Chromogranin A-processing proteinases in purified chromaffin granules: contaminants or endogenous enzymes?
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 rho-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 Ca2+ activated proteinase and a trypsin-like enzyme) are, apparently, true constituents of chromaffin granules.
Laslop
A
A
Department of Pharmacology, University of Innsbruck, Austria.
Fischer-Colbrie
R
R
Kirschke
H
H
Hogue-Angeletti
R
R
Winkler
H
H
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000313:Adrenal Medulla / Q000648:ultrastructure; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002401:Cathepsin B / Q000302:isolation & purification / Q000378:metabolism; D002402:Cathepsin D / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D002845:Chromatography; D053379:Chromogranin A; D002864:Chromogranins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008247:Lysosomes / Q000201:enzymology; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000032:analysis / Q000378:metabolism*; D011480:Protease Inhibitors; D014357:Trypsin / Q000032:analysis / Q000378:metabolism
D053379:Chromogranin A; D002864:Chromogranins; D009419:Nerve Tissue Proteins; D010436:Pepstatins; D011480:Protease Inhibitors; C014195:Streptomyces pepsin inhibitor; D002731:4-Chloromercuribenzenesulfonate; D010447:Peptide Hydrolases; D014357:Trypsin; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D002402:Cathepsin D; D002118:Calcium; C031375:pepstatin