Conversion of cholesterol to adreno-cortical hormone intermediates by subcellular fractions of bovine adrenal cortex
Halkerston, Ian D. K.
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The sequence of reactions concerned in the formation of adrenocortical steroids from cholesterol includes the cleavage of the cholesterol side chain between C-22, 23 to form 5 -pregnenolone. This step is accelerated by adrenocorticotrophic hormone (ACTH) administration, whereas the later hydroxylation of progesterone to form corticosterone and cortisol are not. These hydroxylations are known to be dependent upon triphosphopyridine nucleotide (TPNH), but the cofactor requirement for cholesterol side chain cleavage has not been defined. In view of the dominant role proposed fro TPNH by currently held theories of the mechanism of action of ACTH, it is important to know if the only ACTH responsive step in the corticosteroidogenetic sequence requires TPNH as an essential cofactor. The conversion of cholesterol to c-21 steroids was investigated in subcellular fractions of bovine adrenal cortex tissue incubated with cholesterol-4-c^14 and the extent of conversion estimated by separating the radioactive products from unchanged cholesterol-4-c^14 on silicic acid columns. The radioactive products were tentatively identified on the basis of their chromatographic behavior in paper and partition column systems. The subcellular fractions were prepared by conventional differential centrifugation techniques with 0.44M sucrose, with and without ionic additions, for homogenization and isolation media. Nuclei, mitochondrial, microsomal and soluble fractions were prepared from the ionicsucrose media and in the case of pure sucrose homogenates the "fluffy layer". of the mitochondrial pellet was treated as an additional fraction. The mitochondrial fraction from either homogenate required only Mg++ and fumarate for cholesterol conversion activity. Succinate or a-ketoglutarate could replace fumarate and aerobic conditions were necessary. The requirement for citric acid cycle intermediates could be met by the "extemal" TPNH generating system, glucose-6-phosphate (G6P), its dehydrogenase (G6P+DH) and TPN. Acetone dried mitochondrial fractions from pure sucrose homogenates were fully active with fumarate and Mg++, but after restricted dialysis against phosphate buffer, TPN was required in addition to fumarate. The activity could be obtained in a soluble, non-particulate form by extraction of the acetone dried mitochondrial fraction with M/15 phosphate buffer pH 6.8, followed by centrifugation at 105,000g. The products of cholesterol-4-c^l4 conversion by the dialyzed or soluble extract of acetone dried mitochondrial fractions were largely accounted for by ~5 -pregnenolone and progesterone, with the relative amounts of the two intermediates depending upon the endogenous level of DPN in the preparation or the presence of this cofactor in the medium. Distribution studies based on the activity of acetone dried fractions were run in order to avoid problems connected with unequal mixing of tracer and endogenous cholesterol. The results indicated that approximately equal amounts of the total activity were present in the nuclei, mitochondrial and "fluffy layer" fractions. The microsomal and soluble fractions were inactive. The highest activity per mg. protein was found in the "fluffy layer" with the mitochondrial fraction next, a result possibly related to the greater ease of cholesterol-4-C^14 entry into the disorganized structure of the "fluffy layer". Soluble extracts from the acetone dried fractions were active on addition of Mg++, fumarate and TPN, but only the mitochondrial fraction gave acetone powders active in the absence of TPN. The "bound" cofactor in fresh mitochondrial or "fluffy layer" fractions was not available for TPNH generation by G6P and its dehydrogenase and only reacted to a limited extent in acetone dtted preparations. High concentrations of sucrose inhibited cholesterol conversion by particulate preparations, but not the solubilized activity from acetone powders. The effect was possibly due to a reduced entry of cholesterol-4-c^l4, as the specific activities of the products of conversion were reduced, while TPNH generation by either the "internal" or "external" systems was inhibited to an equal extent. The possible relationships of the findings co ACTH action are discussed. [TRUNCATED]
Thesis (Ph.D.)--Boston University