Identification of β-trace as prostaglandin D synthase

International Colloquium on Beta-Truce: Hayuishi IDENTIFICATION OF P-TRACE AS PROSTAGLANDIN D SYNTHASE Kikuko Watanabe, Osaka Bioscience Institute, Osaka, Japan P-Trace protein is a major polypeptide constituent of human cerebrospinal fluid (CSF). Although it was discovered by immunoelectrophoresis as a protein specific to the CSF in 1961 by Clausen, its function has not yet been determined. Kuruvilla et al. and Zahn et al. reportedthat the amino terminal sequence of p-trace of human CSF is identical with that of glutathione (GSH)independent PGD synthase from human brain. In 1993, Hoffmann et al. demonstrated that the full amino acid sequence of P-trace protein of human CSF is identical with that of human brain PGD synthase without the signal peptide except for S 154T. Furthermore, the N-terminal 33 amino acid sequence of the protein isolated from the urine of a patient with myeloma and associated renal desease is also identical to that of PGD synthase (personal communication, C.Muphy). Human CSF is the richest source of PGD synthase, a key enzyme in sleep regulation, having activity 20-130 nmol/min/mg protein that is almost two order magnitude higher than that of crude rat brain homogenate (2-7 nmol/min/mg protein). PGD synthase was purified from human CSF approximately &fold to apparent homogeneity in a yield of 6% by ammonium sulfate fractionation followed

by

column

chromatographies

on

phenyl-Sepharose and DEAE-cellulose. ‘Ihe purified enzyme displayed enzymatic properties almost identical to those of rat brain PGD synthase in terms of Mr, specific activity, optimum pH, Km value and SH requirement. The purified PGD synthase cross-reacted with an antibody against rat brain PGD synthase and also with that against human p-trace which was kindly donated by Mader. The N-terminal sequences of human PGD synthase, p-trace and purified PGD synthase from human CSF were essentially identical. These results clealy showed that p-trace is structually and enzymologically identical to PGD synthase.

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