John L. McCarthy

Sequence and developmental expression of Cyp18, a member of a new cytochrome P450 family from Drosophila

Preliminary studies of a partial cDNA clone of the Eig17-1 gene from Drosophila melanogaster have shown that it encodes a probable cytochrome P450 of unknown function. To further characterize the Eig17-1 gene product, a full-length cDNA clone was isolated from a late-larval cDNA library and sequenced. Eig17-1 encodes a protein of 538 amino acids. The predicted protein is a cytochrome P450 that has been assigned to a new family, CYP18. The CYP18 protein is most closely related to steroid and xenobiotic metabolizing P450s of family CYP2 (30-33% identity), and to vertebrate steroidogenic P450s of families CYP17 and CYP21 (25-28% identity). Developmental Northern blot analysis revealed five distinct periods of Cyp18 expression during postembryonic development. Each period lasted 12-15 h, and was tightly correlated with reported ecdysteroid pulses in the first, second and third larval instars, at the time of pupariation and in pupae. This pattern of expression is consistent with the known induction of Cyp18 transcription by 20-hydroxyecdysone at the time of pupariation and suggests that ecdysteroids are major regulators of Cyp18 expression throughout postembryonic development. Northern blot analysis of RNA isolated from different prepupal tissues indicates that Cyp18 is differentially expressed in various ecdysteroid-responsive tissues. High Cyp18 expression was observed in body wall and gut while negligible expression was observed in salivary glands and fat body.

THE INTERACTION OF STEROIDS WITH LIVER MICROSOMAL CYTOCHROME P-450—A GENERAL HYPOTHESIS

Abstract The study of the oxidative metabolism of steroids as catalyzed by cytochrome P-450 offers a unique challenge to our understanding of critically important biochemical events in a wide variety of areas of investigation. The approaches currently used in our laboratory are to better define (1) the milieu of the heme iron of cytochrome P-450 for the binding of the substrate and (2) the potential role of organic peroxides as key intermediates in oxygen activation by cytochrome P-450. The present report, although highly speculative, hopefully will stimulate others to expand further and test the hypotheses presented, i.e. the role of steering groups on the substrate molecule which orient the substrate to facilitate hydroxylation at specific sites and the presence of higher valence states for the heme iron-substrate complex of cytochrome P-450 associated with oxygen activation prior to substrate hydroxylation.

A role for elevated plasma corticosterone in modulation of host response during infection with Trichinella pseudospiralis

Summary Suppression of host inflammatory response in mice infected with Trichinella pseudospiralis was associated with host plasma corticosterone levels significantly higher than those seen in uninfecled mice or in mice infected with T. spiralis. Increases in the population of mitochondria and depletion of lipid droplets in cells of the zona fasciculata were seen in the adrenals of mice infected with T. pseudospiralis. Elevations in enteritis, myositis and myocarditis accompanied 100% mortality in adrenalectomized mice infected with T. pseudospiralis, while lower levels of inflammation and no mortality were observed in sham operated or intact animals infected with this parasite. The severe myositis normally accompanying infection with T. spiralis was suppressed by concurrent infection with 1000 or 2000 T. pseudospiralis to levels equivalent to those seen in animals receiving 015 and 0‐41 mg cortisone acetate/25 g mouse/day, respectively.

Induction of 17α-hydroxylase (cytochrome P-45017α) activity by adrenocorticotropin in bovine adrenocortical cells maintained in monolayer culture☆

Abstract Using bovine adrenocortical cells in monolayer culture it has been shown that treatment with adrenocorticotropin (ACTH) causes a dramatic increase in 17α-hydroxylase activity. In postmitochondrial supernatant fractions (PMS) prepared from cells maintained in culture, there was a 15-fold increase in 17α-hydroxylase activity 36 h following initiation of ACTH treatment compared with the activity measured in PMS prepared from control cells. In the continued presence of ACTH, 17α-hydroxylase activity declined; however, even after 60 h of exposure to ACTH, 17α-hydroxylase activity was eight times higher than that present in control cells. The dramatic increase in 17α-hydroxylase activity provides an explanation for the previously observed phenomenon that following initiation of ACTH treatment of bovine adrenocortical cells in monolayer culture there is a shift in the pattern of corticosteroid secretion from approximately equal amounts of cortisol and corticosterone to almost exclusively cortisol. Thus, the modulation of 17α-hydroxylase activity by ACTH action appears to serve a key regulatory role in the pattern of corticosteroid production. Soluble cytosolic factors apparently do not participate in the regulation of 17α-hydroxylase activity in the bovine adrenal cortex. Increases in the magnitude of substrate-induced absorbance changes are indicative that the increase in 17α-hydroxylase activity is due, at least in part, to an elevation of cytochrome P -450 17 α synthesis.

Deduced amino acid sequence of mature chicken testis ferredoxin

The cDNA sequence encoding the complete mature form of the steroidogenic ferredoxin from chicken testis has been determined and the amino acid sequence deduced therefrom has been compared with the sequences of bovine, human and porcine steroidogenic ferredoxins. The chicken sequence is between 84% and 88% identical with those of the other mitochondrial iron-sulfur proteins. Thus, the amino acid structure of steroidogenic ferredoxins which transfer electrons to mitochondrial forms of cytochrome P-450 has been very highly conserved over evolutionary time.

