Isolation and characterization of cathepsin B from bovine brain.
Cathepsin B (EC 3.4.22.1) was purified 746-fold with a 21% recovery from bovine brain by autolysis, fractional precipitation with acetone, carboxy-methyl-Sephadex chromatography, affinity chromatography on a cystamine containing column and gel filtration chromatography. The purified cathepsin B eluted on gel filtration with an apparent molecular weight of 27,000 but was resolved into three bands of 30,000, 25,000 and 5,000 molecular weight by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Antibodies to cathepsin B, raised against the 30,000 dalton band, were shown by immunoblots to react with both the 30,000 and 25,000 dalton proteins with results suggesting that the former predominated as the immunoreactive form in bovine brain homogenates. Isoelectric focusing demonstrated multiple bands, ranging from pH 4.75-5.2 with the major band at pH 5.1-5.2, all of which were capable of degrading N alpha-carbobenzoxy-L-arginyl-L-arginine 4-methoxy-beta-naphthylamide. The cathepsin B activity against N alpha-benzoyl-DL-arginine beta-naphthylamide (BANA) and bovine myelin basic protein (MBP) had a pH optimum of pH 6.0. The Km for the degradation of BANA was 1.0 mM and 5.1 mM when assayed in the presence of 1% and 2.5% dimethylsulfoxide, respectively. Cathepsin B from bovine brain has many properties similar to cathepsin B isolated from other organs. The degradative effect of cathepsin B on MBP suggests a role for this proteinase in inflammatory demyelination.
D000818:Animals; D001587:Benzoylarginine-2-Naphthylamide / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D002401:Cathepsin B; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008970:Molecular Weight; D004676:Myelin Basic Protein / Q000378:metabolism
D004676:Myelin Basic Protein; D001587:Benzoylarginine-2-Naphthylamide; D002403:Cathepsins; D002401:Cathepsin B