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.