Cloning and characterization of bovine cytochrome P-450(11 beta) genes

We isolated 4 different clones of the P-450(11 beta) gene from a bovine genomic library. These genomic clones were highly homologous with each other. Two of the isolated clones were pseudogenes. Determination of its nucleotide sequences indicated that the bovine P-450(11 beta) gene is divided into 9 exons by 8 introns and that it is about 8.5 kb in total length. The number of exons and the locations of intron insertion into the P-450(11 beta) gene are identical with those in the case of P-450(SCC), but different from those of other microsomal P-450s.     

Structural analysis of cloned cDNA for mRNA of microsomal cytochrome P-450(C21) which catalyzes steroid 21-hydroxylation in bovine adrenal cortex

We have isolated cDNA clones of the mRNA for cytochrome P-450 that catalyzes the steroid C-21 hydroxylation (P-450(C21)), which specifically catalyzes 21-hydroxylation of steroids in the microsomes of bovine adrenal cortex by using synthetic oligonucleotides as probes. Sequence determination of the cloned cDNA showed that it contains 2157 nucleotides and a poly(A) chain and that a single open reading frame of 1488 nucleotides codes for a polypeptide of 496 amino acids with a molecular weight of 56,113. The deduced amino acid composition is in agreement with that determined by direct amino acid analysis of purified P-450(C21) and the predicted primary structure contained amino acid sequences of N-terminal region and two internal tryptic fragments of the protein so far analyzed. Comparing the amino acid sequence with those of other forms of P-450 reveals that a conserved amino acid sequence containing a putative heme-binding cysteine is present in the equivalent position, proximate to the COOH terminus of the molecules and that P-450(C21) is phylogenically situated in an intermediate position between steroidogenic mitochondrial cytochrome P-450 which catalyzes the side-chain cleavage of cholesterol (P-450(SCC)) and drug-metabolizing microsomal P-450s. However, the amino acid sequence of P-450(C21) is much closer to that of drug-metabolizing P-450s than to that of P-450(SCC).     

Aldosterone synthase cytochrome P-450 expressed in the adrenals of patients with primary aldosteronism

A human cytochrome P-450 with aldosterone synthase activity was purified from the mitochondria of an aldosterone-producing adenoma. It was recognized by an anti-bovine cytochrome P-450(11 beta) IgG and by a specific antibody raised against a portion of the CYP11B2 gene product, one of the two putative proteins encoded by human cytochrome P-450(11 beta)-related genes (Mornet, E., Dupont, J., Vitek, A., and White, P. C. (1989) J. Biol. Chem. 264, 20961-20967). A similar and probably the same aldosterone synthase cytochrome P-450 was detected in the adrenal of a patient with idiopathic hyperaldosteronism. These aldosterone synthases were distinguishable from cytochrome P-450(11 beta), the product of another cytochrome P-450(11 beta)-related gene, i.e. CYP11B1, by their catalytic, molecular, and immunological properties and also by their localization. The latter enzyme was unable to produce aldosterone and did not react with the specific antibody against the CYP11B2 gene product. It was present both in tumor and non-tumor portions of the adrenals carrying the adenoma and in normal adrenal cortex. On the other hand, aldosterone synthase cytochrome P-450 localized in the tumor portions of the adrenals or in the adrenal of a patient with idiopathic hyperaldosteronism. Thus aldosterone synthase cytochrome P-450, a distinct species from cytochrome P-450(11 beta), is responsible for the biosynthesis of aldosterone in the human, at least in patients suffering from primary aldosteronism.     

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.     

Alterations of heme, cytochrome P-450, and steroid metabolism by mercury in rat adrenal

