Sottrup-Jensen L, Stepanik T M, Jones C M, Lønblad P B, Kristensen T, Wierzbicki D M
J Biol Chem. 1984 Jul 10;259(13):8293-303.
The isolation of the 26 CNBr fragments from the identical Mr = 180,000 subunits of human alpha 2-macroglobulin is described. The fragments have been purified by combinations of gel chromatography, ion-exchange chromatography, high voltage paper electrophoresis, paper chromatography, and high performance liquid chromatography. The complete amino acid sequences of 13 small CNBr fragments have been determined. These fragments include CB1 (residues 1-9), CB3 (residues 79-98), CB4 (residues 99-128), CB9 (residues 442-477), CB10 (residues 478-497), CB13 (residues 644-650), CB14 (residues 651-665), CB15 (residues 666-674), CB16 (residues 675-690), CB19 (residues 937-945), CB20 (residues 946-954), CB24 (residues 1356-1362), and CB25 (residues 1363-1375). The fragments determined account for 200 of the 1451 residues of the subunits of alpha 2-macroglobulin. Most likely, Cys-6 of CB9 is bound to the corresponding residue in CB9 from another subunit, thus forming an interchain disulfide bridge in alpha 2-macroglobulin. Cys-1 of CB15 is bound to Cys-35 of CB12. CB15 contains a pair of Gln residues that can react covalently with amines in a factor XIIIa-catalyzed process (Gln-5 and Gln-6). CB16 contains the primary cleavage sites for proteinases in the bait region of alpha 2-macroglobulin (-Arg7-Val-Gly-Phe-Tyr-Glu-). CB20 contains the residues which in native alpha 2-macroglobulin presumably form an internal reactive beta-cysteinyl-gamma-glutamyl thiol ester (Cys-4 and Glx-7). Partial NH2- and COOH-terminal sequence data are given for the 13 large CNBr fragments. Complete or partial sequence determination of 19 methionine-containing peptides or variants thereof allow the alignment of all the CNBr fragments.
本文描述了从人α2-巨球蛋白相同的Mr = 180,000亚基中分离出26个溴化氰（CNBr）片段的过程。这些片段通过凝胶色谱、离子交换色谱、高压纸电泳、纸色谱和高效液相色谱等多种方法进行了纯化。已确定了13个小CNBr片段的完整氨基酸序列。这些片段包括CB1（第1 - 9位氨基酸残基）、CB3（第79 - 98位氨基酸残基）、CB4（第99 - 128位氨基酸残基）、CB9（第442 - 477位氨基酸残基）、CB10（第478 - 497位氨基酸残基）、CB13（第644 - 650位氨基酸残基）、CB14（第651 - 665位氨基酸残基）、CB15（第666 - 674位氨基酸残基）、CB16（第675 - 690位氨基酸残基）、CB19（第937 - 945位氨基酸残基）、CB20（第946 - 954位氨基酸残基）、CB24（第1356 - 1362位氨基酸残基）和CB25（第1363 - 1375位氨基酸残基）。所确定的这些片段占α2-巨球蛋白亚基1451个氨基酸残基中的200个。很可能，CB9的第6位半胱氨酸（Cys）与另一个亚基的CB9中相应的残基相连，从而在α2-巨球蛋白中形成链间二硫键。CB15的第1位半胱氨酸与CB12的第35位半胱氨酸相连。CB15含有一对谷氨酰胺（Gln）残基，在因子XIIIa催化的过程中可与胺发生共价反应（Gln-5和Gln-6）。CB16包含α2-巨球蛋白诱饵区域中蛋白酶的主要切割位点（-Arg7-Val-Gly-Phe-Tyr-Glu-）。CB20包含在天然α2-巨球蛋白中可能形成内部反应性β-半胱氨酰-γ-谷氨酰硫酯的残基（Cys-4和Glx-7）。给出了13个大CNBr片段的部分氨基末端和羧基末端序列数据。对19个含甲硫氨酸的肽段或其变体进行完整或部分序列测定，使得所有CNBr片段得以比对。
