Purification and characterization of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase from porcine kidney.
A NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) from porcine kidney was purified to homogeneity by acid precipitation, blue agarose affinity chromatography, hydroxyapatite-ultrogel adsorption chromatography, DEAE-Sephadex ion-exchange chromatography and NAD(+)-agarose affinity chromatography. The specific activity of the homogeneous enzyme was 31.2 U/mg. The molecular mass of the native enzyme was estimated to be 55,000 Da, whereas that of SDS-treated enzyme was 29,000 Da indicating that the native enzyme was dimeric. Compared to human placental 15-OH-PGDH, porcine kidney enzyme gave a similar general amino acid residue distribution. Chemical modification of the enzyme with N-ethyl maleimide (3 microM), N-chlorosuccinimide (20 microM) or 2,4,6-trinitrobenzenesulfonic acid (2.5 microM) followed pseudo-first-order inactivation kinetics, and inactivation could be prevented by the presence of NAD+ (1 mM) but not of prostaglandin E1 (140 microM) indicating the involvement of cysteine, methionine and lysine residues in the coenzyme binding site. Inactivation by diethyl pyrocarbonate (1.25 mM) or phenylglyoxal (10 mM) also showed pseudo-first-order kinetics suggesting that histidine and arginine residues were catalytically or structurally important in the native enzyme. These studies provide new insights into the structure and function of 15-OH-PGDH.
Mak
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OT
Department of Biology, National Cheng-Kung University, Tainan, Taiwan, China.
Liu
Y
Y
Tai
H H
HH
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004589:Electrophoresis, Disc; D006911:Hydroxyprostaglandin Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D013552:Swine
D000596:Amino Acids; D009243:NAD; D006911:Hydroxyprostaglandin Dehydrogenases; C035859:15-hydroxyprostaglandin dehydrogenase