Gerbil adrenal 11β- and 19-hydroxylating activities respond similarly to inhibitory or stimulatory agents: Two activities of a single enzyme

A high level of steroid 19-hydroxylation is exhibited by adrenal mitochondria of the gerbil, Meriones, unguiculatus, that accounts for the ability of that species to produce nearly equal amounts of corticosterone and 19-hydroxycorticosterone (Proc. Soc. exp. Biol. Med. 165 (1980) 69-74). Inhibitors of steroidogenesis and a polyclonal antibody against bovine cytochrome P-450(11 beta) were used to determine if the agents would effect differential or parallel suppression of 19- vs 11 beta-hydroxylation by gerbil adrenal mitochondria in vitro. The inhibitors (0.1-60 microM) tested (listed in order of decreasing effectiveness) were imazalil, metyrapone, miconazole and 4-hydroxyandrostenedione. With each inhibitor the degree of suppression of 11 beta-hydroxylation was accompanied by a parallel decline in 19-hydroxylation. The addition of the polyclonal antibody preparation also produced equivalent declines in the rates of the two hydroxylation reactions. The addition of ACTH 1 microM to primary cultures of gerbil adrenal cells brought about nearly equal increases in the secretion of 11 beta- and 19-hydroxylated steroids into the culture media. These results support the hypothesis that the 11 beta-hydroxylase of gerbil adrenal mitochondria has the capacity to carry out 11 beta- and 19-hydroxylations with nearly equal facility.

Discriminatory inhibition of adrenocortical 17α-hydroxylase activity by inhibitors of cholesterol side chain cleavage cytochrome P-450

Two inhibitors of the cholesterol side chain cleavage reaction were tested for their ability to inhibit bovine adrenocortical 17 alpha-hydroxylase and 21-hydroxylase activities. One inhibitor, 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC), was found to be a potent inhibitor of 17 alpha-hydroxylation of either progesterone or pregnenolone but was inactive on 21-hydroxylase activity. 22-ABC was found to be a competitive inhibitor of 17 alpha-hydroxylase (cytochrome P-45017 alpha) activity, having an apparent inhibitor constant of 29 nM when using pregnenolone as the substrate. Spectral binding studies showed that 22-ABC produces a type II difference spectrum when added to a bovine adrenocortical microsomal preparation, due presumably to a coordination of its amine nitrogen atom to the heme-iron of cytochrome P-45017 alpha. The second cholesterol side chain cleavage inhibitor tested, (20R)-20-phenyl-5-pregnene-3 beta,20-diol (20-PPD), was found not to inhibit either the 21- or 17 alpha-hydroxylase activities. It is proposed that the phenyl group projecting from C-20 of 20-PPD prevents this steroid from binding to cytochrome P-45017 alpha. The discriminatory interaction of these two steroids with adrenocortical cytochromes P-450 provides some insight with respect to possible structural features of the active-site regions of these enzymes.

3β-Hydroxysteroid dehydrogenase-isomerase activity in bovine adrenocortical cells in culture: Lack of response to ACTH treatment

Primary cultures of bovine adrenocortical cells (BAC) were used to determine whether the adrenal microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase complex (3 beta-HSD), like the 17 alpha-hydroxylase (17-OHase), responded to ACTH treatment with an increase in activity. Both enzymes influence the steroidogenic path leading to 17 alpha-hydroxyprogesterone formation and thus could affect adrenal androgen biosynthesis. 3 beta-HSD Activity in postmitochondrial supernatant fluid, homogenates or cell monolayers remained unchanged after cells had been maintained in 1 microM ACTH up to 48 h. Since ACTH exposure led to a marked increase in 17-OHase activity over the same time period, it is concluded that, under the conditions used, the 3 beta-HSD-isomerase complex in BAC is nonresponsive to tropic hormone treatment.

Crowding Stress and Adrenal Mitochondrial 11β-Hydroxylation in Vitro

Summary Adrenal mitochondria were obtained from rats subjected to two different crowding regiments in order to test the in vitro capacity for 11 β-hydroxylation (11β-OH) as an index of stress. Compared to controls, 110 cm 2 of floor space per rat, crowding at 30 cm 2 /rat markedly increased 11β-OH. Little change occurred when rats were held at 50 cm 2 /animal. High levels of 11β-OH persisted 3 weeks after the termination of severe crowding. This apparent failure to reverse crowding stress is viewed in terms of pathologic changes that appear to accompany crowding stress. On the basis of measuring 11 β-OH capacity, it is possible to differentiate between the stressful effects of the housing conditions.

The 19-Hydroxylase of the Gerbil Adrenal Gland: A Mitochondrial Enzyme

Abstract The adrenal gland of the Mongolian gerbil produces nearly equal amounts of 19-hydroxy- and of 11β-hydroxycorticosteroids (Oliver and Peron, Steroids 4, 351 (1964)). The intracellular location of the 19-hydroxylase (OHase) was studied by determining the activities of both the 19- and 11β-OHase enzymes in gerbil adrenal homogenate and subcellular fractions. Adrenal preparations were incubated in vitro in the presence of exogenous steroid precursor, androstenedione (A), testosterone (T), or deoxycorticosterone (DOC), plus a source of reducing equivalents. When adrenal homogenate was incubated with A plus isocitrate, the only two products detected were 19-hydroxy A and 11β-hydroxy A; the two were formed at virtually identical rates. With subcellular fractions, the capacity for 19-hydroxylation coincided with that for 11β-hydroxylation; the highest activities were in adrenal mitochondria. For adrenal mitochondria the two hydroxylation reactions were (1) enhanced by the addition of Ca2+ to incubations containing NADPH, (2) supported by either isocitrate or succinate as a source of reducing equivalents, and (3) present at nearly the same levels of activity. The 19- and 11β-OHase activity in other subcellular fractions appeared to be of mitochondrial origin. The cytosol was found to contain a C-17 oxidoreductase catalyzing the interconversion between A and T in the presence of the appropriate NADP(H) cofactor. It was concluded that the 19-OHase of the gerbil adrenal gland is primarily a mitochondrial enzyme. The localization and activities of both the 19- and 11β-OHases account for the relative amounts of the major corticosteroids found in the blood of the gerbil.