The treatment of male rats with Hg2+ resulted in significant alterations in heme and hemoprotein metabolism in the adrenal gland which, in turn, were reflected in abnormal steroidogenic activities and steroid output. Twenty-four hours after the administration of 30 mumol of HgCl2/kg (sc) the mitochondrial heme and cytochrome P-450 concentrations increased by approximately 50%. Also, Hg2+ treatment stimulated a porphyrinogenic response which included an 11-fold increase in the activity of delta-aminolevulinate synthetase. The increase in mitochondrial cytochrome P-450 content was reflected in elevated steroid 11 beta-hydroxylase and cholesterol side-chain cleavage activities. In contrast, Hg2+ treatment resulted in decreased concentrations of microsomal cytochrome P-450 (-75%) and heme (-45%). Similarly, the reduction in the microsomal cytochrome P-450 content was accompanied by reduced steroid 21 alpha-hydroxylase and benzo[alpha]pyrene hydroxylase activities. The mechanisms responsible for the loss of the microsomal cytochrome P-450 content appeared to involve a selective impairment of formation of the holocytochrome as well as an enhanced rate of heme degradation. This suggestion is made on the basis of findings that (a) the decrease in the microsomal cytochrome P-450 content was accompanied by a sevenfold increase in the activity of adrenal heme oxygenase, (b) no decrease in apocytochrome P-450 could be detected in sodium dodecyl sulfate-gel electrophoresis of the solubilized microsomal fractions stained for heme, and (c) the concentration of adrenal microsomal cytochrome b5 was significantly increased in the Hg2+-treated animals. It is suggested that Hg2+ directly caused a defect in adrenal steroid biosynthesis by inhibiting the activity of 21 alpha-hydroxylase. The apparent physiological consequences of this effect included lowered plasma levels of corticosterone and elevated concentrations of progesterone and dehydroepiandrosterone. This abnormal plasma steroid profile is indicative of a 21 alpha-hydroxylase impairment.     

Import and processing of P-450SCC and P-450(11) beta precursors by corpus luteal mitochondria: a processing pathway recognizing homologous and heterologous precursors

Maturation of the precursor forms of bovine cholesterol side-chain cleavage cytochrome P-450 (P-450SCC) and 11 beta-hydroxylase cytochrome P-450 (P-450(11)beta) was investigated using mitochondria from bovine corpus luteum. The results show that both precursors, whose synthesis was directed by bovine adrenocortical RNA, can be imported and proteolytically processed to their corresponding mature forms by bovine corpus luteal mitochondria, even though P-450(11)beta is not expressed in this tissue. Furthermore, the efficiency of processing of pre-P-450(11)beta by corpus luteal mitochondria is similar to that of pre-P-450SCC, an endogenous enzyme of these mitochondria. However, the P-450(11)beta precursor is not processed by mitochondria from a nonsteroidogenic tissue (heart), a result observed previously for the P-450SCC precursor (M. F. Matocha and M. R. Waterman (1984) J. Biol. Chem. 259, 8672-8678). This discriminatory processing of pre-P-450(11)beta by heterologous mitochondria suggests that the precursor forms of P-450SCC and P-450(11)beta are processed via a common pathway in steroidogenic mitochondria and that this pathway is absent in nonsteroidogenic mitochondria.     

In vitro synthesis of mitochondrial cytochromes P-450(scc) and P-450(11-β) and microsomal cytochrome P-450(C-21) by both free and bound polysomes isolated from bovine adrenal cortex

$\text{In}\text{vitro}$ synthesis of mitochondrial cytochromes P-450(scc) and P-450(11-β), and microsomal cytochrome P-450(C-21) programmed by bovine adrenal cortex polysomes was carried out using rat liver cell sap and wheat germ lysate systems. Synthesis of P-450 proteins in the cell-free systems was determined by immunoprecipitation and immunoadsorption using mono-specific antibodies to each species of P-450, and the sizes of the $\text{in}\text{vitro}$ products were analyzed by SDS-polyacrylamide gel electrophoresis. Both free and bound polysomes synthesized these three species of P-450 in the cell-free systems. P-450(scc) and P-450(C-21) were synthesized apparently as the mature size products, whereas P-450(11-β) was synthesized as a putative precursor approximately 5,000 daltons larger than the mature form. Mitochondrial and microsomal P-450 proteins seem to share common sites of synthesis in the cytoplasm of adrenal cortex cells.     

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

Studies were carried out to investigate the effects of prostaglandins (PG) $\text{in vitro}$ on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE₁, PGE₂, PGA₁, PGF_1α or PGF_2α to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (ΔA_385–420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11β-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.     

