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.     

Cytochrome P-450(17alpha) in beta-cells of rat pancreas and its local steroidogenesis

We have found cytochrome P-450(17alpha) in the islets of Langerhans of rat pancreas. Its existence coincided with that of insulin and demarcated those of glucagon and somatostatin, demonstrating the localization in beta-cells. The enzyme has not only 17alpha-hydroxylase activity but also lyase one, which is a prerequisite for androgen biosynthesis. The pancreatic microsomes converted progesterone mainly to androstenedione with a minor production of 17alpha-hydroxyprogesterone. Due to a low activity of the built-in lyase, cytochrome P-450(17alpha) requires a sufficient electron-transfer from P-450 reductase or presence of an activator to promote the C-C bond cleavage. In beta-cells, P-450 reductase was abundant and could efficiently transfer electrons to P-450(17alpha). Actually, inhibition with anti-P-450 reductase or limitation of NADPH preferentially reduced the lyase activity. Androstenedione was accumulated when its further metabolism was suppressed. We also found localization of cytochrome P-450scc and 3beta-hydroxysteroid dehydrogenase in beta-cells. These results indicate that the immediate substrate for androgen formation, progesterone, is intracellularly produced and is converted mainly to androstenedione with support by an efficient electron supply from P-450 reductase. The product was supposed to be further metabolized to the reduced derivatives such as testosterone, 5alpha-androstanedione, and dihydrotestosterone, which would act as local steroids in the islets of Langerhans.     

Kinetic studies on androstenedione production in ovarian microsomes from immature rats.

Androstenedione formation from progesterone by P-450(17 alpha,lyase) was investigated in ovarian microsomes of immature rats treated with pregnant mare serum gonadotropin. Successive monooxygenase reactions in the formation of androstenedione without the intermediate leaving P-450(17 alpha,lyase) were demonstrated by a double-substrate double-label experiment using [14C]progesterone and 17 alpha-[3H]hydroxyprogesterone as substrates and also by specific reduction in the concentration of intermediate 17 alpha-hydroxyprogesterone in the reaction medium by reaction of liposomal P-450C21. A detailed kinetic study on the reactions of P-450(17 alpha,lyase) in microsomes was conducted in the steady state. Kinetic parameters indicated the C17,C20-lyase reaction for 17 alpha-hydroxyprogesterone (Km = 80 nM) to be strongly inhibited by progesterone (Ki = 8 nM). In the presence of a high concentration of progesterone, as in the case of in vivo rat ovary, most androstenedione is concluded to be formed directly from progesterone by successive monooxygenase reactions catalyzed by P-450(17 alpha,lyase). 20 alpha-Dihydroprogesterone competitively inhibited the C17,C20-lyase reaction for 17 alpha-hydroxyprogesterone with Ki = 23 nM, but had only slight effect on progesterone metabolism to androstenedione. 20 alpha-Dihydroprogesterone, thus, cannot be a regulator for androstenedione formation in rat ovary.     

Kinetic control of steroidogenesis by steroid concentration in guinea pig adrenal microsomes

For clarification of the effects of steroid concentration on steroidogenesis of adrenal microsomes, the kinetic parameters, Km and kcat, were determined in the steady-state for progesterone and 17 alpha-hydroxyprogesterone metabolism catalyzed by P-450C21 and P-450(17 alpha lyase) in guinea pig adrenal microsomes. At a high concentration of progesterone, it was equally metabolized by P-450C21 and P-450(17 alpha lyase), while at a low concentration, it was hydroxylated at 17 alpha-position with twice higher rate than at 21-position. 17 alpha-Hydroxyprogesterone is apparently metabolized preferentially by P-450C21 at any concentration. Although the productions of deoxycortisol and androstenedione from 17 alpha-hydroxyprogesterone were strongly inhibited by progesterone, androstenedione formation from progesterone was not inhibited by a high concentration of progesterone. The addition of liposomal P-450C21 to the reaction medium containing adrenal microsomes caused a decrease in the concentration of 17 alpha-hydroxyprogesterone released into the medium in the steady state reaction, but this had no effect on the activity of androstenedione formation from high concentrations of progesterone. It thus follows that androstenedione is produced by successive monooxygenase reactions without the release of 17 alpha-hydroxyprogesterone from P-450(17 alpha lyase) at a high concentration of progesterone, which is the condition of the adrenal microsomes in vivo.     

