Purification and characterization of protease Re, a cytoplasmic endoprotease in Escherichia coli.
Protease Re, a new cytoplasmic endoprotease in Escherichia coli, was purified to homogeneity by conventional procedures, using [3H]casein as the substrate. The enzyme consists of a single polypeptide of 82,000 molecular weight. It is maximally active between pH 7 and 8.5 and is independent of ATP. It has a pI of 6.8 and a Km of 10.8 microM for casein. Since diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride inhibited this enzyme, it appears to be a serine protease. Protease Re was sensitive to inhibition by L-1-tosylamido-2-phenylethylchloromethylketone but not to that by 1-chloro-3-tosylamido-7-aminoheptanone, thiol-blocking reagents, chelating agents, or various peptide aldehydes. Re also degraded [125I]globin, [125I]glucagon, and 125I-labeled denatured bovine serum albumin to acid-soluble products (generally oligopeptides of greater than 1,500 daltons), but it showed no activity against serum albumin, growth hormone, insulin, or a variety of fluorometric peptide substrates. It also hydrolyzed oxidatively inactivated glutamine synthetase (generated by ascorbate, oxygen, and iron) four- to fivefold more rapidly than the native protein. Protease Re appears to be identical to the proteolytic enzyme isolated by Roseman and Levine (J. Biol. Chem. 262:2101-2110, 1987) by its ability to degrade selectively oxidatively damaged glutamine synthetase in vivo. Its role in intracellular protein breakdown is uncertain.
D002364:Caseins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D002364:Caseins; D011480:Protease Inhibitors; D010450:Endopeptidases; D012697:Serine Endopeptidases; C054741:protease Re; D005974:Glutamate-Ammonia Ligase