Adrenocortical Microsomes Research Articles

Two modes of binding of adrenal NADPH–cytochrome P-450 reductase to liposomal membranes

NADPH-cytochrome P-450 reductase, purified from bovine adrenocortical microsomes, was shown to bind in two different modes to liposomal membranes composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a molar ratio of 5:3:1. As demonstrated by Ficoll density gradient centrifugation and HPLC gel filtration, the cholate dialysis method made the reductase bind tightly to the liposomal membranes, while the incubation with the preformed vesicles made the reductase bind loosely to the membranes. From the experiments of electron transfer to P-450 C21 residing at the other vesicles, the loosely bound reductase was found to be transferable between the vesicles, whereas the tightly bound reductase was not readily transferred. The rates of the binding and the release of the loosely bound reductase to and from the membranes were measured with the stopped-flow method by observing the reduction of P-450 C21 embedded in the vesicles. These kinetic studies showed that the rate-limiting step of the reductase transfer between the vesicles was the release of the reductase from the membranes. The reductase in both binding modes well supported the steroid 21-hydroxylase activity.

Studies on the interaction of steroid substrates with adrenal microsomal cytochrome P-450 (P-450C21) in liposome membranes.

Cytochrome P-450 (P-450C21), purified from bovine adrenocortical microsomes, was incorporated into the single bilayer liposomes of egg yolk phosphatidylcholine by gel filtration, using a high pressure liquid chromatography system. Interaction of the steroid substrates, 17 alpha-hydroxyprogesterone and progesterone, with P-450C21 in the liposomes was studied in the equilibrium state by measuring substrate-induced spectral change. The apparent dissociation constant of the P-450C21-substrate complex increased with phosphatidylcholine concentration in the system, showing the substrate to be partitioned between the aqueous and lipid phases. Partition coefficients, determined by equilibrium dialysis and the Hummel-Dreyer method, were 3500 for progesterone and 2000 for 17 alpha-hydroxyprogesterone at 25 degrees C. The binding process of the substrates to P-450C21 in the liposomes and their dissociation were measured by a stopped flow method. The apparent rate of substrate binding to P-450C21 in the liposomes was not effected by substrate partitioning, indicating partitioning to occur much more quickly than substrate binding to P-450C21. Absorption changes observed in the stopped flow experiments were analyzed at a rapid equilibrium of partitioning. Based on these results, the substrate binding site of P-450C21 was concluded to face the lipid phase of the liposome membranes.

Purification and kinetic properties of 3 beta-hydroxysteroid dehydrogenase from bovine adrenocortical microsomes.

3 beta-Hydroxysteroid dehydrogenase was purified from bovine adrenocortical microsomes and its properties were studied. The purified dehydrogenase gave a single homogeneous protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed no steroid delta 5-delta-4 isomerase activity. The molecular weight of the dehydrogenase was estimated to be 41,000 for the monomer and the isoelectric point was determined to be at pH 6.3. The Km values of the dehydrogenase were 6.2 microM for NAD+, 4.9 mM for NADP+, 2.0 microM for pregnenolone, and 5.3 microM for 17 alpha-hydroxypregnenolone. The mechanism of inhibition by trilostane of the dehydrogenase was also examined kinetically. The inhibition was found to be competitive, with Ki values of 0.14 microM for 17 alpha-hydroxypregnenolone and 0.38 microM for pregnenolone.

Effect of Fe 2+-induced lipid peroxidation upon microsomal steroidogenic enzyme activities of porcine adrenal cortex

When porcine adrenocortical microsomes were treated with Fe 2+, enhanced production of malondialdehyde was observed as a result of membrane lipid peroxidation. By treatment of the microsomes with Fe 2+, the activity of Δ 5-3β-hydroxysteroid dehydrogenase coupled with Δ 5-Δ 4 isomerase, concentration of cytochrome P-450 and the activity of the cytochrome-involving enzyme systems such as 17α- and 21-hydroxylases were significantly reduced. 17α-Hydroxylase activity was more effectively decreased by Fe 2+ than that of 21-hydroxylase. On the other hand, activity of NADH- and NADPH-cytochrome c reductases remained unchanged or somewhat increased. Both the induction of lipid peroxidation and the decrease of the enzyme activities were prevented by α-tocopherol and N,N′-diphenyl- p-phenylenediamine.

