The Separation and Partial Purification of the Soluble 17α- and 17β-Hydroxysteroid Dehydrogenases of Rabbit Liver

1974 ◽  
Vol 52 (2) ◽  
pp. 120-125 ◽  
Author(s):  
Sadiq Hasnain ◽  
Denis G. Williamson
Keyword(s):  
Dehydrogenase Activity ◽  
Enzyme Activities ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Hydroxysteroid Dehydrogenase ◽  
Rabbit Liver ◽  
Partial Purification ◽  
17Β Estradiol ◽  
Derivatives Of ◽  
Estradiol 17Β

17-Hydroxysteroid dehydrogenase activity toward 17α-estradiol, 17β-estradiol, and the 3-glucuronide derivatives of these steroids has been demonstrated in the 105 000 × g supernatant of rabbit liver. DEAE-cellulose chromatography of the enzyme activities isolated by calcium phosphate gel fractionation and Sephadex gel filtration yielded a single 17α-hydroxysteroid dehydrogenase activity toward both 17α-estradiol and 17α-estradiol 3-glucuronide. However, separate 17β-hydroxysteroid dehydrogenase activities toward 17β-estradiol and 17β-estradiol 3-glucuronide were obtained. Further purification of the 17α-hydroxysteroid dehydrogenase fraction by isoelectric focusing resulted in multiple peaks of activity toward 17α-estradiol and 17α-estradiol 3-glucuronide. One of these enzyme activities was highly specific for the 3-glucuronide derivative, it being essentially devoid of activity toward 17α-estradiol.

Purification and Properties of the Soluble 17β-Hydroxysteroid Dehydrogenase of Rabbit Uterus

1979 ◽  
Vol 34 (9-10) ◽  
pp. 726-737 ◽  
Author(s):  
Kunhard Pollow ◽  
Walter Eiger ◽  
Herrmann Heßlinger ◽  
Barbara Pollow
Keyword(s):  
Enzyme Activity ◽  
Dehydrogenase Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Hydroxysteroid Dehydrogenase ◽  
Maximal Velocity ◽  
Ammonium Sulfate Precipitation ◽  
Ph Optimum ◽  
Mobility Data ◽  
Purification And Properties

Abstract 17 β-Hydroxysteroid dehydrogenase activity towards estradiol-17 β has been demonstrated in the 105,000 X g supernatant of rabbit uterus. Hydroxylapatite chromatography of the enzyme activity isolated by ammonium sulfate precipitation, gel filtration and DEAE-cellulose chromato­graphy yielded a single 17 β-hydroxysteroid dehydrogenase activity. Further purification of the enzyme preparation by isoelectric focusing resulted in multiple peaks of activity. The molecular weight or the enzyme, calculated from mobility data on Sephadex gel, is approximately 64,000. Some properties of partially purified 17 β-hydroxysteroid dehydrogenase activity have been studied. Estradiol-17 β reacts at a faster rate than testosterone. The Km for estradiol is 4.16X 10-5 mol/1 for the NAD-linked enzyme activity and 4.37 X 10-5 mol/1 when NADP as cofactor was used. The ratio of the maximal velocity for NADP to that for NAD was 1.42. The pH-optimum for estradiol appears between 9.5 and 10.5 and for estrone between 5.5 and 6.5. The enzyme appears to be of the sulfhydryl type.

Uptake of 17β-estradiol and its conjugates by isolated rabbit liver nuclei

1983 ◽  
Vol 61 (7) ◽  
pp. 784-789 ◽  
Author(s):  
J. H. Tong ◽  
E. I. Seper ◽  
D. S. Layne ◽  
D. G. Williamson
Keyword(s):  
Diffusion Process ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
Substrate Concentration ◽  
Rabbit Liver ◽  
17Β Estradiol ◽  
Liver Nuclei ◽  
Layer Chromatography ◽  
Estradiol 17Β ◽  
Uptake And Metabolism

