The enzyme activity of bovine adrenocortical cytochrome P-450 producing pregnenolone from cholesterol: Kinetic and electrophoretic studies on the reactivity of hydroxycholesterol intermediates

1. Changes in certain kinetic properties (Vmax. and apparent Km) of hepatic microsomal mixed-function oxidases have been studied as a function of postnatal development and maturation in male rats. 2. Microsomal cytochrome P-450 content changed only slightly between 1 and 12 weeks of age. 3. Aniline hydroxylase activity (Vmax.) increased abruptly between 1 and 2 weeks of age to greater than adult activities and then returned to a plateau value between 4½ and 12 weeks of age. Ethylmorphine demethylase activity remained low and relatively constant between 1 and 3 weeks of age and then increased markedly (∼100%) between 3 and 4½ weeks. 4. The apparent Michaelis constant (Km) for aniline hydroxylation increased almost linearly with time between 1 and 6 weeks of age and tended to reach a plateau value thereafter. The apparent Km for ethylmorphine demethylation increased between 1 and 3 weeks of age and then decreased abruptly to a constant value between 6 and 12 weeks. 5. The data indicate that developmental changes in the activity of these microsomal oxidases do not correlate temporally with each other or with changes in microsomal cytochrome P-450 content. 6. The most dramatic changes in enzyme activity were associated with early development (1–3 weeks) and weaning (3–4 weeks). 7. Changes in weight of seminal vesicle, a criterion of sexual maturation in male rats, were most prominent between 6 and 8 weeks of age and thus appeared to be separated in time from the prominent changes in enzyme activity.

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Cytochrome P-450-dependent 14 α-demethylation of lanosterol in Candida albicans

Biochemical Journal ◽

10.1042/bj2600549 ◽

1989 ◽

Vol 260 (2) ◽

pp. 549-556 ◽

Cited By ~ 22

Author(s):

C A Hitchcock ◽

S B Brown ◽

E G V Evans ◽

D J Adams

Keyword(s):

Enzyme Activity ◽

Candida Albicans ◽

Fungal Pathogen ◽

Reciprocal Relationship ◽

Sterol Biosynthesis ◽

Assay System ◽

Opportunistic Fungal Pathogen ◽

Cytochrome P 450

A novel assay for cytochrome P-450-dependent 14 alpha-sterol demethylase of the important opportunistic fungal pathogen, Candida albicans, is described. The enzyme was assayed in microsomal preparations (microsomes) by measuring the incorporation of [14C]lanosterol into (4,14)-desmethylated sterols. The efficacy of different cell-breakage methods was compared; desmethylated-sterol biosynthesis was maximal when cells were broken with a Braun disintegrator. The solubilization of [14C]lanosterol with detergent in the assay system was essential for enzyme activity, which was enhanced considerably when microsomes were gassed with O2. Under these conditions, there was a reciprocal relationship between the amount of radioactivity incorporated into desmethylated sterols and that lost from lanosterol. The major radiolabelled desmethylated sterol was ergosterol. The enzyme had an apparent Km of 52.73 +/- 2.80 microM and an apparent Vmax of 0.84 +/- 0.14 nmol/min per mg of protein (n = 3). Enzyme activity was decreased greatly when microsomes were treated with CO or the triazole antifungal ICI 153066.

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An anti-peptide antibody targeted to a specific region of rat cytochrome P-450IA2 inhibits enzyme activity

Biochemical Journal ◽

10.1042/bj2660497 ◽

1990 ◽

Vol 266 (2) ◽

pp. 497-504 ◽

Cited By ~ 25

Author(s):

R J Edwards ◽

A M Singleton ◽

B P Murray ◽

D Sesardic ◽

K J Rich ◽

...

