Corticotropin-induced changes in a soluble desmolase preparation

Following simple homogenization, substantial desmolase activity is recovered in rat adrenal 105 000 × g supernatant. The desmolase complex sediments at 3–4 S on sucrose gradients, is found in the clear cytosol, requires NADPH, is derived from mitochondria and is inhibited by aminoglutethimide and pregnenolone. The lipid fraction contains little or no desmolase activity but greatly enhances pregnenolone synthesis in soluble desmolase preparations, presumably by supplying free cholesterol substrate. Prior adrenocorticotropin (ACTH) administration enhances pregnenolone synthesis in the 105 000 × g supernatant, and cycloheximide, an inhibitor of adrenal protein synthesis, does not block this effect of ACTH (but rather potentiates it). The ACTH effect may be largely explained by an increase in free cholesterol, which enhances the activity of both the lipid fraction and clear cytosol, since: free cholesterol levels are increased by ACTH, particularly with cycloheximide pretreatment; type I and inverted type I difference spectrum changes, indicating greater cholesterol availability for binding to cytochrome P-450, are enhanced by ACTH with or without cycloheximide treatment; cholesterol-rich lipid fraction enhances such spectral changes and obliterates the differences in spectral and pregnenolone-synthesizing activities betwen control and ACTH-stimulated soluble desmolase preparations; and desmolase stimulatory properties of clear cytosol co-chromatographs with [¹⁴C]cholesterol. Since cycloheximide blocks ACTH-induced effects in intact mitochondria but not in the soluble desmolase preparation, it is postulated that the labile protein required during ACTH action functions to overcome a ?restraining influence’ which is present in intact mitochondria but not in the soluble desmolase system. The ‘restraining influence’ may be due to limited cholesterol-desmolase interaction.     

Control of sterol metabolism in rat adrenal mitochondria

Steroidogenesis by adrenal mitochondria from endogenous precursors is stimulated by corticotropin (ACTH) and is sensitive to the protein-synthesis inhibitor cycloheximide. In the present investigation the effect of cycloheximide treatment on the metabolism of a number of analogues of the normal steroidogenic substrate, i.e. cholesterol, by rat adrenal mitochondria was studied. It was observed that the metabolism of analogues such as desmosterol, 26-norcholest-5-en-3β-ol and 5-cholen-3β-ol (that is with non-polar alkyl side chains like cholesterol), was sensitive to cycloheximide treatment. By contrast, the metabolism of those analogues with polar groupings on the side chain, i.e., 20α-, 24-, 25- and 26-hydroxycholesterols was insensitive to pretreatment with cycloheximide. The binding of added sterol to the cytochrome P-450 component of the mitochondrial sterol desmolase was studied. Similar studies on the equilibration time on addition of exogenous sterols to achieve maximum rates of pregnenolone production were also made. Both studies show that cholesterol, a non-polar sterol, penetrated slowly through the mitochondrial milieu to reach the cytochrome P-450 reaction centre whereas 24- and 26-hydroxycholesterols rapidly attained the enzymic environment. The cycloheximide-sensitive process in sterol metabolism appeared related to the transfer of non-polar sterols such as cholesterol within the mitochondria to a region in close proximity to the enzyme. The importance, and possible mechanism of action, of the cycloheximide-sensitive factor in the control of adrenal steroidogenesis is discussed.     

Presence of a low molecular weight inhibitor of succinate-supported cholesterol side chain cleavage in rat ovaries

