. Isolation of the microsomal fraction of the hepatic cell employs a procedure of differential ultracentrifugation. This method is developed for mammalian liver tissue, but also employed to obtain microsomal fractions from a variety of tissues, such as lung, kidney, spleen, adrenals. Other procedures used to isolate microsomes are developed to replace ultracentrifugation for two main reasons, (1) differential ultracentrifugation requires an expensive ultracentrifuge; (2) it is time consuming, requiring 2 hr of ultracentrifugation. Two methods that are used to isolate microsomes from the postmitochondrial fraction are acid precipitation and gel filtration. In recent years, one of the procedures for microsomal isolation that is extensively studied and gaining acceptance in mammalian liver studies is a method involving aggregation of microsomes with calcium. Basically, this involves the addition of Ca 2+ ions to the postmitochondrial supernatant followed by a short centrifugation at speeds obtainable with most refrigerated centrifuges, such as the Sorvall RC-2B.

Diethylaminoethyl 2,2-diphenylvalerate HCl (SKF 525-A)—In vivo and in vitro effects of metabolism by rat liver microsomes—Formation of an oxygenated complex

Abstract Metabolism of diethylaminoethyl 2,2-diphenylvalerate HCl (SKF 525-A) or its primary amine analogue causes formation of a stable oxygenated complex of ferrous P-450. The complex is also generated in vivo and survives preparation of liver microsomes. Formation of the complex requires active metabolism of either SKF 525-A or the N -dealkylated primary amine, SKF 26754A. This new species has an absorption maximum at 455 nm. Oxidation of the hemoprotein with potassium ferricyanide causes loss of the absorption band, but subsequent reduction and oxygenation of the medium restore it. This new complex is believed to be responsible for the observed noncompetitive inhibition of drug metabolism in vitro by SKF 525-A.

Hepatic mixed function oxidase activity in rapidly prepared microsomes

Abstract A new method for rapid isolation of hepatic microsomes has been evaluated with respect to the drug biotransformation system. The new preparation requires approximately one hour from the isolation of liver to a working microsomal suspension. The microsomes obtained by this Ca +2 -aided sedimentation did not differ in drug metabolizing activity or hemoprotein content from the conventionally prepared microsomes. Morphologically, however, the microsomes prepared in the presence of Ca +2 lose their ribosomes, which aggregate.

The role of the mitochondria in rat liver mixed function oxidation reactions

Abstract Recombination experiments indicate a complex role by the mitochondria in controlling drug biotransformation reactions. The use of liver slices and homogenates demonstrates that the effect of Krebs cycle intermediates on aminopyrine N-demethylation is not via an increase in NADH, but probably by a well-defined route from the mitochondria to the endoplasmic reticulum.

Metabolic action of urethan on protein synthesis and drug oxidation in normal and regenerating liver

Abstract Administration of the carcinogen urethan to sham-operated rats resulted in a dose-dependent increase in the synthesis of protein on membrane-bound polyribosomes and to a lesser degree on free polyribosomes 6 hr after drug. Maximal elevation of the specific radioactivity of nascent peptide synthesized on both classes of polyribosomes of sham-operated animals occurred 6–12 hr after the administration of urethan (1 g/kg). Animals injected with urethan 12 hr after partial hepatectomy showed no increase in protein synthesis on membrane-bound polyribosomes above that occurring in saline-treated rats 2–24 hr after urethan and only a slight elevation in peptide synthesis on free polyribosomes 12 hr after the carcinogen. Aniline hydroxylation was elevated 2-fold 12–24 hr after the administration of urethan (1 g/kg) to shamoperated animals, while little or no change occurred in: (1) rate of demethylation of aminopyrine, (2) NADPH cytochrome c reductase activity, and (3) levels of cytochrome b 5 and cytochrome P-450. Mixed-function oxidase activities in partially hepatectomized animals injected with urethan 12 hr after the operation were either unaffected or increased to a lesser degree than in sham-operated animals.

INFLUENCE OF SUBSTRATES OF HEPATIC MIXED FUNCTION OXIDASES ON SPIN EQUILIBRIUM OF CYTOCHROME P-450

Publisher Summary This chapter discusses the influence of substrates of hepatic mixed function oxidases on spin equilibrium of cytochrome P-450. In the study, cytochrome P-450 was purified to a content of 12 nmoles per mg of protein. The enzyme contained 1 nmol of phospholipids per nmol of P-450 and was free of detectable levels of fatty acid. Temperature-difference spectra relative to 9°C were obtained. The chapter illustrates the increase in the magnitude of the spin state change with increasing temperature. In the experiment described in the chapter, Glycerol, which effects conformational changes in some enzymes in the region of the active site, did not affect the spin equilibrium of P-450 between 9°C and 40°C. Thus, like the microbial P-450, the purified hepatic enzyme undergoes temperature-dependent spin equilibration in the total absence of substrates. Purified hepatic cytochrome P-450 undergoes reversible temperature-dependent spin equilibration, in both the presence and absence of substrates, to an extent and direction dependent upon both the substrate and species of hemoprotein. It is also shown that some substrate bound P-450 species are in a temperature-independent spin equilibrium.