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.     

Characterization of insulin receptors in the bovine adrenal cortex and medulla.

In order to identify insulin receptors in the bovine adrenal cortex and medulla, we have studied 125I-porcine insulin binding to the membrane preparations from the bovine adrenal cortex and medulla. 125I-porcine insulin bound not only to the bovine adrenal cortex but to the medulla in time-, temperature-, and pH-dependent manners. The maximum levels of 125I-porcine insulin binding in the two tissues were observed at 4 degrees C for 24 h of incubation, and its optimum pH ranged from 7.6 to 8.0. Under these conditions, at tracer concentration of porcine insulin (200 pg/ml), 10.4% and 6.6% of 125I-porcine insulin added to each reaction tube bound specifically to 10(5) x g-pellet fractions (microsomal membrane) from the cortical tissue (0.3 mg of protein) and from the medullary tissue (2 mg of protein), respectively. 125I-porcine insulin binding was observed predominantly in the microsomal membrane from the bovine adrenal cortex, and in a 15,000 x g- pellet fraction (synaptosomal membrane) from the bovine adrenal medulla. Scatchard analysis of binding data yielded curvilinear plots in each tissue. Analysis of curvilinear plots based on two sites model revealed similar affinity constant between the cortex and medulla. Receptor concentration of the cortex was several times higher than that of the medulla. In the two bovine adrenal tissues, human proinsulin and insulin-like growth factor I (IGF-I) had about 1/100 potency compared to porcine insulin in displacing 125I-porcine insulin binding. Porcine glucagon added with concentration up to 10(-6) M did not inhibit 125I-porcine insulin binding to both the cortex and the medulla.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pregnenolone binding sites in the rat olfactory bulb

High concentrations of pregnenolone and its sulfate have been found in several areas of rat and human brain and seem to be controlled by local mechanisms. In the present experiments we have demonstrated pregnenolone binding sites in the cytosolic fraction of the rat olfactory bulb. The pregnenolone binding component showed a Kd = 2.34 +/- 0.66 x 10(-7) M and Nmax = 7.25 +/- 1.20 pmol/mg protein. Pregnenolone, pregnenolone sulfate and 17OH-pregnenolone competed equally for the binding sites while other steroids were less competitive. Protease and trypsin inhibited binding by 48 and 60% respectively. Sucrose density gradient analysis showed a minor peak at 4.6 s and a major one at 3.6 s. After gel filtration chromatography the pregnenolone binding component appeared as 2 peaks corresponding to molecular weights of approximately 150 and 220 kDa. Heating at 60 degrees C increased binding by 150%. These results indicate that the olfactory bulb pregnenolone binding component is complex in nature. Rat plasma also bound pregnenolone. Plasma binding sites could be partially differentiated from those in the olfactory bulb on the basis of susceptibility to lipoprotein lipase, effect of heating and mobility during polyacrylamide gel electrophoresis.     

Isolation and characterization of subcellular membranes from canine stomach smooth muscle

Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5'-nucleotidase and NADPH-cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18-32% (w/w) sucrose was maximally enriched (13-fold) in 5'-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH-cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32-40% (w/w) sucrose, was maximally enriched in NADPH-cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive. ATP-dependent Ca2+ uptake correlated very well with that of 5'-nucleotidase but less well with NADPH-cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role ih regulating intracellular Ca2+ concentration in canine corpus circular muscle.     

C21 steroid side chain cleavage enzyme from porcine adrenal microsomes. Purification and characterization of the 17 alpha-hydroxylase/C17,20-lyase cytochrome P-450

The properties and the purity of a cytochrome P-450 (17 alpha-hydroxylase) from porcine adrenal microsomes have been examined following a report that the corresponding enzyme from bovine adrenocortical microsomes is inactive as a 17 alpha-hydroxylase and fails to show a high spin spectrum on addition of substrate, once the enzyme has been purified (Bumpus, J. A., and Dus, K. M. (1982) J. Biol. Chem. 257, 12696-12704). The purity of the porcine enzyme was demonstrated by electrophoresis on polyacrylamide with sodium dodecyl sulfate, immunoelectrophoresis, and NH2-terminal amino acid sequence (16 residues). The pure enzyme shows Mr = 54,000, heme content of greater than 0.8 nmol/nmol of protein, and absorption spectra typical of cytochrome P-450. The enzyme is active with both delta 4 (progesterone) and delta 5 (pregnenolone) substrates as a 17 alpha-hydroxylase and with the corresponding 17 alpha-hydroxysteroids as a C17,20-lyase. All four substrates produce typical type I spectra with the enzyme (so-called high spin form). We conclude that: 1) porcine adrenal microsomes contain a 17 alpha-hydroxylase/C17,20-lyase which is a single protein molecule readily purified to an enzymatically active form; 2) the C17,20-lyase activity is largely suppressed in the microsomes; and 3) the enzyme closely resembles that found in testicular microsomes. We propose that this enzyme be referred to as the adrenal C21 steroid side chain cleavage enzyme.     

