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J C Warren

Publications and source records attributed to J C Warren.

At least 19 recordsLinked to original sources
PubMed1993

Molecular cloning of testicular 20 alpha-hydroxysteroid dehydrogenase: identity with aldose reductase.

Complementary DNA (cDNA) clones encoding bovine testicular 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) have been isolated from a bovine testicular lambda gt11 library using polyclonal antibodies against 20 alpha-HSD and DNA probe hybridization. Nucleotide sequencing of three independently isolated clones was used to establish a composite cDNA sequence that encodes the enzyme. It contains a coding sequence of 921 nucleotides, a stop codon, and a 264-nucleotide 3'-noncoding segment which allowed deduction of the amino acid sequence of the enzyme. A computer homology search of the 20 alpha-HSD cDNA performed against the GenBank DNA sequence database revealed it to be identical with bovine lens aldose reductase (alditol:NADPH oxidoreductase; EC 1.1.1.21), and a literature search reveals the deduced amino acid sequence to be identical with that reported for the bovine enzyme. Sequences obtained from the N-terminus of purified testicular 20 alpha-HSD and from random peptides obtained by treatment with endopeptidase Lys-C are all identical with regions of the deduced amino acid sequence of 20 alpha-HSD and/or the published sequence of aldose reductase. Further, the enzyme purified to homogeneity by following activity with 17-hydroxyprogesterone as a substrate was shown to reduce glucose, glyceraldehyde, and benzaldehyde (all classic aldose reductase substrates). Finally, 17-hydroxyprogesterone inhibited the reduction of benzaldehyde and glyceraldehyde. Because aldose reductase has been implicated in the etiology of diabetic complications, acceptance of steroid substrates may offer new implications for therapy.

20-Hydroxysteroid Dehydrogenases↗
PubMed1991

Estradiol 17 beta-dehydrogenase: full enzymatic activity in the absence of zinc.

The precise catalytic mechanism of the steroid interconverting enzyme, human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62, estradiol-17 beta:NAD+ 17-oxidoreductase), is not known. Two general models for the catalytic mechanism of dehydrogenases have been defined. One model requires Zn2+ metal for the catalytic event, as has been shown for horse liver alcohol dehydrogenase (EC 1.1.1.1, alcohol:NAD+ oxidoreductase). Another model has been demonstrated for the 2-hydroxy acid dehydrogenases in which histidine residues are necessary for enzyme activity, without participation of a metal ion. In order to define which mechanism might be operative for the placental enzyme, it became important to determine whether Zn2+, or another metal ion, is associated with the macromolecule. Several homogeneous enzyme preparations, having protein concentrations from 5-80 microM, were extensively dialyzed in a buffer containing EDTA. Atomic absorption analysis of each sample demonstrated that no Zn2+ was present, although the enzymatic activity was maintained. In addition, there was no significant detection of Mg2+ or Mn2+ above background levels. When the isolated enzyme was dialyzed against buffer containing added 0.01-20 microM ZnCl2, no increase in specific activity of the enzyme was seen. The data indicate that the presence of zinc is not required for the catalytic event. These results, together with our previous affinity-labeling studies, which demonstrate a histidine residue in the catalytic region of the active site, allow us to propose that the catalytic mechanism of the human placental estradiol 17 beta-dehydrogenase is similar to that of the 2-hydroxy acid dehydrogenases.

Amino Acid Sequence↗
PubMed1990

Stereospecificity of hydrogen transfer between progesterone and cofactor by human placental estradiol-17 beta dehydrogenase.