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

Adrenocortical mitochondrial cytochrome P-450 specific to the cholesterol side-chain cleavage (desmolase) reaction differs from that for the 11beta-hydroxylation reaction of deoxycorticosterone. The former cytochrome appears to be more loosely bound to the inner membrane than the latter. Upon ageing at 0 degrees C or by aerobic treatment with ferrous ions, the desmolase P-450 was more stable than the 11beta-hydroxylase P-450. By utilizing artificial hydroxylating agents such as cumene hydroperoxide, H2O2, and sodium periodate, the hydroxylation reaction of deoxycorticosterone to corticosterone in the absence of NADPH was observed to a comparable extent with the reaction in the presence of adrenodoxin reductase, adrenodoxin and NADPH. However, the hydroxylation reaction of cholesterol to pregnenolone was not supported by these artificial agents. Immunochemical cross-reactivity of bovine adrenal desmolase P-450 with rabbit liver microsomal P-450LM4 was also investigated. We found a weak but significant cross-reactivity between the adrenal mitochondrial P-450 and liver microsomal P-450LM4, indicating to some extent a homology between adrenal and liver cytochromes P-450.     

Import and processing of the precursor of cytochrome P-450(SCC) by bovine adrenal cortex mitochondria

Isolated bovine adrenal cortex mitochondria imported in vitro synthesized pre-P-450(SCC) and processed it to the mature form. Partial radio-sequencing of the processed P-450(SCC) gave a result identical with that for authentic P-450(SCC). Rat liver mitochondria also imported pre-P-450(SCC) and processed it to the mature form, whereas bovine heart mitochondria were unable to import and process pre-P-450(SCC) although both mitochondrial preparations imported and processed pre-adrenodoxin. The pre-P-450(SCC) processing activity of bovine adrenal cortex mitochondria was associated with the matrix side surface of the inner membrane. The processing protease could be solubilized by sodium cholate and partially purified by ammonium sulfate fractionation. The partially purified processing protease cleaved pre-P-450(SCC) at the correct position. It was also active in processing pre-P-450(11 beta) but inactive toward pre-adrenodoxin. Bovine heart mitochondria lacked the processing activity to pre-P-450(SCC). The localization of pre-P-450(SCC) and mature P-450(SCC) in bovine adrenal cortex mitochondria was examined. Mature P-450(SCC) processed by the mitochondria was found associated with the matrix-side surface of the inner membrane, which is the correct location of P-450(SCC) in the cell. In the presence of o-phenanthroline, pre-P-450(SCC) was imported into the organelles without being processed and remained soluble in the matrix. The incorporation of newly processed mature P-450(SCC) into the inner membrane was also observed when pre-P-450(SCC) was incubated with inner membrane vesicles. Mature P-450(SCC) generated in vitro from pre-P-450(SCC) by the partially purified processing protease was incorporated not only into the inner membrane vesicles but also into bovine adrenal cortex microsomes. These findings suggested that the processing of pre-P-450(SCC) occurred prior to the incorporation of mature-P-450(SCC) into the inner membrane.     

Hepatic microsomes from freshwater fish--I. In vitro cytochrome P-450 chemical interactions

1. Of 87 chemicals tested for their ability to interact with oxidized hepatic cytochrome P-450 from mature male brook trout (Salvelinus fontinalis), 21 formed detectable type I or type II binding spectra. 2. When 8 of these 21 chemicals were tested with cytochrome P-450 of nine other species of freshwater fish, wide species variation in hepatic microsomal cytochrome P-450 was evident, since the spectral size of chemical interactions as related to the carbon monoxide spectrum and the ratio of type II to type I binding were not alike. 3. These spectral data suggest that hepatic microsomal cytochrome P-450 of freshwater fish exists in different forms.     

Purification and properties of cytochrome P-450 (SCC) from pig testis mitochondria

Cytochrome P-450 was purified from pig testis mitochondria to a specific content of 13.1 n mol/mg of protein. The purified preparation was found to contain a single species of P-450, on sodium dodecyl sulfate polyacrylamide gel electrophoresis, with an apparent molecular weight of about 53000 +/- 2000. The cholesterol side chain-cleavage system could be reconstituted by mixing the purified cytochrome P-450, adrenodoxin reductase, adrenodoxin, cholesterol and NADPH. The rate of conversion of cholesterol to pregnenolone was 6.2 n mol/min/n mol of P-450 under the conditions employed. The absorption spectrum of the oxidized cytochrome P-450 had maxima at 416, 530 and 568 nm. The reduced CO-complex of the cytochrome P-450 exhibited an absorption maximum at 448 nm. The purified P-450 was subjected to microsequence analysis and its NH2-terminal amino acid sequence was found to show considerable homology with that of bovine adrenal P-450 (SCC).     