Studies on cytochrome P-450-dependent microsomal enzymes of testicular androgen and estrogen biosynthesis in a urodele amphibian, Necturus

The microsomal fraction isolated from the testis of the urodele amphibian, Necturus maculosus, is very rich in cytochrome P-450 and three cytochrome P-450-dependent steroidogenic enzyme activities, 17 alpha-hydroxylase, C-17, 20-lyase, and aromatase. In this study, we investigated aspects of these reactions using both spectral and enzyme techniques. In animals obtained at different points in the annual cycle, Necturus testis microsomal P-450 concentrations ranged from 0.6-1.8 nmol/mg protein. Substrates for the three enzymes generated type I difference spectra; progesterone and 17 alpha-hydroxyprogesterone appeared to bind to one P-450 species while the aromatase substrates, androstenedione, 19-hydroxyandrostenedione, and testosterone, all bound to another P-450 species. Spectral binding constants (Ks) for these interactions were determined. Michaelis constants (Km) and maximum velocities were determined for progesterone 17 alpha-hydroxylation, 17 alpha-hydroxyprogesterone side-chain cleavage, and for the aromatization of androstenedione, 19-hydroxyandrostenedione, and testosterone. Measured either by spectral or kinetic methods, progesterone, androstenedione, and 19-hydroxyandrostenedione were high affinity substrates (Ks or Km less than 0.3 microM), while 17 alpha-hydroxyprogesterone and testosterone were low affinity substrates (Ks or Km = 0.6-4.8 microM). As evidence for the participation of cytochrome P-450 in these reactions, carbon monoxide was found to inhibit each of the enzyme activities studied. The activity of NADPH-cytochrome c reductase, a component of cytochrome P-450-dependent reactions, was also high in Necturus testis microsomes.     

Specificity of steroid binding to testicular microsomal cytochrome P-450. Relation of steroid structure to type-I spectral responses after correction for hydrophobic association with the membrane

On the basis of the concept that steroids accumulate in the lipid phase of endoplasmic reticulum membranes and approach the active sites of steroidogenic cytochromes P-450 from a hydrophobic environment, we describe a procedure that allows calculation of spectral dissociation constants Ks for steroid interaction with testicular microsomal cytochrome P-450 after correction for hydrophobic association of ligand with the membrane. Maximal type-I spectral responses, apparent Ks, and partition into microsomal lipids were determined for 36 steroids, and corrected Ks values were derived from these primary data. Partition coefficients range from 60 to 62,000, and corrected Ks range from 60 microM to 25 mM steroid concentration in the lipid phase. Full spectral properties depend on a side-chain (1-3 carbon atoms) at the C17-position which may be hydrophobic or may bear a 20-oxo or 20 beta-hydroxy, but not a 20 alpha-hydroxy group. Binding constants are especially sensitive towards modifications of ring A structure (aromatization or 5 beta-, but not 5 alpha-reduction) and of the side-chain length. Androgens, with the exception of those bearing a 17 beta-acetoxy or 17 beta-propionyloxy group, are poorly accommodated by this cytochrome P-450.     

Side-chain cleavage of C21 steroids by testicular microsomal cytochrome P-450 (17 alpha-hydroxylase/lyase): involvement of heme

The two steps in the side-chain cleavage of C21 steroids to give C19 steroids (i.e. 17 alpha-hydroxylation and C17,20 lyase activity) were examined using a highly purified cytochrome P-450 from microsomes of neonatal pig testis to determine the photochemical action spectra for the two reactions. Photochemical action spectra, using either 4-ene (progesterone) or 5-ene (pregnenolone) substrates, showed maximal reversal of inhibition by CO with light of 451 nm. Evidently the heme of cytochrome P-450 is involved in both 17 alpha-hydroxylation and in C17,20-lyase activity as in the case of the side-chain cleavage of cholesterol. Mechanisms proposed to account for enzymatic cleavage of the alpha-ketol side-chain of C21 steroids (C17,20 lyase activity) must be consistent with these findings.     