ブタ副腎ミクロソームのステロイド17α-ヒドロキシラーゼ-C<SUB>17,20</SUB>リアーゼ(チトクロームP-450) : プロゲステロンによるリアーゼ活性の阻害と17α-ヒドロキシプロゲステロンによるヒドロキシラーゼ活性の阻害

Highly purified 17α-hydroxylase-C17, 20 lyase (cytochrome P-450) from porcine adrenocortical microsomes was incubated with [4-14C]-C21-steroid substrate in the presence of cytochrome P-450 reductase and nicotinamide adenine dinucleotide phosphate as a cofactor. On the kinetic analysis, C17, 20 lyase activity was strongly inhibited by progesterone. The inhibition was competitive, and K1 value for progesterone was 0.9μM. In addition, 17α-hydroxylase activity was inhibited by 17α-hydroxyprogesterone. The inhibition was competitive, and K1 value for 17α-hydroxyprogesterone was 7.5μM. These results suggest that both hydroxylase and lyase reactions are catalyzed on a single active site.

Kinetics of cytochrome P-450 reduction: studies in bovine adrenocortical microsomes.

The results of the present study indicate first that in the microsomal preparation, the components of the P-450 reduction system are heterogeneously distributed, comprising dissociable and nondissociable parts. Second, the P-450 reduction curve can be adequately described by a sum of two exponential functions, indicating two concurrent first-order reactions. Third, the two phases can be altered independently. The addition of the substrate increased the extent of the fast phase while it had little or no effect on that of the slow phase. Changes in the interaction of the dissociable and nondissociable components affected the extent of the slow phase while they were without effect on that of the fast phase. Experiments with different steroids indicated that the independence of the two phases is not due to functionally different P-450's and that the cytochrome reduced in both phases is essentially P-450C-21. The results are interpreted as follows: Transformation of P-450 from the low- to the high-spin state controls the total P-450 reduced. The rate and the biphasicity of the reduction are functions of the interaction of P-450 and the reductase.

Interaction between cytochrome P-450 (P-450C21) and NADPH-cytochrome P-450 reductase from adrenocortical microsomes in a reconstituted system.

The interaction between P-450C21 and NADPH-cytochrome P-450 reductase, both purified from bovine adrenocortical microsomes, has been investigated in a reconstituted system with a nonionic detergent, Emulgen 913, by kinetic analysis and gel filtrations. Steady state kinetic data in progesterone 21-hydroxylation showed formation of an equimolar complex between the two enzyme proteins at low Emulgen concentration. Steady state kinetic studies on the electron transfer from NADPH to P-450C21 via the reductase showed that a stable complex formation between the two enzyme proteins was not involved in the steady state electron transfer at high Emulgen concentration. In stopped flow experiments, a time course of the P-450C21 reduction showed biphasic kinetics composed of fast and slow phases. The dependence of kinetic parameters on Emulgen concentration indicates that the fast phase corresponds to the electron transfer within the complex and the slow phase to the electron transfer through a random collision between P-450C21 and the reductase. The stable complex formation between P-450C21 and the reductase has been clearly demonstrated by gel filtration. The stable complex was composed of several molecules of the two enzyme proteins at an equimolar ratio, which was active for progesterone 21-hydroxylation and had a tendency to dissociate at high Emulgen concentration.

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.

