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Biomedical subjects

J C Warren

Publications and source records attributed to J C Warren.

At least 37 records · Page 2Linked to original sources
PubMed1977

Free testosterone concentration in serum: elevation is the hallmark of hirsutism.

A simple, reliable procedure for quantitating the concentration of free (not protein bound and thus biologically active) testosterone in serum has been applied to normal and hirsute patients. Each of 32 hirsute women studied had a significantly elevated level of free testosterone (that is, more than two standard deviations above the mean of that found in normal women), although half of them had a total serum testosterone level within normal limits. Suppression of free testosterone to within normal limits could be accomplished by dexamethasone alone in 47 per cent of the hirsute group. In the remainder of the hirsute group, suppression of free testosterone to within normal limits could be accomplished by addition of Enovid-E. Use of free serum testosterone concentration is clearly superior to the use of total serum testosterone concentration for the identification and rational therapy of simple hirsutism.

Adolescent↗
PubMed1976

Crystallization of human placental estradiol 17beta-dehydrogenase. A new method for crystallizing labile enzymes.

Estradiol 17beta-dehydrogenase from human placenta has been crystallized by a new technique, herein referred to as electrophoretic diffusion. This is the first crystallization of an enzyme from human placenta as well as the first crystallization of any steroid-converting enzyme of human source. A solution of the enzyme (specific activity 7.1 units/mg) in 1.5 ml of Tris-barbituric acid buffer, pH 7.0, containing 20% glycerol as stabilizer, was placed in an electrophoresis tube and the tube was closed at both ends with a dialysis membrane which permits the passage of substances of molecular weight less than 18,000. The tube was placed in a gel electrophoresis apparatus and the reservoirs filled with the Tris-barbituric acid buffer. A potential of 100 V was applied for 12 hours, then raised to 200 V for another 12 hours, and finally to 300 V until opalescence appeared at the bottom of the tube. Activity measurements showed that more than 90% of the enzyme had concentrated in the bottom 0.15-ml portion of the solution. When this section of the solution was removed and kept overnight at 4 degrees, gross and microscopic examination revealed a heavy crop of crystals which possessed a specific activity of 7.2 units/mg. The specific activity remained constant throughout three recrystallizations. The crystalline enzyme displayed a single band by analytical and sodium dodecyl sulfate-polyacrylamide gel analysis. Crystals of enzyme of high specific activity could also be obtained from an enzyme sample initially possessing a specific activity of only 4.5 units/mg. The new technique should be appliable for the crystallization of other labile enzymes and receptor proteins which have so far resisted crystallization by conventional methods.

Crystallization↗
PubMed1975

Purification of human placental estradiol 17 beta-dehydrogenase study of the steroid-binding site.

Human placental estradiol 17 beta-dehydrogenase has been purified by affinity chromatography. The purified enzyme is homogenous by polyacrylamide-gel electrophoresis. To study topography of the steroid-binding site, 16 alpha-bromoacetoxyestradiol 3-methyl ether was synthesized with estriol 3-methyl ether, bromoacetic acid, or [2-3H] bromoacetic acid and dicyclohexylcarbodiimide. The steroid alkylates cysteine, histidine, methionine, and tryptophan under physiologic conditions. Being a substrate of the enzyme, it must bind at the steroid-binding site. The steroid inactivates the enzyme in a time-dependent, irreversible manner. Inactivation of the enzyme by excess 16 alpha-bromoacetoxyestradiol 3-methyl ether follows pseudo--first-order kinetics with t1/2 = 1.5 hours. Amino acid analysis reveals that a histidyl residue is carboxymethylated. Estradiol-17 beta slows inactivation; 2-mercaptoethanol stops it. Previous studies have shown a histidyl residue also present at the catalytic region of the active site of 20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans. It is tempting to consider that these histidyl residues may be an essential component for the dehydrogenation of the steroid substrates.

Binding Sites↗
PubMed1975

Artificial insemination with fresh donor semen.

A total of 141 patients received donor insemination during a 21/2-year period. Pregnancy was achieved by 57 per cent of these patients. When patients who were inseminated for less than three cycles are excluded, the pregnancy rate is 77 per cent. The mean conception time was three treatment cycles. In 69 pregnancies, the occurrence of abortion was that of the general population, and an increased male-to-female ratio was observed in the 36 live-born infants. Questionnaires sent to all couples and returned by 81 per cent indicated that donor insemination was not emotionally harmful to their marriage. Heterologous insemination is a safe, effective therapeutic alternative to adoption for selected couples.

Abortion, Spontaneous↗
PubMed1975

Affinity labeling of steroid binding sites. Study of the active site of 20beta-hydroxysteroid dehydrogenase with 2alpha-bromoacetoxyprogesterone and 11alpha-bromacetoxyprogesterone.

To further characterize the active site of 20beta-hydroxysteroid dehydrogenase (EC 1.1.1.53) from Streptomyced hydrogenans we synthesized 2alpha-bromoacetoxyprogesterone, a substrate for the enzyme in 0.05 M phosphate buffer at 25 degrees, pH 7.0, with Km and Vmax values of 1.90 X 10(-5) M and 6.09 nmol/min/mg of enzyme, respectively. This affinity labeling steroid inactivates 20beta-hydroxysteroid dehydrogenase in an irreversible and time-dependent manner which follows pseudo-first order kinetics with a t1/2 value of 4.6 hours. 2alpha-[2-3H]Bromoacetoxyprogesterone was synthesized and used to radiolabel the enzyme active site. Amino acid analysis of the acid hydrolysate of the radiolabeled enzyme supports a mechanism whereby the steroid moiety delivers the alkylating group to the steroid binding site of the enzyme where it reacts with a methionyl residue. Both 2alpha- and 11alpha-bromoacetoxyprogesterone alkylate a methionyl residue at the active site of 20beta-hydroxysteroid dehydrogenase. The enzyme was inactivated with a mixture containing both 2alpha-[2-3H]Bromoacetoxyprogesterone and 11alpha-2[2-14C]bromoacetoxyprogesterone. Following degradation of separate aliquots of the radiolabeled enzyme by cyanogen bromide or trypsin, the protein fragments were separated by gel filtration and ion exchange chromatography. Resolution of peptides carrying the 3H label from those possessing the 14C label demonstrates that 2alpha-bromoacetoxyprogesterone and 11alpha-bromoacetoxyprogesterone each label a different methionine at the steroid binding site of 20beta-hydroxysteroid dehydrogenase.