Multiple catalytic properties of the purified and reconstituted cytochrome P-450 (P-450sccII) system of pig testis microsomes

The catalytic properties of the testis microsomal P-450, termed P-450sccII, have been studied in a refined assay system which consists of P-450sccII (13 nmol of P-450 heme/mg of protein) and its reductase has been purified extensively from pig testis. The results indicated that P-450sccII was highly active in catalyzing hydroxylation of 11 beta-hydroxyprogesterone at the 17 alpha-position to give 21-deoxycortisol and cleavage of 17 alpha-hydroxyprogesterone at the 17-20 bond to give androstenedione with turnover numbers of 25 and 30 mol/min X mol of P-450, respectively. In contrast, many physiologically important corticosteroids we tested were found to be poor substrates for both the hydroxylase and lyase reactions. The possible reason for the importance of these substrate specificity of P-450sccII in production of both corticosteroids and androgens in the endocrine tissues is discussed. P-450sccII also catalyzed conversion of testosterone to androstenedione, but 18O experiments failed to show incorporation of atmospheric oxygen into the androstenedione formed. However, this does not preclude the possibility that the P-450-bound intermediate gem-diol stereoselectively dehydrates to give the nonlabeled ketosteroid. In addition to these steroid-oxidizing activities, P-450sccII revealed considerable specificities toward various xenobiotics, suggesting that P-450sccII and liver microsomal P-450 are basically similar as regards enzymatic functions and activities.     

The interference of polychlorinated biphenyls (Aroclor 1254) with membrane regulation of the activities of cytochromes P-450C21 and P-450(17) alpha,lyase in guinea-pig adrenal microsomes.

Regulation of cytochromes P-450 21-hydroxylase (P-450C21) and P-450 17 alpha-hydroxylase/C17,20-lyase (P-450(17) alpha,lyase) activities and impairment of this regulation by Aroclor 1254 was studied in guinea-pig adrenal microsomes. In a membrane depleted system, a decrease in the normally predominant, P-450C21 activity and an increase in P-450(17) alpha,lyase activities was observed. The same deviations were observed in intact microsomes with increase in the reaction temperature (0-40 degrees C). Breaks in Arrhenius plots for activities of P-450C21 and P-450(17) alpha,lyase correlate with transition temperatures reported for the microsomal membrane. These results point to: (1) preference of a gel state membrane for catalytic expression of P-450C21 suggesting a clustered organization of this P-450 species with reductase; (2) preference of a fluid membrane for lyase activity suggesting a random collision mechanism for reduction of P-450(17) alpha,lyase. Aroclor 1254 introduced to reaction mixtures containing intact microsomes elicited basically the same changes as caused by depletion of the microsomal membrane or by increase in the incubation temperature. Lack of effect of Aroclor 1254 on P-450C21 and P-450(17) alpha,lyase activities in the membrane depleted system demonstrates that its interference with monooxygenase activities is mediated by the microsomal membrane. The similarities between altered cytochrome P-450 mediated activities in the presence of Aroclor 1254 and the deviations observed in the membrane depleted system or upon increase in the incubation temperature may suggest that this chemical exerts its impacts by influencing membrane fluidity.     

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig.

Studies were carried out to investigate the effects of prostaglandins (PG) in vitro on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE1, PGE2, PGA1, PGF1 alpha or PGF2 alpha to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (delta A385-420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11 beta-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.     

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.     

The rate-determining step in P450 C21-catalyzing reactions in a membrane-reconstituted system

