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Biomedical subjects

W Gibb

Publications and source records attributed to W Gibb.

At least 73 records · Page 4Linked to original sources
PubMed1981

Substrate and nucleotide specificity of placental microsomal 3 beta-hydroxysteroid dehydrogenase.

Recent kinetic studies on the placental microsomal 3 beta-hydroxysteroid dehydrogenase have shown that apparent Km values for 3 beta-hydroxy-5-androsten-17-one (dehydroepiandrosterone) and 3 beta-hydroxy-5-pregnen-20-one (pregnenolone) are 15nM and 40nM respectively, which are orders of magnitude lower than found in earlier studies. The purpose of this study was to investigate the substrate and nucleotide specificity of the 3 beta-hydroxysteroid dehydrogenase, and the ability of various steroids to inhibit the reaction at these lower steroid concentrations. Each steroid inhibited the metabolism of the other competitively, and the Ki values obtained were not significantly different from their respective Km values. The ability of various steroids to inhibit the reaction at concentrations of 100nM was usually less than that found at micromolar concentrations. However, certain steroids showed marked inhibition. For example, estrone and estradiol-17 beta inhibit the oxidation of both substrates competitively with Ki values of between 15 and 24nM. The Km values of dehydroepiandrosterone and pregnenolone with NADP+ as cofactor are higher than those with NAD+ as cofactor and the V values are much lower. These data indicate that in human placental microsomes a single 3 beta-hydroxysteroid dehydrogenase, essentially NAD+ specific, metabolizes dehydroepiandrosterone and pregnenolone.

3-Hydroxysteroid Dehydrogenases↗
PubMed1981

Perinatal activity of the hypothalamic-pituitary-gonadal axis in the lamb. IV. Testicular responsiveness to hCG from 1 through 28 days of life.

Previous studies in this laboratory have shown the existence of an early postnatal activation of the hypothalamic-pituitary-gonadal axis (HPGA) in the male lamb which was present at 2 and 4 weeks of age. In order to define more precisely the time sequence of HPGA activity, we have studied the in vivo and in vitro testicular responsiveness to human chorionic gonadotropin (hCG) of the immature lamb at 1, 3, 7, 14, 21 and 28 days of life. Plasma testosterone (T) increments (delta) after hCG were lower in 1-day-old animals than in other age groups. Testicular concentrations of T, dehydroepiandrosterone and 17-hydroxyprogesterone increased from 1 to 14 days. Testicular 17, 20 lyase activity rose significantly with age but was not influenced by hCG. hCG and dibutyryl cyclic AMP increased significantly the T production by enriched interstitial cell preparation at 1, 3, and 7 days, the greatest response being found at 7 days. hCG also increased significantly the T production at 14 days. These data suggest that the lamb testis has the capacity to respond to hCG in vivo and to various stimuli vitro from the 1st day of life and that the response reaches a plateau from 2 to 4 weeks after birth.

Androstenedione↗
PubMed1980

In vitro conversion of pregnenolone to progesterone by term human fetal membranes.

The conversion of pregnenolone to progesterone by homogenates of term human fetal membranes (38 to 40 weeks' gestation) was quantitated prior to and after labor. Chorion pars reflexa obtained after spontaneous labor by vaginal delivery was more active than that obtained prior to labor by elective cesarean section (p less than 0.05). With homogenates of amnion pars reflexa (n = 18), and pars placentaris (n = 18) obtained prior to labor no conversion or pregnenolone to progesterone was detected. In contrast, in amnion pars reflexa obtained after labor the reaction was detected in five of the 13 tissues examined and was significantly different from tissues obtained prior to labor (p less than 0.01). However, the conversion by the amnion pars placentaris after labor was not significantly different from that to labor. Evidence for inhibition of the reaction in the amnion pars reflexa and pars placentaris obtained prior to labor was found when prolonged washing of these tissues (up to 5 hours) with isotonic KCl resulted in the conversion of pregnenolone to progesterone being easily detected. This study suggests that there may be "activation" of the conversion of pregnenolone to progesterone in fetal membranes during labor and indicates that considerable care should be taken in studying this and perhaps other reactions in fetal membranes as the mode of delivery, the preparation of tissue, and, in the case of the amnion, the area studied can affect the results obtained.

Amnion↗
PubMed1980

Substrate specificity of the placental microsomal aromatase.

Using an accurate and sensitive assay for the human placental aromatase we have found apparent Km values for androstenedione (4-androstene-3,17-dione) and testosterone to be 14 +/- 4.0 nM and 41 +/- 12 nM respectively. These values were significantly different (p < 0.001). Analyses at substrate concentrations 5-10 fold above and below the Km values did not indicate any anomalous kinetic behavior. Mixed substrate experiments were consistent with a single enzyme metabolizing both steroids: each competitively inhibited the aromatization of the other, and the "Ki" values were the same as their apparent Km values. Sodium chloride (1.2M) significantly increased the rate of testosterone aromatization by decreasing its Km value and had no significant effect on the aromatization of androstenedione. However, in the presence of this salt testosterone still inhibited the aromatization of androstenedione competitively with a "Ki" equal to its apparent Km. Our data is therefore consistent with the proposal that human placental microsomes contain a single "high affinity" site for the aromatization of androstenedione and testosterone.

Androstenedione↗
PubMed1980

Relationship of 2,3-diphosphoglycerate and 2,3-diphosphoglycerate mutase in various mammals.

To investigate a possible mechanism involved in the regulation of 2,3-diphosphoglycerate (2,3-DPG) synthesis, 2,3-DPG mutase (DPGM) was measured in different mammals presenting large differences in 2,3-DPG concentration between fetal, neonatal and adult life to see the activity of this enzyme, necessary for 2,3-DPG synthesis, was related to the levels of 2,3-DPG. The data demonstrated that the minimal levels of 2,3-DPG in the adult sheep are likely due to the very low levels of DPGM. Also these findings show that the increases in 2,3-DPG levels, found in the newborn sheep during the 1st week of life, in adult rabbit and guinea pig when compared with their fetuses, are not due to an increase in levels of the DPGM.