Affinity Labels↗
PubMed1975

Synthesis of 16alpha-bromoacetoxyestradiol 3-methyl ether and study of the steroid binding site of human placental estradiol 17beta-dehydrogenase.

Homogeneous estradiol 17beta-dehydrogenase (EC 1.1.1.62) was prepared from human placenta by affinity chromatography and the steroid binding site was studied with affinity-labeling techniques. 16alpha-Bromoacetoxyestradiol 3-methyl ether and the tritated compound were synthesized by condensation of estriol 3-methyl ether with bromoacetic acid or [2-3H]bromoacetic acid in the presence of dicyclohexylcarbodiimide. 16alpha-Bromoacetoxyestradiol 3-methyl ether is stable in 0.01 M phosphate buffer at pH 7.0, 25 degrees, for at least 24 hours. It alkylates cysteine, histidine, methionine, lysine, and tryptophan under physiological conditions. The steroid is a substrate of estradiol 17beta-dehydrogenase, thus it must bind at the steroid binding site. The inactivation of estradiol 17beta-dehydrogenase by 150-fold molar concentrations of 16alpha-bromoacetoxyestradiol 3-methyl ether follows pseudo-first order kinetics with a half-time of 1.5 hours. Estradiol-17beta, NADH, and NADPH slow the rate of inactivation. 2-Mercaptoethanol in molar concentrations 50-fold that of 16alpha-bromoacetoxyestradiol 3-methyl ether stops the inactivation, but does not reverse it. 16alpha-Bromoacetoxyestradiol 3-methyl ether alkylates both NADH and NADPH; the presence of small amounts of enzyme markedly increases the rate of this alkylation. When the enzyme is inactivated with 16alpha-[2-3H]bromoacetoxyestradiol 3-methyl ether, amino acid analysis of acid hydrolysates reveals 3-carboxymethylhistidine and 1,3-dicarboxymethylhistidine. Comparison of 28 and 51% inactivated samples indicates that, as inactivation proceeds, the total amount of 3-carboxymethylhistidine decreases, while 1,3-dicarboxymethylhistidine increases, suggesting that the former is converted to the latter by a second alkylation step. When the enzyme is inactivated in the presence of a large excess of NADPH, only 1,3-dicarboxymethylhistidine is found. From the present study it is concluded that estradiol 17beta-dehydrogenase has a histidyl residue present in the catalytic region of the active site as does the previously studied 20beta-hydroxysteroid dehydrogenase.

Amino Acids↗
PubMed1975

Synthesis and study of the estrogenic activity of 2,4-bis(bromomethyl)estradiol-17 beta 3-methyl ether.

Synthesis of 2,4-bis(bromomethyl)estradiol-17 beta 3-methyl ether (BBE2M) was accomplished by reducing a methanolic solution of 2,4-bis(bromomethyl)estrone methyl ether with sodium borohydride. In 0.5 M phosphate buffer, pH 7.0, 25 degrees, BBE2M readily reacts with Ellman's anion and alkylates cysteine to form a steroid-amino acid conjugate. Stoichiometry of the reaction indicates that the bromosteroid is divalent with cysteine. Tryptophan and histidine react more slowly with the bromosteroid. Estrogenic activity of BBE2M was evaluated in ovariectomized rats by uterine intraluminal administration and quantitation of glucose-6-phosphate dehydrogenase (D-glucose-6-P:NADP+ oxidoreductase, EC 1.1.1.49) activity in the uterus. BBE2M induced glucose-6-phosphate dehydrogenase activity as did estradiol-17 beta or estradiol-17 beta 3-methyl ether (E2M). BBE2M was more persistent in activity than E2M. Histological examination of uterus following BBE2M treatment shows classic estrogenic morphology. BBE2M covalently binds to the cytoplasmic estrogen receptor of calf uterus. Such binding is prevented by pretreatment of the receptor protein with estradiol-17 beta. The covalently bound steroid-receptor complex appears to stimulate RNA synthesis in isolated nuclei from calf endometrium.

Animals↗
PubMed1975

Synthesis and use of affinity-labeling steroids for interceptive purposes.

Synthesis of 17 beta-bromoacetoxy-19-nortestosterone was carried out by reaction of 19-nortestosterone with bromoacetic acid in the presence of dicyclohexycarbodiimide. The steroid was capable of alkylating cysteine, methionine, and histidine under physiologic conditions, indicating its potential as an affinity-labeling steroid. 17beta-Bromoacetoxy-19-nortestosterone interrupted postimplantation pregnancy in the rat when administered into the lumen of the uterus at low doses or subcutaneously at higher doses. Exogenous gonadotropins or steroids in dosages sufficient to maintain pregnancy do not prevent the interceptive action of this steroid. Animals whose first pregnancies were interrupted by this steroid had a subsequent normal pregnancy. The mode of action may be via covalent bonding to the progesterone receptor resulting in exclusion of endogenous progesterone.

Abortifacient Agents↗