Bovine Adrenocortical Microsomes Research Articles

20β-hydroxy-C21-steroid 20β-oxidase activity of cytochrome P450 c21 purified from bovine adrenocortical microsomes

We showed previously that cytochrome P450 c21 (CYP21A1) from bovine adrenocortical microsomes has a putative 20β-oxidase activity for 20β-hydroxyprogesterone leading to a conversion to progesterone [M. Tsubaki, K. Morimoto, S. Tomita, S. Miura, Y. Ichikawa, A. Miyatake, F. Masuya, H. Hori, Biochim. Biophys. Acta 1259 (1995) 89–98]. We have extended the investigation on the 20β-oxidase activity of cytochrome P450 c21. Combination of 17α,20β-dihydroxyprogesterone with purified cytochrome P450 c21 in oxidized form caused an induction of a typical type I difference spectrum (a peak at 390 nm and a trough at 420 nm) with a spectral dissociation constant of 2.3 μM. EPR spectrum at low temperature (15 K) exhibited an appearance of a new low-spin signal at g z =2.42, g y =2.21, and g x =1.966 and an increase in intensity of the high-spin signal ( g=8.06, 3.54) upon formation of the enzyme-steroid complex, as previously found for 17α-hydroxyprogesterone and 20β-hydroxyprogesterone. The enzymatic activity for 17α,20β-dihydroxyprogesterone was confirmed in a reconstituted system consisting of cytochrome P450 c21 and NADPH-cytochrome P450 reductase. 17α,20β-Dihydroxyprogesterone was converted to 17α-hydroxyprogesterone via the 20β-oxidase reaction. The high turnover numbers of the 20β-oxidase activity for 20β-hydroxy-c21-steroids suggests that this activity is likely to have some physiological roles. Cytochrome P450 c21 and 20β-hydroxysteroid dehydrogenase may regulate the androgen biosynthesis catalyzed by cytochrome P450 c17α with controlling the concentration of 20β-hydroxy-C21-steroids.

Direct inhibitions of the activities of steroidogenic cytochrome P-450 mono-oxygenase systems by anticonvulsants.

The effects of anticonvulsants on the activities of cytochromes P-450(17alpha,lyase) (CYP17), P-450arom (CYP19), P-450C21 (CYP21), P-450SCC (CYP11A1), and P-450(11beta) (CYP11B1) mono-oxygenase systems were studied using rat testicular microsomes, human placental microsomes, bovine adrenocortical microsomes, bovine adrenocortical mitochondria and purified cytochrome P-450(11beta). Phenytoin, clonazepam and carbamazepine inhibited the steroidogenesis catalysed by these cytochrome P-450 mono-oxygenase systems and the Ki values for each anticonvulsant were determined. Neither hydantoin nor sodium valproate inhibited the activities of steroidogenic cytochromes P-450. When the activities of cytochromes P-450arom and P-450C21 were measured in the presence of anticonvulsants, the Ki values (0.15 mM) for phenytoin were close to the plasma concentration of phenytoin under therapeutic conditions. Phenytoin, clonazepam and carbamazepine directly inhibited the monooxygenase activities of cytochromes P-450, because they did not affect the activities of NADPH-cytochrome P-450 reductase, NADPH-adrenoferredoxin reductase and adrenoferredoxin.

Electron paramagnetic resonance investigation of cytochrome P-450 c21 from bovine adrenocortical microsomes: a new enzymatic activity

Cytochrome P-450 c21 (CYP21A1) purified from bovine adrenocortical microsomes was investigated by electron paramagnetic resonance (EPR) spectroscopy to clarify the interactions among heme active center, protein surroundings, water molecules and bound substrates or analogues. The low-spin EPR signals of the oxidized enzyme (as purified) consisted of two species; one at g z = 2.39, g z = 2.23, and g x = 1.925 (component A) and the other at g z = 2.42, g y = 2.23, and g x = 1.916 (component B). The component A is probably representing a product-bound form, whereas the component B indicates either occupation of the substrate-binding site with a substrate analogue or absence of steroid at the site. Upon addition of progesterone, the component A signal completely disappeared and the intensity of high-spin signal ( g = 8.06, 3.54) increased. Addition of 17α-hydroxyprogesterone caused a development of a new low-spin signal at g z = 2.42, g z = 2.21, and g x = 1.966 (component C) and a further increase in intensity of the high-spin signal ( g = 8.06, 3.54). Addition of 20β-hydroxyprogesterone caused an increase in intensity of the component C signal (and the g = 8 high-spin signal) even stronger than did 17α-hydroxyprogesterone. These observations suggest that 20β-hydroxyprogesterone binds to the cytochrome P-450 c21 active center in a very similar manner as 17α-hydroxyprogesterone does and, therefore, may be a metabolizable substrate. A new enzymatic pathway catalyzed by cytochrome P-450 c21 was confirmed with a reconstituted enzymatic system consisting of cytochrome P-450 c21 NADPH-cytochrome P-450 reductase and an NADPH-generating system. 20β-Hydroxyprogesterone was converted to progesterone via a putative 20β-oxidase reaction in a comparable turnover number to that of the 21-hydroxylation of 17α-hydroxyprogesterone.

