Purification and structural characterization of placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase. The primary structure reveals the enzyme to belong to the short-chain alcohol dehydrogenase family.
Human placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase was purified to homogeneity according to a five-step method, with chromatography on DEAE-Sepharose, Blue Sepharose, and Mono-Q FPLC as principal steps. Final yield was 23% and purification about 13,000-fold, with a specific activity of 24,000 milliunits/mg. The subunit molecular weight is about 29,000 as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and the native protein molecular weight is about 54,000 as estimated by Sephadex G-100 chromatography, establishing the enzyme to be a dimer of similar-sized protein chains. The subunit N-terminal residue is methionine, and the alpha-amino group is free. The complete primary structure was determined by peptide analysis, based essentially on four different proteolytic treatments (Lys-specific protease, Glu-specific protease, Asp-specific protease, and CNBr). The protein chain is composed of 266 residues, with C-terminal glutamine. A microheterogeneity was detected at position 217, with both Cys and Tyr, in about equal amounts, from a preparation starting with a single placenta. No other subunit heterogeneities were detected. The protein is clearly but distantly related to insect alcohol dehydrogenases, characterized bacterial dehydrogenases of sugar metabolism, and bacterial and eukaryotic steroid dehydrogenases. Together, these results establish that placental 15-hydroxyprostaglandin dehydrogenase is a member of the short-chain nonmetalloenzyme alcohol dehydrogenase protein family. The protein has four cysteine residues (five with the positional microheterogeneity), but there is no evidence for functional importance of any of these residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Krook
M
M
Department of Chemistry I, Karolinska Institutet, Stockholm, Sweden.
Marekov
L
L
Jörnvall
H
H
D000426:Alcohol Dehydrogenase; D000595:Amino Acid Sequence; D000837:Anion Exchange Resins; D002845:Chromatography / Q000379:methods; D005260:Female; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D012117:Resins, Synthetic; D012685:Sepharose / Q000031:analogs & derivatives
D000837:Anion Exchange Resins; D012117:Resins, Synthetic; C042855:Mono Q; D012685:Sepharose; D006911:Hydroxyprostaglandin Dehydrogenases; D000426:Alcohol Dehydrogenase; C035859:15-hydroxyprostaglandin dehydrogenase