Purification of multiple forms of the soluble 17alpha-hydroxy steroid dehydrogenase or rabbit liver.
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ABSTRACT:
SUBMITTER: Hasnain S
PROVIDER: S-EPMC1165472 | biostudies-other | 1975 Jun
REPOSITORIES: biostudies-other
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Project description:The six forms of the 17alpha-hydroxy steroid dehydrogenase purified from rabbit liver cytosol have very similar physical properties. The molecular weights of all the enzymes were within 3% of the average mol.wt of 39 600. Only one of the six enzymes showed a significant difference in amino acid composition. All but one form of the 17alpha-hydroxy steroid dehydrogenases exhibited greater activities towards the androgen, epitestosterone, than towards oestrogen substrates. With oestrogen substrates one enzyme displayed a high specificity towards the substrate oestradiol-17alpha 3-glucuronide. This high activity was lost if the glucuronic acid moiety was removed or replaced by glucose or galacturonic acid. The other enzyme forms had approximately equal activity toward oestradiol-17alpha and its glucuronide or glucoside derivative. However, substitution of galacturonic acid at C-3 of oestradiol-17alpha substantially decreased the activity of all but one enzyme form.
| S-EPMC1164505 | biostudies-other
Project description:The soluble NADP-dependent 17 beta-hydroxysteroid dehydrogenase activity of female rabbit liver increases with the age of the animal, the specific activity of the enzyme in the 56-day-old rabbit being 3 times that of the 28-day-old animal. The increase in activity is accompanied by a change in the molecular heterogeneity of the enzyme. Three forms (enzymes I, II and III) were identified in the liver cytosol of the 56-day-old female rabbit, whereas only one major form (enzyme IIIY) was present in the 28-day-old animal. Peptide maps of the four purified enzymes showed that there were minor differences in structure. The enzyme present in the liver of the 28-day-old rabbit was distinct from the three enzymes of the 56-day-old animal. All of the enzymes exhibited bifunctional activity, having 17 beta-hydroxysteroid dehydrogenase activity towards androgen and oestrogen substrates and 3 alpha-hydroxysteroid dehydrogenase activity towards androgens of the 5 beta-androstane series. The differences in substrate specificity of the enzymes paralleled their differences in structure. The data suggest that one enzyme (enzyme III) may have a special role in steroid metabolism during development in the female rabbit.
| S-EPMC1144602 | biostudies-other
Project description:Multiple forms of the soluble 17 beta-hydroxysteroid dehydrogenase of female rabbit liver were identified. NAD-dependent and NADP-dependent enzyme activities were separated by affinity chromatography on agarose-immobilized Procion Red HE3B, and three forms of the NADP-dependent enzyme activity were purified by chromatofocusing. These three enzyme forms are charge isomers and have no quaternary structure. The enzymes catalysed the C-17 oxidoreduction of oestrogens and androgens; with all enzyme forms the activity towards androgens was higher than that toward oestrogens. The enzymes also exhibited 3 alpha-hydroxysteroid dehydrogenase activity towards androgens of the 5 beta-androstane series. Comparison of the relative activities of the enzymes towards a number of oestrogen and androgen substrates revealed differences among the enzyme forms for both the oxidative and the reductive reactions. In particular, one enzyme form had a significantly lower Km for the 3 alpha-hydroxysteroid substrate and a higher 3 alpha-/17 beta-hydroxysteroid dehydrogenase activity ratio than the other two enzyme forms.
