Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0338671 (Steroids)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Microsomal estrogen synthetase (aromatase) cytochrome P-450 was purified from fresh human placental microsomes by monoclonal anti-aromatase P-450 antibody-Sepharose 4B chromatography. The purified P-450 showed a single band of 55 kDa on SDS-polyacrylamide gel electrophoresis and the aromatase specific activity on reconstitution was 70 nmol/min/mg protein. The purified P-450 was stable with a t 1/2 of approximately 2 years on storage at -90 degrees C and showed Km = 43 nM for androstenedione aromatization. However, it was unstable under spectral measurement conditions in the presence of sodium dithionite and carbon monoxide and the carbon monoxide difference spectra showed a maximum at 450 nm and a specific content of 9.1 nmol of P-450/mg protein, giving a turnover number of approximately 7.7 per min for the purified aromatase. The one-step immunochemical purification method gave a 490-fold increase of specific activity with 55% yield of aromatase activity of the original microsomes. Analysis of androgen metabolism by the purified aromatase and an apparent large kinetic isotope effect found at the secondary positions when using [19(-3)H3, 4(-14)C] androgens revealed metabolic switching from the first 19-hydroxylation to 1 beta- and 2 beta- monohydroxylation by aromatase. Substrate specificity for [19(-3)H3]androstenedione and testosterone was indicated by differences in the extent of metabolic switching (18% and 30%) and in the 2 beta/1 beta ratio (60/40 and 10/90, respectively). The mouse monoclonal antibody used for immunoaffinity purification suppresses aromatase activity of human placenta, but was totally ineffective for aromatase in goldfish brain and rat ovary. Rabbit polyclonal antibodies to human placental aromatase P-450 suppressed both human placental and rat ovarian aromatase but were ineffective for goldfish brain aromatase. The study indicates that they are isozymes of aromatase based on different structures of P-450.
Steroids
PMID:Immunoaffinity purification of aromatase cytochrome P-450 from human placental microsomes, metabolic switching from aromatization to 1 beta and 2 beta-monohydroxylation, and recognition of aromatase isozymes. 314 9

Iododesethyl tamoxifen aziridine (I-Tam-Az), an analog of the estrogen receptor-affinity label tamoxifen aziridine (Tam-Az) in which the ethyl group has been replaced by an iodine, has been prepared by two routes: (a) metallation of a bromotriarylethylene system, followed by reaction with iodine, and aziridinylation, and (b) direct iodination of a trimethylstannyl triarylethylene system that is the immediate precursor of I-Tam-Az. The latter method can be used to prepare [125I]I-Tam-Az rapidly and in good yield, both at carrier-added and no-carrier-added levels; specific activities greater than 200 Ci/mmol have been obtained. In competitive radiometric binding assays with the estrogen receptor, I-Tam-Az has an apparent affinity of ca. 20%, equivalent to that of Tam-Az. It also undergoes rapid and selective time-dependent, irreversible binding to the estrogen receptor. [125I]I-Tam-Az reacts covalently with estrogen receptor in uterine cytosol preparations; its attachment is rapid and efficient, but somewhat less selective than that of Tam-Az. Estrogen receptor in intact MCF-7 human breast cancer cells can also be labeled with [125I]I-Tam-Az, and autoradiographic analysis of salt extracts of labeled nuclear estrogen receptor on SDS-polyacrylamide slab gels shows highly selective labeling of a 65K protein. [125I]I-Tam-Az is an efficient, selective affinity label for the estrogen receptor, available at high specific activity, and should be useful in studies on estrogen receptor structure, dynamics, and chromatin interactions.
Steroids
PMID:[125I]iododesethyl tamoxifen aziridine: synthesis and covalent labeling of the estrogen receptor with an iodine-labeled affinity label. 344 83

Aromatase cytochrome P-450 (P-450arom) was purified from human placental microsomes. Preparations exhibit a single major band of approximately 55 kDa on SDS-polyacrylamide gel electrophoresis and have a specific content of 11-13 nmol P-450/mg protein. The purified enzyme exhibits spectral properties typical of ferric and ferrous forms of cytochromes P-450. Full enzymatic activity can be reconstituted with rabbit liver P-450 reductase, and catalytic characteristics similar to aromatase in microsomes are observed. Rabbit antibodies to purified P-450arom were affinity purified and show high specificity and sensitivity on immunoblots.
Steroids
PMID:Aromatase cytochrome P-450. Purification and characterization of the enzyme from human placenta. 350 64