Immunochemical studies on cytochrome P-450 in adrenal microsomes

An antibody was prepared against electrophoretically homogeneous cytochrome P-450C21 purified from bovine adrenal microsomes. This antibody was used to compare various cytochromes P-450 in bovine and guinea pig adrenal microsomes. In an Ouchterlony double diffusion test, a spur formation was observed between the precipitin lines of the purified bovine cytochrome P-450C21 and guinea pig adrenal microsomes against anti-cytochrome P-450C21 IgG. Anti-cytochrome P-450C21 IgG inhibited 21-hydroxylation both of bovine and guinea pig adrenal microsomes but the inhibition was much more effective in the bovine microsomes than in the guinea pig microsomes. These results suggest that the 21-hydroxylase in the guinea pig microsomes has some molecular similarities to the bovine cytochrome P-450C21 and a part of the antibodies cross-reacts with the 21-hydroxylase in the guinea pig microsomes. Anti-cytochrome P-450C21 IgG did not inhibit the activities of 17 alpha-hydroxylase and C17,20-lyase in the bovine and guinea pig microsomes but stimulated these activities. This result shows that different species of cytochrome P-450 other than cytochrome P-450C21 catalyzes the 17 alpha-hydroxylation and C17,20 bond cleavage. The stimulation of 17 alpha-hydroxylation and C17,20 bond cleavage by blocking 21-hydroxylation indicates that the electron transfer systems for various cytochromes P-450 are intimately linked in adrenal microsomes.     

Spectrophotometric study of cytochrome P-450 (11 beta) interaction with physiological effectors

Using the optical absorbance spectroscopy method, the interaction of a number of biospecific ligands (steroids, adrenodoxin) with homogeneous cytochrome P-450 (11 beta) from bovine adrenal mitochondria was investigated. The parameters of the steroid-protein interaction in a number of substrates and products of the 11 beta- and 18 (19)-hydroxylation with the active site of cytochrome P-450 (11 beta) were determined. A sharp decrease in the cytochrome affinity for steroids upon the insertion of the first hydroxy group was observed, which provides for a predominant formation of monohydroxylated products from the substrate and minimum amounts of dihydroxylated ones, despite the presence of more than one position for the substrate hydroxylation by cytochrome P-450 (11 beta). Some structural elements of the steroid molecule were determined as any alterations in these strongly affect the enzyme affinity for the steroid. These structures are: 1) delta 4-3-oxo structure; 2) either 21-hydroxy group of pregnen steroids or the one fulfilling its functions, 17 beta-hydroxy or 17-oxo group of androsten steroids, and 3) the 11th position of all the substrates under study. It was shown that the binding of various substrates into stoichiometric (1:1) steroid-protein complexes provides a transition to high spin state from 30-40% (cortisol, corticosterone) to 90-95% (11-deoxycorticosterone) of hemoprotein iron. Using the experimental system containing individual cytochrome P-450 (11 beta) and adrenodoxin, as well as the steroid and nonionic detergent Tween 20, it was shown that the parameters of substrate binding and hemoprotein spin equilibrium did not differ from the corresponding parameters of the cytochrome-adrenodoxin dienzyme complex. The peculiarities of the multiligand interactions in the 11 beta-hydroxylase system, involving cytochrome, substrates and ferredoxin demonstrate some analogy with a bacterial camphor hydroxylase system and some differences from the mitochondrial system for the side chain cleavage of cholesterol.     

The relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in adrenal cortex mitochondria

The relationship between NADPH-dependent lipid peroxidation and the degradation of cytochrome P-450 has been studied in bovine adrenal cortex mitochondria. Malondialdehyde formation is accompanied by a corresponding decrease in total cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of P-450. To differentiate between cytochrome P-450(11)beta and P-450scc, steroid-induced difference spectra were used to evaluate P-450 degradation. These measurements provide the first evidence that both P-450's are degraded during NADPH-dependent lipid peroxidation with P-450(11)beta being much more susceptible to this process.     

