Chromogranin A-processing proteinases in purified chromaffin granules: contaminants or endogenous enzymes?

• Published in: Biochimica et biophysica acta Vol. 1033; no. 1; pp. 65 - 72
• Main Authors: Laslop, Andrea, Fischer-Colbrie, Reiner, Kirschke, Heidrun, Hogue-Angeletti, Ruth, Winkler, Hans
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 29.01.1990; Elsevier; North-Holland
• Subjects: 4-Chloromercuribenzenesulfonate - pharmacology; Adrenal Medulla - ultrastructure; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Calcium - pharmacology; Cathepsin B - isolation & purification; Cathepsin B - metabolism; Cathepsin D - isolation & purification; Cathepsin D - metabolism; Cattle; Chromaffin granule; Chromaffin Granules - enzymology; Chromaffin System - enzymology; Chromatography; Chromogranin A; Chromogranins - metabolism; Cysteine Endopeptidases - analysis; Cysteine Endopeptidases - metabolism; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Hydrolases; Immunoblotting; Lysosomes - enzymology; Nerve Tissue Proteins - metabolism; Pepstatins - pharmacology; Peptide Hydrolases - analysis; Peptide Hydrolases - metabolism; Protease Inhibitors; Proteinase; Trypsin - analysis; Trypsin - metabolism; Chromogranin A; PCMPS; Chromaffin granule; Proteinase; Bovine; Gel electrophoresis; Enzyme; Adrenal medulla; HPLC chromatography; Proteins; Substrate; Vertebrata; Mammalia; Chromogranin; Enzymatic activity; Affinity chromatography; Blotting assay; Substrate specificity; Gel filtration; Artiodactyla; Ungulata
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(90)90195-3

It was the purpose of this study to define the chromogranin A-processing proteinases present in highly purified preparations of bovine chromaffin granules. The most active enzyme had a pH optimum of 5.0 and was inhibited by pepstatin. It could be identified immunologically as a cathepsin D-like enzyme and subcellular fractionation established its lysosomal origin. After removal of this enzyme the remaining activity at pH 5.0 was mainly due to a cathepsin B-like proteinase. The presence of this enzyme could also be attributed to lysosomal contamination. In the presence of calcium, a further proteolytic activity became apparent at pH 5.0. This enzyme which was inhibited by p-chloromercuriphenylsulfonic acid was localized in chromaffin granules. A trypsin-like peptidase, most active at pH 8.2, was enriched in a membrane wash of chromaffin granules. Subcellular fractionation indicated that this enzyme is preferentially bound to the membranes of very dense particles probably representing a subpopulation of chromaffin granules. This study establishes that the most active chromogranin A-degrading proteinases present in highly purified chromaffin granules are attributable to lysosomal contamination. Two enzymes with low activity (a Ca 2+ activated proteinase and a trypsin-like enzyme) are, apparently, true constituents of chromaffin granules.