1. Low molecular weight fractions (mol. wt. 3500-10 000) prepared from cytosols of luteinized rat ovaries inhibited succinate-supported cholesterol side chain cleavage by intact ovarian mitochondria utilizing endogenous or exogenous sterol as substrate. 2. The low molecular weight fractions inhibited steroid secretion by collagenase-dispersed ovarian cells stimulated with lutropin or dibutyryl cyclic AMP. 3. Steroidogenesis by intact mitochondria incubated with NADPH was enhanced by the low molecular weight ovarian fraction, but cholesterol side chain cleavage carried out by sonicated mitochondria incubated with NADPH was unaffected. 4. Succinate-supported mitochondrial respiration was stimulated by the low molecular weight factor, apparently by uncoupling of oxidative phosphorylation. The uncoupling seems to be the mechanism by which steroid synthesis is inhibited. 5. The low molecular weight factor was heat-labile and not extracted by activated charcoal. Similar heat-labile material capable of inhibiting succinate-supported mitochondrial steroid synthesis was not found in low molecular weight fractions prepared from rat kidney, liver, spleen, brain, plasma and bovine corpus luteum. 6. Treatment of rats with cycloheximide 1 h before killing resulted in a reduction of inhibitory activity in ovarian low molecular weight cytosolic fractions. 7. We conclude that ovarian cytosols contain a low molecular weight factor, presumably a protein, which inhibits mitochondrial cholesterol side chain cleavage by uncoupling oxidative phosphorylation. The physiological function of this factor remains to be determined.     

The side-chain cleavage of cholesterol sulfate--II. The effect of phospholipids on the oxidation of the sterol sulfate by inner mitochondrial membranes and by a reconstituted cholesterol desmolase system

This study compares the side-chain cleavage of aqueous suspensions of cholesterol sulfate with the side-chain cleavage of cholesterol sulfate which is incorporated into phospholipid vesicles. Three different cholesterol desmolase systems are examined: the membrane-bound cholesterol side-chain cleavage system present in inner mitochondrial membranes isolated from bovine adrenal mitochondria; a soluble, lipid-depleted, reconstituted side-chain cleavage system prepared from cytochrome P-450scc, adrenodoxin and adrenodoxin reductase; a membrane associated side-chain cleavage system prepared by adding phospholipid vesicles, prepared from adrenal mitochondrial, to the reconstituted system. Soluble cholesterol sulfate, in low concentration, is a good substrate for the lipid-depleted reconstituted side chain cleavage system. However, at concentrations above 2 microM, in the absence of phospholipids, the sterol sulfate appears to bind at a non-productive site on cytochrome P-450scc which leads to substrate inhibition. Phospholipids, while inhibiting the binding of cholesterol sulfate to the cytochrome, also appear to prevent non-productive binding of the sterol sulfate to the cytochrome. Thus the addition of phospholipids to the lipid-depleted enzyme system leads to an activation of side-chain cleavage of high concentrations of the sterol sulfate. Soluble cholesterol sulfate is a good substrate for both the native and reconstituted membrane-bound systems and no substrate inhibition is observed when the membrane bound enzyme systems are employed in the assay of side-chain activity. However, the cleavage of cholesterol sulfate, which is incorporated into phospholipid vesicles, by both membrane bound enzyme systems appears to be competitively inhibited by the phospholipids of the vesicles. The results of this study suggest that the regulation of the side-chain cleavage of cholesterol sulfate may be entirely different than the regulation of the side-chain cleavage of cholesterol, if cholesterol sulfate exists intracellularly as a soluble non-complexed substrate. If, on the other hand, cholesterol sulfate is present in the cell in lipid droplets as a complex with phospholipids, its metabolism may be under the same constraints as the side-chain cleavage of cholesterol.     

Protein kinase stimulation of a reconstituted cholesterol side chain cleavage enzyme system in the bovine corpus luteum.

A solubilized preparation of cytochrome P-450, obtained by treatment of mitochondria from bovine corpora lutea with phospholipase A, contained all of the necessary components for the cholesterol side chain cleavage activity. The solubilized cytochrome -450 preparation could be isolated essentially free of endogenous cholesterol side chain cleavage activity by various fractionation techniques. A cholesterol side chain cleavage enzyme system was reconstituted using the isolated cytochrome P-450 preparation and purified adrenodoxin and adrenodoxin reductase (components of the enzyme system purified from the adrenal cortex). Protein kinase was partially purified from the cytosol fraction of bovine corpora lutea. It was purified 43-fold and the activity was highly dependent on cyclic adenosine 3:5-monophosphate (cyclic AMP). When ATP and this partially purified cyclic AMP-dependent protein kinase were added to the reconstituted cholesterol side chain cleavage enzyme assay in which cytochrome P-450 was limiting, a stimulation (20 to 74%) of the conversion of cholesterol into pregnenolone was observed. This stimulation was statistically significant with p value less than 0.001. The stimulatory effect of the protein kinase appeared to be dependent on ATP and was not mimicked by bovine serum albumin, indicating that the effect was specific for protein kinase. Protein kinase caused a phosphorylation of the cytochrome P-450 preparation when large amounts of this preparation were used in the assay. It is concluded from these results that the direct activation of the cytochrome P-450 component of the cholesterol side chain cleavage by protein kinase may be one of the ways by which cyclic AMP mediates the effect of luteinizine.     

