Microsomal coumarin 7-hydroxylase activity is regulated differently from several other monooxygenase enzymes, at least in mice [Wood, A. W. and Conney, A. H. (1974) Science (Wash. DC) 612-6141. Recently we found that in D2 mice this activity is strongly and selectively induced by pyrazole [Juvonen, R. O., Kaipainen, P. K. and Lang, M. A. (1985) Eur. J . Biochem. 152,. This paper describes the purification of the pyrazoleinducible cytochrome P-450 isoenzyme. Because of the lability of the protein, a special procedure for the purification was developed. The procedure is based on a combination of hydrophobic and ion-exchange chromatography and the presence of 100 FM coumarin in the preparations throughout the whole purification.Coumarin effectively protected the P-450 from degradation and also converted the pyrazole-inducible P-450 to its high-spin state. This enabled us to choose only those fractions for further purification where the P-450(s) was in its high-spin state (rather than measuring the content of the total P-450). As a result the purified protein had an apparent molecular mass of 49.7 kDa, a specific content of 19.9 nmol/mg protein and a very high affinity and metabolic capacity for coumarin.It has been suggested that the Coh locus regulates the 7-hydroxylation of coumarin (Coh for coumarin hydroxylase). This is based on the fact that the liver microsomal coumarin 7-hydroxylase activity varies among inbred strains of mice and is differently regulated from other liver microsomal P-450-dependent reactions [l]. A high microsomal coumarin 7-hydroxylase activity has also been found in rabbits, guinea-pigs, hamsters, pigs and humans [2-41. As a structural gene product of this locus, a so-far unknown cytochrome P-450 isoenzyme, capable of catalyzing the 7-hydroxylation of coumarin, was suggested to exist [5]. In 1985 Kaipainen et al. [6] from our laboratory gave the first direct evidence for the existence of this protein by purifying a cytochrome P-450 isoenzyme from liver microsomes of phenobarbital-pretreated D2 mice with a high affinity and specificity for coumarin 7-hydroxylation.Recently we found [7] that microsomal coumarin 7-hydroxylase is strongly increased in mice by pyrazole, a compound better known as an inhibitor of alcohol dehydrogenase. This induction is interesting in at least three different respects. First, the effect of pyrazole is much stronger than that of phenobarbital, the only inducer so far known to induce coumarin 7-hydroxylase. Second, the induction was strong only in D2 mice and weak or absent in B6 or AKR mice. Third, the effect on microsomal P-450-catalyzed reactions was selective, unlike that of phenobarbital, since only coumarin 7-hydroxylation (and to a lesser extent 7-ethoxycoumarin 0-deethylase) was increased by the compound while several other P-450-Correspondence to M. A. Lang, Farmakologian Laitos ja Toksikologian Laitos, Kuopion Yliopisto, Postilokero 6, SF-7021 1 Kuopio, Finland dependent reactions, in addition to the total microsomal P-450 content, were either not affe...

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(3/4) This review summarizes data about structural and functional organization of steroidogenic P450-dependent enzymatic systems. Problems of catalysis of steroid substrate transformation, special features of mitochondrial type P450scc topogenesis, and abilities of some microbial electron transport proteins to support P450 activity in vitro and in vivo are considered. Principal steps in the creation and catalytic properties of transgenic strains of Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica expressing both mammalian steroidogenic P450s and the corresponding electron transport proteins are also described. Achievements and prospects of using such transgenic strains for biotechnological synthesis and pharmacological screening are considered.

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The effect of ultrasound (US) (26.4 kHz, 26 W/cm2) on the activation process of a mixture of chymotrypsinogen and trypsinogen was studied. US led to a significant decrease in proteolytic activity, as well as inhibition of the activation process in general. It was shown that inhibition of proteinase activity under US influence was a consequence of inhibition of chymotrypsinogen-chymotrypsin transformation and the complete proteolytic trypsin degradation in the proenzymes mixture.

The fluorescent probes Nile Red (nonsteroidal dye) and 25-{N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino}-27-norcholesterol (25-NBD-cholesterol) (a cholesterol analog) were evaluated as novel substrates for steroid-converting oxidoreductases. Docking simulations with AUTODOCK showed that Nile Red fits well into the substrate-binding site of cytochrome P450 17a-hydroxylase/17,20-lyase (CYP17A1) (binding energy value of À8.3 kcalÁmol À1 ). Recombinant Saccharomyces cerevisiae and Yarrowia lipolytica, both expressing CYP17A1, were found to catalyze the conversion of Nile Red into two N-dealkylated derivatives. The conversion by the yeasts was shown to increase in the cases of coexpression of electron-donating partners of CYP17A1. The highest specific activity value (1.30 AE 0.02 min À1 ) was achieved for the strain Y. lipolytica DC5, expressing CYP17A1 and the yeast's NADPH-cytochrome P450 reductase. The dye was also metabolized by pure CYP17A1 into the N-dealkylated derivatives, and gave a type I difference spectrum when titrated into low-spin CYP17A1. Analogously, docking simulations demonstrated that 25-NBD-cholesterol binds into the active site of the microbial cholesterol oxidase (CHOX) from Brevibacterium sterolicum (binding energy value of À5.6 kcalÁmol À1 ). The steroid was found to be converted into its 4-en-3-one derivative by CHOX (K m and k cat values were estimated to be 58.1 AE 5.9 lM and 0.66 AE 0.14 s À1 , respectively). The 4-en-3-one derivative was also detected as the product of 25-NBD-cholesterol oxidation with both pure microbial cholesterol dehydrogenase (CHDH) and a pathogenic bacterium, Pseudomonas aeruginosa, possessing CHOXs and CHDHs. These results provide novel opportunities for investigation of the structure-function relationships of the aforementioned oxidoreductases, which catalyze essential steps of steroid bioconversion in mammals (CYP17A1) and bacteria (CHOX and CHDH), with fluorescence-based techniques. -methyl]amino}-27-norcholesterol; ABC, ATP-binding cassette; AdR, adrenodoxin reductase; Adx, adrenodoxin; CHDH, microbial cholesterol dehydrogenase; CHOX, microbial cholesterol oxidase; Nile Red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one; CPR, NADPH-cytochrome P450 reductase; CYP, cytochrome P450; CYP17A1, cytochrome P450 17a-hydroxylase/17,20-lyase; HPLC-FL, HPLC with fluorometric detection; HSD, hydroxysteroid dehydrogenase; PDB, Protein Data Bank. Abbreviations

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