NEUROCHEMICAL CHARACTERIZATION OF PREPROTACHYKININ B(50-79) IMMUNOREACTIVITY IN THE RAT

Preprotachykinin B (PPT-B) contains two peptide sequences which are fl anked by pairs of dibasic amino acids: the decapeptide neurokinin B an d a 30 amino acid non-tachykinin peptide consisting of the amino acids 50-79 of PPT-B. Whereas the existence of neurokinin B is well establi shed in brain and peripheral tissues, native PPT-B(50-79) has not been identified so far. We have previously studied the distribution of PPT -B(50-79)-immunoreactivity in the rat brain using antibodies directed against synthetic PPT-B(50-79). Now we adapted a radioimmunoassay for characterizing neurochemically PPT-B(50-79)-immunoreactivity in the ra t. In the brain concentrations ranging from 2 to 180 fmol/mg wet tissu e weight were measured using synthetic PPT-B(50-79) as standard. The h ighest concentrations were observed in the interpeduncular nucleus and in the hypothalamus (180 and 90 fmol/mg tissue, respectively). Interm ediate concentrations (15 to 60 fmol/mg tissue) were present in cortic al areas, in the hippocampus, the spinal cord and in the olfactory bul b. Modest levels were detected in the cerebellum. Considerably lower c oncentrations of PPT-B(50-79)-immunoreactivity were observed in periph eral tissues. They were highest in the adrenal medulla and in the urin ary bladder (3.0 and 1.2 fmol/mg tissue, respectively). This distribut ion, as observed by radioimmunoassay, correlated to that previously re vealed by immunocytochemistry. Tissue concentrations of total PPT-B(50 -79) immunoreactivity, however, were slightly higher than those of neu rokinin B. Gel filtration chromatography on Sephadex G50 and reversed phase HPLC revealed at least three PPT-B(50-79) immunoreactive peaks. About 90% of the PPT-B(50-79)-immunoreactivity was contained within 2 peaks of apparently higher molecular weight than PPT-B(50-79). A minor portion of PPT-B(50-79)-immunoreactivity comigrated with the syntheti c peptide, suggesting that only minor amounts of PPT-Bf are formed in vivo. The processing enzyme(s) cleaving protachykinin B at the pair of basic amino acids (Lys(80)-Arg(81)) located between PPT-B(50-79) and neurokinin B may not be acting at the Arg(48)-Arg(49) Site (followed b y -Leu(50)) at the amino terminal end of PPT-B(50-79).