The present study compares the uptake and metabolism of 17β-estradiol, 17β-estradiol 3-glucoside, and 17β-estradiol 3-glucuronide by a highly purified preparation of rabbit liver nuclei. The uptake of the three estrogens was rapid and equilibration was reached within 60 s. The order of uptake was 17β-estradiol (64 fmol∙mg protein−1) > 17β-estradiol 3-glucoside (10 fmol∙mg protein−1) > 17β-estradiol 3-glucuronide (6.5 fmol∙mg protein−1). Thin-layer chromatography of the estrogens taken up by rabbit liver nuclei indicated the presence of a β-glucosidase activity associated with the nuclear preparation. The apparent Km value of this enzyme for 17β-estradiol 3-glucoside (3.5 μM) was about 10-fold higher when compared with the cytosolic enzyme. The uptake of the three estrogens was linearly proportional to the substrate concentration from 1 to 100 nM. No competition for uptake was observed among the steroids and the presence of diethylstilbestrol did not reduce the uptake of the steroids. These findings suggest that 17β-estradiol, 17β-estradiol 3-glucoside, and 17β-estradiol 3-glucuronide are taken up by nuclei by a nonsaturable diffusion process. The effect of cytosol on the uptake of estrogens by purified nuclei was also investigated. It was observed that cytosol reduced the uptake of 17β-estradiol but had little effect on that of its conjugates.

scholarly journals Age-dependent changes in the multiple forms of the soluble 17 β-hydroxysteroid dehydrogenase of female rabbit liver

1985 ◽  
Vol 225 (2) ◽  
pp. 391-398 ◽  
Author(s):  
G R Antoun ◽  
D G Williamson
Keyword(s):  
Dehydrogenase Activity ◽  
Specific Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Rabbit Liver ◽  
Molecular Heterogeneity ◽  
Special Role ◽  
Female Rabbit ◽  
Age Dependent ◽  
Androstane Series ◽  
Peptide Maps

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.

scholarly journals Aspartate transcarbamoylase from Phaseolus aureus. Partial purification and properties

1972 ◽  
Vol 129 (3) ◽  
pp. 571-581 ◽  
Author(s):  
B. L. Ong ◽  
J. F. Jackson
Keyword(s):  
Gel Filtration ◽  
Deae Cellulose ◽  
Aspartate Transcarbamoylase ◽  
Partial Purification ◽  
Saturation Curve ◽  
Phaseolus Aureus ◽  
Phosphate Binding ◽  
Carbamoyl Phosphate ◽  
Sequential Reaction ◽  
Substrate Saturation

1. Aspartate transcarbamoylase from 4-day-old radicles of Phaseolus aureus was purified 190-fold by (NH4)2SO4 fractionation, DEAE-cellulose and DEAE-Sephadex chromatography and Sephadex-gel filtration. The partially purified enzyme, which required Pi for maximum stability, had an apparent molecular weight of 83000±5000. 2. Uridine nucleotides were found to inhibit the activity; UMP was the most potent inhibitor, followed by UDP and UTP. No other nucleotide was found to affect the enzyme, nor could UMP inhibition be overcome by adding another nucleotide. Aspartate gives a hyperbolic substrate-saturation curve, both with and without UMP. The nucleotide inhibitor is non-competitive with respect to this substrate. Carbamoyl phosphate also yields a hyperbolic substrate-saturation curve in the absence of feedback inhibitor, but when UMP is added a sigmoidal pattern results, and the inhibition is competitive with carbamoyl phosphate. 3. The degree of inhibition by UMP is not affected by p-chloromercuribenzoate, urea, mild heat pretreatment or change in pH over the range 8.5–10.5, but is affected by temperature. 4. The aspartate analogue, succinate, both activates and inhibits the reaction, depending on the concentrations of aspartate and succinate used. 5. Kinetic studies with the partially purified enzyme showed that the Km for carbamoyl phosphate (0.091 mm) is much lower than that for aspartate (1.7mm). A sequential reaction mechanism was inferred from product-inhibition kinetics, with carbamoyl phosphate binding to the enzyme before aspartate, and the product, carbamoylaspartate, being released ahead of Pi. Initial-velocity studies gave a set of parallel reciprocal plots, compatible with an essentially irreversible step occurring before the binding of aspartate.

Solubilisierung, Anreicherung und Separierung zweier 17β-HSDAktivitäten nach Phospholipase-Behandlung / Microsome-Associated 17β-Hydroxysteroid Dehydrogenases of Human Placenta, I Solubilization, Enrichment and Separation of Two 17β-HSD-Activities after Phospholipase-Treatment

1975 ◽  
Vol 30 (1-2) ◽  
pp. 4-16b ◽  
Author(s):  
Kunhard Pollow ◽  
Wilfried Runge ◽  
Barbara Pollow
Keyword(s):  
Human Placenta ◽  
Gel Filtration ◽  
Hydroxysteroid Dehydrogenase ◽  
Phospholipase A ◽  
Membrane Phospholipids ◽  
Molecular Weights ◽  
Ammonium Sulphate Precipitation ◽  
Isoelectric Points ◽  
Hydrolysis Of ◽  
Estradiol 17Β