Mechanism of corticotropin action on adrenal protein synthesis. The effects of inhibitors of protein synthesis and calcium chelators

We have recently shown that beside a general stimulation of most adrenal proteins, corticotropin induces a marked increase in a specific adrenal cytosolic protein, protein E, in intact and hypophysectomized rats. To further clarify the mechanisms by which corticotropin exerts its trophic action we have investigated the effects of cycloheximide, calcium and calcium chelator administration on intact and hypophysectomized animals. These substances were injected in rats with or without corticotropin, and slices of adrenal glands from control and treated animals were removed 5 h later, incubated with [14C]- or [3H]-leucine for 2 h, and cytosolic proteins analyzed by polyacrylamide gel electrophoresis using a dual labelling technique. When high doses of cycloheximide (higher than 500 micrograms) were injected in rats, incorporation of labelled leucine in adrenal slices of control and corticotropin-treated animals was inhibited. With 500 micrograms cycloheximide per rat, incorporation of labelled leucine in adrenal slices of control animals was normal, but the corticotropin stimulation of both protein E and total protein synthesis was inhibited. Lower doses of cycloheximide (100 micrograms per rat) completely inhibited the stimulatory effect of corticotropin on total protein synthesis but did not affect protein E synthesis, while after 50 micrograms per rat both stimulatory effects were preserved. The two higher doses of cycloheximide (500 and 100 micrograms per rat) could not completely block the steroidogenic effect of the hormone. The effects of calcium and calcium chelators were studied in 1-day hypophysectomized rats. Calcium alone or injected simultaneously with corticotropin has no effect. Calcium chelators injected simultaneously with corticotropin partially inhibited the stimulatory effects of corticotropin on steroidogenesis but totally inhibited stimulation of total protein synthesis, while the stimulation of protein E persisted. Our results show that after corticotropin, stimulation of protein E synthesis correlates better with steroidogenesis than with total protein synthesis.     

Inhibition of protohaem ferro-lyase in experimental porphyria. Isolation and partial characterization of a modified porphyrin inhibitor.

Rat adrenal 105,000 g supernatant contains two lipid moieties, 'lipid-I' and 'lipid-II' which contain non-esterified cholesterol and stimulate cholesterol side-chain cleavage in soluble or mitochondrial enzyme systems. Lipid-I contains relatively large low-density heat-stable particles, whereas lipid-II particles are smaller, more dense and heat-labile. Lipid-I and lipid-II can be separated from clear cytosol by ultracentrifugation and gel filtration respectively. Corticotropin plus cycloheximide treatment increases the non-esterified cholesterol concentrations in the lipid fractions, and stimulatory effects of lipids on cholesterol side-chain cleavage appear to correlate with non-esterified cholesterol concentrations therein. On addition of saturating amounts of cholesterol-rich lipid, pregnenolone synthesis and cholesterol binding to cytochrome P-450 are stimulated more in mitochondria from corticotropin-stimulated adrenals than in mitochondria from control or corticotropin-plus cycloheximide-stimulated adrenals. These results support the contention that the corticotropin-induced increase in mitochondrial cholesterol side-chain cleavage involves an increase in cholesterol utilization as well as an increase in cholesterol availability.     

In vitro activation of a soluble cholesterol esterase from bovine adrenals by a cAMP-dependent protein kinase.

