Roche P A, Strickland D K, Enghild J J, Pizzo S V
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1988 May 15;263(14):6715-21.
Digestion of human alpha 2-macroglobulin-methylamine (alpha 2M-CH3NH2) with papain prior to gel filtration resulted in the resolution of three distinct peaks. The material in peak I (Mr approximately 600,000) and peak II (Mr approximately 55,000) did not have any receptor binding ability as determined by in vivo clearance studies and in vitro competitive binding studies using mouse peritoneal macrophages. In contrast, the material in peak III (Mr approximately 20,000) bound to macrophage alpha 2-macroglobulin (alpha 2M) receptors with a Kd of 250 nM. This represents a 500-fold decrease in affinity relative to undigested alpha 2M-CH3NH2. Sequence analysis demonstrated that this material constituted the carboxyl-terminal fragment (COOH-terminal fragment) of alpha 2M. alpha 2M is known to possess a methionyl residue which is susceptible to modification by cis-dichlorodiammineplatinum (II) (cis-DDP) with the result being a loss of receptor binding ability by alpha 2M. For this reason, experiments were performed to determine if the platinum-reactive methionyl residue is located in the COOH-terminal receptor binding fragment of alpha 2M. The results of this investigation demonstrate that cis-DDP is not reactive with either the isolated COOH-terminal fragment or the COOH-terminal fragment isolated from alpha 2M-CH3NH2 which had been pretreated with cis-DDP. In addition, the COOH-terminal fragment did not bind to monoclonal antibody 7H11D6, a monoclonal antibody which binds to the platinum-reactive epitope of the alpha 2M-CH3NH2 receptor recognition site. In contrast, the 55-kDa fragment of alpha 2M bound approximately 1 mol platinum/mol of 55-kDa fragment and also bound to monoclonal antibody 7H11D6. Since the COOH-terminal fragment retains some receptor binding ability, the results of this investigation demonstrate that this fragment is not the complete receptor recognition site and suggest that a platinum-reactive methionyl residue located in the 55-kDa fragment of alpha 2M is another component of this site.
在进行凝胶过滤之前，用木瓜蛋白酶消化人α2-巨球蛋白-甲胺（α2M-CH3NH2），得到了三个不同的峰。通过体内清除研究和使用小鼠腹腔巨噬细胞的体外竞争性结合研究确定，峰I（Mr约为600,000）和峰II（Mr约为55,000）中的物质没有任何受体结合能力。相比之下，峰III（Mr约为20,000）中的物质与巨噬细胞α2-巨球蛋白（α2M）受体结合，Kd为250 nM。这表示相对于未消化的α2M-CH3NH2，亲和力降低了500倍。序列分析表明，该物质构成了α2M的羧基末端片段（COOH末端片段）。已知α2M含有一个甲硫氨酰残基，该残基易受顺式二氯二氨铂（II）（顺式-DDP）修饰，结果是α2M失去受体结合能力。因此，进行了实验以确定铂反应性甲硫氨酰残基是否位于α2M的COOH末端受体结合片段中。这项研究的结果表明，顺式-DDP与分离的COOH末端片段或从经顺式-DDP预处理的α2M-CH3NH2中分离的COOH末端片段均无反应性。此外，COOH末端片段不与单克隆抗体7H11D6结合，7H11D6是一种与α2M-CH3NH2受体识别位点的铂反应性表位结合的单克隆抗体。相比之下，α2M的55-kDa片段每摩尔55-kDa片段结合约1摩尔铂，并且也与单克隆抗体7H11D6结合。由于COOH末端片段保留了一些受体结合能力，这项研究的结果表明该片段不是完整的受体识别位点，并表明位于α2M的55-kDa片段中的铂反应性甲硫氨酰残基是该位点的另一个组成部分。
