Solubilization and partial purification of cytochrome P-450 from rat lung microsomes

Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino-$\text{n}$-octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome $\text{c}$ reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found.     

Solubilization and partial purification of human placental cytochromes P-450

Human placental mitochondrial and microsomal cytochrome P-450 (P-450) was solubilized with sodium cholate in the presence of Emulgen 911 and the solubilized preparations purified by phenyl-Sepharose and DEAE-cellulose column chromatography. The final preparations exhibited specific activities between 3.0 and 7.0 nmol P-450/mg protein implying a 30–115-fold purification over the starting material. Androstenedione exhibited type I spectral interaction with the microsomal P-450 and pregnenolone a reverse type I spectrum with mitochondrial P-450. Hydrophobic column chromatography proved to be a rapid and efficient initial purification step for placental P-450s.     

Induction by Triacetyloleandomycin and partial purification of a LM3 form of cytochrome P-450 from rabbit liver microsomes

Treatment of rabbits with Triacetyloleandomycin (a currently used antibiotic in human therapy) at 1 mmol per kg of body weight daily for 5 days results in a significant induction of liver microsomal cytochrome P-450, (2.6 nmol/mg proteins). Electrophoresis in SDS polyacrylamide gels shows this increase in P-450 is associated to the appearance of a strong band in a zone located between the major bands of microsomes induced by phenobarbital and β-naphtoflavone (LM₃ forms in Coon's terminology). Partial purification of this P-450 LM₃ (TAO) was undertaken by chromatographic procedures (CM cellulose and hydroxylapatite). Its subunit molecular weight is 52 000; the absolute spectra in the oxidized, ferrous and CO-ferrous forms present maxima at 417, 536, and 570 nm; 415 and 548 nm; 450 and 555 nm respectively. Monooxygenase activity of LM₃ (TAO) was compared with that of LM₂ and LM₄ in a reconstituted system containing NADPH cytochrome P-450 reductase and phosphatidylcholine; the activity of P-450 LM₃ (TAO) was higher than that of LM₂ and LM₄ with chlorcyclizine as a substrate. According to these observations, LM₃ (TAO) resembles LM₃ (b), a constitutive form of untreated rabbit liver microsomes.     

Spectroscopic quantitation of cytochrome P-450 in human lung microsomes

The cytochrome P-450 content of human lung microsomes was measured by difference spectroscopy of the carbon monoxide-complexed hemoprotein. These measurements were only possible after the microsome preparation had been subjected to centrifugation over a discontinuous sucrose gradient, to remove an opaque black contaminant. The specific concentration of total cytochrome P-450 in human lung microsomes is essentially identical to that of microsomes prepared under identical conditions from untreated baboon lungs, but is only 0.7% of the specific content found in lung microsomes from untreated rabbits. These measurements correspond well to the observed metabolic capacities of the various microsome samples.     

Occurrence of cytochrome P-450 with prostaglandin omega-hydroxylase activity in rabbit placental microsomes

The microsomes of placenta and uterus from pregnant rabbits have been found to catalyze the omega-hydroxylation of PGE1, PGE2, PGF2 alpha, and PGA1 as well as the omega- and (omega-1)-hydroxylation of palmitate and myristate in the presence of NADPH. These activities were greatly inhibited by carbon monoxide, indicating the involvement of cytochrome P-450. The apparent Km for PGE1 was 2.38 microM and 2.1 microM with the placental and uterus microsomes, respectively. Cytochrome P-450 has been solubilized with 1% cholate from the placental microsomes, and partially purified by chromatography on 6-amino-n-hexyl Sepharose 4B, DEAE-Sephadex A-50 and hydroxylapatite columns. The partially purified cytochrome P-450 efficiently catalyzed the omega-hydroxylation of various prostaglandins such as PGE1, PGE2, PGF2 alpha, PGD2, and PGA1 in a reconstituted system containing NADPH-cytochrome P-450 reductase, cytochrome b5, and phosphatidylcholine. The reconstituted system also hydroxylated palmitate and myristate at the omega- and (omega-1)-position, but could not hydroxylate laurate. These catalytic properties resemble those of a new form of cytochrome P-450 highly purified from the lung microsomes of progesterone-treated rabbits (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., and Kusunose, M. (1984) J. Biochem. 96, 593-603). This type of cytochrome P-450, viz., cytochrome P-450 with high prostaglandin omega-hydroxylase activity may play a role in the regulation of prostaglandin levels in pregnancy.     