Properties and partial purification of the bovine adrenal cholesterol esterase from the 100000 X g supernatant fraction were investigated. Variations of the enzyme activity with time-dependent (enzymatic) and time-dependent (non enzymatic) effects have been demonstrated. Mg2 has been proved to inhibit the enzyme activity by a non-enzymatic effect in 50mM Tris/HCl buffer, pH 7.4. A time-dependent inactivation of the cholesterol esterase has been observed in the same buffer. The enzyme could be protected from this enzymatic inactivation by its substrate, cholesterol oleate. cAMP, ATP and Mg2 cuase a time-dependent stimulation of the enzyme in 50mM Tris/HCl buffer, pH 7.4. This result suggests that corticotropin activates the soluble cholesterol esterase from bovine adrenals via cAMP-dependent protein kinase. This view is strengthened by the incorporation of 32P radioactivity from [gamma-32P] ATP into the protein fraction of the 100,000 X g supernatant. The protein-bound 32P radioactivity could be co-purified with the enzyme activity during the partial purification of the soluble cholesterol esterase.     

Cholesterol side chain cleavage in rat adrenal supported by outer mitochondrial membrane NADH-semidehydroascorbate reductase

Rat adrenal mitochondria have an active rotenone-insensitive outer mitochondrial membrane NADH-semidehydroascorbate (NADH-SDA) reductase which supports cholesterol side chain cleavage at a rate equal to that supported by malate. Side chain cleavage activity supported by both of these electron donor systems is equally inhibited by cycloheximide. Catalase or butylated hydroxyanisole are required for the NADH-SDA reductase-supported cholesterol side chain cleavage. This requirement can be removed by briefly subjecting the mitochondrial preparations to -20 degrees C. Ascorbic acid alone or with malate is either inhibitory or has no effect on side chain cleavage activity. These observations demonstrate that outer mitochondrial membrane NADH-SDA reductase in rat adrenal functions to provide cytoplasmic reducing equivalents to intramitochondrial cytochrome P-450scc and provides a new explanation for the function of ascorbic acid in corticosteroidogenesis.     

Inbition of steroidogenic response to corticotropin in mouse adrenal tumor cells (Y-1 by the ionophore A23187 Role of protein biosynthesis

Addition of the ionophore A123187 to Y-1 mouse adrenal tumor cells in monolayer culture inhibits steroidogenesis and the steroidogenic response to corticotropin (50% inhibition at 1 · 10^?7 M). inhibition is rapid in onset and is not overcome by addition of external Ca²⁺. The ionophore also inhibits stimulation of steroid synthesis by cyclic AMP. A23187 inhibits incorporation of the amino acid lysine into protein by Y-1 cells and the dose dependence of this inhibition closely resembles that of the inhibition of the steroidogenic response to corticotropin. Addition of A23187 to a subcellular system for protein synthesis prepared from Y-1 cells, inhibits incorporation of the amino acid phenylalanine into protein and this effects and this effect is not overcome by high concentrations of Ca²⁺. The inhibitory effect of A23187 on the response to corticotropin, like that response itself, takes place at some part of steriod synthesis after entry of cholesteriol into the cells and before the side-chain cleavage of cholesterol. These studies confirm the importance of protein synthesis in the response to corticotropin and demonstrate that the effect of protein synthesized under the influence of corticotropin is exerted at some point in the events which bring substrate (cholesterol) to the mitochondrial side-chain cleavage enzyme system. It is also shown that A23187 inhibits protein synthesis, and hence the response to corticotropin, by a mechanism which is independent of the concentration of available Ca²⁺.     

Biosynthesis of rat liver transhydrogenase in vivo and in vitro

The biosynthesis of pyridine dinucleotide transhydrogenase, a homodimeric inner mitochondrial membrane redox-linked proton pump, has been studied in isolated rat hepatocytes. Newly synthesized transhydrogenase, having an apparent molecular weight identical to the enzyme of isolated liver mitochondria, was selectively immunoprecipitated from detergent extracts of isolated hepatocytes which were labeled with [35S]methionine. That the enzyme is a nuclear gene product is indicated since 1) synthesis was inhibited by cycloheximide, but not by chloramphenicol and 2) no synthesis could be demonstrated in hepatocyte ghosts which are competent only in mitochondrial translation. In addition to the mature form of the enzyme, a species about 2000 daltons larger was also immunoprecipitated from pulse-labeled cells. The half-life of the larger form during a subsequent chase at 37 degrees C was about 2 min, whereas the mature form was not degraded. The relationship between the two forms of the enzyme was established by in vitro studies. A protein approximately 2000 daltons larger than mature transhydrogenase was immunoisolated from a rabbit reticulocyte lysate system programmed with sucrose gradient fractionated rat liver mRNA. This protein was converted to a species having the same size as mature enzyme after incubation with either intact rat liver mitochondria or a soluble matrix fraction derived from mitoplasts. These studies indicate that transhydrogenase is synthesized in the cytoplasm as a higher molecular weight precursor which is post-translationally processed to the mature protein by a soluble matrix protease during or after membrane insertion.     