Keyword(s):

Enzyme Activity ◽

Amino Acid Sequences ◽

Enzyme Inactivation ◽

Enzyme Linked Immunosorbent Assay ◽

Variable Regions ◽

Aryl Hydrocarbon ◽

Hepatic Microsomes ◽

Peptide Antibody ◽

A Site ◽

Cytochrome P 450

An anti-peptide antibody has been produced which binds to and specifically inhibits the activity of cytochrome P-450IA2 in rat hepatic microsomes. This was achieved by raising an antibody against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn), the sequence of which occurs in cytochrome P-450IA2 at positions 290-296. The selection of this region of cytochrome P-450IA2 was based on several criteria, including prediction of surface and loop areas, identification of variable regions between cytochromes P-450IA2 and P-450IA1, and consideration of a site on cytochrome P-450IA1 where chemical modification has been shown to cause substantial enzyme inactivation. The specificity of antibody binding was determined by enzyme-linked immunosorbent assay and by immunoblotting using hepatic microsomal preparations and purified cytochrome P-450 isoenzymes. This showed that the antibody binds specifically to rat and mouse cytochrome P-450IA2 and to no other cytochrome P-450, as was predicted from the amino acid sequences of the peptide and the cytochromes P-450. The effect of the antibody upon enzyme activity was studied in hepatic microsomes from rats treated with 3-methylcholanthrene. The antibody was shown to inhibit specifically the activity of reactions catalysed by cytochrome P-450IA2 (phenacetin O-de-ethylase and 2-acetylaminofluorene activation), but had no effect on aryl hydrocarbon hydroxylase activity, which is catalysed by cytochrome P-450IA1, or on aflatoxin B1 activation.

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Ovarian aromatase cytochrome P-450 mRNA levels correlate with enzyme activity and serum estradiol levels in anestrous, pregnant and lactating rats

Molecular and Cellular Endocrinology ◽

10.1016/0303-7207(92)90259-9 ◽

1992 ◽

Vol 85 (3) ◽

pp. 205-214 ◽

Cited By ~ 21

Author(s):

Edwin D. Lephart ◽

Evan R. Simpson ◽

Michael J. McPhaul

Keyword(s):

Enzyme Activity ◽

Mrna Levels ◽

Serum Estradiol ◽

Cytochrome P 450 ◽

Estradiol Levels

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Quantification of NADPH: cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique

Biochemical Journal ◽

10.1042/bj2110333 ◽

1983 ◽

Vol 211 (2) ◽

pp. 333-340 ◽

Cited By ~ 38

Author(s):

E A Shephard ◽

I R Phillips ◽

R M Bayney ◽

S F Pike ◽

B R Rabin

Keyword(s):

Enzyme Activity ◽

Rat Liver ◽

Liver Microsomes ◽

Fold Increase ◽

Molar Ratio ◽

Nadph Cytochrome ◽

Specific Radioimmunoassay ◽

Microsomal Membranes ◽

Cytochrome P 450

We have developed a specific radioimmunoassay to quantify NADPH: cytochrome P-450 reductase. The assay is based on the use of 125I-labelled NADPH: cytochrome P-450 reductase as the radiolabelled antigen and can detect quantities of this protein in amounts as low as 30 pg. The results of the radioimmunoassay demonstrates that the 2.7-fold increase in enzyme activity in rat liver microsomal membranes after phenobarbital treatment is due to increased amounts of the protein. beta-Naphthoflavone treatment, however, did not alter the activity or the quantity of this enzyme in microsomes. The quantification of NADPH: cytochrome P-450 reductase in the microsomes isolated from control and phenobarbital- and beta-naphthoflavone-treated animals permits the calculation of the ratio of this protein to that of total cytochromes P-450. A molar ratio of 15:1 (cytochromes P-450/NADPH: cytochrome P-450 reductase) was calculated for control and phenobarbital-treated animals. This ratio increased to 21:1 after beta-naphthoflavone treatment. Thus the molar ratio of these proteins in liver microsomes can vary with exposure of the animals to particular xenobiotics.

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Correction: Cobalt Induction of Hepatic Heme Oxygenase; with Evidence that Cytochrome P-450 is not Essential for this Enzyme Activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.72.2.772 ◽

1975 ◽

Vol 72 (2) ◽

pp. 772-772

Keyword(s):

Enzyme Activity ◽

Heme Oxygenase ◽

Cytochrome P 450

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Ecdysone 20-mono-oxygenase in the desert locust, Schistocerca gregaria

Biochemical Journal ◽

10.1042/bj2230837 ◽

1984 ◽

Vol 223 (3) ◽

pp. 837-847 ◽

Cited By ~ 38

Author(s):

D R Greenwood ◽

H H Rees

Keyword(s):