Incorporation of bovine adrenal 3β-hydroxy-5-ene steroid dehydrogenase/ Δ5- Δ4 isomerase into phospholipid vesicles

3β-Hydroxysteroid dehydrogenase/ Δ 5- Δ 4 isomerase (3β-HSD/I) and cytochrome P-450 C21 were co-purified from bovine adrenocortical microsomes by an improved method. The 3β-HSD/I was successfully incorporated into liposomal membranes in which the enzyme activity was greatly stabilized. Enzymatic activities and kinetic parameters of the 3β-HSD/I proteoliposomes were almost the same as those of the solubilized form.

Membrane topology of bovine adrenocortical cytochrome P-450C21: structural studies by trypsin digestion in vesicle membranes.

Purified adrenocortical microsomal P-450C21 was incorporated into vesicle membranes composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a molar ratio of 5:3:1. Trypsinolysis of the incorporated P-450C21 resulted in the formation of 30-, 25-, and 20-kDa fragments. Similar fragment formation was observed by trypsinolysis of bovine adrenocortical microsomes with Western blotting using anti-P-450C21 IgG. In the detergent-solubilized state, trypsin cleaved P-450C21 into very small peptides. Washing of the trypsin-treated vesicles with 500 mM Na2CO3 failed to cause these fragments to separate from membranes. N-Terminal amino acid sequencing of these fragments showed that trypsin cleaved the 267 Arg-268Val and 332Arg-333Val bonds of P-450C21. The time course of fragment formation indicated that trypsin cleaved the 267Arg-268Val bond first to produce 30- and 25-kDa fragments and subsequently the 332Arg-333Val bond in the 25-kDa fragment to produce the 20-kDa fragment. Neither 21-hydroxylase activity, the reduced CO difference spectrum, nor the EPR spectrum of digested P-450C21 differed from those of undigested P-450C21. Heat treatment at 50 degrees C for 20 min did not cause any decrease in activity of digested P-450C21, when the substrate progesterone was present. This high stability toward heat treatment was not observed in the solubilized state. Rotational diffusion experiments on P-450C21 showed that the size of the molecule holding the heme was not changed significantly after digestion. On the basis of these results, P-450C21 is concluded to be deeply embedded in the vesicle membranes.

Absorbance spectral change of the cytochrome P-450 s21-phenylisocyanide complex upon binding of reduced NADPH-cytochrome- P-450 reductase

Reduction of cytochrome P-450 s21 (SF) (SF, substrate-free; purified from bovine adrenocortical microsomes) with sodium dithionite (Na 2S 2O 4) in the presence of phenylisocyanide produced a ferrous cytochrome P-450 s21 (SF)-phenylisocyanide complex with Soret absorbance maxima at 429 and 456 nm. On the other hand, when a preformed ferric cytochrome P-450 s21(SF)-NADPH-cytochrome- P-450 reductase (Fp2) complex was reduced chemically or enzymatically under the same conditions, the absorbance spectrum of the ferrous cytochrome P-450 s21 (SF)-phenylisocyanide complex changed drastically, as characterized by an increase in absorbance intensity at 429 nm and a decrease at 456 nm. Similar spectral changes were observed by addition of reduced Fp2 to the preformed ferrous cytochrome P-450 s21 (SF)-phenylisocyanide complex. Experiments to reduce a ferric cytochrome P-450 s21 (SF)-phenylisocyanide complex with sodium dithionite in the presence of various amounts of Fp2 showed that; (1), the spectral change reached maxima for both absorption increase at 429 nm and decrease at 456 nm when cytochrome P-450 s21 and Fp2 were previously mixed at the cytochrome P-450 s21: Fp2 ratio of 1:5; (2), the spectral change was suppressed in 300 mM potassium phosphate buffer (pH 7.4). These results suggest that the absorbance spectral change is due to a conformational change around the heme moiety induced by association with reduced Fp2.

Relationship between agonist- and thapsigargin-sensitive calcium pools in adrenal glomerulosa cells. Thapsigargin-induced Ca2+ mobilization and entry.