Effects of a supernatant protein activator on microsomal squalene-2,3-oxide-lanosterol cyclase.

A soluble protein termed "supernatant protein factor" (SPF) that stimulates microsomal squalene epoxidase has been isolated in this laboratory (Ferguson, J.B., and Bloch, K. (1977) J. Biol. Chem. 252, 5381-5385). We now show that the purified protein also stimulates microsomal squalene-2,3-oxide leads to lanosterol cyclase but has no effect on the subsequent conversion of lanosterol to cholesterol. Phospholipid, specifically phosphatidylglycerol or phosphatidylethanolamine, is required for maximal stimulation of the cyclase by purified SPF. The response of microsomal squalene epoxide-lanosterol cyclase to SPF was abolished by pretreatment of the membranes with phospholipase A2 or by low concentrations of deoxycholate, indicating that an intact membrane system is required. Digestion of intact microsomes with trypsin had no effect on the SPF-stimulated cyclase activity. However, in the presence of 0.4% deoxycholate, trypsin completely inhibited microsomal squalene epoxide-lanosterol cyclase. We conclude that the cyclase is located on the luminal side of the microsomal membrane. SPF also significantly enhances the formation of lanosterol from squalene-2,3-oxide already bound to microsomes. This finding is constant with the proposal that SPF influences intramembrane events.     

Inhibition of steroidogenic response to corticotropin in mouse adrenal tumor cells (Y-1) by the ionophore A23187. Role of protein biosynthesis.

Addition of the ionophore A23187 to Y-1 mouse adrenal tumor cells in monolayer culture inhibits steroidogenesis and the steroidogenic response to corticotropin (50% inhibition at 1 . 10(-7)M). Inhibition is rapid in onset and is not overcome by addition of external Ca2+. The ionophore also inhibits stimulation of steroid synthesis by cyclic AMP. A23187 inhibits incorporation of the amino acid lysine into protein by Y-1 cells and the dose dependence of this inhibition closely resembles that of the inhibition of the steroidogenic response to corticotropin. Addition of A23187 to a subcellular system for protein synthesis prepared from Y-1 cells, inhibits incorporation of the amino acid phenylalanine into protein and this effect is not overcome by high concentrations of Ca2+. The inhibitory effect of A23187 on the response to corticotropin, like that response itself, takes place at some part of steroid synthesis after entry of cholesterol into the cells and before the side-chain cleavage of cholesterol. These studies confirm the importance of protein synthesis in the response to corticotropin and demonstrate that the effect of protein synthesized under the influence of corticotropin is exerted at some point in the events which bring substrate (cholesterol) to the mitochondrial side-chain cleavage enzyme system. It is also shown that A23187 inhibits protein synthesis, and hence the response to corticotropin, by a mechanism which is independent of the concentration of available Ca2+.     

Synergistic stimulation of pregnenolone synthesis in rat adrenal mitochondria by n-hexane and cardiolipin

