Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation. I. Purification and spectral properties

A form of cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation (tentatively called "P-450(14)DM") was purified from microsomes of semi-anaerobically grown cells of Saccharomyces cerevisiae to gel electrophoretic homogeneity. An apparent monomeric Mr = 58,000 was estimated for the purified cytochrome by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both optical and EPR spectra of oxidized P-450(14)DM are characteristic of low spin ferric heme proteins, and its reduced CO complex showed a Soret absorption peak at 447 nm. As in the case of hepatic microsomal cytochromes P-450, the ethyl isocyanide complex of reduced P-450(14)DM was in a pH-dependent equilibrium between two states having Soret peaks at 429 and 453 nm, the equilibrium being considerably shifted toward the 453-nm state. Oxidized P-450(14)DM was peculiar in that in its CD spectrum there was a negative shoulder at 425 nm and the 350- and 414-nm troughs possessed larger and relatively smaller [theta] values, respectively, than those reported for other low spin ferric cytochromes P-450. Lanosterol was the only compound which caused a Type I spectral change in oxidized P-450(14)DM. The lanosterol-induced low to high spin state change was, however, only slight even at saturating concentrations of the sterol, indicating that the lanosterol-P-450(14)DM adduct was in a spin state equilibrium.     

Separation of two constitutive forms of cytochrome P-450 active in aminopyrine N-demethylation from rabbit intestinal mucosa microsomes

Two constitutive forms of cytochrome P-450, designated P-450ib and P-450ic, were purified from intestinal mucosa microsomes of untreated rabbits. P-450ib and P-450ic have minimal molecular weights of 56 000 and 49 000, respectively, as determined by calibrated sodium dodecyl sulphate polyacrylamide gel electrophoresis. The CO-reduced difference spectral maximum of cytochrome P-450ib is at 450 nm and P-450ic is at 451 nm. Both the cytochromes preferentially demethylate aminopyrine, benzphetamine and N,N-dimethylaniline in the presence of NADPH-cytochrome P-450 reductase. Cytochrome P-450ib has absorption maxima at 417, 535 and 573 nm in the oxidized form, indicating that this cytochrome is in a low-spin state. Ouchterlony double-diffusion studies show that cytochrome P-450ib does not cross-react with antisera against liver cytochrome P-450LM2 purified from phenobarbital-treated rabbits, but P-450ic cross-reacts with spur formation. Unlike cytochrome P-450ib, P-450ic is very similar, if not identical, to liver cytochrome P-450LM2 on the basis of its molecular weight, spectral properties, catalytic activities and immunochemical properties.     

Participation of cytochrome P-450 in nicotine oxidation

Addition of nicotine to phenobarbital-inducible cytochrome P-450 caused a shift of maximum of Soret peak toward the red approximately 3 nm. The difference spectrum produced by nicotine showed a type 2 spectral change with a peak at 427 nm and a trough at 393 nm. A spectral dissociation constant of phenobarbital-inducible cytochrome P-450 was found to be 0.16 mM for nicotine. Nicotine oxidation in the reconstituted system depended on cytochrome P-450, NADPH-cytochrome P-450 reductase and NADPH. These results indicate that phenobarbital-inducible cytochrome P-450 participates in nicotine oxidation.     

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus.

A soybean flour-induced, soluble cytochrome P-450 (P-450soy) was purified 130-fold to homogeneity from Streptomyces griseus. Native cytochrome P-450soy is a single polypeptide, with a molecular weight of 47,500, in association with one ferriprotoporphyrin IX prosthetic group. Oxidized P-450soy exhibited visible absorption maxima at 394, 514, and 646 nm, characteristic of a high-spin cytochrome P-450. The CO-reduced difference spectrum of P-450soy had a Soret maximum at 448 nm. When reconstituted with spinach ferredoxin and spinach ferredoxin:NADP+ oxidoreductase, purified cytochrome P-450soy catalyzed the NADPH-dependent oxidation of the xenobiotic substrates precocene II and 7-ethoxycoumarin. In vitro proteolysis of cytochrome P-450soy generated a stable and catalytically active cytochrome P-450, designated P-450soy delta.     

