Abstract
Cholesterol sulfate inhibits (K1/2, 6 microM) the side chain cleavage of exogenous cholesterol in intact rat adrenal mitochondria. Inhibition is at a site other than cytochrome P-450scc: the spin state of the hemoprotein is not perturbed, and its activity is unaffected as judged by the failure to inhibit the metabolism both of 25-hydroxycholesterol and of endogenous cholesterol in a mitochondrial "steroidogenic pool." In contrast, 25-hydroxycholesterol, known to interact with the cytochrome, prevented the cleavage of both endogenous and exogenous cholesterol and produced the expected optical changes in the hemoprotein. Inhibition was specific, since a variety of related compounds including pregnenolone sulfate were not effective. Metabolic conversion to other species was insufficient to account for inhibition, indicating that cholesterol sulfate is the effective molecule. A hallmark of an inhibitor of a transport system is that disruption of the barrier to transport eliminates inhibition. Sonic disruption of mitochondria abated by 70% the effect of cholesterol sulfate, but did not affect inhibition by 25-hydroxycholesterol. Thus, the cholesterol sulfate appears to inhibit an intramitochondrial cholesterol translocation system that functions to move cholesterol into a steroidogenic pool. The high content of cholesterol sulfate in adrenal cortex (Drayer, N.M., Roberts, K.D., Bandi, L., and Lieberman, S. (1964) J. Biol. Chem. 239, 3112-3114) suggests a possible regulatory role for this molecule.
Highlights
Cholesterol sulfate inhibits(&, 6 /AM)the side chain (ACTH),’ a pituitary peptide hormone released in response cleavage of exogenous cholesterol in intact rat adrenal to stress
The cycloheximide-inhibitable, ACTH-regulated step by the failure to inhibit the metabolism both of 25- is expressed in the mitochondrion [9], since the activation hydroxycholesterol and of endogenous cholesterol in a state is preserved in isolated mitochondria from pretreated mitochondrial ”steroidogenic pool.”
Recent interest in ACTH regulation has focused on two major areas: 1) the identity and properties of the cycloheximide-inhibitable steroidogenic factor, and 2) the mechanism for inhibition, indicatingthat cholesterol sulfate is the of activation of steroidogenesis within adrenal mitochondria
Summary
Mitochondrial Preparation-Adrenals were dissected from male rats (Sprague-Dawley,approximately 200 g) decapitated following 10 min of ether anesthesia. An aliquot of dichloromethane was dried in a conical reaction vial, and samples were derivatized by addition of 25 pl of TMS reagent followed by incubation at room temperature for at least 2 h This method allowed simultaneous quantitation of both pregnenolone and cholesterol. Cholesterol sulfate side chain cleavage activity of the purified beef cytochrome P-450,, was determined as described above, except thatthe incubation contained the following components: 0.2 pM cytochrome P-450,, 2 p~ adrenodoxin reductase, 10p~ adrenodoxin, 10 pg of egg phosphatidylcholine, 1pg of beef heart cardiolipin, and 2 units of glucose-6-phosphate dehydrogenase in 1ml buffer containing 100 m M NaCl plus 10 mM Hepes, pH 7.2. Assay for CholesterolSulfatase Activity-Generation of free cholesterol and pregnenolone from radiolabeled cholesterol sulfate (22 pM containing 1.2 X lo dpm) was determined as described above using the HPLC assay of Tribble et al [31]. Absorbance Spectra-Difference absorbance spectra were recorded at various added steroid concentrations using an Aminco DW2a UVvisible Spectrophotometer in the split beam mode
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