Isolation and identification of intact chromogranin A and two N-terminal processing products, vasostatin I and II, from bovine adrenal medulla chromaffin granules by chromatographic and mass spectrometric methods.
Chromogranin A (CGA) is the most abundant protein of the bovine adrenal 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 peptide hormones and neurotransmitters to granules of the regulated pathway. However, its complete primary structure and proteolytic processing have not yet been identified. This study describes a rapid and efficient procedure for the high yield isolation of bovine CGA and its N-terminal processing products, vasostatin I and II. Using the lysate from bovine adrenal medulla chromaffin granules, the soluble proteins were purified by three consecutive HPLC steps, thereby avoiding the use of buffer solutions. The protein fractions were isolated and characterized by SDS-PAGE and Western blot analysis as well as by mass spectrometry. In the latter analysis, the efficiency of matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) was demonstrated, enabling the unequivocal and sensitive characterization of proteins from crude mixtures. Sufficient amounts of pure protein were obtained by the present procedure to form the basis for detailed structural studies by spectroscopic methods and X-ray crystallography.
Bauer
S H
SH
Faculty of Chemistry, University of Konstanz, Germany.
Zhang
X Y
XY
Liang
F
F
De Potter
W P
WP
Claeys
M
M
Przybylski
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D019439:Chromaffin Cells / Q000737:chemistry* / Q000648:ultrastructure; D002837:Chromaffin Granules / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D053379:Chromogranin A; D002864:Chromogranins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000235:genetics; D014665:Vasodilator Agents / Q000032:analysis / Q000378:metabolism
D053379:Chromogranin A; D002864:Chromogranins; D010446:Peptide Fragments; D014665:Vasodilator Agents; C102106:vasostatin II