Organ-specific properties of cytochromes P-450 s21 (steroid 21-hydroxylases) of liver and adrenocortical microsomes

Organ specificities of cytochrome P-450 s21 (steroid 21-hydroxylase) were investigated. The substrate specificity of the liver cytochrome P-450 s21 was found to be different from that of adrenocortical cytochrome P-450 s21. The steroid 21-hydroxylase activity of liver microsomes decreased with treatment by sodium phenobarbital or β-naphthoflavone and was inhibited by anti-cytochrome b 5 immunoglobulin, although that of adrenocortical microsomes was not. The liver cytochrome P-450 s21 was immunochemically similar to adrenocortial cytochrome P-450 s21. The bovine liver and adrenocortical cytochromes P-450 s21 showed immunoprecipitin lines against antibody to bovine adrenocortical cytochrome P-450 s21. The molecular masses of the bovine liver and adrenocortical cytochromes P-450 s21 were 60 ± 1 and 50 ± 1 kDa, respectively. The content of cytochrome P-450 s21 and its percentage for the total microsomal cytochrome P-450 were immunochemically determined using the antibody to the bovine adrenocortical cytochrome P-450 s21.

Steroid 21-hydroxylase (cytochrome P-450) from porcine adrenocortical microsomes: microsequence analysis of cysteine-containing peptides.

The steroid 21-hydroxylase cytochrome P-450 from porcine adrenocortical microsomes was purified to homogeneity. The protein exhibited two NH2-terminal sequences, one of which was identical with the first but lacking the NH2-terminal methionine. The sequence was extremely hydrophobic but had little homology to the 17 alpha-hydroxylase/C17,20-lyase isolated from neonatal porcine testes or to rat or rabbit liver microsomal cytochromes P-450. The cysteine-containing fragments of the S-carboxymethylated protein were purified by high-performance liquid chromatography and sequenced. Three of the cysteine-containing peptides exhibited significant sequence homology with peptides from the major phenobarbital-induced rat liver cytochrome P-450 (P-450b) and two with peptides from cytochrome P-450cam (camphor methylene hydroxylase from Pseudomonas putida). The presence of conserved regions in the primary sequences of these proteins appears likely to provide clues to the nature of their heme-binding domains.

Effect of spin state on reduction of cytochrome P-450 ( P-450 c21) from bovine adrenocortical microsomes

The effect of spin state on cytochrome P-450 reduction was studied with a reconstituted system consisting of P-450 c21 and NADPH-cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) purified from bovine adrenocortical microsomes. The absolute high spin contents of substrate-free, progesterone-bound and 17α-hydroxyprogesterone-bound P-450 c21 were estimated from the analysis of thermally induced difference spectra to be 25, 78 and 94% at 25°C, respectively, in 50 mM potassium phosphate buffer (pH 7.2) containing 20% glycerol, 0.1 mM EDTA and 0.5% Emulgen 913. The effect of the high spin content on P-450 c21 reduction by NADPH in the reconstituted system was analyzed by a steady-state method and by a stopped-flow method at 25°C. The steady-state results showed that the rate of P-450 c21 reduction was not affected by the high spin content of substrate-bound P-450 c21 but was very slow without a steroid substrate. Biphasic reduction of P450 c21 containing two first-order processes was observed in the stopped-flow experiment in the presence of either of the steroid substrates, but the reduction was very slow without the substrate. There were no significant differences in the rate and the amount of the fast phase of reduction between 17α-hydroxyprogesterone-bound and progesterone-bound P-450 c21. Both kinetic studies indicate that the spin state does not control the electron transfer from NADPH to P-450 c21 via NADPH-cytochrome P-450 reductase but the presence of substrate is essential for the reduction of P-450 c21.

