ISOLATION OF 2 PEPTIDES FROM RAT GONADOTROPH-CONDITIONED MEDIUM DISPLAYING AN AMINO-ACID-SEQUENCE IDENTICAL TO FRAGMENTS OF SECRETOGRANIN-II

Pituitary cells from 14-day-old rats were separated by unit gravity ve locity sedimentation, and a highly enriched population of gonadotrophs was established in reaggregate cell culture in serum-free and serum a lbumin-free defined culture media. The medium conditioned by these agg regates was concentrated, ultrafiltrated, and the concentrated substan ces consecutively separated by two reversed-phase HPLC steps, a gel fi ltration step and an additional reversed-phase purification step. Two peptides could be isolated that displayed an N-terminal amino acid seq uence identical to fragments of rat presecretogranin II: one was cleav ed at the dibasic amino acid residues K-182-R(183) and the other at th e dibasic amino acid residues K-569-R(570). The latter peptide was com pletely purified and separated into three variants with the same N-ter minal amino acid sequence. From an analysis by electrospray ionization mass spectrometry, it was deduced that the three forms extended up to amino acid residue 612 (A), which is, in presecretogranin II, also fl anked by two basic amino acids (K(613)R(614)). Two variant forms had a molecular mass that was 17 Da higher. The present data support the hy pothesis that secretogranin II is a precursor of putative regulatory p eptides.