Inactivation of porcine calcitonin by rat kidney microsome.

Biological activity of porcine calcitonin was most actively inactivated by the rat kidney homogenate than by other tissue homogenates. Among the various subcellular fractions of the rat kidney homogenate examined, microsome fraction was most active in the in vitro inactivation of porcine calcitonin. Inactivation of porcine calcitonin by the rat kidney microsome was dependent on pH and temperature. Inactivating activity of the rat kidney microsome was inhibited by 1 X 10(-3) M monoiodoacetate and 1 X10(-5) M p-chloromercuribenzoate. These results suggest that porcine calcitonin is probably inactivated by a SH-enzyme in the rat kidney microsomes. However, the participation of other enzymes cannot be ruled out, since the inactivating activity of the rat kidney microsome fraction is also inhibited by 1 X 10(-4) M diisopropylfuorophosphate.     

Cholesterol sulfate is a naturally occurring inhibitor of steroidogenesis in isolated rat adrenal mitochondria

We previously reported (Lambeth, J. D., Xu, X. X., and Glover, M. (1987) J. Biol. Chem. 262, 9181-9188) that exogenously added cholesterol sulfate inhibits the conversion of cholesterol to pregnenolone in isolated adrenal mitochondria, and does so by affecting intramitochondrial cholesterol movement but not its subsequent metabolism to pregnenolone by cytochrome P-450scc. We now report that a major kinetic component of the inhibition is noncompetitive with respect to cholesterol, consistent with an allosteric effect at a site other than the substrate binding site of cytochrome P-450scc. We now also report that cholesterol sulfate is present as an endogenous compound in preparations of adrenal mitochondria. Its content varied from 0.05 to 0.8 nmol/mg protein. Cholesterol sulfate level correlated inversely with the mitochondrial cholesterol side-chain cleavage activity. Endogenous cholesterol sulfate thus appeared to account for the variable rates of pregnenolone synthesis which were seen in different mitochondrial preparations. Cholesterol sulfate was metabolized to pregnenolone sulfate by a mitochondrial side-chain cleavage system, but proved to be a relatively poor substrate for an extramitochondrial steroid sulfatase activity present in adrenal cortex. Confirming a role as a naturally occurring inhibitor, removal of endogenous mitochondrial cholesterol sulfate by metabolism to pregnenolone sulfate correlated with a 3-fold activation of cholesterol side-chain cleavage. We suggest that cholesterol sulfate functions in steroidogenic tissues to regulate the magnitude of the steroidogenic response.     

Subcellular fractionation of porcine neutrophils by nitrogen cavitation and sucrose-density-gradient centrifugation

Neutrophils, isolated in large quantities from porcine blood were disrupted by nitrogen cavitation and separated by differential centrifugation into a nuclear fraction and a post-nuclear supernatant. The latter was subfractionated by sucrose density gradient centrifugation into cytosol, a fraction consisting of membrane vesicles and two granule-rich fractions. The membrane fraction accounted for 1.9% of the protein in the post-nuclear supernatant, the light granule fraction for 2.2% and the dense granule fraction for 4.2%. Catalase, lactate dehydrogenase and malate dehydrogenase were largely confined to the cytosol. The dense granule fraction contained the highest quantities of the hydrolytic enzymes, although the membrane fraction was also rich in alkaline and acid phosphatase and gamma-glutamyl transpeptidase activities. Electron microscopy of the membrane fraction showed intact membrane vesicles, whereas the granular fractions consisted of electron-dense, membrane-bound granules. Two granular fractions were isolated which contained granules of differing size and density. 3H-labeled wheat germ agglutinin bound to the surface of intact neutrophils and when these were disrupted and fractionated the membrane fraction showed a specific binding activity 16-times greater than that of the cavitated sample. The membrane fraction interacted with the detergent digitonin and as a result underwent density perturbation increasing from 1.13 g X cm-3 to 1.18 g X cm-3. Dodecylsulphate-polyacrylamide gel electrophoresis showed the membrane fraction to consist of at least 40 protein bands, with relative molecular masses ranging from 200 000-16 000. The granule fractions contained less protein bands, with a protein composition quite distinct from that of the membrane fraction.     

Extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex. II. ACTH-induced changes in subcellular corticosterone

Zona fasciculata-reticularis subcellular structures were implicated in corticosterone transport and secretion by noting changes in subcellular corticosterone during a 30-min period following ACTH stimulation. Six decapsulated adrenal homogenate subcellular fractions separated by gradient centrifugation were characterized cytochemically and morphologically. Predominant components in each of six fractions were: floating lipid droplets, 0.125 M sucrose (no organelles), cytosol (0.25 M sucrose supernatant with 0.25-1.2 micron electron dense granules), microsomes (interface between 0.5 M and 1.1 M sucrose layers), mitochondria (boundary between 1.1 M and 2.2 M sucrose layers) and nuclei (centrifuge pellet). Whole glands and most subcellular fractions showed peak corticosterone levels 10 to 15, and 30 min after stimulation. Sucrose and cytosolic fractions contained about 75% of the total corticosterone, responded to stimulation most significantly, and were rich in protein. In these two fractions only cytosol contained structures; these consisted of 0.15-1.2 micron electron dense granules.     

Adrenal Chromaffin Cell Calmodulin: Its Subcellular Distribution and Binding to Chromaffin Granule Membrane Proteins

Bovine adrenal medullae were homogenized in the presence or in the absence of EGTA and different subcellular fractions were prepared by differential and density gradient centrifugations. In the presence of the chelating agent, 69% of the total calmodulin, measured by radioimmunoassay, was present in the cytosol; the rest was bound to different membrane-containing fractions (nuclei, microsomal, and crude granule fraction). When the chelating agent was omitted, 43% of the calmodulin was present in the cytosol, the remaining calmodulin being membrane-bound. Further resolution of the crude granule fraction by sucrose density centrifugation demonstrated that the distribution of calmodulin in the density gradient was similar to the distribution of chromaffin granules rather than to that of mitochondria, Golgi elements, and lysosomes. In this case, there was also more calmodulin bound to chromaffin granules when EGTA was omitted from the density gradient. Experiments with 125I-calmodulin indicated the presence of high-affinity binding sites (KD = 1.3 X 10(-8) M; Bmax = 30 pmol/mg protein) for calmodulin in chromaffin granule membranes. Further, photoaffinity crosslinking experiments with 125I-calmodulin followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography indicated the presence of three calmodulin-binding polypeptide complexes (84,000; 41,000; and 38,000 daltons) in chromaffin granule membranes. These polypeptides were not labelled when either Ca2+ was omitted or an excess of nonradioactive calmodulin was present in the photolysis buffer, indicating the Ca2+ dependency and the specificity of the interaction. On the basis of the results described, it is suggested that the cellular levels of Ca2+ control the cellular distribution of calmodulin and its binding to specific chromaffin granule membrane proteins. Further, it is also suggested that the interactions between calmodulin and granule proteins might play a role in stimulus-secretion coupling.     

Use of a simple fractionation method to evaluate binding, internalization and intracellular distribution of oligonucleotides in vascular smooth muscle cells

Antisense oligonucleotides (ODN) are potent molecules that could be used to inhibit the synthesis of a protein specifically if delivered to the appropriate compartments (cytoplasm and nucleus) of the cell under study. We present here a simple method providing access to the fractions of internalized ODN available in the cytosolic and nuclear compartments. Cells are incubated with appropriately labeled ODN, either naked or vectorized. They are then washed and treated with pronase to remove species bound to the surface of the cell. Digitonin is added at a low concentration to induce leakage of the cytosol, which is collected. Endosomes and lysosomes are then lysed with Triton X100, and their contents, recovered by centrifugation. The crude nuclei comprising the pellet are purified by ultracentrifugation through a 2M sucrose cushion. Lactate dehydrogenase, fluorescent transferrin and cathepsin B are used as cytosolic, endosomal and lysosomal markers respectively. For vascular smooth muscle cells, the use of digitonin under optimal conditions (0.008% w/v, 4 degrees C for 5 min) resulted in more than 88% plasma membrane permeabilization, with less than 12% of endosomes and 5% of lysosomes lysed. We mainly studied a 3'-tritiated 20-mer ODN sequence complementary to the AUG region of the mRNA for the insulin-like growth factor 1 receptor, with either a phosphodiester (PO-ODN) or a phosphorothioate (PS-ODN) backbone. Cellular processing was evaluated with and without 25 kDa polyethylenimine (PEI) as a carrier. After 2.5 h of incubation at 37 degrees C, 100 times as much naked PS-ODN as naked PO-ODN was bound to the cell surface and internalized. Complexation with PEI dramatically increased both binding, by a factor of 10 and internalization by a factor of 80 of PO-ODN and, to a lesser extent, of PS-ODN. The intracellular distributions of naked PO-ODN and PS-ODN were similar. The radioactivity accumulated in nuclei accounted for about 15-20% of an intracellular radioactivity. A large proportion (about 60%) of intracellular radioactivity remained associated with the endocytic compartment. Complexation with PEI completely changed intracellular distributions: the nuclear fraction increased to 70% for PS-ODN. The fractionation method proposed, facilitating study of the subcellular distribution of the ODN, could also be used under appropriate circumstances, to study variations in cytosolic ODN content.     

