Characterization of proenkephalin-cleaving proteinases in bovine adrenal chromaffin granules using [35S]proenkephalin copolymerized into sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Proteinases capable of cleaving proenkephalin into smaller peptides have been identified in bovine adrenal chromaffin granules using [35S]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-CH2Cl, and D-Val-Phe-Lys-CH2Cl 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 Ca2+ activated and inhibited by EDTA but not diisopropyl fluorophosphate, soybean trypsin inhibitor, p-chloromercuriphenylsulfonic acid, 1,10-phenanthroline, or pepstatin.
Roberts
S F
SF
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Irvine
J W
JW
Lindberg
I
I
D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002837:Chromaffin Granules / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004745:Enkephalins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011108:Polymers; D011498:Protein Precursors / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D004745:Enkephalins; D011108:Polymers; D011498:Protein Precursors; D013462:Sulfur Radioisotopes; C029992:proenkephalin; D010450:Endopeptidases