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Characterization of Proenkephalin‐Cleaving Proteinases in Bovine Adrenal Chromaffin Granules Using [ 35 S]Proenkephalin Copolymerized into Sodium Dodecyl Sulfate‐Polyacrylamide Gel Electrophoresis
Author(s) -
Roberts Steven F.,
Irvine Joseph W.,
Lindberg Iris
Publication year - 1992
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1992.tb09760.x
Subject(s) - diisopropyl fluorophosphate , chemistry , sodium dodecyl sulfate , biochemistry , pepstatin , gel electrophoresis , phenylmethylsulfonyl fluoride , pmsf , leupeptin , chromatography , polyacrylamide gel electrophoresis , molecular mass , enzyme , kunitz sti protease inhibitor , trypsin , protease
Proteinases capable of cleaving proenkephalin into smaller peptides have been identified in bovine adrenal chromaffin granules using [ 35 S]methionine‐labeled recombinant rat proenkephalin as a selective substrate in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis proteinase radiozymography. This technique was used for the screening of subcellular fractions, general characterization of pH optima, and the mechanistic characterization of proteinases with both reversible and irreversible inhibitors. Two enzymes with approximate molecular masses of 76 and 30 kDa were shown to be localized to the highest‐density fractions of chromaffin granules by sucrose density gradient fractionation. Both were enriched in a 1 M NaCl wash of purified chromaffin granule membranes, were active at high pH, and were characterized as serine proteinases based on inhibition by soybean trypsin inhibitor. The 30‐kDa enzyme was also inhibited by diisopropyl fluorophosphate. d ‐Phe‐Pro‐Arg‐CH 2 Cl, and d ‐Val‐Phe‐Lys‐CH 2 Cl and appeared to be the previously described adrenal trypsin‐like enzyme. A third enzyme, of 66 kDa, was also associated with the 1 M NaCl wash of purified chromaffin granule membranes but was not localized exclusively to chromaffin granules in sucrose gradients. This proteinase was found to be Ca 2+ activated and inhibited by EDTA but not diisopropyl fluorophosphate, soybean trypsin inhibitor, p ‐chloromercuriphenylsulfonic acid, 1,10‐phenanthroline, or pepstatin.