Araki T, Yamamoto T, Torikata T
Department of Biochemistry, School of Agriculture, Kyushu Tokai University, Kumamoto, Japan.
Biosci Biotechnol Biochem. 1998 Feb;62(2):316-24. doi: 10.1271/bbb.62.316.
Soft-shelled turtle egg-white lysozyme was purified and sequenced. Lysozyme was reduced and carboxymethylated to fragment it with trypsin, V8 protease and CNBr. The peptides yielded were purified by RP-HPLC and sequenced. Every trypsin peptide was overlapped by V8 protease peptides and CNBr fragment. The amino acid sequence was compared with other lysozymes. This lysozyme has an extra Gly residue at N-terminus, which was found in pheasant lysozyme. Further, this lysozyme has an insertion of a Gly residue between 47 and 48 residues when compared with chicken lysozyme, as found in human lysozyme, therefore it proved that this lysozyme has the largest number of amino acids (131 aa) in chicken type lysozymes. The amino acid substitutions were found at subsites E and F. Namely Phe34, Arg45, Thr47, and Arg114 were replaced by His, Tyr, Arg, and Tyr, respectively. The time course using N-acetylglucosamine pentamer as a substrate showed a reduction of the rate constant for glycosidic cleavage and increase of binding free energy for subsites E and F, which proved the contribution of amino acids mentioned above for substrate binding at subsites E and F.
对甲鱼蛋清溶菌酶进行了纯化和测序。将溶菌酶进行还原和羧甲基化处理，以便用胰蛋白酶、V8蛋白酶和溴化氰对其进行片段化。产生的肽段通过反相高效液相色谱法（RP-HPLC）进行纯化并测序。每个胰蛋白酶肽段都与V8蛋白酶肽段和溴化氰片段重叠。将氨基酸序列与其他溶菌酶进行比较。这种溶菌酶在N端有一个额外的甘氨酸残基，这在雉鸡溶菌酶中也有发现。此外，与鸡溶菌酶相比，这种溶菌酶在第47和48个残基之间插入了一个甘氨酸残基，这与人类溶菌酶的情况相同，因此证明这种溶菌酶在鸡型溶菌酶中氨基酸数量最多（131个氨基酸）。在亚位点E和F发现了氨基酸替换。具体来说，苯丙氨酸34、精氨酸45、苏氨酸47和精氨酸114分别被组氨酸、酪氨酸、精氨酸和酪氨酸取代。以N-乙酰葡糖胺五聚体为底物的时间进程显示糖苷键裂解的速率常数降低，亚位点E和F的结合自由能增加，这证明了上述氨基酸对亚位点E和F底物结合的贡献。
