On the requirement for protein synthesis during corticotropin-induced stimulation of cholesterol side chain cleavage in rat adrenal mitochondrial and solubilized desmolase preparations.

Following simple homogenization, significant amounts of mitochondrial-derived, cholesterol side chain cleaving enzyme (desmolase) activity are recovered in rat adrenal 105 000 X g-supernatant fraction. Corticotropin administration enhances soluble desmolase activity, and cycloheximide potentiates this effect. The lipid droplet fraction which has no desmolase activity markedly enhances pregnenolone synthesis in the soluble desmolase preparations, presumably by supplying free cholesterol substrate. Corticotropin particularly with cycloheximide pretreatment, enhances lipid fraction activity. Thus increased cholesterol availability may largely explain the corticotropin effect on the soluble desmolase system. Since protein synthesis is required for corticotropin activity in intact mitochondria, but not in calcium-swollen mitochondria or the soluble enzyme system, the labile protein apparently required during corticotropin action may function to overcome a "barrier" which exists only in the intact mitochondria and restrains cholesterol side chain cleavage.     

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.     

Cytosol stimulation of pregnenolone synthesis by isolated adrenal mitochondria

Hypophysectomized male rats were injected with either ACTH or saline (control) and killed 15 min later. Mitochondrial and 235, 000 x g supernatant (cytosol) fractions were prepared from the adrenal glands. When cytosol from ACTH-treated animals was mixed with isolated mitochondria from the control animals, a dose-dependent increase in pregnenolone production occurred. Liver cytosol caused no increase in the production of pregnenolone. Thus, ACTH elicits a soluble adrenal factor(s) which activates the mitochondrial cholesterol side-chain cleavage system which is the rate-limiting step in steroidogenesis. The cytosol stimulatory factor was found to be nondialyzable, heat-sensitive, and resistant to trypsinization.     

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.     

Sterol carrier protein2. Identification of adrenal sterol carrier protein2 and site of action for mitochondrial cholesterol utilization

Addition of homogeneous rat liver sterol carrier protein2 (SCP2) or an adrenal cytosolic fraction enhanced pregnenolone production by adrenal mitochondria. Pretreatment of SCP2 or adrenal cytosol with anti-SCP2 IgG abolished the stimulatory effect of both preparations on mitochondrial pregnenolone output. Incubation of mitochondria with aminoglutethimide, which blocks interaction of cholesterol with inner membrane cytochrome P-450scc, resulted in decreased pregnenolone production and a decreased level of mitoplast cholesterol. Addition of SCP2 to the incubation media caused an almost 2-fold increase in cholesterol associated with the mitoplast, but did not enhance mitochondrial pregnenolone production. Studies with reconstituted cytochrome P-450scc in phospholipid vesicles also suggested that SCP2 did not affect interaction of cholesterol with the hemoprotein. Treatment of rats with cycloheximide alone or with adrenocorticotropic hormone resulted in a dramatic increase in mitochondrial cholesterol. However, these mitochondria did not exhibit increased levels of pregnenolone output under control incubation conditions. When SCP2 was included in the mitochondrial incubation media, pregnenolone production was significantly increased over that observed with adrenal mitochondria from untreated or adrenocorticotropic hormone-treated rats. The results imply that SCP2 enhances mitochondrial pregnenolone production by improving transfer of mitochondrial cholesterol to cytochrome P-450scc on the inner membrane, but does not directly influence the interaction of substrate with the hemoprotein.     