We isolated testosterone-estradiol-binding globulin TeBG rapidly and in high yield from pooled pregnancy plasma. It showed two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Both bands stained with three different monoclonal antibodies to TeBG, thus demonstrating their immunological similarity. Freshly drawn, individual sera, from men, women, and pregnant patients were submitted to microaffinity chromatography, a procedure which partially purifies TeBG in approximately 4 hr. The partially purified plasma was submitted to SDS PAGE, followed by immunoblotting. The blotted TeBG exhibited the same two bands seen in the isolated, purified protein. The size heterogeneity observed in TeBG purified to: proteolysis occurring during isolation; a peculiarity of pregnancy plasma; or heterogeneity attendant upon the use of pooled plasma for isolation.
Steroids 1985 May
PMID:Size isomers of testosterone-estradiol-binding globulin exist in the plasma of individual men and women. 383 62

The soluble enzyme, estradiol-17 beta dehydrogenase from human term placenta, appears to co-purify with a second soluble enzyme, 20 alpha-hydroxysteroid dehydrogenase. The enzyme, which had been partially purified by affinity chromatography, fractionated on a preparative electrophoresis gel to a homogeneous preparation containing both estradiol-17 beta dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities in a ratio of approximately 100:1. Analytical polyacrylamide disc-gels resolved this homogeneous preparation as a single band by both protein and activity staining techniques. Homogeneous enzyme inactivated and affinity-radioalkylated by 16 alpha-[2'-14C]bromoacetoxyprogesterone or 16 alpha-[2'-14C] bromoacetoxyestradiol 3-methyl ether, and when analyzed by SDS disc-gel electrophoresis, gave a single protein band which corresponded identically to the radioactivity peaks. These observations support the hypothesis that estradiol-17 beta dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase represent dual oxidoreductase activity in one enzyme. Preparative disc-gel electrophoresis, a technique which has not been previously adapted to purification of these human placental enzyme activities, was useful to rapidly (3 days) effect a 15-fold enrichment of the estradiol-17 beta dehydrogenase specific activity from "heat-treated cytosol". Thus, laboratory-scale preparative disc-gel electrophoresis is useful for rapid, small-scale enrichment of this soluble enzyme.
Steroids 1981 Jun
PMID:Study of human placental estradiol-17 beta dehydrogenase/20 alpha-hydroxysteroid dehydrogenase by preparative disc-gel electrophoresis. 694 48

Preincubation of female rat liver microsomal preparations with [2'-32P]2N3-NADP+ followed by photolysis with UV light (254 nm) and analysis by SDS-PAGE/autoradiography showed incorporation of 32P into at least 3 major protein bands in the molecular weight range of 14-97 Kd. Labeling of a 26 kD band, the apparent molecular weight of 5 alpha-reductase in liver microsomes, was accompanied by a loss of enzyme activity, consistent with its covalent modification. The inclusion of 20-fold excess NADP+ (100 microM) completely inhibited the incorporation of [2'-32P]2N3-NADP+ and preserved the enzyme activity, whereas excess NAD+ (100 microM) failed to protect 5 alpha-reductase (5 alpha R) activity. Similar results were obtained with the detergent-solubilized form of 5 alpha R. Polyethylene glycol (PEG) fractionation of detergent-solubilized preparations of 5 alpha R showed that all the 5 alpha R activity could be recovered in the 6.5% pellet with a 3-4-fold increase in the specific activity. photolysis of this fraction with [2'-32P]2N3-NADP+ resulted in approximately 2-fold increase in 32P labeling of the 5 alpha R band. Increasing photolysis time and concentration of the [2'-32P]2N3-NADP+ indicated that the half-life for photoincorporation and the apparent Kd were 1.0 min and 2 microM, respectively. These results suggest that 2N3-NADP+ is an effective probe of the NADP(H) binding site of 5 alpha R, and is a useful marker during purification of the enzyme.
Steroids 1994 Nov
PMID:Photoaffinity labeling of rat liver microsomal steroid 5 alpha-reductase by 2-azido-NADP+. 770 39

We have previously shown that [2'-32P]-2-azido-NADP+ is an effective probe of the NADP-(H) binding site of rat liver microsomal 5 alpha-reductase (5 alpha R-1) [Bhattacharyya et al. (1994) Steroids 59, 634-641]. PEG-fractionated (6.5%) detergent-solubilized preparations (40 mg) containing 5 alpha R-1 activity were UV-photolyzed with [32P]-2-azido-NADP+ and subjected to preparative gel electrophoresis on 8% SDS-PAGE. Fractions corresponding to the second major [32P]-labeled peak following the dye-front were analyzed by 10% SDS-PAGE and showed a single [32P]-labeled species with an apparent molecular mass of approximately 26 kDa (5 alpha R-1). TCA precipitation (13.6%) of the labeled fractions resulted in recovery of > 70% of the total radioactivity in the protein pellet. Trypsin digestion of the resuspended pellet followed by immobilized-Al3+ affinity chromatography indicated that > 90% of the radioactivity remained bound to the affinity column. The [32P]-2N3-NADP(+)-labeled peptide was eluted with potassium phosphate, concentrated, and further purified by reverse-phase (C8) HPLC. Sequence analysis of the purified peptide indicated that it consisted of 11 amino acids with the sequence N-L-R-K-P-G-E-T-G-Y-K, corresponding to residues 170-180 of the rat 5 alpha R-1 sequence [Andersson et al. (1989) J. Biol. Chem. 264, 16249-16255].
 ...
PMID:Identification of the NADP(H) binding site of rat liver microsomal 5 alpha-reductase (isozyme-1): purification of a photolabeled peptide corresponding to the adenine binding domain. 789 62