Purification and characterization of adrenal cortex mitochondrial cytochrome P-450 specific for cholesterol side chain cleavage activity.

Cytochrome P-450 was purified from bovine adrenal cortex mitochondria by affinity chromatography using an octylamine-substituted Sepharose column. The resulting optically clear preparation was stable at -20 degrees for months. The specific concentration of cytochrome P-450 in the preparation was about 5 nmol of heme per mg of protein. The preparations were free of adrenodoxin, adrenodoxin reductase, phospholipids, and other heme contaminations. Polyacrylamide gel electrophoresis of the purified cytochrome P-450 preparation treated with sodium dodecyl sulfate and mercaptoethanol showed a single major band with a molecular weight of about 60,000. The optical absorption spectra of the preparation exhibited Soret maxima at 416, 416, and 448 nm for the Fe3+, Fe2+ and the C.Fe2+ complex, respectively. The EPR spectrum showed the characteristic features of the low spin form of ferric cytochrome P-450 with principal components 1.914, 2.241, and 2.415 of the g-tensor. The circular dichroism spectrum revealed two large negative ellipticities at 412 and 350 nm. Fluorescence spectra showed an excitation maximum at 285 nm and an emission maximum at 305 nm with a shoulder at 330 nm as the cytochrome P-450 molecule is excited at 285 nm, or an emission maximum at 335 nm when the cytochrome molecule is excited at 305 nm. After reconstitution with adrenodoxin and its reductase, this cytochrome P-450 was highly active for cholesterol desmolase with an NADPH-generating system as electron donor but was not active for steroid 11beta-hydroxylase.     

Spectral properties of a novel cytochrome P-450 of a Saccharomyces cerevisiae mutant SG1. A cytochrome P-450 species having a nitrogenous ligand trans to thiolate

An altered cytochrome P-450 (SG1 P-450) was partially purified from Saccharomyces cerevisiae mutant SG1 which is defective in lanosterol 14 alpha-demethylation. Oxidized SG1 P-450 showed a Soret peak at 422 nm and the alpha peak was lower than the beta peak. This spectrum was considerably different from those of known low-spin P-450s, indicating a unique ligand structure of SG1 P-450. The absorption spectrum of ferric SG1 P-450 was superimposable on that of the imidazole complex of ferric P-450, suggesting the presence of a nitrogenous ligand such as histidine of the apoprotein at the 6th coordination position. SG1 P-450 was immunochemically indistinguishable from cytochrome P-450 of S. cerevisiae catalyzing lanosterol 14 alpha-demethylation (P-45014DM) but had no lanosterol 14 alpha-demethylase activity.     

Effects of detergent on substrate binding and spin state of purified liver microsomal cytochrome P-450LM2 from phenobarbital-treated rabbits

Spectral changes accompanying the binding of the nonionic detergent n-octyl beta-D-glucopyranoside (n-octyl glucoside) to cytochrome P-450LM2 purified from liver microsomes of phenobarbital-treated rabbits have been compared to changes in catalytic activity obtained in a reconstituted system consisting of various levels of detergent, P-450LM2, and NADPH-cytochrome P-450 reductase. In the absence of substrate and reductase, addition of n-octyl glucoside to 2-3 mM resulted in a difference spectrum (detergent-bound minus detergent-free cytochrome) characterized by a small maximum at 390 nm and a minimum at 410 nm, suggestive of a slight stabilization of the high-spin (S = 5/2) state of the cytochrome. As the detergent concentration was increased to 4-8 mM (corresponding to maximal activity and pentameric or hexameric P-450), a new peak appeared at 427 nm while the minimum remained at 410 nm. Between 10 and 30 mM n-octyl glucoside (conditions which produced catalytically inactive and monomeric P-450) the minimum in the difference spectrum shifted to 390 nm and the maximum to 425 nm, characteristic of a shift in spin equilibrium toward low-spin (S = 1/2) cytochrome. At low and high detergent concentrations, substrate [d-benzphetamine with n-octyl glucoside or cyclohexane with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)] was bound to P-450LM2 with formation of high-spin P-450, although the increase in high-spin cytochrome was less at high detergent levels than at low. The affinity of P-450 for substrate decreased by 2-3-fold at high detergent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Error in assay due to time dependency of carbon monoxide difference spectrum of reduced yeast cytochrome P-450: Slow reduction caused by Triton X-100 present

