Subject(s)
Disorders of Sex Development/metabolism , Molybdenum/pharmacology , Receptors, Androgen/metabolism , Receptors, Steroid/metabolism , Adolescent , Adult , Androgen-Insensitivity Syndrome/metabolism , Cells, Cultured , Centrifugation, Density Gradient , Child , Child, Preschool , Cytosol/metabolism , Dihydrotestosterone/metabolism , Disorders of Sex Development/genetics , Drug Stability , Fibroblasts/metabolism , Gynecomastia/metabolism , Hot Temperature , Humans , Infant , Infertility, Male/metabolism , Male , Middle Aged , Receptors, Androgen/drug effects , SyndromeABSTRACT
Steroid 5 alpha-reductase in skin fibroblasts varies in two ways: 1) mean activity in uncloned fibroblasts corresponds to the activity in skin from which fibroblasts are derived (high in genital skin and low in nongenital skin); 2) activity in normal genital skin fibroblasts varies over a wide range (more than 200-fold). Studies of activity in fibroblast clones provides an explanation for this variability. Activity in clones of genital skin fibroblasts varies from the limits of detectability to very high levels, whereas activity in fibroblasts cloned from nongenital skin is uniformly low. The mean value of 5 alpha-reductase in genital skin clones is similar to the value in uncloned fibroblasts from the same explant. This suggests that the numbers of high and low activity cells in a given explant influence the overall enzyme activity. Variability among normal genital skin strains is also due to inherent differences in the range of enzyme activities. Furthermore, high activity genital skin clones give rise to a mixture of both high and low-activity subclones, whereas low activity genital skin and nongenital skin clones give rise to populations with uniformly low activity. Thus, with time genital skin gives rise to high activity fibroblasts that continually undergo shift in phenotypic expression to low activity.
Subject(s)
3-Oxo-5-alpha-Steroid 4-Dehydrogenase/genetics , Genetic Variation , Oxidoreductases/genetics , Skin/enzymology , 17-Hydroxysteroid Dehydrogenases/metabolism , 3-Oxo-5-alpha-Steroid 4-Dehydrogenase/metabolism , Adenosine Deaminase/metabolism , Clone Cells , Female , Fibroblasts/enzymology , Humans , Male , Organ Specificity , PhenotypeABSTRACT
Ecdysone 3-epimerase was partially purified by ammonium sulfate fractionation from the 100,000 g supernate of Manduca sexta midguts. The enzyme converts ecdysone and 20-hydroxyecdysone to their respective 3-epimers, requires NADH or NADPH and O2 for this reaction, and has the following kinetic parameters: for ecdysone, Km = 17.0 +/- 1.4 microM, Vmax = 110.6 +/- 14.6 pmol min-1 mg-1; for 20-hydroxyecdysone, Km = 47.3 +/- 7.5 microM, Vmax = 131.0 +/- 3.5 pmol min-1 mg-1: for NADPH, Km = 85.4 +/- 10.6 microM; for NADH, Km = 51.3 +/- 1.3 microM. The reaction is irreversible and can be inhibited by various ecdysteroids.
Subject(s)
Ecdysone , Intestines/enzymology , Isomerases/isolation & purification , Lepidoptera/enzymology , Steroid Isomerases/isolation & purification , Animals , Chromatography, High Pressure Liquid , NAD/metabolism , NADP/metabolism , Steroid Isomerases/antagonists & inhibitorsABSTRACT
NADPH-cytochrome c (P-450) reductase (EC 1.6.2.4) was purified to apparent homogeneity from microsomes of house flies, Musca domestica L. The purification procedure involves column chromatography on three different resins. The key step in the purification scheme is the chromatography of the enzyme mixture on an affinity column of agarose-hexane-nicotinamide adenine dinucleotide phosphate. The enzyme has an estimated molecular weight of 83,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 mol each of FAD and FMN per mol of enzyme. The enzyme exhibited a Bi Bi ping-pong kinetic mechanism with NADPH and cytochrome c. The Vmax and Km for cytochrome c were 42.3 mumol min-1 mg-1 and 12.7 muM, respectively. Turnover numbers based on micromoles of enzyme were 2,600 min-1. NADP+ and 2'-AMP both inhibited the reductases with apparent Ki values of 6.9 and 187 muM, respectively. These preparations of NADPH-cytochrome c reductase were found to reduce purified house fly cytochrome P-450 in the presence of NADPH.
