Yamagami T, Funatsu G
Laboratory of Protein Chemistry & Engineering, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
Biosci Biotechnol Biochem. 1994 Feb;58(2):322-9. doi: 10.1271/bbb.58.322.
The complete amino acid sequence of rye seed chitinase-a (RSC-a) has been analyzed. RSC-a was cleaved with cyanogen bromide and the resulting three fragments, CB1, CB2, and CB3, were separated by gel filtration. The amino acids of the N-terminal fragment CB1 were sequenced by analyzing the peptides produced by digestion with trypsin, lysylendopeptidase, or pepsin of reduced S-carboxymethylated or S-aminoethylated CB1. The sequences of fragments CB2 and CB3 were established by sequencing the tryptic peptides from reduced S-carboxymethylated CB2 and CB3, and by aligning them with the sequence of rye seed chitinase-c (RSC-c) to maximize sequence homology. The complete amino acid sequence of RSC-a was established by connecting these three fragments. RSC-a consists of 302 amino acid residues including hydroxyproline residues, and has a molecular mass of 31,722 Da. RSC-a is basic protein with a cysteine-rich amino terminal domain, indicating that this enzyme belongs to class I chitinases. The amino acid sequence of RSC-a showed that the sequence from Gly60 to C-terminal Ala302 in this enzyme corresponds to that of RSC-c belonging to class II chitinases with 92% identity, and that RSC-a has high similarity to other plant class I chitinases but a longer hinge region and an extra disulfide bond.
已对黑麦种子几丁质酶-a（RSC-a）的完整氨基酸序列进行了分析。用溴化氰裂解RSC-a，所得的三个片段CB1、CB2和CB3通过凝胶过滤进行分离。通过分析用胰蛋白酶、赖氨酰内肽酶或胃蛋白酶消化还原的S-羧甲基化或S-氨乙基化的CB1所产生的肽段，对N端片段CB1的氨基酸进行测序。通过对还原的S-羧甲基化的CB2和CB3的胰蛋白酶肽段进行测序，并将它们与黑麦种子几丁质酶-c（RSC-c）的序列进行比对以最大化序列同源性，从而确定片段CB2和CB3的序列。通过连接这三个片段确定了RSC-a的完整氨基酸序列。RSC-a由包括羟脯氨酸残基在内的302个氨基酸残基组成，分子量为31,722道尔顿。RSC-a是一种具有富含半胱氨酸的氨基末端结构域的碱性蛋白，表明该酶属于I类几丁质酶。RSC-a的氨基酸序列显示，该酶中从Gly60到C端Ala302的序列与属于II类几丁质酶的RSC-c的序列具有92%的同一性，并且RSC-a与其他植物I类几丁质酶具有高度相似性，但具有更长的铰链区和一个额外的二硫键。