Triton X-100, added to yeast Saccharomyces cerevisiae for the purpose of stabilization or solubilization affects the carbon monoxide difference spectrum of reduced cytochrome P-450 and consequently the measurement of cytochrome P-450. Eight minutes is needed for 450-nm peak to reach its maximum height. Triton X-100 is shown to behave as a Type II substrate (absorption maximum at 418 nm and minimum at 390 nm) and to modulate the spin state of cytochrome P-450 from high to low form. Low-spin yeast cytochrome P-450 is reduced more slowly than the high-spin form.     

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).     

Isolation and characterization of an altered cytochrome P-450 from a yeast mutant defective in lanosterol 14 alpha-demethylation

A cytochrome P-450 (P-450SG1) was purified from a lanosterol 14 alpha-demethylase (P-450(14DM)) defective mutant of Saccharomyces cerevisiae, strain SG1, by a method similar to that used in the purification of the wild type enzyme (Yoshida, Y., and Aoyama, Y. (1984) J. Biol. Chem. 259, 1655-1660). P-450SG1 had the same apparent Mr as and was immunochemically identical to P-450(14DM). Peptide maps of P-450SG1 made by limited proteolysis with Staphylococcus aureus V8 proteinase, chymotrypsin, or papain followed by gel electrophoresis were identical to corresponding peptide maps of P-450(14DM). However, P-450SG1 showed no lanosterol 14 alpha-demethylase activity and its mode of interaction with diniconazole [(E)-1-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-y1)-1- penten-3- o1], a specific inhibitor of P-450(14DM), was fundamentally different from that of P-450(14DM). The absorption spectrum of ferric P-450SG1 was unusual for a native low-spin cytochrome P-450 and was superimposable on that of 1-methylimidazole complex of P-450(14DM), indicating that P-450SG1 has a histidine 6th ligand trans to the thiolate 5th ligand, while the 6th ligand of other ferric low-spin cytochrome P-450s is a water molecule or a hydroxyl group of an oxyamino acid. It is concluded that P-450SG1 is an altered P-450(14DM). Difference in the primary structure between P-450SG1 and P-450(14DM) may be slight and was not detected by peptide mapping. However, the alteration caused significant change in the substrate site and heme environments of the cytochrome. P-450SG1 is the first example of a cytochrome P-450 having a histidine axial ligand trans to thiolate and of a genetically altered cytochrome P-450 isolated in a homogeneous state.     

Induction of multiple cytochrome P-450 species in housefly microsomes--SDS-gel electrophoresis studies.

1. Microsomal fractions isolated from various housefly strains have been characterized with respect to multiple forms of cytochrome P-450 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2. Susceptible NAIDM houseflies were pretreated with known inducers of cytochrome P-450, and their microsomal electrophoretic profiles were compared to control NAIDM microsomes, using as standards partially purified cytochrome P-450s from noninduced NAIDM houseflies. 3. Tentatively, at least five different species of cytochrome P-450 may exist in the NAIDM housefly strain. 4. A comparison of the microsomal electrophoretic profile of different housefly strains also indicates the presence of at least two additional cytochrome P-450 species. 5. Induction with alpha-pinene and phenobarbital was expressed by a shift of the maximum absorbance at 452 nm in the CO-difference spectrum to lower wavelengths in the NAIDM strain; whereas, beta-naphthoflavone, although increasing the amount of cytochrome P-450, did not change the wavelength of maximum absorbance. 6. Cytochromes of the P-452 type appear to predominate in the susceptible NAIDM strain, while cytochromes of the P-450 and P-448 types predominate in resistant strains.     

Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid

Cytochrome P-450j has been purified to electrophoretic homogeneity from isoniazid-treated adult male rats; and this enzyme appears to be a major protein induced in hepatic microsomes after administration of isoniazid, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hemoprotein has a minimum molecular weight of approximately 51,500, and the ferrous-carbonyl complex of cytochrome P-450j has a Soret maximum at 451-452 nm. The oxidized heme iron appears to be predominately in the high spin state as deduced from the Soret maximum at 395 nm. Ethylisocyanide binds to ferrous cytochrome P-450j to yield spectral maxima at approximately 458 and 430 nm with a resultant 458/430 ratio of 0.7 at pH 7.4. Cytochrome P-450j has no measurable catalytic activity for the metabolism of benzo[a]pyrene (3- and 9-hydroxylation), hexobarbital, testosterone, and 5 alpha-androstane-3 alpha,17 beta-diol-3,17-disulfate. Low, but detectable, catalytic activity is obtained for the metabolism of 7-ethoxycoumarin, benzphetamine, p-nitroanisole, zoxazolamine, and 2-hydroxylation of 17 beta-estradiol. In contrast, cytochrome P-450j effectively catalyzes p-hydroxylation of aniline with a turnover of 12.7 nmol/min/nmol cytochrome P-450j. Hydroxyl radical scavengers, Fe-EDTA, superoxide dismutase, and catalase have no effect on aniline p-hydroxylation catalyzed by cytochrome P-450j. Cytochrome P-450j is distinct from nine other rat hepatic microsomal cytochromes P-450 (P-450a-P-450i) previously purified in this laboratory, as well as different isozymes described by other investigators, based on several parameters including minimum molecular weight, spectral properties, and catalytic activity. In Ouchterlony double diffusion plates, antibodies against cytochromes P-450a-P-450f show no cross-reaction with cytochrome P-450j. Structural differences among cytochromes P-450a-P-450j are apparent from the NH2-terminal sequence of cytochrome P-450j, as well as the electrophoretic profiles of proteolytic digests of the hemoproteins.     

Purification and characterization of cholesterol 7 alpha-hydroxylase cytochrome P-450 of untreated rabbit liver microsomes

Cytochrome P-450 which catalyzes the 7 alpha-hydroxylation of cholesterol was purified from liver microsomes of untreated rabbits. The minimum molecular weight of the cytochrome P-450 was estimated to be 48,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preparation contained 7 nmol of cytochrome per mg of protein. The oxidized form of the P-450 showed absorption maxima at 568, 535, and 417 nm, which are characteristic of a low spin hemoprotein, while the reduced form showed maxima at 545 and 413 nm. The carbon monoxide complex of the reduced form showed maxima at 550 and 447 nm. The cholesterol 7 alpha-hydroxylase system of untreated rabbit liver microsomes was reconstituted with the purified P-450, NADPH-cytochrome P-450 reductase, and cytochrome b5. The P-450 catalyzed the 7 alpha-hydroxylation of cholesterol 500 times more efficiently than the starting microsomes. The reconstituted hydroxylase system showed a substantial salt dependency. In the presence of cytochrome b5 the activity was maximum at 0.4 M KCl (4.55 nmol product formed/mg of protein per min), whereas in the absence of cytochrome b5 the activity was marginal (0.65 nmol product formed/mg of protein per min) and inhibited by KCl. Thus, cytochrome b5 stimulated the hydroxylase activity by one order of magnitude. These results indicate that cytochrome b5 is an essential component of the cholesterol 7 alpha-hydroxylase system of untreated rabbit liver microsomes.     