Purification and characterization of human placental aromatase cytochrome P-450

Aromatase cytochrome P-450, which catalyzes the conversion of androgens to estrogens, was purified from human placental microsomes. The enzyme was extracted with sodium cholate, fractionated by ammonium sulfate precipitation, and subjected to column chromatography in the presence of its substrate, androstenedione, and the nonionic detergent, Nonidet P-40. The preparation exhibits a single major band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a specific content of 11.5 nmol of P-450/mg of protein. The purified enzyme displays spectroscopic properties typical of the ferric and ferrous forms of cytochrome P-450. Full enzymatic activity can be reconstituted with rabbit liver microsomal cytochrome P-450 reductase and Nonidet P-40. Purified aromatase cytochrome P-450 displays catalytic characteristics similar to the enzyme in intact microsomes in the aromatization of androstenedione, 19-hydroxyandrostenedione and 19-oxoandrostenedione. Testosterone and 16 alpha-hydroxytestosterone are aromatized at maximal rates similar to androstenedione, and all substrates exhibit relative affinities corresponding to those observed in microsomes. We have raised rabbit antibodies to the purified enzyme which show considerable specificity and sensitivity on immunoblots.     

Immunoaffinity purification of aromatase cytochrome P-450 from human placental microsomes, metabolic switching from aromatization to 1 beta and 2 beta-monohydroxylation, and recognition of aromatase isozymes

Microsomal estrogen synthetase (aromatase) cytochrome P-450 was purified from fresh human placental microsomes by monoclonal anti-aromatase P-450 antibody-Sepharose 4B chromatography. The purified P-450 showed a single band of 55 kDa on SDS-polyacrylamide gel electrophoresis and the aromatase specific activity on reconstitution was 70 nmol/min/mg protein. The purified P-450 was stable with a t 1/2 of approximately 2 years on storage at -90 degrees C and showed Km = 43 nM for androstenedione aromatization. However, it was unstable under spectral measurement conditions in the presence of sodium dithionite and carbon monoxide and the carbon monoxide difference spectra showed a maximum at 450 nm and a specific content of 9.1 nmol of P-450/mg protein, giving a turnover number of approximately 7.7 per min for the purified aromatase. The one-step immunochemical purification method gave a 490-fold increase of specific activity with 55% yield of aromatase activity of the original microsomes. Analysis of androgen metabolism by the purified aromatase and an apparent large kinetic isotope effect found at the secondary positions when using [19(-3)H3, 4(-14)C] androgens revealed metabolic switching from the first 19-hydroxylation to 1 beta- and 2 beta- monohydroxylation by aromatase. Substrate specificity for [19(-3)H3]androstenedione and testosterone was indicated by differences in the extent of metabolic switching (18% and 30%) and in the 2 beta/1 beta ratio (60/40 and 10/90, respectively). The mouse monoclonal antibody used for immunoaffinity purification suppresses aromatase activity of human placenta, but was totally ineffective for aromatase in goldfish brain and rat ovary. Rabbit polyclonal antibodies to human placental aromatase P-450 suppressed both human placental and rat ovarian aromatase but were ineffective for goldfish brain aromatase. The study indicates that they are isozymes of aromatase based on different structures of P-450.     

Novel properties of human placental aromatase as cytochrome P-450: purification and characterization of a unique form of aromatase

Aromatase has been purified to homogeneity from human placental microsomes based on detection of its catalytic activities in the eluates from columns of octylamino-Sepharose 4B, hydroxylapatite, Mono S, hydroxylapatite HCA, and Mono Q. The purified preparation shows only one band corresponding to the apparent subunit molecular weight of 51,000 daltons on sodium dodecyl sulfate-polyacrylamide gel. The aromatase in the presence of NADPH and NADPH-cytochrome P-450 reductase converts testosterone to 17 beta-estradiol with the high specific activity of 103 nmol/min/mg of protein. However, whether the preparation is reduced by sodium dithionate chemically or by NADPH and the reductase enzymatically, its reduced, CO-difference spectrum has no peak at about 450 nm and has only a small peak at about 420 nm, probably due to its inactivation in spite of the catalytically full activity in the same preparation. The absolute spectrum of the aromatase exhibits a Soret peak at 423 nm in the absence of testosterone and addition of testosterone to the aromatase sample makes its absorption peak shift gradually from 423 to 393 nm (high spin type peak), which is a usual characteristic in the spectrum of cytochrome P-450. The reconstituted aromatase system efficiently catalyzes aromatization of 4-androstenedione, 19-hydroxy-4-androstenedione as well as testosterone. 16 alpha-Hydroxy-4-androstenedione and 16 alpha-hydroxytestosterone are also aromatized less efficiently and 19-nortestosterone is aromatized least efficiently. The reconstituted aromatase could scarcely oxidize various xenobiotics examined, suggesting a strict and narrow substrate specificity of this enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of cytochrome P-450 in testis microsomes

