The amino acid sequences, structure comparisons and inhibition kinetics of sheep cathepsin L and sheep stefin B.
Cathepsin L and stefin B were isolated from sheep liver, the cathepsin L being isolated by a low pH homogenisation method, which increases the proportion of the two-chain form of the enzyme, thus facilitating sequencing. The amino acid sequences of the isolated cathepsin L and stefin B were determined. The two-chain form of cathepsin L contains 217 amino acid residues and has an M(r) of 23,627. The sequence was obtained by sequencing the native active enzyme, the light and heavy chains and the peptides generated by cyanogen bromide cleavage. These peptides were aligned with peptides obtained by hydrolysis with endoproteinase Lys-C, glycyl endopeptidase and endoproteinase Glu-C. Sheep liver cathepsin L exhibits a high degree of sequence identity to human cathepsin L. Sheep stefin B consists of 98 amino acid residues and its calculated M(r) is 11,150. The inhibitor has its NH2-terminal amino acid residue blocked. Its amino acid sequence was determined by sequencing the peptides obtained by cleavage with cyanogen bromide and peptides obtained by hydrolysis with endoproteinase Glu-C and endoproteinase Lys-C. Sheep stefin B shows a high degree of sequence identity with bovine and human stefin B. The kinetics of the interaction between sheep cathepsin L and stefin B were determined, with the interaction of stefin B with papain used as a benchmark to compare with other published results. Despite the considerable homology between bovine and sheep stefin B, the kinetics of their interaction with papain and cathepsin L differed markedly, possibly due to the differences in the so-called "trunk" region of the cystatin molecule.
Ritonja
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Department of Biochemistry and Molecular Biology, J. Stefan Institute, Jamova, Ljubljana, Slovenija. Anka.Ritonja@IJS.SI
Coetzer
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TH
Pike
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Dennison
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C
D000595:Amino Acid Sequence; D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D002417:Cattle; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry* / Q000494:pharmacology; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D010450:Endopeptidases; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010206:Papain / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012756:Sheep
C524994:CSTB protein, human; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D055313:Cystatin B; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; D010206:Papain