The relationships between agonist-sensitive calcium pools and those discharged by the Ca(2+)-ATPase inhibitor thapsigargin were studied in intact bovine adrenal glomerulosa cells and a subcellular adrenocortical membrane fraction. In Fura-2-loaded glomerulosa cells, angiotensin II (AII) stimulated a rapid increase in cytoplasmic Ca2+ concentration ([Ca2+]i) followed by a smaller plateau phase that was dependent on extra-cellular Ca2+. In such cells thapsigargin caused a sustained and dose-dependent increase in [Ca2+]i which was diminished in Ca(2+)-deficient medium. The contribution of an influx component to the thapsigargin-induced [Ca2+]i response was demonstrated by measurement of 45Ca influx rate in glomerulosa cells. Thapsigargin-induced Ca2+ entry was significantly less than that evoked by AII, and its kinetics were similar to those of the concomitant increase in [Ca2+]i. The rate of emptying of the agonist-responsive Ca2+ pool after thapsigargin treatment, as indicated by the progressive decrease in the size of the AII-induced Ca2+ transient, showed a rapid initial (t1/2 = 1.7 min) component that accounted for about 80% of the response and a slowly decreasing phase with t1/2 = 112 min. The latter thapsigargin-resistant component was abolished by the removal of extracellular Ca2+. Pretreatment with AII dose-dependently attenuated but did not abolish the subsequent Ca2+ response to thapsigargin and also increased the rate of the Ca2+ rise induced by thapsigargin. In bovine adrenocortical microsomes, thapsigargin inhibited the ATP-dependent filling of Ca2+ pools and caused a dose-dependent rise in extravesicular Ca2+ levels when added to previously loaded microsomes. The thapsigargin-releasable Ca2+ pool in adrenal microsomes was larger than the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ pool but only slightly greater than the GTP-releasable pool. Ins(1,4,5)P3-induced Ca2+ release was reduced markedly when ATP-dependent Ca2+ loading of the microsomes was prevented by prior addition of thapsigargin. However, the subsequent Ca2+ response to Ins(1,4,5)P3 was consistently better preserved after the addition of thapsigargin to microsomes preloaded with Ca2+. This difference suggests that although Ca2+ uptake by the Ins(1,4,5)P3-responsive pool is also sensitive to thapsigargin, once filled, this pool shows a slower passive leakage than other thapsigargin-sensitive pools. These findings indicate that thapsigargin increases [Ca2+]i by inhibiting Ca2+ uptake into multiple intracellular Ca2+ pools and by also promoting entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

A quantitative investigation into the dependence of Ca2+ mobilisation on changes in inositol 1,4,5-trisphosphate levels in the stimulated neutrophil

1. The coupling of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) receptor stimulation to Ca2+ mobilisation has been investigated in the human neutrophil by measuring the concentration-effect curves for inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ mobilisation. 2. fMet-Leu-Phe-dependent mobilisation of intracellular Ca2+ has been monitored in fluo-3-loaded human neutrophils by measuring increases in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in the presence of extracellular EGTA. Fluo-3 was used in preference to fura-2 because it was found to be more sensitive to the high Ca2+ levels seen in stimulated neutrophils. 3. fMet-Leu-Phe induced a rapid mobilisation of intracellular Ca2+ (EC50 = 2.9 +/- 0.1 nM) and increased [Ca2+]i to a maximum of 1286 +/- 184 nM. 4. The amount of IP3 in fMet-Leu-Phe-stimulated neutrophils was determined by competition with [3H]-IP3 for a specific IP3 binding protein isolated from bovine adrenocortical microsomes. Basal IP3 levels of 13.3 +/- 2.0 pmol per 10(7) cells were increased nearly 4 fold by maximally effective concentrations of fMet-Leu-Phe. 5. The EC50 for the IP3 response (95 +/- 18 nM) was much higher than that for mobilisation of intracellular Ca2+, such that only a doubling in the concentration of IP3 was required to fully mobilise intracellular Ca2+. 6. As a result of this relationship IP3 production was more sensitive than Ca2+ mobilisation to inhibition by demethoxyviridin, an inhibitor of phospholipase activation.

On the Solvent Accessibility of Substrate Binding Site of Cytochrome P450c-21in Bovine Adrenocortical Microsomes

The present study offers evidence indicating that acrylamide a polar molecule inhibits substrate-binding to P450C-21 in a competitive manner and quenches tryptophanyl fluorescence in bovine adrenocortical microsomes, similar to that in the purified lipid-free enzyme. Resolution of tryptophanyl fluorescence of the microsomes revealed an acrylamide quenching constant (K2 = 9.9M, is the association constant for the quencher-fluorophore complex) which was similar to the reciprocal of its inhibition constant (1/Kj = Ka = 8.3 +- 0.9M) for substrate-binding. The substrate inhibited the fluorescence quenching by acrylamide which was in accordance with partial competition. In addition the substrate dissociation, acrylamide inhibition and fluorescence quenching constants and tryptophanyl fluorescence maximum (340-342nm) were essentially the same in the microsomes and the purified enzyme. These results suggest that, similar to that in the purified enzyme, a tryptophan in a polar environment in the membrane-bound P450, may serve as a reporter group for the substrate binding site and the site in the membrane-bound enzyme, is accessible to the substrate in aqueous phase.

