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At least 91 records · Page 5Linked to original sources
PubMed1997

Synthesis of novel 21-trifluoropregnane steroids: inhibitors of 17 alpha-hydroxylase/17,20-lyase (17 alpha-lyase).

Novel 21-trifluoropregnenolone (6), 21-trifluoroprogesterone (7) and related compounds 4a and 8 have been synthesized in high yields from 3 beta-acetoxyandrost-5-ene-17 beta-carbaldehyde (3). The key reaction was the conversion of 3 into the 21-trifluoromethyl-20-alcohol as a diastereomeric mixture (4) by trifluoromethyltrimethylsilane (TMS-CF3) in the presence of tetrabutylammonium fluoride (TBAF). All compounds, including 6 and 7, were unambiguously characterized by IR, 1H and 19F NMR, high-resolution mass spectrometry (HRMS), and elemental analysis. On this basis, we concluded that the only report of an earlier synthesis of 6 and 7 is erroneous. Enzyme inhibition studies showed that 20 xi-hydroxy-21-trifluoropregn-4-en-3-one (8) is a potent inhibitor (IC50 value = 0.6 microM) of rat 17 alpha-hydroxylase/17,20-lyase.

Animals↗
PubMed1993

Expression and purification of functional human 17 alpha-hydroxylase/17,20-lyase (P450c17) in Escherichia coli. Use of this system for study of a novel form of combined 17 alpha-hydroxylase/17,20-lyase deficiency.

Enzymatically active human 17 alpha-hydroxylase cytochrome P450 (P450c17) has been expressed in and purified from Escherichia coli. The cDNA containing modifications within the amino-terminal eight codons which are favorable for expression in E. coli, as well as codons for 4 histidine residues at the carboxyl terminus, was placed in the pCWori+ expression vector. The modified human P450c17 was detected spectrophotometrically (400 nmol of P450c17/liter culture) and was found to be integrated into E. coli membranes. This previously inaccessible human P450 was purified to electrophoretic homogeneity (10.7 nmol of P450/mg) from solubilized bacterial membranes using two sequential chromatographic steps, nickel nitrilotriacetate followed by hydroxylapatite. The expected enzymatic activities of human P450c17 were reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase, giving turnover numbers of 8.0 nmol/min/nmol P450 for pregnenolone, 6.5 nmol/min/nmol P450 for progesterone, 0.06 nmol/min/nmol P450 for 17 alpha-hydroxypregnenolone, and no detectable activity for 17 alpha-hydroxyprogesterone. This system was utilized to study the molecular basis of a novel form of combined 17 alpha-hydroxylase, 17,20-lyase deficiency resulting from compound heterozygous mutations, a missense point mutation Tyr64(TAT)--> Ser (TCT), and an Ile112 duplication (ATCATC). Upon expression of these mutant proteins in E. coli, the Tyr64 mutant has 15% of the wild type 17 alpha-hydroxylase activity, whereas the Ile112 duplication shows no activity, results consistent with the observed clinical phenotype.

Adrenocorticotropic Hormone↗
PubMed1972

l-Phenylalanine Ammonia-lyase (Maize): Partial Purification and Response to Gibberellic Acid and Cycloheximide of l-Phenylalanine and l-Tyrosine Ammonia-lyase Activities.

Extracts of maize leaf sheath tissue deaminate both l-phenylalanine and l-tyrosine. The activities with both substrates are enhanced by treating the plant with gibberellic acid. Both activities decrease rapidly at the same rate when tissue is incubated in a moist atmosphere, and this decrease can be slowed by treatment with cycloheximide. The ratio of the activities was constant throughout a series of purification steps which included acetone and ammonium sulfate precipitation, and passage through an agarose column. The two activities could not be separated by isoelectric focusing. These results support our earlier conclusion that both activities occur at the same catalytic site.

Journal Article↗
PubMed1986

Polysaccharide lyases.

Polysaccharide lyases (or eliminases) are a class of enzymes (EC 4.2.2.-) that act to cleave certain activated glycosidic linkages present in acidic polysaccharides. These enzymes act through an eliminase mechanism, rather than through hydrolysis, resulting in unsaturated oligosaccharide products. Acidic polysaccharides are ubiquitous and so are the lyases that degrade them. This review article examines lyases that act on acidic polysaccharides of plant, animal, and microbial origin. These lyases are predominantly of microbial origin and come from a wide variety of both pathogenic and nonpathogenic bacteria and fungi. The lyases discussed include alginate lyase (EC 4.2.2.3), pectin lyase (EC 4.2.2.10), pectate lyase (EC 4.2.2.2), oligogalacturonide lyase (EC 4.2.2.6), exopolygalacturonate lyase (EC 4.2.2.9), chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5), hyaluronate lyase (EC 4.2.2.1), heparin lyase (EC 4.2.2.7), heparan lyase (EC 4.2.2.8), and other unclassified lyases. This review examines the sources, regulation, purification, and properties of these polysaccharide lyases.

Animals↗
PubMed1985

Kinetic studies on ovarian C-17,20-lyase activity: effect of luteinizing hormone surge.

It has been shown that 3 h after the preovulatory gonadotropin surge, an abrupt decrease occurs in follicular C-17,20-lyase (lyase) activity which causes a decrease in C19-steroid production. To determine the reason for the reduced lyase activity, we used rats that were induced to ovulate by means of PMSG administration. In these rats, a 54% decrease in lyase activity occurred at the peak of the LH surge. When the gonadotropin surge and ovulation were blocked by pentobarbitone the decrease was prevented. Administration of LH to the pentobarbitone-blocked rats reduced lyase activity to well below the level reached after the endogenous gonadotropin surge. In cycling proestrous rats as well, human CG (hCG) decreased the lyase activity, as measured in isolated follicles 3 h after hCG administration. Out of three potential inhibitory steroids for lyase activity; progesterone, 3 alpha,17 alpha-dihydroxy-5 alpha-pregnen-20-one, and 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, only the last compound inhibited competitively ovarian lyase activity. The inhibition constant (Ki) value was 29 microM. In order to explore which of the two activities of the lyase complex is regulated by the gonadotropin, a double label double substrate experiment was conducted using [14C]progesterone with [3H]17 alpha-hydroxyprogesterone (17 alpha-OHP). With this assay procedure, we could determine in the same experiment the site of stimulation, the preferred substrate, and the amount of conversion. The conversion of progesterone to 17 alpha-OHP, as well as the conversion to androstenedione were significantly inhibited throughout the reaction by the gonadotropin. Thus, the changes in ovarian lyase after hCG mimic those of 17 alpha-hydroxylase. The labeling pattern of androstenedione showed that the ovarian lyase complex catalyzed the conversion preferentially from progesterone. Whereas the 3H/14C ratio in androstenedione varied between 0.29 to 0.76, the ratios in the 17 alpha-OHP were from 5 to 22. Thus, the exogenous 17 alpha-OHP did not equilibrate with the product formed from progesterone. The effect of the hCG was to decrease the preference of progesterone over 17 alpha-OHP as substrate. It is concluded that: the LH of the surge inhibits both the 17 alpha-hydroxylase and lyase activities. The ovarian lyase complex shows a preference for progesterone as a substrate rather than 17 alpha-OHP. 17 alpha-OHP is not an obligatory intermediate in androstenedione production in ovarian tissue. hCG affects the ovarian lyase complex by shifting the relative preference of substrates towards 17 alpha-OHP.

17-alpha-Hydroxyprogesterone↗