Lactate dehydrogenase activity in the mitochondrial fraction of chicken liver: enzyme binding and kinetic behavior of soluble and bound enzyme

Chicken liver crude mitochondrial fraction showed lactate dehydrogenase activity (6.5% of cytoplasmic enzyme). Most of the mitochondrial lactate dehydrogenase was solubilized by sonication of the mitochondrial fraction in 0.15 M NaCl, pH 6. Total extracted lactate deshydrogenase activity was 3-fold higher than the initial pellet activity. Different isoenzymatic compositions were observed for cytosoluble and mitochondrial extracted lactate dehydrogenase. The pI, values of the 5 lactate dehydrogenase isoenzymes were found to be independent of their origin. The cytosoluble lactate dehydrogenase and the separated H4,H3M and H2M2 isoenzymes were able to bind to the chicken liver mitochondrial fraction in 5 mM sodium phosphate buffered medium, and could be solubilized afterwards with 0.15 M NaCl, pH 6. The enzyme bound to the mitochondrial fraction was less active than the soluble one. Particle saturation by the bound enzyme occurred with all mitochondrial fractions assayed. According to the Langmuir isotherm, the non-sonicated mitochondrial fractions contain a single type of binding sites for lactate dehydrogenase; in contrast, the sonicated mitochondrial fraction should contain different binding sites. Chicken liver crude or sonicated active mitochondrial fractions showed a hyperbolic behavior with respect to NADH and a non-hyperbolic one with respect to pyruvate. This mechanism is different from the bi-bi compulsory order mechanism of the soluble enzyme. With hydroxypyruvate as the substrate, the active mitochondrial fraction fit a sequential mechanism but lost the rapid-equilibrium characteristics of the soluble enzyme.     

Binding of anti-H-Y monoclonal antibodies to separated X and Y chromosome bearing porcine and bovine sperm

Studies designed to answer the question whether or not H-Y antigen is preferentially expressed on Y chromosome bearing sperm have resulted in conflicting results. This is probably due to the absence of reliable methods for estimating the percentage of X and Y chromosome bearing sperm in fractions, enriched or depleted for H-Y antigen positive sperm. In recent years a reliable method for separating X and Y chromosome bearing sperm has been published. With this method, separation is achieved by using a flow cytometer/cell sorter, which detects differences in DNA content. This technique provided the first opportunity for testing anti-H-Y antibody binding to fractions enriched for X and Y chromosme bearing sperm, directly. A total of 7 anti-H-Y monoclonal antibodies were tested using sorted porcine sperm and in one experiment also sorted bovine sperm. All monoclonal antibodies bound only a fraction of the sperm (20 to 50%). However, no difference in binding to the X and Y sperm enriched fractions was found. Therefore, the present experiments do not yield evidence that H-Y antigen is preferentially expressed in Y chromosome bearing sperm. © 1993 Wiley-Liss, Inc.     

Studies on the nuclear binding of steroid hormone-receptor complex; characteristics of binding of nuclei from fetal rat liver to 3H-dexamethasone-liver cytoplasmic receptor complex

Binding of 3H-dexamethasone (Dex)-rat liver cytoplasmic receptor complex to nuclei from fetal rat livers in vitro exhibited a high-affinity and saturable nature (Kd=1.5 X 10- M, maximal binding sites=470 fmole/mg DNA), and the binding was inhibited competitively by prior injection of Dex in vivo. While binding of 3H-Dex-receptor complex to nuclei from adult rat liver was in low affinity and unsaturable, and injection of Dex prior to the sacrifice of animals did not influence the nuclear binding to 3H-Dex-receptor complex in vitro. Differential salt-extraction with KCl solution of the nuclear bound 3H-Dex receptor complex revealed the presence of salt-extractable and residual forms of bound receptors. The amount of the fraction extracted with 0.3 M KCl reached its maximum at 10 min after the start of incubation, while the 1.0 M KCl-extractable and residual fractions reached their maximum plateaus after 30 min of the incubation. Scatchard analysis revealed that the binding of the receptor complex to the 0.3M and 1.0M KCl fractions was saturable, while the residual fraction did not show any tendency of saturation under the experimental conditions employed in the present study. The results obtained in this work were compared to those which have been reported by other investigators.     