Adrenic acid content in rat adrenal mitochondrial phosphatidylethanolamine and its relation to ACTH-mediated stimulation of cholesterol side chain cleavage reaction

We have isolated various phospholipids from adrenal mitochondria of adrenocorticotropic hormone (ACTH)-treated (stimulated) and cycloheximide/ACTH-treated (unstimulated) rats. When the effects of these phospholipids were examined on the formation of pregnenolone from endogenous cholesterol by adrenal mitochondria of unstimulated rats, phosphatidylethanolamine and phosphatidylserine from stimulated mitochondria were effective in enhancing the cleavage reaction in unstimulated mitochondria, whereas these phospholipids from unstimulated mitochondria were all ineffective. Cardiolipins from both stimulated and unstimulated mitochondria were effective. When the compositional changes in fatty acid moiety of phospholipids were examined, a significant increase in C22:4 (adrenic) acid was observed only for phosphatidylethanolamine under the influence of ACTH. A linear relationship between the contents of C22:4 acid in various phospholipids and respective steroidogenic activities was obtained (r = 0.880), suggesting an important role of this fatty acid moiety. The separation of active phosphatidylethanolamine by high performance liquid chromatography revealed that a fraction containing 25% C22:4 acid was most effective in the activation. Based on these results, it is most likely that 1-stearoyl-2-adrenoyl phosphatidylethanolamine is an active species. C22:4 acid was liberated together with C20:4 acid from adrenal triglycerides by the action of ACTH but the liberation was insensitive to cycloheximide inhibition. Finally, cardiolipin which enhances the transfer of cholesterol to cytochrome P-450scc may not be a physiological mediator of ACTH action.     

Rat adrenal corticosteroidogenesis; effect of ACTH, cycloheximide and sterol carrier protein.

Corticosterone formation was determined in the reconstructed rat adrenal system which consisted of the mitochondria and post-mitochondrial supernatant fraction (PM-fraction) supported by l-malate, and effect of ACTH and cycloheximide in vivo and cycloheximide, Ca++ and sterol carrier protein (SCP) in vitro were examined. Mitochondria isolated from adrenals of rats which received ACTH 15 min before sacrifice showed an elevated corticosterone formation. Cycloheximide administration 15 min prior to ACTH injection completely blocked the effect of ACTH but in vitro addition of this drug to the incubation mixture did not modify the rate of corticosterone production even at higher concentrations. Since the PM-fraction isolated from adrenals of rats received ACTH or cycloheximide or both did not change the mitochondrial capacity for corticosterone formation, factor(s) which influenced by ACTH administration seemed to be localized in mitochondria. The SCP-bound cholesterol was utilized for corticosterone formation more efficiently than the free cholesterol when added to the incubation mixture, and this might be due to, at least in part, higher rate of binding to the mitochondrial inner membrane of the SCP-bound cholesterol.     

Factors affecting the adrenocorticotropic hormone stimulation of rabbit adrenal 17α-hydroxylase activity

Adrenocorticotropic hormone (ACTH)-stimulated 17α-hydroxylase activity of rabbit adrenal tissue has been shown to be associated with the subcellular fractions sedimented from 0.25 M sucrose at 33 000 × g for 60 min and at 105 000 × g for 60 min. The fraction sedimenting at 9000 × g for 20 min (mitochondria) contained the majority of the 11β-hydroxylase activity but also had a significant amount of 17α-hydroxylase activity. All subcellular 17α-hydroxylase activity showed an apparent preference for pregnenolone over progesterone. A 1 : 1 mixture of wholehomogenates of adrenal tissue from control and ACTH-stimulated rabbits incubated with[4-¹⁴C]pregnenolone synthesized as much 17α-hydroxylated corticosteroids as homogenate from the ACTH-stimulated tissue alone. However, the mixed homogenate synthesized only 1/4th–1/5th as much 17-deoxycorticosteroids as control, non-stimulated tissue, suggesting that the control tissue contained no inhibitor of 17α-hydroxylation, whereas ACTH-stimulated tissue may contain an inhibitor of 17-deoxycorticoid formation. 24-h dialysis of whole homogenates and subcellular fractions of adrenal tissue from control and ACTH-stimulated animals showed that 17α-hydroxylation was not activated in control tissue and somewhat inactivated in ACTH-stimulated tissue by this treatment. On the other hand, dialysis activated 17-deoxycorticoid formation by whole homogenates, but not in subcellular fractions, of both ACTH-stimulated and control adrenal tissue. Injection of 5 mg/kg cycloheximide prior to the first of 2 daily ACTH injections caused an average of 270 g body weight loss while not affecting the increase in adrenal weight effected by the ACTH. Adrenal tissue homogenates from cycloheximide injected animals produced only 50% as much 17α-hydroxycorticosteroids as homogenates of tissue from animals injected with ACTH alone and produced an amount of17-deoxycorticoids intermediate between homogenates of control and ACTH-stimulated tissue, suggesting the requirement of protein synthesis for 17α-hydroxylation stimulating activity of ACTH.     

Extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex. III. ACTH-induced temporal subcellular redistributions of steroid precursors to corticosterone

Cholesterol, pregnenolone, progesterone, 11-deoxycorticosterone (11-DOC) and corticosterone were quantitated in subcellular fractions isolated from in vivo adrenocorticotropin (ACTH)-stimulated rat adrenal zona fasciculata/reticularis. Six adrenal subcellular fractions separated by discontinuous sucrose gradient centrifugation (lipid, 0.125 M sucrose, cytosolic, microsomal, mitochondrial and nuclear) were extracted with alkaline ether/ethanol and assayed by high pressure liquid chromatography (HPLC). Lipid fractions contained the major cholesterol stores, while most pregnenolone and progesterone was found in lipid, microsomal and mitochondrial fractions. The 0.125 M sucrose and cytosol fractions together contained approximately 75% of the total 11-DOC and corticosterone. The five steroids were only present in small amounts in organelle fractions containing steroidogenic enzymes. Homogenate and lipid fraction cholesterol decreased between 10 and 15 min and again 30 min after ACTH injection. In the homogenate, lipid, microsomal and mitochondrial fractions, pregnenolone and progesterone were increased after ACTH injection; peak pregnenolone and progesterone concentrations were often measured in adrenal gland sucrose, cytosolic, microsomal and mitochondrial fractions 15 to 20 min after rats were injected with ACTH. Although ACTH increased 11-DOC and corticosterone in all but the mitochondrial and nuclear fractions, the sucrose, cytosolic and microsomal 11-DOC, and cytosolic corticosterone increased most dramatically. In many fractions, peak 11-DOC and corticosterone concentrations were most often observed between the 10 and 15 min periods and again at 30 min.     

The role of the cytoskeleton in the regulation of steroidogenesis

The slow step in steroid synthesis involves the transport of cholesterol from lipid droplets in the cytoplasm to the first enzyme in the pathway—the cytochrome P450 that converts cholesterol to pregnenolone (P450scc) which is located in the inner mitochondrial membrane. ACTH stimulates this intracellular transport of cholesterol in adrenal cells (Y-1 mouse adrenal tumour cells and cultured bovine fasciculata cells) and this effect of the trophic hormone is inhibited by cytochalasins, by anti-actin antibodies and DNase I suggesting that the response to ACTH requires a pool of monomeric (G-) actin that can be polymerized to F-actin. Recent studies have shown that lipid droplets and mitochondria of adrenal cells are both attached to intermediate filaments. Moreover ACTH reorganizes the cytoskeleton and changes the shape of the cell. These observations suggest a mechanism for transport of cholesterol that involves reorganization and contraction of actin microfilaments which may, in turn, cause movement of droplets and mitochondria together through their common attachment to intermediate filaments.     