We have previously shown that human placental estradiol-17 beta dehydrogenase (EC 1.1.1.62; 17 beta-EDH) catalyzes the conversion of estradiol-17 beta to estrone and stereospecifically reduces NAD+ to [4-pro-S]NADH, [( 4-B]NADH). Subsequently, this enzyme was found to reduce the ketone function at C-20 of progesterone, and evidence indicates that both activities reside at the same active site. This study was done to further elucidate spatial arrangements of cofactor and the 21-carbon substrate as they bind at the active site. The cofactor, [4B-3H]NADPH, was generated with homogeneous 17 beta-EDH from term human placenta, utilizing [17 alpha-3H]estradiol-17 beta and NADP+. The resulting [4B-3H]NADPH was then purified by ion exchange chromatography and was separately incubated (24.4 microM) with a large molar excess of progesterone (150 microM) as substrate in the presence of the enzyme. Following incubation, the steroid reactants and products were extracted, separated by high-performance liquid chromatography and quantitated as to mass and tritium content. Oxidized and reduced cofactor were separated by ion-exchange chromatography and similarly quantitated. In all incubations, equimolar amounts of 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OHP) and NADP+ were obtained. Radioactivity was stoichiometrically transferred from [4B-3H]NADPH to the steroid product [( 3H]20 alpha-OHP). These results further substantiate a single active site for both 17 beta- and 20 alpha-dehydrogenation enzyme activities. In addition, the enzyme is B-side specific, catalyzing the transfer of the 4B-hydrogen from the dihydronicotinamide moiety of the cofactor, for both C-18 and C-21 steroid substrates. Since the 20 alpha-dehydrogenation by other enzyme sources has always been demonstrated to be an A-side specific reaction, this observation represents an important exception to the Alworth-Bentley rules of enzyme stereospecificity.

Binding Sites↗
PubMed1989

Stereospecificity of hydrogen transfer by bovine testicular 20 alpha-hydroxysteroid dehydrogenase.

The stereospecificity of hydrogen transfer between steroid (17-hydroxyprogesterone) and both natural cofactors by bovine testicular 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) has been determined. Cofactors used in these studies, [4-pro-S-3H]NADH ([4B-3H]NADH) and [4-pro-S-3H]NADPH ([4B-3H]NADPH) were generated with human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) utilizing [17 alpha-3H]estradiol-17 beta and NAD+ or NADP+, respectively. The resulting [4B-3H]NADH and [4B-3H]NADPH were purified by ion-exchange chromatography and separately incubated with molar excess of 17-hydroxyprogesterone as substrate in the presence of 20 alpha-HSD. Following incubation, steroid reactant and product were extracted, separated by HPLC and quantitated as to mass and content of tritium. The oxidized and reduced cofactors were separated by ion-exchange chromatography and quantitated as to mass and tritium content. In all incubations, equimolar amounts of 17,20 alpha-dihydroxy-4-pregnen-3-one and oxidized cofactor were obtained. Further, all recovered radioactivity remained with cofactor and none was found in the steroid product. In additional experiments, both reduced cofactors were separately incubated with glutamate dehydrogenase, an enzyme known to transfer from the B-side of the nicotinamide ring. Here radioactivity was present only in the unreacted cofactor fractions and in the product, glutamic acid. The results indicate that bovine testicular 20 alpha-HSD catalyzes transfer of the 4A-hydrogen from the dihydronicotinamide moiety of the reduced cofactor. Finally, this work described modifications that represent considerable improvement in the purification and assay of bovine 20 alpha-HSD as originally described.

20-Hydroxysteroid Dehydrogenases↗
PubMed1988

Human placental estradiol 17 beta-dehydrogenase: evidence for inverted substrate orientation ("wrong-way" binding) at the active site.

Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity labeled with 17 alpha-estradiol 17-(bromo[2-14C]acetate) (10 microM) or 17 beta-estradiol 17-(bromo[2-14C]acetate) (10 microM). The steroid bromoacetates competitively inhibit the enzyme (against 17 beta-estradiol) with Ki values of 90 microM (17 alpha bromoacetate) and 134 microM (17 beta bromoacetate). Inactivation of the enzyme followed pseudo-first-order kinetics with a t1/2 = 110 min (17 alpha bromoacetate) and t1/2 = 220 min (17 beta bromoacetate). Amino acid analysis of the affinity radioalkylated enzyme samples from the two bromoacetates revealed that N pi-(carboxy[14C]methyl)histidine was the modified amino acid labeled in each case. Digestion with trypsin produced peptides that were isolated by reverse-phase high-performance liquid chromatography and found to contain N pi-(carboxy[14C]methyl)histidine. Both the 17 alpha bromoacetate and also the 17 beta bromoacetate modified the same histidine in the peptide Phe-Tyr-Gln-Tyr-Leu-Ala-His(pi-CM)-Ser-Lys. Previously, the same histidine had been exclusively labeled by estrone 3-(bromoacetate) and shown not to be directly involved in catalytic hydrogen transfer at the D-ring of estradiol. Therefore, this histidine was presumed to proximate the A-ring of the bound steroid substrate. The present results suggest that the 17 alpha bromoacetate and 17 beta bromoacetate D-ring analogues of estradiol react with the same active site histidine residue as estrone 3-(bromoacetate), the A-ring analogue of estrone.(ABSTRACT TRUNCATED AT 250 WORDS)

17-Hydroxysteroid Dehydrogenases↗
PubMed1986

Human placental estradiol 17 beta-dehydrogenase: sequence of a histidine-bearing peptide in the catalytic region.

The amino acid sequence of an octapeptide from the catalytic site of human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was established by affinity-labeling techniques. The enzyme was inactivated separately by 12 beta-hydroxy-4-estrene-3,17-dione 12-(bromo[2-14C]acetate) and 3-methoxyestriol 16-(bromo[2-14C]acetate) at pH 6.3. The inactivations, in both cases, followed pseudo-first-order kinetics with half-times for the 12 beta and 16 alpha derivatives being 192 and 68 h, respectively. Both derivatives are known substrates that inactivate in a time-dependent, irreversible manner and that modify cysteine residues to form (carboxymethyl)cysteine and histidine residues to form either N tau- or N pi-(carboxymethyl)histidine. The inactivated enzyme samples were separately reduced, carboxymethylated, and digested with trypsin. The tryptic digests were applied to Sephadex G-50 and the radioactive N tau- and N phi-(carboxymethyl)histidine-bearing peptides identified. The peptides were further purified by cation-exchange chromatography and gel filtration. Final purification was achieved by HPLC prior to sequencing. It was determined that both steroid derivatives modified either of the two histidine residues in the peptide Thr-Asp-Ile-His-Thr-Phe-His-Arg. These histidines are different from a histidine that was previously shown to be alkylated by estrone 3-(bromoacetate) and that was presumed to proximate the A ring of the bound steroid. It is concluded that the two histidine residues identified in the present study proximate the D ring of the steroid as it binds at the active site and may participate in the hydrogen transfer effected by human placental estradiol 17 beta-dehydrogenase.

17-Hydroxysteroid Dehydrogenases↗
PubMed1985

Purification and characterization of 20 alpha-hydroxysteroid dehydrogenase from bull testis.

20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSD) from bull testis has been purified to homogeneity and characterized in terms of apparent molecular weight, lack of subunit composition, substrate and cofactor specificity and certain kinetic parameters. The enzyme activity is localized in the 105,000 g supernatant and is stable at 4 degrees C in the presence of glycerol and dithiothreitol. Purification was achieved by ammonium sulfate precipitation followed by affinity chromatography on reactive red 120-agarose and subsequent gel filtration. The apparent molecular weight of the homogeneous enzyme, as determined by gel filtration on Sephacryl S-300 is 34,000. The mobility of the enzyme in sodium dodecyl sulfate (SDS) gel electrophoresis corresponds to a mol. wt of 40,000. These observations indicate that the enzyme is a single-stranded, monomeric polypeptide. The enzyme catalyzes the reduction of the 17-hydroxyprogesterone to 17,20 alpha-dihydroxy-4-pregnene-3-one in the presence of NADPH, the preferred cofactor. Homogeneous 20 alpha-HSD has an SA of 115 mIU/mg, and has been purified 14,000-fold with an overall 68% recovery. It exhibits a pH optimum at 5.6 and appears to be highly specific for 17-hydroxyprogesterone with an apparent Km-value of 7.3 X 10(-5) M. Androstenedione and corticosterone do not serve as substrates under the described experimental conditions. The enzyme does not possess 17 alpha- or 17 beta-HSD activity.