| S-EPMC1144601 | biostudies-other
Project description:BACKGROUND: Epi-testosterone (epiT) is the 17alpha-epimer of testosterone. It has been found at similar level as testosterone in human biological fluids. This steroid has thus been used as a natural internal standard for assessing testosterone abuse in sports. EpiT has been also shown to accumulate in mammary cyst fluid and in human prostate. It was found to possess antiandrogenic activity as well as neuroprotective effects. So far, the exact pathway leading to the formation of epiT has not been elucidated. RESULTS: In this report, we describe the isolation and characterization of the enzyme 17alpha-hydroxysteroid dehydrogenase. The name is given according to its most potent activity. Using cells stably expressing the enzyme, we show that 17alpha-HSD catalyzes efficienty the transformation of 4-androstenedione (4-dione), dehydroepiandrosterone (DHEA), 5alpha-androstane-3,17-dione (5alpha-dione) and androsterone (ADT) into their corresponding 17alpha-hydroxy-steroids : epiT, 5-androstene-3beta,17alpha-diol (epi5diol), 5alpha-androstane-17alpha-ol-3-one (epiDHT) and 5alpha-androstane-3alpha,17alpha-diol (epi3alpha-diol), respectively. Similar to other members of the aldo-keto reductase family that possess the ability to reduce the keto-group into hydroxyl-group at different position on the steroid nucleus, 17alpha-HSD could also catalyze the transformation of DHT, 5alpha-dione, and 5alpha-pregnane-3,20-dione (DHP) into 3alpha-diol, ADT and 5alpha-pregnane-3alpha-ol-20-one (allopregnanolone) through its less potent 3alpha-HSD activity. We also have over-expressed the 17alpha-HSD in Escherichia coli and have purified it by affinity chromatography. The purified enzyme exhibits the same catalytic properties that have been observed with cultured HEK-293 stably transfected cells. Using quantitative Realtime-PCR to study tissue distribution of this enzyme in the mouse, we observed that it is expressed at very high levels in the kidney. CONCLUSION: The present study permits to clarify the biosynthesis pathway of epiT. It also offers the opportunity to study gene regulation and function of this enzyme. Further study in human will allow a better comprehension about the use of epiT in drug abuse testing; it will also help to clarify the importance of its accumulation in breast cyst fluid and prostate, as well as its potential role as natural antiandrogen.
| S-EPMC1185520 | biostudies-literature
Project description:Treatment of pregnant rats with human chorionic gonadotrophin, luteotrophin (luteinizing hormone), luteotrophin-releasing hormone, prostaglandin F2alpha, aminoglutethimide, or by foetoplacental removal or hysterectomy achieved a common multiple-response pattern, namely increased activity of luteal 20alpha-hydroxy steroid dehydrogenase with decreased activity of delta5-3beta-hydroxy steriod dehydrogenase and release of delta4-3-oxo steroids in vitro. 2. Similar effects of foetoplacental removal are noted in pregnant mice. 3. Gonadotrophin induced lower activities of 20alpha-hydroxy steroid dehydrogenase, except at the very end of pregnancy, and partly inhibited the induction caused by foetoplacental removal. 4. The results suggest that existence of a placental factor that restrains these changes until the end of normal pregnancy, which is produced in amounts proportional to the number of placentae and is conveyed to the ovary via the blood. 5. This factor was not replaced by prolactin. 6. It is argued that neither placental lactogen nor pituitary luteotrophin participate in the induction of 20alpha-hydroxy steroid dehydrogenase at late pregnancy in the rat. 7. Aminoglutethimide induced 20alpha-hydroxy steroid dehydrogenase only in late pregnancy. This was partly reversed by progesterone, wholly reversed by progesterone plus oestrogen, and did not involve the pituitary.
| S-EPMC1172495 | biostudies-other
Project description:1. beta-Glucuronidase (EC 3.2.1.31) was purified from rabbit liver by a procedure involving autolysis, (NH(4))(2)SO(4) fractionation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration, sedimentation in a sucrose gradient, and isoelectric focusing. 2. Electron microscopy revealed ferritin as the major contaminant in later stages of purification and also showed aggregates of enzyme molecules. Particular attention was paid to the removal of ferritin. 3. The purified enzyme was homogeneous in polyacrylamide-gel electrophoresis both in non-dissociating conditions and in the presence of sodium dodecyl sulphate, and in Ouchterlony gel diffusion and immunoelectrophoresis against polyspecific antisera. 4. Sedimentation in sucrose gradients gave a molecular weight of 300000, whereas gel filtration indicated 440000. 5. Subunits of 75000 molecular weight were observed in gel electrophoresis in the presence of sodium dodecyl sulphate and in gel filtration in the presence of urea. 6. The K(m) value for p-nitrophenyl beta-d-glucuronide was 0.6mm, and the enzyme was extremely sensitive to lactone inhibitors. It was also inhibited by Hg(2+) ions. 7. Multiple forms were observed in the pure enzyme by isoelectric focusing, with pI values of 4.5-5.8. Subunits showed similar heterogeneity. The origin of the multiple forms was investigated in detail, and the possibility of artifact generation largely excluded. Some of the forms of lowest pI disappeared after neuraminidase digestion. The nature of the residual heterogeneity remains to be elucidated.