n-Hexane and cardiolipin each stimulate pregnenolone production by isolated rat adrenal mitochondria. Following corticotropin (ACTH) stimulation, mitochondrial cholesterol metabolism exhibits a fast phase lasting 2 min, followed by a 10-fold slower metabolism. ACTH suppression by dexamethazone or cycloheximide (CX) treatment removes this fast phase. n-Hexane, at concentrations approaching 80% of the aqueous solubility limit (approximately 0.08 mM), selectively stimulates the slow phase of metabolism, while cardiolipin (100 microM) stimulates only the fast phase. Other alkanes and ethers are effective. The effect of n-hexane is dependent on mitochondrial integrity, as evidenced by decreased effects in hypoosmotically shocked mitochondria (outer membrane disrupted) and ineffectiveness in sonicated mitochondria (both membranes disrupted). n-Hexane apparently enhances the transfer of outer membrane cholesterol to inner membrane P-450scc. Stimulation by cardiolipin is retained by disrupted mitochondria and may involve enhanced availability of P-450scc to inner membrane cholesterol. When added together, these agents produce more than additive effects on cholesterol metabolism. Preincubation with n-hexane did not increase reactive cholesterol, suggesting that enhanced cholesterol transport occurs only in concert with metabolism of inner membrane cholesterol. Uptake of alkanes into mitochondrial membranes may effect structural changes that facilitate outer to inner membrane cholesterol transfer, but major changes are excluded by the effectiveness of isocitrate as a reductant for P-450scc. In combination, n-hexane and cardiolipin reproduce the effect of the ACTH-sensitive sterol regulatory peptide on mitochondria [R. C. Pedersen and A. C. Brownie (1983) Proc. Natl. Acad. Sci. USA 80, 1882-1886], suggesting that peptide action on adrenal mitochondria may resolve into two analogous components.     

Fatty acid synthesis in cell-free system from rabbit aorta

The objectives of this study were to identify the subcellular fraction responsible for fatty acid synthesis in rabbit aorta and to determine the effect of cholesterol feeding on the system. A method for homogenization of aorta is described which permitted the isolation of subcellular components of aorta, including mitochondria that were morphologically and functionally intact. Mitochondria were identified as the major site of fatty acid synthesis in this tissue. Cofactor requirements and products showed that the synthetic system operates by chain elongation. Mitochondria from atherosclerotic aortas incorporated acetate into fatty acids faster than did mitochondria from control aortas. It is concluded that cholesterol feeding leads to alterations of aortic mitochondrial function and accelerates the fatty acid elongation pathway.     

An early steroidogenic defect in hormone-induced Leydig cell desensitization

To define the nature of the lesion of the early steroidogenic pathway (prior to pregnenolone formation) in gonadotropin-induced desensitization of rat testicular Leydig cells, we evaluated cholesterol side-chain cleavage activity in isolated mitochondria by measurement of pregnenolone synthesis and [14C]isocaproic acid formation from [26-14C]cholesterol. The enzyme activity was shown to be reduced after in vivo treatment with 10 micrograms hCG when compared to that of mitochondria from control animals only when measured in the presence of limiting NADPH concentrations (100 microM). Sonication of mitochondria from control and hCG-treated rats caused complete loss of cholesterol side-chain cleavage activity. When acetone-powdered adrenal cell mitochondria were employed as the source of the enzyme, the addition of sonicated Leydig cell mitochondria from control and hCG-treated animals caused the same differences as those observed with intact Leydig cell mitochondria in the presence of low concentration of NADPH. The Km value of the adrenal enzyme for NADPH incubated with Leydig cell mitochondria increased from 0.111 mM in control to 0.37 mM after hCG, with no changes in Vmax. Moreover, cholesterol side-chain cleavage activity of adrenal mitochondria assayed in the presence of 100 microM cholesterol was progressively inhibited by increasing amounts of acetone powder from Leydig cell mitochondria of control and hCG-treated rats, with ID50 of 500 and 280 micrograms protein, respectively. The inhibiting factor was not a lipid or steroid but a heat-labile protein, with an approximate Stokes radius of 4.8 nm and an isoelectric point of 5.05 +/- 0.23 SD (n = 8). The inhibitory effect was confined to the Leydig cell mitochondrial membrane, and was not related to changes in oxidative phosphorylation. NADPH was not directly oxidized or immobilized by the mitochondrial factor, and this inhibiting substance was not adsorbed on 2',5' ADP-Sepharose 4B. These results have demonstrated that a heat-labile inhibiting protein factor is present in mitochondria from normal Leydig cells and is markedly activated or increased by hCG treatment. This substance that competitively modulates cholesterol side-chain cleavage activity could contribute to the early steroidogenic lesion, and also serve as an endogenous modulator of steroid hormone biosynthesis.     

