Ritonja A, Coetzer T H, Pike R N, Dennison C
Department of Biochemistry and Molecular Biology, J. Stefan Institute, Jamova, Ljubljana, Slovenija.
Comp Biochem Physiol B Biochem Mol Biol. 1996 Jun;114(2):193-8. doi: 10.1016/0305-0491(96)00010-7.
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.
组织蛋白酶L和丝抑蛋白B是从羊肝中分离得到的。组织蛋白酶L采用低pH匀浆法进行分离，该方法可增加酶的双链形式比例，从而便于测序。测定了分离得到的组织蛋白酶L和丝抑蛋白B的氨基酸序列。组织蛋白酶L的双链形式含有217个氨基酸残基，相对分子质量为23,627。通过对天然活性酶、轻链和重链以及溴化氰裂解产生的肽段进行测序获得了该序列。这些肽段与用内肽酶Lys-C、甘氨酰内肽酶和内肽酶Glu-C水解得到的肽段进行了比对。羊肝组织蛋白酶L与人类组织蛋白酶L具有高度的序列同一性。羊丝抑蛋白B由98个氨基酸残基组成，其计算相对分子质量为11,150。该抑制剂的氨基末端氨基酸残基被封闭。通过对溴化氰裂解得到的肽段以及用内肽酶Glu-C和内肽酶Lys-C水解得到的肽段进行测序，确定了其氨基酸序列。羊丝抑蛋白B与牛和人类丝抑蛋白B具有高度的序列同一性。测定了羊组织蛋白酶L与丝抑蛋白B之间相互作用的动力学，以丝抑蛋白B与木瓜蛋白酶的相互作用作为基准，与其他已发表的结果进行比较。尽管牛和羊丝抑蛋白B之间存在相当大的同源性，但它们与木瓜蛋白酶和组织蛋白酶L相互作用的动力学却明显不同，这可能是由于半胱氨酸蛋白酶抑制剂分子所谓的“主干”区域存在差异所致。
