A synthetic peptide, representing a portion of the 16K (16,000 dalton)-fragment sequence within the pro-adrenocorticotropin/endorphin precursor molecule, potentiates the steroidogenic action of the 1 to 24 portion of adrenocorticotropin [ACTH(1-24)] on the rat adrenal cortex. The peptide has 27 amino acid residues and consists of gamma-melanotropin with a carboxyl terminal extension. It affects both the inner and outer adrenocortical zones of hypophysectomized animals, as evidenced by a synergistic augmentation of corticosterone and aldosterone production, respectively. The peptide can be distinguished from adrenocorticotropin by its activation of cholesterol ester hydrolase and its failure to stimulate cholesterol side-chain cleavage.

Cholesterol side-chain cleavage has been studied in intact adrenal mitochondria from rats subjected to stress by ether anaesthesia as a means of raising the plasma adrenocorticotrophin levels. Control rats were either injected with cycloheximide or kept in a quiescent state. After initiation of cholesterol side-chain cleavage by addition of isocitrate, pregnenolone formation from endogenous cholesterol in intact rat-adrenal mitochondria follows a biphasic time-course of formation with an initial rapid phase lasting 3-5 min followed by a much slower rate of formation.Pregnenolone formation from [4-14C]cholesterol is linear if the tracer substrate is added to the mitochondria together with isocitrate. Preincubation of the mitochondria with [4-14C]cholesterol prior to addition of isocitrate results in a biphasic [4-14C]pregnenolone formation; the rate of the initial rapid phase depending on the duration of preincubation.The effect of stress is to increase 2-3 times the rate of pregnenolone formation in the initial phase compared to the rates observed in mitochondria from quiescent or cycloheximide-treated rats.Depletion of cholesterol from adrenal mitochondria follows a similar pattern to pregnenolone formation, but the initial cholesterol content of the mitochondria is not affected by the pretreatment.On the basis of these results it is suggested that only a fraction of the total mitochondrial cholesterol is readily available for cholesterol side-chain cleavage and that this pool is increased by stress.The cytochrome P450 type I1 spectral changes induced by addition of pregnenolone and isocitrate to the intact rat adrenal mitochondria have also been studied. The effect of stress was to increase pregnenolone-induced difference spectrum in adrenal mitochondrial two-to three-fold compared with cycloheximide-treated animals. Similar results were found for the isocitrateinduced type I1 difference spectrum.The spectral changes are interpreted to mean that stress causes an increase in the cholesterol complex of side-chain cleavage cytochrome P460. Metabolism of the remainder of the cholesterol in the mitochondria is limited by the rate of transport, or binding, to this reactive centre. It is proposed that the acute effect of stress, mediated by adrenocorticotrophin, is to increase the proportion of mitochondrial cholesterol in the readily available form perhaps by an increase in the rate of transport or binding of cholesterol to sites from which it can be readily metabolised.Side-chain cleavage of cholesterol is the first step in the production of corticosteroids in the adrenal cortex. The initial products of the reaction are pregnenolone and a six-carbon fragment usually isolated as isocaproic acid side-chain is thought to involve hydroxylation a t (3-20 and C-22 [2,3] but the exact nature and sequence of formation of intermediates still has to be established [4,5].I n common with other hydroxylases, cholesterol side-chain cleavage requires NADPH and molecular oxygen [6]. The enzyme system occurs in the mitochondria of...

Five peptides derived from pro-corticotropin/endorphin (pro-ACTH/endorphin), the pituitary corticotroph cell rohormone, were bioassayed with isolated.rat adrenocortica cells: a-and fl-melanotropin, P-lipotropin, P-endorphin, and the amino-terminal region of pro-ACTH/ endo hin known as "16k fragment." The effect of each on steroidogenesis was measured at tentially physiological concentrations (0.01-1 nM) in both the absence and presence of varying concentrations of Corticotropin (ACTH) is initially synthesized in the rat pituitary as a pro-ACTH/endorphin precursor (1-4). This glycoprotein prohormone also contains the amino acid sequences for (3-lipotropin (f3-LPH) and for a third region at the amino-terminal end of the molecule, which has been designated "16k fragment" in the literature (1,5,6). Herein, we shall use the term "16k fragment" to mean the amino-terminal region of pro-ACTH/endorphin that precedes the ACTH sequence and has an apparent molecular weight of tu16,000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Trypsin-like proteolytic cleavage at pairs of basic amino acid residues within the prohormone structure apparently generates these three pro-ACTH/endorphin products. In turn, ACTH and f3-LPH contain the sequences for a group of smaller peptides, including a-melanotropin (a-MSH), f3-melanotropin (f3-MSH), and ,B-endorphin. No physiological role for many of the pro-ACTH/endorphin derivatives, including 16k fragment, has been identified.Stimulated by our previous observation that a corticotroph peptide other than ACTH may account for stress-induced activation of cholesterol ester hydrolase (cholesterol esterase; sterol-ester acylhydrolase, EC 3.1.1.13) activity in the rat adrenal cortex (7), we have investigated the effect of various peptides derived from pro-ACTH/endorphin on adrenal steroidogenesis. We report here that trypsin treatment of low concentrations of 16k fragment in vitro generates a peptide(s) that is capable of substantially augmenting the stimulation of corticosterone production by ACTH-(1-24) both in isolated rat adrenocortical cells and in vivo and of increasing adrenocortical cholesterol ester hydrolase activity independently of ACTH. METHODS AND MATERIALSIsolation and Incubation of Adrenocortical Cells. Isolated rat adrenocortical cells were prepared by a modification of the method of Ramachandran and Suyama (8). Female rat adrenals were enucleated in situ and the inner zones were minced and washed with medium 199 (GIBCO). Tissue dispersion by repeated passage through a plastic pipet tip was carried out at 37°C in a mixture of medium 199, Earle's salts, 25 mM Hepes (pH 7.4), 4 mg of collagenase I per ml (Worthington), 25 ,tg of DNAse I per ml (Sigma), and an adjusted Ca2+ concentration of 2.55 mM (9). After dispersion and washing, the cells were resuspended in medium 199/Hepes containing 1 mg of lima bean trypsin inhibitor per ml (Sigma) and preincubated for 30 min at 37°C. Cells were counted with a hemacytometer and viability was determined by using trypan...

BY A. C. BROWNIE AND J. K. GRANT ' cyclophorase' system (Green, Loomis & Auerbach, 1948). 'Cyclophorase' preparations, however, are not cytoplasmically homogeneous (Harman, 1950). Credit for showing that 1 1l,-hydroxylating enzymes are associated with adrenal-cell particles which by their method of preparation were mainly

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