Setlow P, Ozols J
J Biol Chem. 1979 Dec 10;254(23):11938-42.
The complete covalent structure of Protein A, a protein degraded during bacterial spore germination, has been determined. The intact protein was cleaved with a highly specific spore protease into two peptides, residues 1 to 21 and 22 to 61. The larger peptide was further cleaved into two fragments with either cyanogen bromide or by trypsin cleavage following arginine modification with cyclohexanedione. The peptides derived from cyanogen bromide fragmentation encompassed residues 22 to 53 and 54 to 61 while trypsin hydrolysis yielded overlapping fragments comprising residues 22 to 48 and 49 to 61. Automated sequenator analysis together with carboxypeptidase Y digestion of the intact protein and the peptide fragments provided data from which the following unique amino acid sequence was deduced. NH2-Ala-Asn-Thr-Asn-Lys-Leu-Val-Ala-Pro-Gly10-Ser-Ala-Ala-Ala-Ile-Asp-Gln-Met-Lys-Tyr20-Glu-Ile-Ala-Ser-Glu-Phe-Gly-Val-Asn-Leu30-Gly-Pro-Glu-Ala-Thr-Ala-Arg-Ala-Asn-Gly40-Ser-Val-Gly-Gly-Glu-Ile-Thr-Lys-Arg-Leu50-Val-Gln-Met-Ala-Glu-Gln-Gln-Leu-Gly-Gly60-Lys-COOH.
已确定在细菌孢子萌发过程中会降解的A蛋白的完整共价结构。完整的蛋白质被一种高度特异性的孢子蛋白酶切割成两个肽段,即第1至21位残基的肽段和第22至61位残基的肽段。较大的肽段在用溴化氰处理或用环己二酮对精氨酸进行修饰后再用胰蛋白酶切割,进一步被切割成两个片段。溴化氰裂解产生的肽段包括第22至53位残基和第54至61位残基,而胰蛋白酶水解产生的重叠片段包括第22至48位残基和第49至61位残基。通过自动测序仪分析以及对完整蛋白质和肽段进行羧肽酶Y消化,得到了相关数据,据此推导出了以下独特的氨基酸序列。NH2-丙氨酸-天冬酰胺-苏氨酸-天冬酰胺-赖氨酸-亮氨酸-缬氨酸-丙氨酸-脯氨酸-甘氨酸10-丝氨酸-丙氨酸-丙氨酸-丙氨酸-异亮氨酸-天冬氨酸-谷氨酰胺-甲硫氨酸-赖氨酸-酪氨酸20-谷氨酸-异亮氨酸-丙氨酸-丝氨酸-谷氨酸-苯丙氨酸-甘氨酸-缬氨酸-天冬酰胺-亮氨酸30-甘氨酸-脯氨酸-谷氨酸-丙氨酸-苏氨酸-丙氨酸-精氨酸-丙氨酸-天冬酰胺-甘氨酸40-丝氨酸-缬氨酸-甘氨酸-甘氨酸-谷氨酸-异亮氨酸-苏氨酸-赖氨酸-精氨酸-亮氨酸50-缬氨酸-谷氨酰胺-甲硫氨酸-丙氨酸-谷氨酸-谷氨酰胺-谷氨酰胺-亮氨酸-甘氨酸-甘氨酸60-赖氨酸-COOH。