The Ins(1,4,5)P3 binding site of bovine adrenocortical microsomes: function and regulation

Adrenocortical microsomes possess a single population of Ins(1,4,5)P3-specific binding sites [IC50 5.9 +/- 0.9 nM; Palmer, Hughes, Lee & Wakelam (1988) Cell. Signalling 1, 147-156]. Competition studies showed that Ins(1:2-cyclic,4,5)P3 exhibits a 21-fold lower affinity for the site than Ins(1,4,5)P3 (IC50 124 +/- 16 nM). The affinity of the binding sites for Ins(1,4,5)P3 was not influenced by the non-hydrolysable GTP analogues GTP gamma S and Gpp[NH]p or by preincubation of the binding protein with a preparation of partially purified protein kinase C in the presence of ATP and TPA (12-O-tetradecanoylphorbol 13-acetate). These observations are discussed with reference to the identify and function of the Ins(1,4,5)P3 binding site.

Development of a novel, Ins(1,4,5)P 3-specific binding assay: Its use to determine the intracellular concentration of Ins(1,4,5)P 3 in unstimulated and vasopressin-stimulated rat hepatocytes

The binding of [ 3H]Ins(1,4,5)P 3 to bovine adrenocortical microsomes has been shown to be rapid, reversible and saturable. The microsomal preparation contained a single population of high affinity sites ( K D = 6.82 +/− 2.3 nM, B max = 370 +/− 38 fmol/mg protein). The binding site was shown to exhibit positional specificity with respect to inositol trisphosphate binding, i.e., Ins(2,4,54)P 3 was able to complete with [ 3H]Ins(1,4,5)P 3 whereas Ins(1,3,4)P 3 was not. Ins(1,3,4,5)P 4 showed a similar affinity for the receptor as Ins(2,4,5)P 3 whereas the other inositol phosphates tested, ATP, GTP and 2,3-DPG, were poor competitors. [ 3H]Ins(1,4,5)P 3-binding was independent of free Ca 2+ concentrations. The adrenocortical microsomal preparation has been incorporated into an assay which has been used to determined the basal and vasopressin-stimulated content of neutralised acid extracts of rat hepatocytes. Intracellular concentrations of Ins(1,4,5)P 3 were calculated to be 0.22 +/− 0.15 μM basal and 2.53 +/− 1.8 μM at peak stimulation. This assay provides a simple, specific and quantitative method for the measurement of Ins(1,4,5)P 3 concentratiosn in the picomolar range.

Ca2+ uptake in bovine adrenocortical microsomes: formation of phosphorylated intermediate of Ca2+ dependent ATPase.

Bovine adrenocortical microsomes were prepared and partially purified by discontinuous sucrose density gradient. Light fractions of the microsomes at the interface between 15 and 30% sucrose solution, exhibited ATP dependent Ca2+ uptake. The Ca2+ uptake was dependent on temperature and stimulated by free Ca2+ (the concentration for half maximal activation = 1.0 microM) and Mg2+. The Ca2+ uptake was inhibited by ADP but not affected by 10 mM NaN3 or 0.5 mM ouabain. Calcium release from the microsomes was accelerated by a Ca2+ ionophore, A23187, but not by a Ca2+ antagonist, diltiazem. A microsomal protein with a molecular weight of 100-110 kDa was phosphorylated by [gamma-32P]ATP in the presence of Ca2+, and the Ca2+ dependency was over the same range as the Ca2+ uptake (the concentration for half maximal activation = 3.0 microM). The phosphorylated protein (EP) was stable at acidic pH but labile at alkaline pH and sensitive to hydroxylamine. The rate of EP formation at 0 degrees C in the presence of 1 microM ATP and 10 microM Ca2+ (half time = 0.2 s) was less than that in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (half time = 0.1 s). The rate of EP decomposition at 0 degrees C after adding EGTA was about 6.7 times slower (rate constant: kd = 4.3 X 10(-3) s-1) than that of SR. It was suggested that adrenocortical microsomes contain a Ca2+ dependent ATPase which function as a Ca2+ pump with similar properties to that of SR.

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.

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.

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.