Species variations amongst lysosomal cysteine proteinases.
Cathepsin B was purified from rabbit, human, ox and sheep liver. SDS-polyacrylamide gel electrophoresis after reaction of the purified cathepsin B samples with the active site directed inhibitor, L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido- ([3H]acetamido)-butane ([3H]Ac-Ep-459), showed that the enzyme exists as either a two-chain form (approx. Mr 25,000 and 4,000), a single-chain form (approx. Mr 30,000) or both. The active site was found in the light chain of the two-chain forms. The two-chain and single-chain forms of ox cathepsin B were separated by ion exchange chromatography and shown to have similar catalytic activities against the substrates Z-Phe-Arg-4-methyl-7-coumarylamide (Z-Phe-Arg-NHMec) and Z-Arg-Arg-NHMec and rates of inhibition by L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4- guanidino)butane (E-64). Cathepsin L was purified from the same four species, and compared with the enzyme from rat liver. SDS-polyacrylamide gel electrophoresis after reaction of the purified cathepsin L preparations with the active site directed inhibitor, [3H]Ac-Ep-459, showed that cathepsin L from each species consists of two chains; a light chain of approx. Mr 5,000 and a heavy chain of approx. Mr 25,000, which contained the active site cysteine. All species variants of cathepsin L were recognized by the antibody to the human enzyme. With Z-Phe-Arg-NHMec as substrate, kinetic constants were found to be similar for all five species (Km 1-4 microM and and kcat 10-30 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D000818:Animals; D002401:Cathepsin B / Q000032:analysis / Q000302:isolation & purification; D056668:Cathepsin L; D002403:Cathepsins / Q000032:analysis / Q000302:isolation & purification; D002417:Cattle; D003546:Cysteine Endopeptidases; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000032:analysis*; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D012756:Sheep; D013045:Species Specificity
D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; C534280:Ctsl protein, rat