Adrenal cytochrome P450 C21 in a membrane-reconstituted system catalyzed 21-hydroxylation of 17alpha-hydroxyprogesterone at a rate higher than that for progesterone in the steady state at 37 degrees C. The rate of product formation in the steady state increased with the concentration of the complex between P450 C21 and the reductase in the membranes. The complex formation was independent of the volume of the reaction, showing that the effective concentrations of the membrane proteins should be defined with the volume of the lipid phase. The rates of conversion of progesterone and 17alpha-hydroxyprogesterone to the product in a single cycle of the P450 C21 reaction were measured with a reaction rapid quenching device. The first-order rate constant for the conversion of progesterone by P450 C21 was 4.3 +/- 0.7 s(-)1, and that for 17alpha-hydroxyprogesterone was 1.8 +/- 0.5 s(-)1 at 37 degrees C. It was found from the analysis of kinetic data that the rate-determining step in 21-hydroxylation of progesterone in the steady state was the dissociation of product from P450 C21, whereas the conversion to deoxycortisol was the rate-determining step in the reaction of 17alpha-hydroxyprogesterone. The difference in the rate-determining steps in the reactions for the two substrates was clearly demonstrated in the pre-steady-state kinetics.     

Dynamic structures of adrenocortical cytochrome P-450 in proteoliposomes and microsomes: protein rotation study.

Purified adrenocortical microsomal cytochromes P-45017 alpha,lyase and P-450C21 were reconstituted with and without NADPH-cytochrome P-450 reductase in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles at a lipid to P-450 ratio of 35 (w/w) by cholate dialysis procedures. Trypsinolysis revealed that a considerable part of each P-450 molecule is deeply embedded in the lipid bilayer, on the basis of the observation of no detectable digestion for P-45017 alpha,lyase and the proteolysis-resistant membrane-bound heavy fragments for P-450C21. Rotational diffusion was measured in proteoliposomes and adrenocortical microsomes by observing the decay of absorption anisotropy, r(t), after photolysis of the heme-CO complex. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The absorption anisotropy decayed within 1-2 ms to a time-independent value r3. Coexistence of a mobile population with an average rotational relaxation time phi of 138-577 microseconds and immobile (phi > or = 20 ms) populations of cytochrome P-450 was observed in both phospholipid vesicles and microsomes. Different tilt angles of the heme plane from the membrane plane were determined in proteoliposomes to be either 47 degrees or 63 degrees for P-45017 alpha,lyase from [r3/r(0)]min = 0.04 and either 38 degrees or 78 degrees for P-450C21 from [r3/r(0)]min = 0.19, when these P-450s were completely mobilized by incubation with 730 mM NaCl. Very different interactions with the reductase have been observed for the two P-450s in proteoliposomes. In the presence of the reductase, the mobile population of cytochrome P-450C21 was increased significantly from 79% to 96% due to dissociation of P-450 oligomers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate binding site of microsomal cytochrome P-450 directly faces membrane lipids

Cytochrome P-450, purified from liver microsomes of phenobarbital-treated rabbits, was incorporated into dimyristoylphosphatidylcholine liposomes. The binding of benzphetamine to the liposome-bound cytochrome P-450 was examined by measuring the benzphetamine-induced spectral change at various temperatures. The van't Hoff plot of the apparent spectral dissociation constant showed a distinct break at the temperature of phase transition of the synthetic lipid. On the other hand, no such break was observed for benzphetamine binding to microsomal bound cytochrome P-450. These results suggest that the substrate binding site of cytochrome P-450 is embedded in the apolar interior of phospholipid bilayer membranes.     

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-450C21 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-450C21 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.     

Specific accumulation of 17 alpha-hydroxyprogesterone in microsomal membranes during the process of cytochrome P-450(C-17)-catalysed androgen biosynthesis. A dynamic study of intermediate formation and turnover.

A complete dynamic analysis of cytochrome P-450(C-17)-catalysed androgen biosynthesis from a single dose of progesterone and 17 alpha-hydroxyprogesterone in a double-label double-substrate experiment was performed in order to elucidate the controversial intermediacy of 17 alpha-hydroxyprogesterone. Label distribution within the steroid fractions as well as in the membrane and buffer compartments yields direct evidence that the endogenously formed 17 alpha-hydroxyprogesterone (which is in an 'intermediate state') accumulates to a higher degree in microsomal membranes than does the exogenously added 17 alpha-hydroxyprogesterone (which is in a 'substrate state') under certain conditions. It is also demonstrated that endogenously formed 17 alpha-hydroxyprogesterone may partly leave the membrane compartment (in terms of a 'leakage' or 'overflow' phenomenon) and is then able to equilibrate with the pool of exogenously added 17 alpha-hydroxyprogesterone. Since only the label distribution in the membrane-associated (but not always in the aqueous) 17 alpha-hydroxyprogesterone pool corresponds to the label distribution in the androgen fraction, it is concluded that only the membrane-associated 17 alpha-hydroxyprogesterone pool is directly accessible to cytochrome P-450(C-17)-catalysed conversion into androgens.     