Enzyme Activity ◽

Schistocerca Gregaria ◽

Specific Activity ◽

Monochromatic Light ◽

Malpighian Tubule ◽

Malpighian Tubules ◽

Lower Sensitivity ◽

Marker Enzyme ◽

Energy Dependent ◽

Cytochrome P 450

The enzyme catalysing the hydroxylation of ecdysone to 20-hydroxyecdysone, ecdysone 20-mono-oxygenase (EC 1.14.99.22), was investigated in the Malpighian tubules of fifth-instar locusts, Schistocerca gregaria. Enzyme activity was optimal at 35 degrees C and pH 6.8-8.0. Under these conditions the mono-oxygenase exhibited an apparent Km for ecdysone of 7.1 × 10(-7) M, a maximal specific activity of 1.1 nmol/h per mg of protein and was competitively inhibited by 20-hydroxyecdysone with an apparent Ki of 6.3×10(-7) M. Enzyme activity was decreased in the presence of Ca2+, Mg2+, EDTA and non-ionic detergents. The Malpighian tubule ecdysone 20-mono-oxygenase was localized primarily in the subcellular fraction sedimenting at 7500 g and, on the basis of marker enzyme profiles, was assigned mainly to the mitochondria. NADPH was required for activity, although addition of NADH together with NADPH had a synergistic effect. NADP+-dependent isocitrate dehydrogenase (EC 1.1.1.42) and an energy-dependent NAD(P) transhydrogenase (EC 1.6.1.1.) appeared to be the major sources of reducing equivalents, with the contribution from the ‘malic enzyme’ (EC 1.1.1.40) being less important. The monooxygenase was characterized as a cytochrome P-450-containing mixed-function oxidase from the inhibition patterns with metyrapone, CO and cyanide; CO inhibition was reversible with monochromatic light at 450 nm. However, the ecdysone 20-mono-oxygenase shows much lower sensitivity to CO inhibition and to photodissociation of the CO-inhibited complex than do vertebrate cytochrome P-450-dependent hydroxylation systems. The concentration of cytochrome P-450 in the Malpighian tubule mitochondria was 30 pmol/mg of protein. The properties of the mono-oxygenase are discussed in relation to hydroxylation enzymes from other sources.

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Corticosterone 6 beta-hydroxylase in A6 epithelia: a steroid-inducible cytochrome P-450

AJP Cell Physiology ◽

10.1152/ajpcell.1990.258.3.c480 ◽

1990 ◽

Vol 258 (3) ◽

pp. C480-C488 ◽

Cited By ~ 19

Author(s):

W. M. Grogan ◽

V. M. Phillips ◽

E. G. Schuetz ◽

P. S. Guzelian ◽

C. O. Watlington

Keyword(s):

Enzyme Activity ◽

Epithelial Cells ◽

Rat Liver ◽

Time Course ◽

Effective Concentration ◽

Enzyme Protein ◽

Inducer Concentration ◽

A6 Epithelia ◽

Phenobarbital Sodium ◽

Cytochrome P 450

We found microsomal corticosterone 6 beta-hydroxylase (6 beta-OHase) from cultured A6 kidney epithelial cells to be a cytochrome P-450 enzyme with both similarities to and differences from the rat liver steroid 6 beta-OHase P-450p. Enzyme activity was inhibited by CO, alpha-naphthoflavone, metyrapone, and clotrimazole, well-known inhibitors of P-450 enzymes, and increased by known inducers of P-450 enzymes, including dilantin, phenobarbital sodium, and corticosteroids. Moreover, some additional, relatively specific inducers of P-450p (troleandomycin and pregnenolone-16 alpha-carbonitrile) also induced the A6 6 beta-OHase, whereas inducers of other forms of P-450 (aroclor, spironolactone, and isosafrole) appeared to repress the A6 enzyme. The time course of increase in enzyme activity and increased cellular cytochrome P-450 content were consistent with increased levels of enzyme protein. Induction of 6 beta-OHase by the substrate (corticosterone), the metabolite (6 beta-OH-corticosterone), dexamethasone, and aldosterone was biphasic as a function of inducer concentration, with approximate 50% effective concentration (EC50) values of 10(-8)-10(-9) M and 10(-5)-10(-6) M for the respective components of induction. Cortisol also induced the enzyme at 10(-8)-10(-6) M; however, its metabolite 6 beta-OH-cortisol was ineffective or decreased activity at higher concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

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