Abstract Treatment of human placenta microsomes with phospholipase A or D inhibits the 17β-hydroxysteroid dehydrogenase [17β-HSD] activity, parallel with the hydrolysis of membrane phospholipids. The 17β-HSD activity of phospholipase treated microsomes is reactivated by synthetic phospholipids. The distribution of 17β-HSD activity in subfractions of original microsomes and of phospholipase treated microsomes obtained by zonal centrifugation was studied. Solubilization of the microsomal 17β-HSD was achieved by phospholipase A treatment. Two 17β-HSD were solubilized from human placenta microsomes by phospholipase A treatment and were further purified by ammonium sulphate precipitation, gel filtration on BioGel A-0.5 m, DEAESephadex chromatography and by isoelectric focusing. The enzymes were purified 25.8 and 17.4 times. The isoelectric points and molecular weights of the two 17β-HSD were determined. Both enzymes are of a 17β-HSD type. One of the 17β-HSD, however, was sensitive to estradiol- 17β, the other to testosterone. The question of whether the two enzymes constitute a monomer and a dimer of the same 17β-HSD or are completely different enzymes, is discussed.

Metabolism of [4-14C]estrone by sheep erythrocytes around the time of parturition

1976 ◽  
Vol 54 (7) ◽  
pp. 666-669 ◽  
Author(s):  
Charles P. W. Tsang
Keyword(s):  
Dehydrogenase Activity ◽  
Blood Cells ◽  
Phosphate Buffer ◽  
Incubation Medium ◽  
Late Pregnancy ◽  
Hydroxysteroid Dehydrogenase ◽  
Internal Standards ◽  
17Β Estradiol ◽  
Acetate Derivative

The metabolism of [4-14C]estrone in vitro by red blood cells of sheep in late pregnancy and after parturition has been studied. [14C]estrone (600 ng) was incubated with 0.5 ml erythrocytes plus 0.5 ml of Krebs–Ringer phosphate buffer, pH 7.4, for 2 h at 37 °C in an atmosphere of air. After incubation, [3H]estrogens were added to the incubation medium as internal standards for identification and for correction for procedural losses. Metabolites were isolated and purified by chromatography, acetate derivative formation, and recrystallization to a constant 3H/14C ratio. Approximately 20% and 2% of added estrone were converted to 17β-estradiol and 17α-estradiol, respectively. The remainder was recovered unchanged. Daily measurements of 17β-hydroxysteroid dehydrogenase activity in erythrocytes of five ewes, over the period 8 days prepartum to 4 days postpartum, showed no significant change in activity.

scholarly journals Prolinase and non-specific dipeptidase of human kidney

1985 ◽  
Vol 231 (3) ◽  
pp. 689-694 ◽  
Author(s):  
D A Priestman ◽  
J Butterworth
Keyword(s):  
Active Site ◽  
Enzyme Activities ◽  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Human Kidney ◽  
Competitive Inhibitor ◽  
Dipeptidase Activity ◽  
Single Enzyme

Human kidney prolinase, assayed with Pro-Ala, and non-specific dipeptidase, assayed with Gly-Leu, were purified by using DEAE-cellulose, gel-filtration, metal-ion-chelate, hydrophobic and adsorption chromatography and chromatofocusing. Both enzymes gave single peaks of activity that were congruent and the ratio of their activities was constant throughout the purification. Gel filtration indicated an Mr of 100 000 and chromatofocusing a pI of 5.4. Ni2+-chelate chromatography demonstrated the presence of exposed histidine residues on the enzyme and was an effective separative procedure. Polyacrylamide-gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. Both enzyme activities decayed at the same rate at 53 degrees C and were inhibited to the same extent by p-hydroxymercuribenzoate. Of six non-specific dipeptidase substrates tested Gly-Leu gave the highest activity, and of six prolinase substrates Pro-Leu had the highest activity. Gly-Leu was hydrolysed at double the rate of Pro-Leu. Pro-Ala was a competitive inhibitor of activity towards Gly-Leu, and Gly-Leu was a competitive inhibitor of activity towards Pro-Ala. Mixed-substrate studies strongly suggested that Gly-Leu and Pro-Ala were hydrolysed at a common active site. The data are consistent with prolinase and non-specific dipeptidase activity in human kidney being due to a single enzyme.

Sign in / Sign up

Export Citation Format

Share Document