20-Hydroxysteroid Dehydrogenases↗
PubMed1984

Purification and characterization of epimeric estradiol dehydrogenases (17 alpha and 17 beta) from equine placenta.

Estradiol 17 alpha-dehydrogenase (EC 1.1.1.148) and estradiol 17 beta-dehydrogenase (EC 1.1.1.62) from horse placenta have been purified to homogeneity. Both enzymes are localized in the microsomal fraction and are solubilized in 1.5% sodium cholate. The 17 alpha- and 17 beta-dehydrogenases are separated by selective elution from hydroxylapatite with 0.5 and 1.0 M potassium phosphate, respectively. Subsequent purification is achieved by two affinity-absorption steps using reactive blue 2-agarose and estriol hemisuccinate-Sepharose. Homogeneous estradiol 17 alpha-dehydrogenase has a specific activity of 10 IU/mg and has been purified 5900-fold with an 87% recovery. Homogeneous estradiol 17 beta-dehydrogenase has a specific activity of 10.6 IU/mg and has been purified 15 000-fold with an apparent recovery of 100%. Each enzyme exhibits a single band on polyacrylamide disc gel electrophoresis and on sodium dodecyl sulfate (SDS) gel electrophoresis. The mobilities of the two on SDS gels are identical and correspond to subunit molecular weights of 33000. The apparent molecular weight of the undenatured, active enzyme, as determined by gel filtration on Sephacryl S-300, is 52000 in the case of the 17 alpha-dehydrogenase and 68500 for the 17 beta-dehydrogenase. Both enzymes exhibit pH optima at 9.0-9.5; both prefer NAD+ over NADP+ but utilize both cofactors. Both are highly specific for their respective epimers of estradiol with apparent Km values of 1.7 microM at pH 9.5.

17-Hydroxysteroid Dehydrogenases↗
PubMed1983

Human placental estradiol 17 beta-dehydrogenase. Identification of a single histidine residue affinity-labeled by both 3-bromoacetoxyestrone and 12 beta-bromoacetoxy-4-estrene-3,17-dione.

Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity-labeled at pH 6.3 by 3-bromo[2'-14C]acetoxyestrone and 12 beta-bromo-[2'-14C] acetoxy-4-estrene-3,17-dione (both are substrates) in separate incubations. The affinity-alkylated enzyme samples were then treated separately as described below. Amino acid compositions of both samples revealed radioactive 3-carboxymethylhistidine. Tryptic digests of each sample were prepared, applied to Sephadex G-50, and 3-carboxymethylhistidine-bearing fractions identified. These peptides were further purified by cation exchange chromatography, gel filtration, and paper electrophoresis. The purified, 3-carboxymethylhistidine-bearing peptides labeled by the two steroids had identical electrophoretic mobilities at pH 6.5, 3.5, and 1.9. The amino acid sequence of the radioactive peptide alkylated by 3-bromo[2'-14C]acetoxyesterone was determined as: Leu-Ala-3-[14C]CmHis-Ser-Lys. The smaller quantity of peptide obtained from the inactivation with 12 beta-bromo[2'-14C]acetoxy-4-estrene-3,17-dione precluded the determination of its complete sequence. However, the first 3 residues were found to be Leu-Ala-3-[14C]CmHis and the amino acid composition showed that serine and lysine were also present. It is concluded that the steroid-binding site of human placental estradiol 17 beta-dehydrogenase contains a histidine residue which proximates the upper A-ring region of the steroid as it undergoes the reversible binding step.

17-Hydroxysteroid Dehydrogenases↗
PubMed1982

Spatial relationship of steroid and cofactor at the active site of human placental estradiol 17 beta-dehydrogenase.