Studies on the role of cytochrome P-450 in mouse, rat, and chick testis microsomes showed that this CO-binding hemoprotein is involved in the activity of the 17α-hydroxylase. A 70–80% inhibition by CO of the 17α-hydroxylase activity was detected in rat and chick testis microsomes. In the mouse testis, the level of the enzyme activity is ten times greater than that of the rat. This partly explains why an acceleration of NADPH oxidation by progesterone can be observed in mouse but not in rat testis microsomes. In rat testis microsomes, type I binding spectra of cytochrome P-450 was observed with pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione, and testosterone. The apparent K_s values for progesterone and 17-hydroxyprogesterone were 0.50 and 1.00 μm, respectively.When NADPH is used to measure cytochrome P-450 levels in rat testis microsomes, CO formation resulting from a stimulation in lipid peroxidation by phosphate or Fe²⁺ was sufficient to bind with 50% of the total amount of cytochrome P-450. Substitution of phosphate by Tris reduced the amount of lipid peroxidation to minimal levels. On a comparable basis, no CO formation was observed in avian testis microsomes.An increase in the testicular levels of cytochrome P-450 resulted upon the administration of HCG and cyclic-AMP to 1-day-old chicks. The lack of stimulation of the cytochrome P-450 levels by progesterone and pregnenolone suggest that the hormonal stimulation of the P-450 levels is not due to substrate induction.     

Isolation and partial purification of cytochrome P-450 from uninduced rat liver

Carbon-13 Fourier transform nmr has been used to make the first observation of a carbon-13-iron-57 spin-spin coupling constant in a protein, sperm whale carbonyl myoglobin enriched to 90% in both iron-57 and carbon-13. The coupling constant, 27.1±0.2 Hz, is found to be essentially identical to that of a model compound, supporting the view that the carbonyl is not tilted with respect to the heme plane in solution. Such carbon-13-iron-57 couplings, and the resultant iron-57 chemical shifts obtained from decoupling experiments, should provide valuable new tools for studying the different affinity states of tetrameric hemoglobins.     

Purification and immunological characterization of human placental mitochondrial cytochrome P-450

Human placental mitochondrial cytochrome P-450 was purified to electrophoretic homogeneity by hydrophobic, anion exchange and cation exchange column chromatography. The specific content of the purified protein was 15.7 nmol/mg protein and it showed a single band mol. wt 48,000 D in SDS-gel electrophoresis. When reconstituted with bovine adrenal adrenodoxin reductase and adrenodoxin it converted cholesterol to pregnenolone (cholesterol side-chain cleavage activity, CSCC) at the rate of 1 pmol/min/pmol P-450. Antibodies against the purified protein were raised in rabbits. Inhibition studies demonstrated 85% inhibition of placental CSCC activity at an antibody/protein ratio of 10:1. Placental microsomal aromatase activity was inhibited by 47% at the same antibody/protein ratio. The antibody inhibited bovine mitochondrial CSCC activity by 87% at the same antibody/protein ratio. Placental microsomal 7-ethoxycoumarin O-deethylase, aryl hydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase activities were not significantly inhibited by the antibody. The results indicate that the purified protein catalyzes cholesterol side-chain cleavage reaction, human placental microsomal aromatase and bovine adrenal mitochondrial P-450scc may share common antigenic determinants with placental P-450scc, but the placental microsomal xenobiotic-metabolizing cytochrome(s) is (are) distinctly different.     

Purification and properties of cytochrome P-450 from untreated monkey liver microsomes

Untreated monkey liver cytochrome P-450 (monkey P-450) has been purified to a specific content of 14.9 n mole/mg protein. The purified preparation was apparently homogeneous and the minimum molecular weight was estimated to be 50,000 by SDS-PAGE. Absolute spectrum of the oxidized form showed peaks at 565, 535 and 417 nm. The monkey P-450 was active in the mixed function oxidation of benzphetamine, aminopyrine, ethylmorphine, aniline and 7-ethoxycoumarin in the presence of rat liver NADPH-cytochrome P-450 reductase and DLPC. Anti monkey P-450 IgG could not inhibit rat P-450s (PB P-450, MC P-448(1) and MC P-448(2] catalyzed 7-ethoxycoumarin O-deethylation activities.     