The role of sterol carrier protein 2 in stimulation of steroidogenesis in rat adrenal mitochondria by adrenal cytosol

Cholesterol side-chain cleavage (CSCC) in isolated rat adrenal mitochondria is enhanced by prior corticotropin (ACTH) stimulation in vivo (8-fold). Part of this stimulation is retained in vitro by addition of cytosol from ACTH-stimulated adrenals to mitochondria from unstimulated rats (2.5- to 6-fold). In vivo cycloheximide (CX) treatment fully inhibits the in vivo response and resolves the in vitro cytosolic stimulation into components: (i) ACTH-sensitive, CX-sensitive; (ii) ACTH-sensitive, CX-insensitive; and (iii) ACTH-insensitive, CX-insensitive. These components contribute approximately equally to stimulation by ACTH cytosol. Components (i) and (iii) most probably correspond to previously identified cytosolic constituents steroidogenesis activator peptide and sterol carrier protein 2 (SCP2). SCP2, as assayed by radioimmunoassay or ability to stimulate 7-dehydrocholesterol reductase, was not elevated in adrenal cytosol or other subcellular fractions by ACTH treatment. Complete removal of SCP2 from cytosol by treatment with anti-SCP2 IgG decreased cytosolic stimulatory activity by an increment that was independent of ACTH or CX treatment. Addition of an amount of SCP2, equivalent to that present in cytosol, restored activity to SCP2-depleted cytosol but had no effect alone or when added with intact cytosol, suggesting the presence of a factor in cytosol that potentiates SCP2 action. Pure hepatic SCP2 stimulated CX mitochondrial CSCC 1.5- to 2-fold (EC50 0.7 microM) but was five times less potent than SCP2 in adrenal cytosol. Two pools of reactive cholesterol were distinguished in these preparations characterized, respectively, by succinate-supported activity and by additional isocitrate-supported activity. ACTH cytosol and SCP2 each stimulated cholesterol availability to a fraction of mitochondrial P450scc that was reduced by succinate but failed to stimulate availability to additional P450scc reduced only by isocitrate.     

Some characteristics of adrenal steroidogenesis and their possible relationships to the action of the adrenocorticotropic hormone.

An attempt to define in quantitative terms the characteristics of the biphasic rate curve for pregnenolone synthesis in cell-free systems from the adrenal using male Sprague-Dawley rats is reported. When adrenocorticotropic hormone (ACTH) was used 2 units of .2 ml of .9% saline were injected ip 15 minutes before killing the rats. The effect of ACTH on adrenal steroidogenesis is in the stimulation of the rate of conversion of cholesterol to pregnenolone. This reaction sequence is thought to occur in the mitochondria. Methods of preparing subcellular fractions are described. Incubation of pregnenolone with mitochondria for 20 minutes at 20 degree C resulted in a 70% disappearance of the pregnenolone. This loss does not occur if the mitochondria are boiled, indicating an enzymatic process. The rate of pregnenolone synthesis characteristically shows a biphasic curve with a rapid primary rate and a slower secondary rate. ACTH administration in vivo increased both rates but the percentage increase was greater for the secondary rate. In addition an increase in the duration of the primary rate resulted. Different explanations are offered for these characteristics. Pregnenolone may act as an inhibitor of its own synthesis from cholesterol but not from 20alpha-hydroxycholesterol. Substances that cause mitochondria to swell may stimulate pregnenolone synthesis. Another theory proposes that the limiting ACTH-sensitive step is the rate at which mitochondrial cholesterol is transported to or binds to the cholesterol side-chain cleavage enzyme. The possible role of an inhibitor in the regulation of steroidogenesis is indicated. Data are consistent with the observation that the transition from the primary rate to the slower secondary rate shows the accumulation of an inhibitory substance. The action of ACTH would then be to modify the structure of the cholesterol side-chain cleavage enzyme so that there is a decreased susceptibility of the enzyme to the inhibitor.     

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.     

