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Characterization of Microsomal Methyl Sterol Demethylase in Two Morris Hepatomas¹

Section of Biochemistry, Molecular and Cell Biology, and the Division of Nutritional Sciences, Cornell University, Savage Hall, Ithaca, New York 14853 [M. T. W., J. L. G.]; and Department of Biochemistry, Cancer Research Unit, Howard University College of Medicine, Washington, D. C. 20059 [H. P. M.]

Previously, we reported that the rate of metabolism of methyl sterol intermediates of cholesterol biosynthesis by broken-cell preparations of Morris hepatoma 7777 is very slow, whereas the intact tumors are known to synthesize cholesterol quite efficiently. Active preparations have now been obtained by substitution of pyrophosphate for phosphate buffer. Although substitution of pyrophosphate buffer markedly enhances microsomal methyl sterol demethylation rates 3- to 4-fold in hepatoma 7777, other microsomal enzymes and electron carriers in either liver or a more slowly growing hepatoma appear to be unaffected by pyrophosphate.

Several properties of the active microsomal methyl sterol demethylase have now been compared for control rat liver, host liver, tumor 7777, and tumor 5123C. Conditions necessary for the assay of initial velocities of enzymic reactions in the tumor microsomes have been established with respect to the amount of protein, time-course, concentrations of cofactors and substrate, pH, and other variables. The K'_m and the responses to the variables studied above are very similar for methyl sterol demethylase of microsomes isolated from control liver, host liver, tumor 5123C, and tumor 7777. The multienzymic demethylase in the various preparations has been found to be inhibited similarly by in vitro additions of cyanide, cytochrome c, and bile salts. Thus, the enzymes of the microsomal-bound 4-methyl sterol demethylase of cholesterol biosynthesis appear to be very similar in liver and these 2 Morris hepatomas.

When xenobiotic inducers of microsomal oxidases, such as phenobarbital and methylcholanthrene, are administered to normal and tumor-bearing rats, elevated rates of methyl sterol demethylation are observed with isolated liver microsomes obtained from both normal and tumor-bearing rats. Similar increases are not observed in the tumors. Further-more, daily administration of an intestinal bile acid sequestrant elevates hepatic methyl sterol demethylase, but statistically significant changes were not observed in tumors 7777 and 5123C. Since the enzymes of methyl sterol demethylase appear to be grossly similar in liver and these hepatomas, regulation of the activity of the multienzymic system contained in the tumors may be altered. On the other hand, these agents in vivo simply may not affect liver and the hepatomas similarly, due to a lack of uptake of the foreign substances by the tumor that has been transplanted to the thighs.

¹ This work was supported in part by USPHS Research Grants CA-10729 and CA-14454, from the National Cancer Institute; AM-10767, from the National Institute of Arthritis, Metabolism, and Digestive Diseases; and in part by funds made available through the State University of New York. These results have been presented in part at the 1975 FASEB Annual Meeting and at the Fifth Biannual Hepatoma Conference, Silver Spring, Md., 1975.

² Predoctoral trainee partially supported by a Smith Kline and French Fellowship.

Received 7/ 7/75. Accepted 10/22/75.