Molecular organization (topography) of cytochrome P-450(11)beta in mitochondrial membrane and phospholipid vesicles as studied by trypsinolysis

Cytochrome P-450(11)beta from adrenal cortex is an intrinsic membrane protein embedded in the inner mitochondrial membrane. Topography of the protein inside a phospholipid bilayer was examined using controlled proteolysis of purified cytochrome P-450(11)beta following its integration into artificial liposomes. Inclusion of the protein into phospholipid vesicles led to a marked stabilization of the cytochrome activity. Trypsin treatment of the liposome-integrated cytochrome resulted in the rapid disappearance of the native protein moiety (47 kDa), while a major 34 kDa peptide component was formed. This peptide core retained the heme moiety and part of the cytochrome steroid-11 beta hydroxylase activity. Very similar observations were obtained when inside-out vesicles prepared from isolated adrenocortical mitoplasts were examined with the same approach. It is thus suggested that adrenocortical cytochrome P-450(11)beta is embedded in the inner mitochondrial membrane as well as in artificial liposomes by a major hydrophobic domain associated with the heme moiety while a limited domain remains accessible on the matrix side of the membrane surface. The previous described phosphorylation of the cytochrome P-450(11)beta on a serine residue, by the cAMP-dependent protein kinase is suggested to occur in the protein domain oriented toward the membrane surface, the phosphorylation site being lost under mild proteolytic digestion of the membrane-integrated protein.     

Regulation of rat adrenal messenger RNA and protein levels for cytochrome P-450s and adrenodoxin by dietary sodium depletion or potassium intake

The effect of low sodium and high potassium intake on rat adrenal zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR) were studied during a 7-day period, by analyzing mRNA and protein levels of various enzymes involved in aldosterone synthesis. In ZG significant increases in cytochrome P-450scc, P-450c21, P-450(11 beta), adrenodoxin mRNA and protein levels were observed after 2 days with either diet, and at day 7 these levels were further increased. The largest mRNA induction was observed at day 7 in sodium-depleted rats for P-450(11 beta), with a 4-fold increase, followed by 2.7- and 2.0-fold increases for P-450scc and P-450c21, respectively. A pattern similar to those of P-450scc and P-450(11 beta) was observed for adrenodoxin with a 2.1-fold increase after 7 days of Na+ restriction. In K(+)-loaded rats mRNA levels for P-450scc, P-450(11 beta), P-450c21, and adrenodoxin were also increased by 2.2-, 2.1-, 1.5-, and 1.9-fold respectively. Protein levels of these enzymes were also measured in ZG and showed increases similar to those of their respective mRNAs for both treatments. On the other hand, mRNA levels of P-450scc, P-450(11 beta), P-450c21, and adrenodoxin in ZFR were found significantly lower than in ZG, although they were slightly increased for both treated groups of rats as compared with controls. In addition, ZFR protein levels of corresponding enzymes did not fluctuate significantly under both ionic regimens. In conclusion, both low sodium and high potassium intakes act primarily on ZG. Their action on plasma aldosterone seems to be mediated by increasing both mRNA and protein and levels of steroidogenic enzymes, especially at the early step (cytochrome P-450scc) and even more at the late steps (cytochrome P-450(11 beta]. In addition, a close relationship appears to exist between the two mitochondrial P-450s and their electron donor adrenodoxin, since their mRNA and protein levels were similarly enhanced for both diets used.     

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.     

A novel method for the preparation of substrate-free cytochrome P-450(11) beta

A new method for the removal of the stabilizing substrate, deoxycorticosterone, from adrenal cytochrome P-450(11) beta, has been developed. Dextran coated charcoal is used for the adsorption of the steroid and the adsorbed steroid is separated from the cytochrome P-450-preparation by low speed centrifugation. The substrate-free enzyme, obtained in this manner, has all the characteristic spectral properties of low-spin cytochrome P-450(11) beta and may be converted to the high-spin form by the addition of deoxycorticosterone. The dextran coated charcoal method has the following advantages over the previously used method of substrate removal. It does not require the addition of the cofactors for cytochrome P-450-dependant hydroxylation of deoxycorticosterone, small amounts of enzyme may be prepared in a short time and the enzyme preparation is not diluted to any great extent during the process.