A method for the isolation of lipid droplet fractions from decapsulated rat adrenals

Ultrastructural and cell fractionation studies implicate lipid droplets in the storage of cholesterol and in the secretion of steroids. To evaluate the role of the lipid droplet in steroidogenesis, a discontinuous gradient centrifugation method has been developed for the isolation of both lipid droplet and non-lipid fractions from decapsulated rat adrenal homogenates. Steroids were extracted from the fractions with chloroform/methanol; the cholesterol ester, cholesterol and corticosterone in each extract were purified using a single chromatogram and the purified steroid and sterols were assayed fluorometrically. The lipid droplet fraction contained 85% of the esterified cholesterol and 32% of the free cholesterol found in whole gland extracts. Although adrenal lipid droplet fractions isolated from non-stimulated control animals contained 65–79% of the total corticosterone assayed in extracts of the whole gland, $\text{in vivo}$ injections of ACTH did not increase corticosterone 1n this fraction. On the other hand, the corticosterone measured in non-lipid fraction extracts increased significantly following ACTH treatment. These results suggest that the synthesis/release mechanism for corticosterone is not associated with the lipid droplets but may involve specific components in the non-lipid fraction. The function of lipid droplet corticosterone is unknown.     

Mechanism of glucocorticoid enhancement of the responsiveness of ovine adrenocortical cells to adrenocorticotropin

The present studies were undertaken to precise the mechanism through which glucocorticoids enhance the responsiveness of ovine adrenocortical cells to ACTH. Experiments using intact cells and crude adrenal membranes have shown that, at the level of the adenylate cyclase system, dexamethasone increases the number of ACTH receptors without modification of the catalytic subunit or of the GTP binding regulatory components Gs and Gi. Cells cultured with dexamethasone secreted more pregnenolone and more corticosteroids in response to 8-BrcAMP than did control cells. By contrast, dexamethasone did not increase corticosterone secretion by cells incubated in the presence of 22-(R)-hydroxycholesterol or of exogenous pregnenolone. Dexamethasone neither affected the incorporation of [14C] acetate into cellular cholesterol nor the amount of cholesterol present in mitochondria of unstimulated cells. However, dexamethasone-treated cells incubated in the presence of both 8-BrcAMP and aminoglutethimide exhibited higher amounts of mitochondrial cholesterol than control cells. These data indicate that dexamethasone enhances the number of cellular ACTH receptors together with increasing the cAMP-induced translocation of cholesterol from the cytoplasm into mitochondria and/or mitochondrial storage of cholesterol.     

Cytochrome P-450 from mitochondria of bovine adrenal cortex Comparison of cholesterol side-chain cleavage P-450 with steroid 11β-hydroxylation P-450 and immunochemical cross-reactivity between adrenal mitochondrial and liver microsomal cytochromes P-450

Adrenocortical mitochondrial cytochrome P?450 specific to the cholesterol side-chain cleavage (desmolase) reaction differs from that for the 11β-hydroxylation reaction of deoxycorticosterone. The former cytochrome appears to be more loosely bound to the inner membrane than the latter. Upon ageing at 0°C or by aerobic treatment with ferrous ions, the desmolase P-450 was more stable than the 11β-hydroxylase P-450. By utilizing artificial hydroxylating agents such as cumene hydroperoxide, H₂O₂, and sodium periodate, the hydroxylation reaction of deoxycorticosterone to corticosterone in the absence of NADPH was observed to a comparable extent with the reaction in the presence of adrenodoxin reductase, adrenodoxin and NADPH. However, the hydroxylation reaction of cholesterol to pregnenolone was not supported by these artificial agents.Immunochemical cross-reactivity of bovine adrenal desmolase P-450 with rabbit liver microsomal P-450_LM4 was also investigated. We found a weak but significant cross-reactivity between the adrenal mitochondrial P-450 and liver microsomal P-450_LM4, indicating to some extent a homology between adrenal and liver cytochromes P-450.     