Conformational change of the heme moiety of the ferrous cytochrome P-450scc-phenyl isocyanide complex upon binding of reduced adrenodoxin

Reduction of cytochrome P-450scc(SF) (SF, substrate free) purified from bovine adrenocortical mitochondria with sodium dithionite (Na2S2O4) or with beta-NADPH mediated by catalytic amounts of adrenodoxin and adrenodoxin reductase in the presence of phenyl isocyanide produced a ferrous cytochrome P-450scc(SF)-phenyl isocyanide complex with Soret absorbance maximum at 455 nm having a shoulder at 425 nm. On the other hand, when a preformed cytochrome P-450scc(SF)-adrenodoxin complex was reduced chemically or enzymatically under the same conditions, the absorbance spectrum showed drastic changes, i.e., an increase in intensity at 425 nm and a concomitant decrease in intensity at 455 nm. Similar spectral changes could be produced by addition of the same amount of reduced adrenodoxin afterward to the ferrous cytochrome P-450scc(SF)-phenyl isocyanide complex. Titration experiments with adrenodoxin showed that (1) a 1:1 stoichiometric saturation of the spectral change was obtained for both the absorbance increase at 425 nm and the absorbance decrease at 455 nm, (2) there was no spectral change in the presence of 0.35 M NaCl, and (3) there was no spectral change for cytochrome P-450scc(SF) whose Lys residue(s) essential to the interaction with adrenodoxin had been covalently modified with PLP. These results suggest that ternary complex formation of ferrous cytochrome P-450scc(SF)-phenyl isocyanide with reduced adrenodoxin caused a conformational change around the ferrous heme moiety. By analysis of temperature and pH dependencies of the spectral change of the ternary complex, it was suggested that this conformational change may reflect the essential step for electron transfer from reduced adrenodoxin to the ferrous-dioxygen complex of cytochrome P-450scc.     

Purification and NH2-terminal sequence of cytochrome P-450 from kidney microsomes of untreated male rats

The major form of cytochrome P-450, P-450K-5, was purified from kidney microsomes of untreated male rats with high-performance liquid chromatography with anion-exchange and hydroxylapatite columns. The monomeric molecular weight of P-450K-5 was 52000 on SDS-polyacrylamide gel electrophoresis and the CO-reduced absorption maximum was at 452 nm. P-450K-5 catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was inefficient in the N-demethylation of benzphetamine and the O-dealkylation of 7-ethoxycoumarine. The NH2-terminal sequence of P-450K-5 was quite different from cytochrome P-450s purified from rat hepatic microsomes.     

Purification and characterization of a previously unreported form of cytochrome P-448 from the liver of 3-methylcholanthrene-pretreated rats.

At least four hepatic isoenzymes of cytochrome P-450 were purified and characterized from rats treated with 3-methylcholanthrene. A monoclonal antibody developed against one of the forms (designated cytochrome P-450 MC-B) and polyclonal antibodies against others were used to demonstrate that form MC-B is immunologically distinct from other methylcholanthrene-inducible forms. Limited N-terminal amino acid sequencing showed that cytochrome P-450 MC-B has a primary structure that differs from the N-terminal sequences of other established rat isoenzymes. Cytochrome P-450 MC-B has a minimum Mr of 53,000, a CO-reduced spectral maximum at 448 nm, a Soret maximum of 417 nm in the absolute oxidized spectrum and a pattern of substrate preferences that differs from those of the other methylcholanthrene-induced forms. The other forms (MC-A, MC-C and MC-D) share characteristics with isoenzymes previously reported by other investigators.     