Two affinity-labeling steroids (2-bromo[2'-14C]acetamidoestrone methyl ether and 16 alpha-bromo[2'-14C]acetoxyestradiol 3-methyl ether) which bear their reagent groups on the A- and D-ring of the molecule, respectively, and which are both substrates, have been used to elucidate spatial relationships of steroid and cofactor as they undergo the reversible binding step at the active site of human placental estradiol 17 beta-dehydrogenase. The 2-derivative alkylates its evolutive cofactor (NADH) in the presence of the enzyme but not in the absence of enzyme. The rate of cofactor alkylation increases with increasing quantities of enzyme and is slowed by estrone which competes for the enzyme-active site. To the contrary, the 16 alpha-derivative does not detectably alkylate its evolutive cofactor (NAD+). The product of cofactor alkylation by 2-bromoacetamidoestrone methyl ether was treated to effect hydrolytic removal of the adenine moiety from the remainder of the cofactor, reduction of the steroid 17-keto group, and crystallization. The final crystalline product has been identified as 2-[2'-6N-adenyl]acetamidoestradiol 3-methyl ether (IUPAC name: N-(3-methoxy-17 beta-hydroxy-1,3,5(10)-estratrien-2-yl)-2-(purin-6-ylamino) acetamide) by ultraviolet, infrared, NMR, and mass spectral analysis.

17-Hydroxysteroid Dehydrogenases↗
PubMed1982

Isolation of histidyl peptides of the steroid-binding site of human placental estradiol 17 beta-dehydrogenase.

Human placental estradiol 17 beta-dehydrogenase (E.C. 1.1.1.62) was inactivated at pH 6.3 by 3-bromo [2'-14C] acetoxy-1,3,5(10) estratrien-17-one, a know substrate. The affinity-alkylated enzyme was then hydrolyzed by trypsin. Radioactive peptides were initially isolated by gel filtration and identified according to which residue was alkylated. Tryptic peptides containing radioactive 3-carboxymethylhistidyl residues were further purified by cation-exchange chromatography. The population of these peptides varied, depending upon the conditions of enzyme inactivation. With 60 microM 3-bromo[2'-14]acetoxy-1,3,5 (10) estratrien -17-one four major peptides (a,b,c,d) each containing radioactive 3-carboxymethylhistidine, were eluted from the cation-exchange column. The alkylation of all of these peptides was completely suppressed when the enzyme was inactivated in the presence of excess estradiol-17 beta. The presence of equimolar NADPH during incubation greatly enhanced the alkylation of all four peptides. In the presence of NADPH, estradiol-17 beta most significantly decreased the formation of peptide d. Peptide d was the only peptide identified when the concentration of the alkylating steroid was lowered to 6 microM, a value approaching the Km. These observations indicate that peptide d is a histidyl-bearing peptide from the steroid-binding site which proximates the steroid A-ring. They further suggest that with the affinity labeling steroid at higher concentrations other nonspecific, hydrophobic sites on the enzyme are occupied and labeled.

17-Hydroxysteroid Dehydrogenases↗
PubMed1980

Synthesis of 2-bromoacetamidoestrone methyl ether and study of the steroid-binding site of human placental estradiol 17 beta-dehydrogenase.

To characterize further the active site of human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62), we have synthesized 2-bromoacetamidoestrone methyl ether. The affinity-labeling steroid is a substrate for the homogeneous enzyme. It inactivates the enzyme in a time-dependent, irreversible manner which follows pseudo-first order kinetics. Further, inactivation conducted with varying steroid concentration displays saturation kinetics. When 1.7 x 10(-6) M enzyme is inactivated by 2.6 x 10(-4) M 2-bromoacetamidoestrone methyl ether, the presence of an equimolar concentration of estradiol or 5.2 x 10(-4) M concentrations of NAD+, NADP+, or NADPH markedly slow the rate of inactivation. Bromoacetate (2.6 x 10(-4) M) does not inactivate the enzyme. After inactivation with 2-bromo[2'-14C]acetamidoestrone methyl ether, amino acid analysis reveals carboxymethylated derivatives of cysteine, histidine, and lysine containing 65, 25, and 8%, respectively, of the total incorporated carboxymethyl groups. The presence of estradiol, NADP+, or NADPH clearly inhibits alkylation of cysteinyl and histidyl residues and slows the rate of enzyme inactivation. Protection of these residues by both estradiol and NADPH suggests that they may actually be in the cofactor region of the active site, that this region is close to the steroid A-ring, and that binding of cofactor, by physical interposition, denies the reagent-bearing steroid access to these residues.

