Isolation, purification and properties of cathepsin B from buffalo liver.
Cathepsin B was isolated from buffalo liver by salt fractionation, ion-exchange resin treatment, gel filtration and repeated ion-exchange chromatography using a linear salt gradient. The enzyme showed activity, against denatured hemoglobin (or ovalbumin), alpha-N-benzoyl-DL-arginine p-nitroanilide (BAPNA), and alpha-benzoyl-DL-arginine-naphthylamine (BANA). It inactivated buffalo muscle aldolase with a half life period of 21 min. The pH-activity profiles obtained for the digestion of hemoglobin (or ovalbumin) and aldolase inactivation by the enzyme were found to be different. The enzyme (mol wt 27,800 by SDS-PAGE) eluted in gel filtration with a molecular weight of 27,000 and a Stokes radius of 2.31 nm. The results showed buffalo cathepsin B to be a single-chain molecule. The N- and C-terminal amino acids of the enzyme were found to be leucine and aspartic acid, respectively. It contained 0.7% concanavalin A reactive neutral carbohydrate. The amino acid composition of buffalo cathepsin B was found to be similar to that of human liver cathepsin B. The optical properties of the buffalo enzyme were found consistent with its aromatic amino acid content. The isoionic pH of the enzyme was found to be 5.70 and the intrinsic viscosity was 3.48 ml/g whence the frictional ratio, f/f0 was computed to be 1.10 suggesting that the native enzyme conformation is compact and is globular in solution.
Salahuddin
A
A
Department of Biochemistry, J N Medical College, Aligarh, India.
Siddiqui
F A
FA
Salahuddin
P
P
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002020:Buffaloes / Q000378:metabolism*; D002401:Cathepsin B / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014783:Viscosity
D000596:Amino Acids; D004791:Enzyme Inhibitors; D002401:Cathepsin B; D005634:Fructose-Bisphosphate Aldolase