David H. Schroeder

Enzymic and biochemical composition of smooth and rough microsomal membranes from monkey, guinea pig and mouse liver.

Abstract Smooth and rough microsomal membranes, prepared by density gradient centrifugation from livers of mice, guinea pigs and monkeys, were compared on the basis of biochemical composition, enzyme distribution, and fine structure. Phospholipid and cholesterol distribution varied considerably between smooth and rough membranes and between animal species. The following components of microsomal drug metabolism were studied: ethylmorphine demethylase, l -benzphetamine demethylase, aniline hydroxylase, NADPH cytochrome c reductase, cytochrome P-450, and spectral changes resulting from microsome-substrate interaction. Expressed per milligram of protein, all these components were concentrated about 2-fold in the smooth membranes of monkey and guinea pig liver, but were approximately evenly distributed between smooth and rough membranes of mouse liver. The results support the concept of heterogeneity among microsomal membranes. In addition to the well recognized species differences in microsomal drug metabolism, our data indicate important species differences in submicrosomal membrane composition and enzyme distribution.

Further studies on the submicrosomal distribution of drug-metabolizing components in liver: Localization in fractions of smooth microsomes

Abstract Hepatic microsomes from male guinea pigs were fractionated into three populations—rough, smooth I (S-I) and smooth II (S-II)—by centrifugation in discontinuous sucrose gradients in the presence of Cs + and Mg 2+ The specific activities of several components of the microsomal drug-metabolizing system were unevenly distributed among the three fractions. Activities in smooth membranes were two to three times higher than in rough membranes. Comparison of the two smooth membrane fractions revealed a statistically significant concentration of drug-metabolizing components in S-II microsomes. Thus, ethylmorphine demethylase, aniline hydroxylase, NADPH-cytochrome c reductase, and cytochrome P-450, all expressed per milligram of protein, were 20–30 per cent higher in S-II than in S-I membranes. Morphologic differences between the two smooth membrane fractions were revealed by electron microscopy. Pretreatment of guinea pigs with phenobarbital for 4 days increased the specific activities of ethylmorphine demethylase, aniline hydroxylase, NADPH-cytochrome c reductase, and cytochrome P-450. The increase for each parameter was of approximately the same magnitude in each of the three microsomal fractions, but the four parameters increased disproportionately. Phenobarbital treatment produced significant increases in protein and phospholipid (per gram of liver) in S-I microsomes without accompanying changes in S-II or rough microsomes.

Effect of Temperature on the Relationship Between Hepatic Microsomal Cytochrome P-450 Reduction and Ethylmorphine Demethylation

The rate of reduction of hepatic microsomal cytochrome P-450 (NADPH-cytochrome P-450 reductase activity) was studied at temperatures ranging from 15 to 50°C. Ethylmorphine stimulated the initial , rate of cytochrome P-450 reduction at all temperaturesexamined. Parallel studies of the temperature dependence of microsomal ethylmorphine N-demethylase revealed a proportionality between ethylmorphine N-demethylase and cytochrome P-450 reductase activities at temperatures below 40°C. This finding is in accord with the view that the rate of microsomal P-450 reduction by NADPH is the rate-limiting step in the oxidation of drugs by microsomal enzymes.