| S-EPMC1166225 | biostudies-other
Project description:The behaviour of various C(19) and C(18) steroids as substrates for crystalline preparations of cortisone reductase (EC 1.1.1.53) is described. 3alpha(Axial,3R)-, 3alpha(equatorial,3R)- and 3beta(axial,3S)-hydroxy steroid-NAD oxidoreductase activities are demonstrated. Four pairs of the substrates differed only in the shape of the a/b ring junction, three pairs differed only in substitution at C-10, and four pairs differed only in substitution in ring d. The shape of the substrate molecule and certain substituents (e.g. 10beta-methyl, 17beta-hydroxy, 16-oxo or 17-oxo) altered substrate behaviour, but steroids differing considerably in shape nevertheless acted as substrates, suggesting the possibility of a large or flexible binding site. K(m) values varied about 10-fold, many being approx. 140mum. V(max.) values covered a greater range (about 200-fold) and the good substrates had high V(max.) values rather than low K(m) values.
| S-EPMC1165908 | biostudies-other
Project description:D-3-Phosphoglycerate dehydrogenase (EC 1.1.1.95) was purified from rabbit liver by (NH4)2SO4 fractionation, DEAE-Sephacel chromatography, affinity chromatography on AMP-agarose and molecular-sieve h.p.l.c. The purified enzyme was homogeneous as judged by SDS/polyacrylamide-slab-gel electrophoresis. On the basis of molecular-sieve h.p.l.c. and SDS/polyacrylamide-gel electrophoresis, the enzyme is a tetramer composed of subunits of Mr 60,000.
| S-EPMC1147222 | biostudies-other
Project description:Preimplantation embryo manipulations during standard assisted reproductive technologies (ART) have significant repercussions on offspring. However, few studies to date have investigated the potential long-term outcomes associated with the vitrification procedure. Here, we performed an experiment to unravel the particular effects related to stress induced by embryo transfer and vitrification techniques on offspring phenotype from the foetal period through to prepuberal age, using a rabbit model. In addition, the focus was extended to the liver function at prepuberal age. We showed that, compared to naturally conceived animals (NC), offspring derived after embryo exposure to the transfer procedure (FT) or cryopreservation-transfer procedure (VT) exhibited variation in growth and body weight from foetal life to prepuberal age. Strikingly, we found a nonlinear relationship between FT and VT stressors, most of which were already present in the FT animals. Furthermore, we displayed evidence of variation in liver function at prepuberal age, most of which occurred in both FT and VT animals. The present major novel finding includes a significant alteration of the steroid biosynthesis profile. In summary, here we provide that embryonic manipulation during the vitrification process is linked with embryo phenotypic adaptation detected from foetal life to prepuberal age and suggests that this phenotypic variation may be associated, to a great extent, with the effect of embryo transfer.
| S-EPMC7696440 | biostudies-literature
Project description:Cytosolic, detergent-solubilized and membrane-bound growth hormone (GH) receptors from rabbit adipose tissue and liver were tested for reactivity with a panel of monoclonal antibodies (MAbs). The cytosolic and detergent-solubilized forms of adipose tissue and liver GH receptors were identically reactive with four precipitating and two hormone-binding-site-directed MAbs. However, the membrane-bound form of the adipose receptor was 1000-fold less reactive with one binding-site-directed MAb (MAb 7) than the membrane-bound liver GH receptor. Reactivity with another inhibitory MAb (MAb 263) was identical for adipose tissue and liver membrane GH receptors. The relative potency of 22,000-Mr and 20,000-Mr forms of human GH was identical in assays with liver and adipose tissue membrane receptors. Thus, contrary to earlier suggestions, the discrepancy between the growth-promoting and insulin-like activities of 20,000-Mr human GH cannot be rationalized by a difference in the affinity of this hormone for 'somatogenic' and 'metabolic' receptors when the comparison is made in the same species. Cross-linking studies showed that the major GH-binding subunit of liver and adipose tissue GH receptors had the same Mr (54,000 +/- 5000, reduced). The ligand-binding subunits of liver and adipose tissue receptors are identical by several criteria, but one epitope on the adipose tissue receptor appears to be masked upon membrane insertion, possibly by close association with a tissue-specific component. Tissue specificity may be determined by association of a ubiquitous GH-binding subunit with tissue-specific membrane components, rather than by differences in amino acid sequence.
| S-EPMC1131313 | biostudies-other