Acetone-inducible cytochrome P-450: purification, catalytic activity, and interaction with cytochrome b5

A procedure was developed for the purification of an acetone-inducible form of cytochrome P-450 (P-450ac) to electrophoretical homogeneity from liver microsomes of acetone-treated rats. The P-450ac preparation containing 16.0 to 16.5 nmol P-450/mg protein moved as a single protein band with an estimated molecular weight of 52,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate. The ferric P-450ac showed an absorption maximum at 394 nm at 25 degrees C, suggesting that it exists mainly in the high-spin form. It also existed in the low-spin form, especially at lower temperatures, as indicated by the absorption maximum in the 412-nm region. Upon reconstitution with NADPH: cytochrome P-450 reductase and phospholipid, P-450ac efficiently catalyzed both the demethylation and denitrosation of N-nitrosodimethylamine (NDMA) showing Vmax values of 23.8 and 2.3 nmol min-1 nmol P-450-1, respectively. The catalytic activity of P-450ac was greatly affected by cytochrome b5 which decreased the Km values of these reactions by a factor of 10 and increased the Vmax values. Cytochrome b5 appeared to interact with P-450 at a molar ratio of 1:1 and an intact cytochrome b5 structure was required for such interaction. Among the substrates studied, the demethylation of NDMA was affected the most by cytochrome b5 and showed the highest rate. P-450ac also catalyzed the oxygenation of N-nitrosomethylethylamine and aniline and the activity was enhanced slightly by cytochrome b5. Cytochrome b5 did not enhance the P-450ac-catalyzed metabolism of other drug substrates such as benzphetamine, aminopyrine, and ethylmorphine. P-450ac appeared to be similar in property to the previously studied rat P-450et (ethanol-inducible), rat P-450j (isoniazid-inducible), and rabbit P-450LM3a (ethanol-inducible). These P-450 species represent a new class of P-450 isozymes that are important in the metabolism of many endobiotics and xenobiotics.     

Heterogeneity of liver microsomal cytochrome P-450: the spectral characterization of reactants with reduced cytochrome P-450.

The aerobic metabolism of benzphetamine by liver microsomes, during a cytochrome P-450-catalyzed mixed-function oxidation reaction, results in the formation of an easily detected spectral complex with an absorption band maximum at 456 nm. Electron paramagnetic resonance studies, as well as studies with the chemical reductant, sodium dithionite, or the oxidant, potassium ferricyanide, indicate that the spectral complex results from the formation of a product adduct with reduced cytochrome P-450. The spectral properties of this product complex of cytochrome P-450 have been compared to those observed with carbon monoxide, metyrapone, and ethylisocyanide. The reaction of these reagents to specific pools of microsomal cytochrome P-450 permits the identification of at least two major and two minor types of cytochrome P-450 in liver microsomes prepared from phenobarbital-treated rats.     

Induction and substrate specificity of the lanosterol 14 alpha-demethylase from Saccharomyces cerevisiae Y222.

The potential inducibility of the lanosterol 14 alpha-demethylase (P-45014DM) from Saccharomyces cerevisiae Y222 by xenobiotics was investigated. This enzyme and NADPH-cytochrome P-450 reductase were unaffected by a number of compounds known to induce mammalian and some yeast cytochrome P-450 monooxygenases. Furthermore, dibutyryl cyclic AMP did not affect P-45014DM or P-450 reductase levels, while growth at 37 degrees C resulted in a slight decrease. P-45014DM was found to be specific for lanosterol and did not metabolize a number of P-450 substrates including benzo[a]pyrene.     

Purification and characterization of a form of cytochrome P-450 with high specificity for aflatoxin B1 from 3-methylcholanthrene-treated hamster liver

A form of cytochrome P-450 highly active in inducing mutagenicity of aflatoxin B1 was purified to a specific content of 15.1 nmol/mg of protein from 3-methylcholanthrene-treated hamster liver. This species of cytochrome P-450, having its absorption maximum at 448.5 nm in carbon monoxide-complex of reduced form and low spin ferric ion, is of molecular weight of 56,000 and distinctly different in physicochemical and catalytic properties from major forms of cytochrome P-450 purified from phenobarbital- or 3-methylcholanthrene-treated rat liver. In the induction of aflatoxin B1 mutagenicity, this hamster cytochrome P-450 is 50 times more potent than those from rat liver.