The effect of ether stress and cycloheximide treatment on cholesterol binding and enzyme turnover of adrenal cortical cytochrome P450scc.

Relative rate constants for the formation of pregnenolone from cytochrome P450scc bound cholesterol in adrenal cortical mitochondria of stressed, stressed plus cycloheximide treated and dexamethasone treated rats were calculated from the ratios of initial rates of pregnenolone formation and the pregnenolone induced difference spectrum. In mitochondria from adrenals removed under aerobic conditions in vivo, the rate constant for the enzyme in stressed rats is twice a high as the rate constant for the enzyme from the stressed plus cycloheximide group, and four times as high as that for the enzyme from dexamethasone treated rats. Anoxia for 5 min in the intact gland increases the rate constant in all groups. Pregnenolone difference spectra are higher in mitochondria from stressed plus cycloheximide treated rats than in mitochondria from stressed rats, when adrenals are removed aerobically. It is concluded that ACTH increases cholesterol binding to cytochrome P450scc, by increasing either the enzymes affinity for its substrate or the availability of cholesterol and in addition promotes turnover of the enzyme. Both of these effects of ACTH are inhibited by cycloheximide.

Mechanism of energy coupling for transport of deoxycytidine, uridine, uracil, adenine and hypoxanthine in Escherichia coli.

The transport processes for uridine, deoxycytidine, uracil, adenine and hypoxanthine require an energy source and are active under anaerobic or aerobic conditions. Inhibitory effects of cyanide, arsenate, carbonylcyanide m-chlorophenylhydrazone, 2,4-dinitrophenol and N,N'-dicyclohexylcarbodiimide on the transport of uridine and deoxycytidine differ from the corresponding effects on the transport of uracil, adenine and hypoxanthine. The nature of these inhibitory effects supports the conclusion that uridine and deoxycytidine transport is energized either by electron transport or by ATP hydrolysis via (Ca2+ + Mg2+)-ATPase. The transport or uracil, adenine and hypoxanthine is dependent upon ATP or some high energy phosphate derivative of ATP, but is independent of (Ca2+ + Mg+)-ATPase and electron transport. Uptake of the ribose moiety of uridine by a mutant of Escherichia coli B, which lacks the transport system for uracil and intact uridine, is neither stimulated by energy sources nor inhibited by various inhibitors of energy metabolism under either aerobic or anaerobic conditions.

Deoxycytidine transport in the presence of a cytidine deaminase inhibitor and the transport of uracil in Escherichia coli B.

Tetrahydrouridine, a cytidine deaminase inhibitor, prevents periplasmic degradation of deoxycytidine by Escherichia coli B. It does not inhibit deoxycytidine transport and therefore allows an accurate determination of deoxycytidine transport. Data obtained using tetrahydrouridine show that deoxycytidine is transported in E. coli B as the intact nucleoside by an active transport process, with a K-m of 6 times 10-minus 6 M. Cytidine and deoxyadenosine inhibit transport competitively, whereas guanosine has no effect on transport. Arsenate or KCN greatly reduces transport. In a mutant resistant to the nucleoside antibiotic, showdomycin, the active transport of deoxycytidine is lost, and residual slow uptake occurs by passive diffusion. Uracil is accumulated in E. coli B by an active transport process with a K-m of 5 times 10-minus 7 M.