Characterization of the porcine ACTH receptor with the aid of a monoclonal antibody

Monoclonal antibodies were raised against the ACTH receptor by immunization of BALB1 c mice with porcine adrenal cortex cell membranes. Competition and binding experiments confirmed that one of these antibodies, IV/14/9, reacted with the ACTH receptor in competition with ACTH and caused a definite ACTH-like effect. This antibody was used to characterize the hormone receptor of the porcine adrenal cortex. The number of antibody binding sites per cell was calculated from a Scatchard plot to be 124,100 +/- 10,000. The curve was linear indicating the existence of a single class of receptors. The finding that a high concentration of IV/14/9 totally suppressed maximally ACTH-induced steroidogenesis confirms the view that only a single class of ACTH receptors exists. The antibody IV/14/9 neither reacted with intact porcine thymus cells nor with normal porcine lymphocytes but was bound to the mouse adrenal tumor cell line Y1 and to normal rat adrenal cortex cells with a low affinity. Two dimensional electrophoresis of lysates of porcine adrenal cortex cells and subsequent blotting of the proteins on nitrocellulose, using IV/14/9 as a primary and anti-mouse Ig as a secondary reagent, permitted the detection of three forms of the receptor, two of which had an identical apparent molecular mass of about 85,000 Da (isoelectric points: 6.14 and 6.27). The third was somewhat larger (94,000 Da and pI = 6.21).     

Subcellular distribution and properties of carbonyl reductase in guinea pig lung

On subcellular fractionation, carbonyl reductase (EC 1.1.1.184) activity in guinea pig lung was found in the mitochondrial, microsomal, and cytosolic fractions; the specific activity in the mitochondrial fraction was more than five times higher than those in the microsomal and cytosolic fractions. Further separation of the mitochondrial fraction on a sucrose gradient revealed that about half of the reductase activity is localized in mitochondria and one-third in a peroxidase-rich fraction. Although carbonyl reductase in both the mitochondrial and microsomal fractions was solubilized effectively by mixing with 1% Triton X-100 and 1 M KCl, the enzyme activity in the mitochondrial fraction was more highly enhanced by the solubilization than was that in the microsomal fraction. Carbonyl reductases were purified to homogeneity from the mitochondrial, microsomal, and cytosolic fractions. The three enzymes were almost identical in catalytic, structural, and immunological properties. Carbonyl reductase, synthesized in a rabbit reticulocyte lysate cell-free system, was apparently the same in molecular size as the subunit of the mature enzyme purified from cytosol. These results indicate that the same enzyme species is localized in the three different subcellular compartments of lung.     

Effect of Ca2+, ruthenium red and ageing on pregnenolone production by mitochondrial fractions from normal and luteinizing hormone treated rat testes.

The effect of Ca2+ in vitro on pregnenolone production rates under various incubation conditions by mitochondrial fractions isolated from testes of normal rats and of rats after in vivo treatment with luteinizing hormone has been investigated. Concentrations of Ca2+ in the range of 0.1-0.5 mM stimulated succinate supported pregnenolone production in mitochondrial fractions from both control and luteinizing hormone treated testes. When mitochondrial fractions were isolated in 0.25 M sucrose without additions, Ca2+ in vitro increased succinate supported pregnenolone production rates in mitochondrial fractions isolated from control testes to a greater extent than in mitochondrial fractions, from luteinizing hormone treated testes. Production rates in control mitochondrial fraction, incubated in the presence of initial Ca2+ concentrations of 0.7 mM and higher were almost similar to production rates in relevant luteinizing hormone treated mitochondria.     

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.     

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.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in beef adrenal cortex.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) activity (mevalonate:NADP+ oxidoreductase )CoA-acylating) EC 1.1.1.34) was demonstrated in beef adrenal cortex. Most of the HMG-CoA reductase activity is in the microsomal fraction while a small percentage of the activity is associated with the mitochondria, Mitochondria purified on a linear sucrose gradient are enriched in HMG-CoA reductase and cytochrome c oxidase activities. The reductase present in microsomal preparations from the whole adrenal cortex has an apparent Km of 5.6 X 10(-5) M for (R,S)-HMG-CoA. Reductase activities found in the microsomal fractions from the zona glomerulosa, the zona fasciculata, and the zona reticularis were 1.32, 7.37, and 9.74 nmol mevalonate formed per milligram protein in 30 min respectively.