The cytosol as site of phosphorylation of the cyclic AMP-dependent protein kinase in adrenal steroidogenesis

The mitochondria, the microsomes and the cystosol have been described as possible sites of cAMP-dependent phosphorylation. However, there has been no direct demonstration of a cAMP-dependent kinase associated with the activation of the side-chain cleavage of cholesterol. We have investigated the site of action of the cAMP-dependent kinase using a sensitive cell-free assay. Cytosol derived from cells stimulated with ACTH or cAMP was capable of increasing progesterone synthesis in isolated mitochondria when combined with the microsomal fraction. Cytosol derived from cyclase or kinase of negative mutant cells did not. Cyclic AMP and cAMP-dependent protein kinase stimulated in vitro a cytosol derived from unstimulated adrenal cells. This cytosol was capable of stimulating progesterone synthesis in isolated mitochondria. Inhibitor of cAMP-dependent protein kinase abolished the effect of the cAMP. ACTH stimulation of cytosol factors is a rapid process observable with a half maximal stimulation at about 3 pM ACTH. The effect was also abolished by inhibitor of arachidonic acid release. The function of cytosolic phosphorylation is still unclear. The effect of inhibitors of arachidonic acid release, and the necessity for the microsomal compartment in order to stimulate mitochondrial steroidogenesis, suggest that the factor in the cytosol may play a role in arachidonic acid release.     

The effects of cytochalasin B and vinblastine on movement of cholesterol in rat adrenal glands.

Vinblastine (antimicrotubular agent) and cytochalasin B (antimicrofilament agent) block the build up of adrenal mitochondrial cholesterol seen in the presence of AMG. ACTH stimulated steroidogenesis is inhibited $\text{in vivo}$ by both agents via a reduction in the transfer of intra-adrenal cholesterol to adrenal mitochondria, resulting in a decrease in the synthesis of adrenal steroids. Both inhibitors also decrease ACTH stimulated formation of cholesterol cytochrome P450_SCC complex in adrenal mitochondria, as determined by difference spectroscopy. The effects of these inhibitors contrast with the actions of protein synthesis inhibitors which decrease cholesterol binding to P450_SCC while increasing mitochondrial cholesterol content.     

Cholesterol exchange between microsomal, mitochondrial and erythrocyte membranes and its enhancement by cytosol.

1. Cholesterol exchanges between isolated rat liver microsomes and mitochondria and between erythrocytes and microsomes or mitochondria during incubation in vitro. The exchange process is temperature dependent and is no accompanied by a net movement of sterol. 2. cholesterol exchange between the membranes was enhanced by the addition of 105 000 x g supernatant fraction (S105) from rat liver. The degree to which sterol exchange was enhanced was dependent on the amount of this supernatant fraction present in the incubation. 3. enhancement of sterol exchange was not observed with heated S105 fraction, but activity was retained after dialysis or aging at 10 degrees C; these results suggest the presence of a cholesterol-exchange protein in the cytosol from rat liver.     

Hartmanella culbertsoni: biochemical changes in the brain of the meningoencephalitic mouse.

Meningoencephalitis was produced in albino mice by intranasal inoculation of Hartmanella culbertsoni. In the infected brain phosphatidyl choline (PC), phosphatidyl ethanolamine + phosphatidyl serine (PE + PS), sphingomyelin and cholesterol registered decrease, lysophospholipids and free fatty acids accumulated, and part of the cholesterol was esterified. Phospholipase A (EC 3.1.1.4) and sphingomyelinase (EC 3.1.4.12) were elaborated in the postmitochondrial supernatant fraction. The levels of lipid peroxides, in brain as well as the capacity of brain homogenates to form lipid peroxides in vitro, was higher in the infected animals as compared to the uninfected. The activities of succinate dehydrogenase (EC 1.3.99.1) and cytochrome c oxidase (EC 1.9.3.1) in the mitochondrial fraction of the infected brain decreased while adenosine triphosphatase (EC 3.6.1.3) was stimulated. Addition of cytosol fraction (105,000g supernatant) of infected brain to the mitochondrial fraction of uninfected brain caused inhibition of succinate dehydrogenase and cytochrome c oxidase and stimulation of adenosine triphosphatase. This shows that some toxic substance(s) which inhibits mitochondrial function in brain is produced in the cytosol during infection. This inhibitor was nondialyzable and heat stable.