Localization of the active center of microsomal cytochrome P-450

To solve the problem of localization of the active center of cytochrome P-450 in microsomal membranes, new bifunctional compounds (I-IV), which contain pyridine radical, aliphatic chain of variable length and diphosphonic acid ("floating" molecules) have been applied. These compounds inhibit oxidation and binding of the substrates of cytochrome P-450 (aminopyrine and aniline), inhibition being of a competitive character. Measurements of distribution coefficients between water and membranes of microsomes and liposomes from egg phosphatidylcholine evidence that the microsomal proteins are necessary for providing effective interaction of I-IV with microsomal membrane. The 1H-NMR method has demonstrated compounds to be incorporated into lipid bilayer so that the non-polar part is in the inner membrane volume. The results obtained confirm our previous conclusion (Krainev A.G., Weiner L.M., Alferyev I.S., Slynko N.M. (1985) Biochim. Biophys. Acta, 818, 96-104) about localization of the active center of microsomal cytochrome P-450 at the depth of approximately 18 A from the hydrophilic surface of a membrane.     

Rotation and interaction with epoxide hydrase of cytochrome P-450 in proteoliposomes

Purified rat liver cytochrome P-450MC or P-450PB was co-reconstituted with epoxide hydrase in liposomal vesicles made of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a lipid to protein weight ratio of 5 by the cholate dialysis procedure. Rotational diffusion of the cytochromes was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane-normal" model. The measurements were used to investigate interactions of cytochrome P-450MC or P-450PB with epoxide hydrase. Different rotational mobilities of the two cytochromes were observed. The amount of mobile molecules was 78% for cytochrome P-450MC and 91% for P-450PB, and the rest was immobile within the experimental time range of 1 ms. In the presence of epoxide hydrase 85% of cytochrome P-450MC and 96% of P-450PB were mobile. Cross-linking of epoxide hydrase by anti-epoxide hydrase antibodies resulted in a drastic immobilization of the cytochromes, reducing the mobile population to 49% for P-450MC and to 60% for P-450PB. The rotational relaxation times phi of the mobile populations ranged from 210 to 283 microseconds. These results imply that both cytochromes P-450MC and P-450PB transiently associate with epoxide hydrase in liposomal membranes. Further analysis of the data showed that the angle between the heme plane of P-450MC and the membrane is 48 degrees or 62 degrees, different from the value of 55 degrees reported previously for P-450PB (Gut, J., Richter, C., Cherry, R. J., Winterhalter, K. H., and Kawato, S. (1983) J. Biol. Chem. 258, 8588-8594).     

Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect

In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, [1,2,6,7,16,17-3H]progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting [1,7,16-3H]progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of 3H2O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of [1,7,16-3H]progesterone and [4-14C]progesterone was employed.     

Study of the structural organization of cytochrome P-450 oligomers using immobilized isoenzyme LM2

Subunit interactions of highly purified hexameric cytochrome P-450 LM 2 has been studied using covalent binding of one of the six protomers to an insoluble matrix. Immobilized cytochrome was catalytically active in monooxygenase reactions and retained the spectral characteristics of cytochrome P-450 LM 2. High ionic strength, large scale pH changes and addition of guanidine chloride were without effect on the aggregation state of the immobilized hemoprotein. However, several detergents induced hexamer dissociation. The crucial role of hydrophobic forces in hexamer subunit interaction was demonstrated. Incubation of the immobilized cytochrome P-450 LM 2 with sonicated liposomes composed of various phospholipids did not result in oligomer dissociation and protein translocation from the matrix to the lipid phase, although the catalytic activity of the immobilized cytochrome significantly increased in the presence of liposomes. The data suggest that cytochrome P-450 LM 2 may be of hexameric structure in the native membranes.