Isolation of rat renal NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.
Rat renal NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase was purified to apparent homogeneity in the present study. The purification included ammonium sulfate precipitation, DEAE-Sepharose CL-6B column chromatography, Blue Sepharose CL-6B column chromatography, Sephadex G-100 column chromatography and Mono-P isoelectrofocusing column chromatography. Among the chromatographies used, Mono-P chromatofocusing column of fast protein liquid chromatography gave the most powerful resolution. The enzyme was eluted at pH 6.75 on chromatofocusing column. It indicated that the rat renal enzyme is an acidic protein. Its molecular weight in SDS-polyacrylamide gel electrophoresis was 28 Kd, and the molecular weight of the enzyme having enzyme activity detected by gel filtration column chromatography was 55 Kd. For comparing the characteristics of rat renal enzyme with that of human placental enzyme, polyclonal antibodies and a monoclonal antibody against human placental enzyme were used. The rat renal enzyme did not react with the polyclonal antibodies of human placental enzyme, however, it reacted with a monoclonal antibody of human placental enzyme. The results indicated that the antigenicity of rat renal NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase is different from that of human placental enzyme. The amino acid composition of rat renal enzyme was also different from that of human placental enzyme. Nine tyrosine molecules were observed in the subunit of rat renal enzyme, while no tyrosine has been reported in human placental enzyme.
Chang
W C
WC
Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.
Wu
H L
HL
Hsu
S Y
SY
Chen
F S
FS
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003429:Cross Reactions; D005260:Female; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000276:immunology / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D007668:Kidney / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity
D000596:Amino Acids; D006911:Hydroxyprostaglandin Dehydrogenases; C035859:15-hydroxyprostaglandin dehydrogenase