Nuclear membrane bound testosterone 5 alpha-reductase solubilized in active form from human prostatic tissue by 0.5% n-octyl beta-D-glucopyranoside was purified by a four-step chromatographic procedure including DEAE-Trisacryl ion exchange, hydroxylapatite adsorption, testosterone-Sepharose affinity and Sepharose 4B gel filtration. A purification of approximately 30-fold was achieved judging from the increase in the specific enzymatic activity. We have purified the acidic pH-optimum 5 alpha-reductase type 2 isoenzyme. The apparent molecular weight of the purified enzyme was estimated as 42,000 by SDS-PAGE. At the same time we isolated a 38 kDa protein characterized by a real affinity for testosterone and by a possible association to the 5 alpha-reductase enzyme.
Steroids 1994 Dec
PMID:Purification of testosterone 5 alpha-reductase from human prostate by a four-step chromatographic procedure. 790 Jan 70

Functional studies in extra-renal, nonepithelial cells such as smooth muscle cells and more recently circulating human lymphocytes have provided increasing evidence that aldosterone produces not only classical genomic effects, but also rapid non-genomic effects on transmembrane electrolyte movements. These involve activation of the sodium/proton-exchanger of the cell membrane at very low, physiological concentrations of aldosterone with an acute onset within 1-2 minutes. A second messenger cascade involved is the inositol-1,4,5-trisphosphate/calcium pathway which responds over the same rapid time course. Such changes clearly cannot be explained by genomic mechanisms, which are responsible for later effects than the membrane-related rapid responses. In addition to its rapid time course the unique characteristics of this new pathway for steroid action include a 10000-fold selectivity for aldosterone over cortisol and the ineffectiveness of spironolactones, classical mineralocorticoid antagonists, as antagonists of the response. Subsequently binding sites have been demonstrated in the plasma membrane of human lymphocytes which show pharmacological (aldosterone specificity) and kinetic (high turnover) properties identical with those of the rapid aldosterone effects in the same cells. SDS-PAGE analysis of the receptor protein has shown a molecular weight of approximately 50 kD. The present paper reviews the data supporting a new, two-step model for non-genomic and genomic aldosterone effects. It also suggests a novel specificity-conferring mechanism for mineralocorticoid action at the membrane level.
Steroids 1994 Feb
PMID:Novel aldosterone receptors: specificity-conferring mechanism at the level of the cell membrane. 819 47

The glycoprotein corticosteroid-binding globulin (CBG) migrates as doublet bands in PAGE and SDS-PAGE, and as numerous bands in isoelectric focusing (IEF). This study deals with the origin of this heterogeneity. Desialation of rat CBG with neuraminidase does not abolish the doublet in either PAGE or SDS-PAGE, indicating that the doublet does not arise as a result of differences in sialic acid residues. Treatment of the separated upper and lower variants of native CBG with N-glycosidase F (PNGase-F) shows a differential pattern of deglycosylation over time indicating either differences in the number, type, or location of sugars attached to each of the variants. Rate of deglycosylation is quicker and more extensive for the upper variant when compared to the lower variant. PNGase-F treatment of 1% SDS-denatured CBG does not abolish the CBG doublet seen in SDS-PAGE, indicating that there is variation in the protein moiety. Sugars could not be detected on PNGase-F treated CBG using either wheat germ aglutinin horse radish peroxidase conjugate, concavilin-A HRP conjugate, or the digoxigenin glycan detection system. While the results clearly show differences in glycosylation between the CBG variants, differences in the protein moiety may also occur to give rise to the heterogeneity seen in CBG. The latter is supported by the fact that desialated CBG migrates as two bands in IEF. Migration in IEF is based solely on charge, and since only sialic acid residues are charged in N-linked glycosylation, any heterogeneity seen for the desialated glycoprotein must reside within the protein moiety itself. The presence of O-glycosylation containing an N-acetylgalactosamine with a beta 1-3 linkage to galactose could not be demonstrated using O-glycosidase. N-terminal blockage could not account for the variation, as both the upper and lower variants were able to be sequenced resulting in identical sequences for the first 13 amino acids. The data presented supports the hypothesis that the differences in the sugar as well as the protein moiety are responsible for the heterogeneity seen for CBG.
Steroids 1995 Nov
PMID:Studies on the role of glycosylation in the origin of the electrophoretic variants for rat corticosteroid-binding globulin. 858 98


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