Study of the ultrastructural and functional expression of the hepatocyte genome under conditions of prolonged depression of protein biosynthesis by cycloheximide. III]

Dynamics of changes in mtRNA synthesis and mitochondria ultrastructure is strictly dependent on the level of inhibition of biosynthesis of cytoplasm proteins and "soluble" proteins of mitochondria by cycloheximide in hepatocytes: 1-6 hrs later a progressive weakening of protein synthesis is accompanied by a drop in mtRNA synthesis and essential destruction of mitochondria; from 12 to 24 hrs a partial restoration of protein biosynthesis induces the processes of the above-mentioned indexes normalization.     

Ultrastructural changes in rat adrenal cortical cells after chronic stimulation with β1–24-corticotropin

Summary Adult rats were given 15 daily subcutaneous injections either of synthetic ^1–24-corticotropin or of the corresponding placebo (controls) and were sacrificed 1 h after the final injection. In stimulated animals, the adrenal glands were increased in weight as compared to those of controls. Stereological analysis at light microscopic level of the outer zona fasciculata cells showed moderate volumetric increases of nuclei, cytoplasm and capillaries and a marked volumetric increase of lipid droplets in stimulated animals. Stereologic analysis of electron micrographs confirmed the marked increase in relative volume and surface density of lipid droplets, while volume fractions alone were increased for the Golgi apparatus and decreased for the endoplasmic reticulum and mitochondria. Biochemical analysis of the whole adrenal gland showed that the corticotropin injections produced a moderate increase in protein concentration, a marked increase in triglycerides and no appreciable changes in either phospholipid or cholesterol concentrations. The synthetic polypeptide therefore appears to have stimulating trophic effects on adrenal cortical cells, as shown by the increase in protein and cell size. However, it depresses the activity of the two types of organelle, endoplasmic reticulum and mitochondria, which have a major functional role in steroid synthesis. The increase of lipid droplets was interpreted as being primarily due to neutral fat accumulation, and secondarily to a diminished utilization of cholesterol for steroid synthesis. These findings suggest that, using this regime of administration, synthetic ^1–24 corticotropin, unlike native ACTH, inhibits steroid synthesis.     

Evidence for sterol carrier protein2-like activity in hepatic, adrenal and ovarian cytosol

Purified sterol carrier protein2 (SCP2) from rat liver stimulated utilization of endogenous cholesterol for pregnenolone synthesis by adrenal mitochondria. Cytosolic preparations of rat liver, adrenal and luteinized ovary were also stimulatory in mitochondrial pregnenolone synthesis to different extents. Treatment of all preparations with rabbit anti-rat SCP2 IgG neutralized the stimulatory effects, and immunoprecipitated proteins gave similar patterns on SDS-gradient polyacrylamide gel electrophoresis. Treatment with rabbit pre-immune IgG had no effect on these parameters. Thus, proteins which are immunochemically compatible with hepatic SCP2 appear to be present in steroidogenic tissues and may play a role in control of mitochondrial cholesterol side chain cleavage activity.     

Protein synthesis in relation to ripening of pome fruits

Protein synthesis by intact Bartlett pear fruits was studied with ripening as measured by flesh softening, chlorophyll degradation, respiration, ethylene synthesis, and malic enzyme activity. Protein synthesis is required for normal ripening, and the proteins synthesized early in the ripening process are, in fact, enzymes required for ripening. ¹⁴C-Phenylalanine is differentially incorporated into fruit proteins separated by acrylamide gel electrophoresis of pome fruits taken at successive ripening stages. Capacity for malic enzyme synthesis increases during the early stage of ripening. Fruit ripening and ethylene synthesis are inhibited when protein synthesis is blocked by treatment with cycloheximide at the early-climacteric stage. Cycloheximide became less effective as the climacteric developed. Ethylene did not overcome inhibition of ripening by cycloheximide. The respiratory climacteric is not inhibited by cycloheximide. It is concluded that normal ripening of pome fruits is a highly coordinated process of biochemical differentiation involving directed protein synthesis.