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J Z Jin

Publications and source records attributed to J Z Jin.

6 recordsLinked to original sources
PubMed1999

Human estrogenic 17beta-hydroxysteroid dehydrogenase: predominance of estrone reduction and its induction by NADPH.

Human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) plays a crucial role in the last step of the synthesis of estrogens. A detailed kinetic study demonstrated that the enzyme shows about 240 fold higher specificity towards estrone reduction than estradiol oxidation at physiological pH using tri-phosphate cofactors. The kcat/Km values are 96 +/- 10 and 0.4 +/- 0.1 s-1 (microM)-1 respectively for the above two reactions. However, it has been shown that this difference is closely linked to the use of NADPH and NADP cofactors. A binding study using equilibrium dialysis indicated similar KD (equilibrium dissociation constant) of 11 +/- 1 and 4.7 +/- 0.9 microM for estrone and estradiol, respectively. The binding affinity of 17beta-HSD1 to estrone was significantly increased with a KD of 1.6 +/- 0.2 microM in the presence of NADP, the latter used as an analogue of the NADPH. The results of binding studies agree with the steady-state kinetics, which showed that the Km of estrone is 12-fold lower when using NADPH as a cofactor than when using NADH. These results strongly suggest that the cofactor plays a crucial role in the stimulation of the specificity for estrogen reduction.

17-Hydroxysteroid Dehydrogenases↗
PubMed1995

Human 17 beta-hydroxysteroid dehydrogenase: optical properties of its complex with NADP+.

The apoenzyme of the human placental 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) and its complex with NADP+ were prepared from two alternative procedures. The apoenzyme (Form I) has an absorption maximum at about 279 nm, and an absorption ratio at 280 and 260 nm of 1.65 +/- 0.1; whereas the complex (Form II) has a broad absorption peak between 268-278 nm, and a 280 to 260 nm ratio of 1.1 +/- 0.05. Upon addition of the substrate estradiol to the complex, an absorption increase at 340 nm and a fluorescence emission at 450 nm, following NADPH formation, were produced. Both changes indicate that one cofactor is tightly bound to the 17 beta-HSD molecule in this complex. No significant optical change can be produced in this way for the apoenzyme. Convenient analyses of cofactor content of the enzyme are thus provided. The optical analyses and the homogeneous apo- or holo-enzyme preparations are important in the study of the enzyme's function and crystallization. This is the first human steroid converting enzyme which has yielded X-ray quality crystals.

17-Hydroxysteroid Dehydrogenases↗
PubMed1994

Human 17 beta-hydroxysteroid dehydrogenase: overproduction using a baculovirus expression system and characterization.

Estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) plays a pivotal role in the synthesis of estrogens. We overproduced human placental estrogenic 17 beta-HSD using a baculovirus expression system for the study of the enzyme mechanism. A cDNA encoding the entire open reading frame of human 17 beta-HSD was inserted into the genome of Autographa californica nuclear polyhedrosis virus and expressed in Spodoptera frugiperda (Sf9) insect cells. Metabolic labeling and Western blot analysis using polyclonal antibodies raised against native human 17 beta-HSD indicated that a molecule with an apparent mass of 35 kDa was maximally expressed 60 h after infection. At that time interval, intracellular 17 beta-HSD activity reached 0.26 U/mg of protein in crude homogenate, about 70 times the level measured in human placenta. Purification of recombinant 17 beta-HSD was achieved by a single affinity fast liquid protein chromatography step yielding 24 mg of purified 17 beta-HSD protein per liter of suspension culture, with a specific activity of about 8 mumol/min/mg of protein for conversion of estradiol into estrone, at pH 9.2. In addition, the recombinant protein purified from infected Sf9 cells was assembled as a dimer with molecular mass and specific activity identical to those of the enzyme purified directly from placenta. The present data show that the baculovirus expression system can provide active 17 beta-HSD that is functionally identical to its natural counter-part and easy to purify in quantities suitable for its physico-chemical studies.

17-Hydroxysteroid Dehydrogenases↗
PubMed1993

Preparation of the apoenzyme and holoenzyme forms of human 17 beta-hydroxysteroid dehydrogenase.

The apoenzyme and holoenzyme (NADP+ complex) of human placental 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) were prepared from affinity chromatography using various elutions by column liquid chromatography. The apoenzyme was obtained using NAD+ elution in a Blue-Sepharose column, followed by NAD+ separation on a Phenyl-Superose hydrophobic-interaction or a Mono Q anion-exchange column. The 17 beta-HSD-NADP+ complex was prepared using NADP+ elution in a Blue-Sepharose column. The two forms have different A280/A260 ratios and are suitable for further study of enzyme-cofactor interactions.

17-Hydroxysteroid Dehydrogenases↗
PubMed1992

Subunit identity of the dimeric 17 beta-hydroxysteroid dehydrogenase from human placenta.

Human placental 17 beta-hydroxysteroid dehydrogenase has been purified with a new rapid procedure based on fast protein liquid chromatography, yielding quantitatively a homogeneous preparation with high specific activity catalyzing the oxidation of 7.2 mumol of estradiol/min/mg of enzyme protein at 23 degrees C, pH 9.2. This preparation was shown to have a subunit mass of 34.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis while having a molecular mass of 68 kDa by both Superose-12 gel-filtration and native pore gradient gel electrophoresis. When 17 beta-hydroxysteroid dehydrogenase was expressed in HeLa cells or overproduced in insect cells using the baculovirus expression system, both from its cDNA encoding a protein of 34 kDa, the enzyme had the same migration in native and sodium dodecyl sulfate-gel electrophoresis as the purified one from human placenta and eluted from the Superose-12 column at the same elution volume. Moreover, all the above forms of this enzyme have similar specific activity. These results clearly demonstrate the identity of the three enzyme forms. The enzyme produced from the cDNA is expressed as a dimer, and its two subunits are identical. 17 beta-Hydroxysteroid dehydrogenase subunit identity is thus proved. The NH2-terminal analysis revealed a unique sequence of Ala-Arg-Thr-Val-Val-Leu-Ile for the purified enzyme from placenta, further confirming the above conclusion.

17-Hydroxysteroid Dehydrogenases↗