2,3-Diphosphoglycerate↗
PubMed1979

Kinetic analysis of the placental microsomal 3 beta-hydroxysteroid dehydrogenase activity.

A sensitive accurate assay for the placental microsomal 3 beta-hydroxysteroid dehydrogenase (E.C.1.1.1.51) has been developed using tritiated substrates. Kinetic analysis of the enzyme with 3 beta-hydroxy-5-androsten-17-one and 3 beta-hydroxy-5-pregnen-20-one indicates that the apparent Km values for these substrates are orders of magnitude less than previously described. Analyses were carried out with microsomal preparations from two different placentas. For placenta 1 the apparent Km value for 3 beta-hydroxy-5-androsten-17-one was 14 nM and for 3 beta-hydroxy-5-pregnen-20-one was 36 nM; for placental 2 apparent Km values were 19 nM and 42 nM respectively. The analyses were performed over wide ranges of substrate concentration (about 200 fold), both above and below the Km values and no deviation from linearity of Eadie-Hoftsee plots was observed.

3-Hydroxysteroid Dehydrogenases↗
PubMed1978

3beta-Hydroxysteroid dehydrogenase activity in human fetal membranes.

A 3beta-hydroxysteroid dehydrogenase (3betaHSD) was demonstrated in term human fetal membranes (chorion and amnion) with both dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) and pregnenolone (3beta-hydroxy-5-pregnen-20-one as substrates, and the subcellular distribution substrate and nucleotide specificity of the enzyme was studied. In both membranes the microsomal fraction (particles which sedimented at 105,000 g after 90 min) had the highest specific activity. The chorion was more active than the amnion but the enzyme in both tissues had similar substrate and nucleotide specificity. NAD was the preferred cofactor, and pregnenolone was a better substrate than dehydroepiandrosterone in the presence of NAD. However, with NADP as cofactor both steroids were equally good substrates. When the 3beta-hydroxysteroid dehydrogenase activity of chorion microsomes was compared with that of placental microsomes, the specific activities were found to be of the same order of magnitude, and the substrate, nucleotide specificity and steroid binding properties were almost identical.

3-Hydroxysteroid Dehydrogenases↗
PubMed1976

The specificity of the 3beta-hydroxysteroid dehydrogenase activity of bovine ovaries toward dehydroepiandrosterone and pregnenolone: evidence for multiple enzymes.

The 3beta-hydroxysteroid dehydrogenase activity in whole bovine ovaries was systematically studied using dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) and pregnenolone (3 beta-hydroxy-5-pregnen-20-one) as substrates, in order to determine whether, in this tissue, the same or different 3beta-hydroxysteroid dehydrogenases metabolize these steroids. The majority of the activity, with both substrates was found in the microsomes. Detergent extraction of the microsomes indicated that more than one enzyme was present in this fraction. A number of experiments on the Triton X-100 extract of the microsomes (the stability of the activity, its nucleotide specificity and kinetic analyses) were most simply explained by a single enzyme metabolizing both steroids. However, the stereospecificity of hydride-ion transfer from pregnenolone to NAD+ (B transfer) was different than that from dehydroepiandrosterone to NAD+ (A and B transfer). Thus, as no single enzyme is known to catalyze the transfer of hydride-ion to both sides of NAD+, it is proposed that there are at least two 3beta-hydroxysteroid dehydrogenases in the Triton X-100 extract.

Animals↗
PubMed1975

The altered specificity of cortisone reductase with certain retroandrostan-3-one substrates.

The retro steroids 17beta-hydroxy-5beta,9beta,10alpha-androstan-3-one and 5beta,9beta,10alpha-androstane-3,17-dione were good substrates for cortisone reductase in the presence of NADH, and the products corresponded to the respective 3beta-hydroxy compounds, in which the 3beta-hydroxyl group is axial and the absolute configuration is 3S. The analogous natural steroids 17beta-hydroxy-5beta,9alpha,10beta-androstan-3-one and 5beta,9alpha,10beta-androstane-3,17-dione were very poor substrates, and gave the corresponding 3alpha(equatorial,3R)-hydroxy compounds, and, in the latter case, also an appreciable amount of 3beta(axial, 3S)-hydroxy-5beta,9alpha,10beta-androstan-17-one. The natural steroids 17beta-hydroxy-5alpha,9alpha,10beta-androstan-3-one and 5alpha,9alpha,10beta-androstane-3,17-dione were better substrates than the retro steroid 17beta-hydroxy-5alpha,9beta,10alpha-androstan-3-one, but were not such good substrates as the retro steroids 17beta-hydroxy-5beta,9beta,10alpha-androstan-3-one and 5beta,9beta,10alpha-androstane-3,17-dione. Unlike these retro steroid 5beta,9beta,10alpha-androstan-3-ones, the natural steroids 17beta-hydroxy-5alpha,9alpha,10beta-androstan-3-one and 5alpha,9alpha,10beta-androstane-3,17-dione gave the corresponding 3alpha(axial,3R)-hydroxy compounds. The retro steroid 17beta-hydroxy-5alpha,9beta,10alpha-androstan-3-one was not a good substrate, and the product of reaction corresponded to the 3alpha(axial,3R)-hydroxy compound. The nature of substrate recognition by this enzyme is discussed in the light of these structure-activity relationships.

Androstane-3,17-diol↗
PubMed1973

3-Hydroxy steroid dehydrogenase activities of cortisone reductase.

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.

Acetylation↗