ISOLATION AND IDENTIFICATION OF INTACT CHROMOGRANIN-A AND 2 N-TERMINAL PROCESSING PRODUCTS, VASOSTATIN-I AND VASOSTATIN-II, FROM BOVINE ADRENAL-MEDULLA CHROMAFFIN GRANULES BY CHROMATOGRAPHIC AND MASS-SPECTROMETRIC METHODS

Chromogranin A (CGA) is the most abundant protein of the bovine adrena l medulla and plays an important role as precursor protein of several peptides that act as modulators for endocrine cell secretory activity. Furthermore, it is presumed to play a role in the targeting of peptid e hormones and neurotransmitters to granules of the regulated pathway. However, its complete primary structure and proteolytic processing ha ve not yet been identified. This study describes a rapid and efficient procedure for the high yield isolation of bovine CGA and its N-termin al processing products, vasostatin I and II. Using the lysate from bov ine adrenal medulla chromaffin granules, the soluble proteins were pur ified by three consecutive HPLC steps, thereby avoiding the use of buf fer solutions. The protein fractions were isolated and characterized b y SDS-PAGE and Western blot analysis as well as by mass spectrometry. In the latter analysis, the efficiency of matrix-assisted laser desorp tion ionization-mass spectrometry (MALDI-MS) was demonstrated, enablin g the unequivocal and sensitive characterization of proteins from crud e mixtures. Sufficient amounts of pure protein were obtained by the pr esent procedure to form the basis for detailed structural studies by s pectroscopic methods and X-ray crystallography.