Purification and reconstitution of the steroid 21-hydroxylase system (cytochrome P-450-linked mixed function oxidase system) of bovine adrenocortical microsomes

The steroid 21-hydroxylase system was purified from bovine adrenocortical microsomes. The physicochemical properties of the cytochrome P-450 s21 were studied. The properties of NADPH-cytochrome P-450 reductase (EC 1.6.2.4) of bovine adrenocortical microsomes have been reported previously (Hiwatashi, A. and Ichikawa, Y. (1979) Biochim. Biophys. Acta 580, 44–63). The steroid 21-hydroxylase system was reconstituted by cytochrome P-450 s21 and NADPH-cytochrome P-450 reductase in the presence of detergent. The substrate specificity of the cytochrome P-450 s21 was examined with the reconstituted system. The enzyme system was active in the 21-hydroxylations of 17 α- hydroxyprogesterone and progesterone, and the N-demethylation of (+)-benzphetamine. The cytochrome P-450 s21 purified to as high as 16–17 nmol per mg protein was an electrophoretically pure glycoprotein. The molecular weight of the cytochrome P-450 s21 was estimated to be 47 500 by SDS-polyacrylamide gel electrophoresis. Although cytochrome P-450 scc or cytochrome P-450 11β of bovine adrenocortical mitochondria did not react with antibody to cytochrome P-450 BPA of bovine liver microsomes, the cytochrome P-450 s21 formed an immunoprecipitin line against the antibody to cytochrome P-450 BPA in the Ouchterlony double diffusion test.

Purification and organ-specific properties of cholecalciferol 25-hydroxylase system: Cytochrome P-450 D25-linked mixed function oxidase system

Summary A cholecalciferol 25-hydroxylase(vitamin D3 25-hydroxylase) system was purified from beef liver microsomes by affinity chromatography of sepharose with a ligand of vitamin D3. The hydroxylase system was composed of two components of NADPH-cytochrome P-450 reductase and cytochrome P-450D25. The molecular weight of cytochrome P-450D25, a glycoprotein, was 48,000. The molecular weight of NADPH-cytochrome P-450 reductase was 79,000, a value which differed slightly from that of the beef adrenocortical microsomes. The optical and physicochemical properties of cytochrome P-450D25 and NADPH-cytochrome P-450 reductase were investigated. The reconstituted vitamin D3 25-hydroxylase system was not accelerated by the NADH-dependent electron transport system of NADH-cytochrome b5 reductase and cytochrome b5. The NADPH-cytochrome P-450 reductase as well as cytochrome P-450D25 were chemically and immunochemically different from the steroid 21-hydroxylase system of beef adrenocortical microsomes.

Constraint on the substrate cytochrome [formula omitted] binding reaction in bovine adrenocortical microsomes at physiological temperature

The addition of cholate to the microsomes at 37.5°C resulted in a striking decrease in the apparent substrate dissociation constant ( K′ s ) and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the K s and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome P-450 is considerably restrained from expressing its maximum substrate binding potential at physiological temperature. In addition, the results indicate that the majority of the lipids apparently do not play a significant role in imposing constraint on the substratecytochrome P-450 binding reaction and in the temperature dependency of the K s .

Physicochemical properties of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from bovine adrenocortical microsomes

Adrenocortical NADPH-cytochrome P-450 reductase (EC. 1.6.2.4) was purified from bovine adrenocortical microsomes by detergent solubilization and affinity chromatography. The purified cytochrome P-450 reductase was a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being electrophoretically homogeneous and pure. The cytochrome P-450 reductase was optically a typical flavoprotein. The absorption peaks were at 274, 380 and 455 nm with shoulders at 290, 360 and 480 nm. The NADPH-cytochrome P-450 reductase was capable of reconstituting the 21-hydroxylase activity of 17α-hydroxyprogesterone in the presence of cytochrome P-450 21 of adrenocortical microsomes. The specific activity of the 21-hydroxylase of 17α-hydroxyprogesterone in the reconstituted system using the excess concentration of the cytochrome P-450 reductase, was 15.8 nmol/min

Immunohistochemical studies on electron transport proteins associated with cythochromes P-450 in steroidogenic tissues II. Microsomal NADPH-cytochrome c reductase in the rat adrenal