17-Hydroxysteroid Dehydrogenases↗
PubMed1979

Some characteristics of 17 beta-estradiol dehydrogenase from bovine placenta.

The activity of 17 beta-estradiol dehydrogenase (E.C. 1.1.1.62) was measured, and its distribution in the subcellular fractions of bovine placenta was compared. Assay of activity was based on the formation of radioactive estrone from 17 beta[4(-14)C]-estradiol. Either NAD+ or NADP+ can serve as cofactor for the enzyme. The nuclear and microsomal fractions of the placental homogenate exhibited the highest specific enzymatic activities before and after treatment with Triton X-100. Electron micrographs of these two fractions prior to treatment with Triton X-100 showed satisfactory purity. 17 beta-estradiol dehydrogenase from bovine placenta exhibits a pH optimum of about 9.5-10.5, and is activated by 5 x 10(-6)M ZnCl2; comparable concentrations of CaCl2 and MgCl2 inactivate the enzyme. The apparent Michaelis constants, Km, for 17 beta-estradiol and NAD+ are 1.4 x 10(-6)M and 5.5 x 10(-5)M respectively. No 17 alpha-estradiol dehydrogenase activity was demonstrable when using 17 alpha-estradiol as substrate.

17-Hydroxysteroid Dehydrogenases↗
PubMed1978

Free testosterone concentration in serum: a method for determination.

Two procedures for quantifying serum levels of testosterone-estradiol-binding globulin (TeBG) have been evaluated. Both procedures utilize indirect methods for assessing the extent of protein-steroid interaction for the purpose of devising a clinically feasible procedure for estimating serum free testosterone. In the first method serum protein concentration was varied by dilution until 50 per cent of steroid binding was obtained at a given concentration of steroid. Dextran-coated charcoal was used for separating TeBG-bound steroid from the unbound fraction. In the second procedure DEAE-cellulose filtration was used to isolate the TeBG-steroid complex for quantitation. Having a sufficiently accurate measure of TeBG levels together with a knowledge of the total endogenous testosterone permitted the estimation of serum free testosterone based on the binding affinities of serum proteins. The preferred DEAE-cellulose filtration method has proved useful for routine clinical evaluation of physiologically effective androgen levels.

Charcoal↗
PubMed1977

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

We recently demonstrated that human placental estradiol-17beta-dehydrogenase possesses a histidyl residue in the catalytic region of the active site by affinity-labeling studies with 16alpha-bromoacetoxyestradiol-3-methyl ether. We now report the synthesis of 12beta-bromoacetoxy-4-estrene-3,17-dione and its use in affinity labeling of the enzyme. The steroid was synthesized by incubation of 4-estrene-3,17-dione with Colletotrichum gloesporioides. The product was recrystallized from ethanol and structure assured by IR and NMR spectroscopy. The steroid is a substrate, which indicates that it binds at the active site. When the enzyme is incubated with a 150-fold molar excess of 12beta-bromoacetoxy-4-estrene-3,17-dione in potassium phosphate buffer at pH 7.0, the enzyme is inactivated in a time-dependent, irreversible manner. Inactivation follows pseudo-first-order kinetics with a half life of 18 hours. Analysis of a hydrolysate of the enzyme after inactivation with 12beta-bromo[2'-3H]acetoxy-4-estrene-3,17-dione reveals tritiated 1-, 3-, and 1,3-dicarboxymethylhistidine. The affinity labeling of a histidyl enzyme residue by both 16alpha- and 12beta-bromoacetoxy steroids localizes that residue near the point of catalysis and suggests that it may participate in the catalytic event.

17-Hydroxysteroid Dehydrogenases↗