Purification and reconstitution properties of human placental aromatase. A cytochrome P-450-type monooxygenase

The hemoprotein component of human placental aromatase (estrogen synthetase) has been purified to a high degree of homogeneity by a combination of affinity and adsorption chromatography on aminohexyl-Sepharose, concanavalin-A-Sepharose, and hydroxyapatite. The monomeric form of the enzyme has an Mr of 55000 +/- 1000 as estimated by sodium dodecyl sulfate gel electrophoresis. Its absolute spectrum shows a high-spin Soret band at 394 nm while its reduced, CO-difference spectrum has a maximum at 447 +/- 1 nm. Full reconstitution of aromatase activity was obtained when it was recombined with a homogeneous preparation of the higher-Mr form of either human placental, or bovine hepatic NADPH-cytochrome P-450 reductase. Critical factors for purification of the very unstable, membrane-bound hemoprotein with good retention of activity were, besides the chromatographic sequence, the use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) during the solubilization, and the stabilizing effect of the aromatase substrate, 4-androstene-3,17-dione, throughout the procedure. In the presence of NADPH, the reconstituted enzyme system smoothly aromatizes 19-oxoandrostenedione, 19-hydroxyandrostenedione and androstenedione in this order of reactivity. The same reconstituted system also aromatized testosterone, but it was inactive towards 19-norandrostenedione. Known cytochrome P-450 inhibitors decreased its activity. We conclude: (a) the terminal oxidase of human placental aromatase is indeed a cytochrome P-450-type monooxygenase; (b) the multistep aromatization reaction of C19 androstenes is catalyzed by a single enzyme; (c) aromatization of 19-norsteroids reported by other authors must be due to a different aromatase. Experimental data obtained with the reconstituted enzyme are fully compatible with the concept of a reaction mechanism for the aromatization sequence involving an all-trans, antiparallel elimination of the 19-methyl group, the 2 beta proton and the 1 alpha proton, rather than the 1 beta proton, as generally assumed.     

Isolation and properties of cytochrome P-450 from rabbit liver microsomes]

A purified low-spin form of cytochrome P-450 was isolated from phenobarbital-induced rabbit liver microsomes. The preparation was functionally active and free from cytochromes b5 and P-420 and phospholipids. The specific content of the cytochrome was 18 nmoles per mg of protein. At the molecular weight of the hemoprotein of 50,000, it corresponds to 90% of purification. The purified hemoprotein binds substrates of type II and some substrates of type I. The complexes formed reveal spectral properties, similar to those for the complexes of these substrates with the microsomal form of cytochrome P-450.     

Isolation of rat intestinal microsomes: partial characterization of mucosal cytochrome P-450

A procedure is presented for the isolation of subcellular fractions from small intestinal mucosal cells in the rat. The mucosal cells were detached by a scraping procedure resulting in an almost complete harvest of all types of cells as judged by light microscopy. Homogenization using a Potter-Elvehjem Teflon-glass device at high speed with ensuing sonication was found to be necessary for complete disruption of the cells. The subcellular fractions obtained after differential centrifugation--10,000g pellet, 105,000g pellet (microsomal fraction), and supernatant--were characterized with respect to different marker enzymes. The highest yield of 7-ethoxyresorufin-O-deethylase and NADPH-cytochrome c reductase activity in the microsomal fraction was achieved after resuspension and recentrifugation of the 10,000g pellet. Addition of anti-P-450 beta-naphthoflavone (BNF)-B2 antibodies to the incubation mixture resulted in almost complete inhibition of the O-deethylation of 7-ethoxyresorufin whereas addition of anti-P-450 phenobarbital (PB)-B2 had no effect. The presence of BNF-inducible isozymes was demonstrated by the Western blotting technique not only in intestinal microsomes from BNF-treated rats, but also in microsomes from untreated rats. Anti-P-450 BNF-B2 was also used in the peroxidase-antiperoxidase method for studies on the localization of cytochrome P-450. No BNF-inducible cytochrome P-450 could be detected in untreated rats, whereas BNF treatment resulted in a general staining of the whole villus.