NADPH-cytochrome c reductase (NADPH : ferricytochrome oxido-reductase, EC 1.6.2.4), the flavoprotein which mediates the NADPH-dependent reduction of cytochromes P-450 in adrenocortical microsomes, has been localized immunohistochemically at the light microscopic level in rat adrenal glands. Localization was achieved through the use of sheep antiserum procued against purified, trypsin-solubilized rat hepatic microsomal NADPH-cytochrome c reductase in both an unlabeled antibody peroxidase-antiperoxidase techniques and an indirect fluorecent antibody method. The sheep antibody to rat hepatic microsomal NADPH-cytochrome c reductase concomitantly inhibited the NADPH-cytochrome c reductase and progesterone 21-hydroxylase activities catalyzed by isolated rat adrenal microsomes. When sections of rat adrenal glands were exposed to the reductase antiserum in both immunohistochemical procedures, positive staining for NADPH-cytochrome c reductase was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. The intensity of staining, however, was found to differ among the three cortical zones, with the most intense staining being found in the zona fasciculata and the least in the zona glomerulosa. The intensity of staining was also found differ among cells within the zona fasciculata. These immunohistochemical observations demonstrate that microsomal NADPH-cytochrome c reductase is not distributed uniformly throughout the rat adrenal cortex.

Inhibition of lipid peroxidation by calcium ions and their protection of steroid hydroxylase activity from peroxidative damage.

Lipid peroxidation of adrenocortical mitochondria and microsomes was greatly stimulated by addition of 1.0 mM or less ferric ions. In the presence of NADPH-yielding system, the formation of corticosterone from endogeneous cholesterol and exogeneous deoxycorticosterone was inhibited as the concentrations of iron increased. Of interest is the fact that 0.5 mM ferric ion-mediated lipid peroxidation was completely abroagated upon addition of 2 mM calcium ions. Accordingly, protected from the peroxidative damage.

Purification and optical studies of cytochrome P-450 from bovine adrenocortical microsomes

Purification and optical studies of cytochrome P-450 from bovine adrenocortical microsomes

Solubilization and separation of Δ 5,3β-hydroxysteroid dehydrogenase and 3-oxosteroid-Δ 4–Δ 5-isomerase from bovine adrenal cortex microsomes

A physical separation of Δ 5,3β-hydroxysteroid dehydrogenase and 3-oxosteroid Δ 4–Δ 5-isomerase solubilized from bovine adrenocortical microsomes is described for the first time. The solubilization as well as the separation was carried out with a mixture of a detergent: a substituted betaine (Empigen BB/P) and sodium cholate. This latter detergent protects isomerase from complete inactivation by Empigen and is necessary for the recovery of a significant amount of soluble isomerase. Separation of dehydrogenase and isomerase was successfully accomplished by the use of a DEAE-Biogel A anion-exchanger. Dehydrogenase activity was eluted, while the isomerase was retained. Measurements of dehydrogenase activity with androst-5-en-3β-ol-17-one, pregnen-3β-ol-20-one and pregn-5-en-(3β,17α)-diol-20-one and of isomerase activity with androst-5-en-(3,17)-dione and pregn-5-en-(3,20)-dione suggested that more than one isomerase and more than one dehydrogenase form were present.

Thermotropic transitions in fluidity of bovine adrenocortical microsomal membrane and substrate-cytochrome P-450 binding reaction

Temperature dependence of the substrate-cytochrome P-450 binding reaction is investigated in the adrenocortical microsomes using 17-hydroxyprogesterone as the substrate. The apparent substrate dissociation constant (‘ K s ’) increases with increase in temperature. Van't Hoff s plot of ‘ K s ’ shows breaks at 21 and 31°C. These temperatures correlate well with the lipid phase transition temperatures as determined by the technique of fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene as the probe. The results indicate that the affinity of the substrate depends upon the temperature. In addition the results suggest that changes in the physical state of the lipids is a factor responsible for the temperature dependency of the reaction.