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Separation of a new alpha n benzoyl arginine beta naphthylamide hydrolase from cathepsin b 1 ec 3.4.22.1 purification characterization and properties of both enzymes from rabbit lung/



Separation of a new alpha n benzoyl arginine beta naphthylamide hydrolase from cathepsin b 1 ec 3.4.22.1 purification characterization and properties of both enzymes from rabbit lung/



Journal of Biological Chemistry 253(12): 4319-4327



Two proteolytic enzymes, each capable of hydrolyzing .alpha.-N-benzoyl-DL-arginine-.beta.-naphthylamide (BANA) were isolated from rabbit lung. The enzymes, cathepsin B1 and BANA hydrolase, were purified to apparent homogeneity by acid precipitation, ammonium sulfate fractionation, gel filtration, organomercurial-Sepharose affinity chromatography, CM-Sephadex chromatography and preparative isoelectric focusing. These 2 proteolytic enzymes appeared to be different in a number of their properties. Cathepsin B1 was inhibited by leupeptin, rapidly inactivated aldolase, and was only slightly inhibited by an extract of lung cytosol. This enzyme readily hydrolyzed the more specific cathepsin B1 substrate, .alpha.-N-benzyloxycarbonyl-L-Ala-L-Arg-L-Arg-4-methoxy-.beta.-naphthylamide (Cbz-Ala-Arg-Arg-4MeO.beta.NA) and did not hydrolyze L-Leu-.beta.-naphthylamide (Leu-.beta.NA), thus exhibiting only endopeptidase activity. In addition, cathepsin B1 was shown to consist of 4 isozymes with isoelectric points ranging from pH 5.0-5.5, was irreversibly inactivated by exposure to mild alkaline pH, and its pH and temperature optima were 6.2 and 42.5.degree. C, respectively, with BANA or with Cbz-Ala-Arg-Arg-4MeO.beta.NA as substrates. In contrast, BANA hydrolase was not inhibited by leupeptin, only slightly inactivated aldolase, and was strongly inhibited by an extract of rabbit lung cytosol. This enzyme exhibited very slight activity with Cbz-Ala-Arg-Arg-4MeO.beta.NA as substrate but rapidly hydrolyzed Leu-.beta.NA, in addition to BANA, thus, exhibiting strong exopeptidase and endopeptidase activities. This new enzyme, BANA hydrolase, was shown to consist of 6 isozymes with isoelectric points ranging from pH 5.8-6.5, was stable on exposure to mild alkaline pH, and its pH and temperature optima were 6.5 and 49.degree. C with BANA and 7.2 and 38.5.degree. C with Leu-.beta.NA as substrate. In all of the above properties, cathepsin B1 and BANA hydrolase are different. However, the 2 enzymes show some similarities in that both hydrolyze BANA and .alpha.-N-benzoyl-DL-arginine-p-nitroanilide (BAPA), have somewhat similar MW (26,000-29,000), are sulfhydryl enzymes, and require EDTA and thiol compounds for full activation. With each enzyme, the most effective reducing agents were .beta.-mercaptoethylamine and dithioerythritol. However, .beta.-mercaptoethanol was a good activator for BANA hydrolase but was not especially effective with cathepsin B1.

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Accession: 006391244

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