Jindal H K, Chaney W G, Anderson C W, Davis R G, Vishwanatha J K
Department of Biochemistry, University of Nebraska Medical Center, Omaha 68198.
J Biol Chem. 1991 Mar 15;266(8):5169-76.
Primer recognition proteins (PRP) stimulate the activity of DNA polymerase alpha on DNA substrates with long single-stranded template containing few primers. Purified PRP from HeLa cells and human placenta are composed of two subunits of 36,000 (PRP 1) and 41,000 (PRP 2) daltons. By amino acid sequence homology, we have identified PRP 2 as the glycolytic enzyme 3-phosphoglycerate kinase. Here we present data that establishes PRP 1 to be the protein-tyrosine kinase substrate, calpactin I heavy chain. Amino acid sequence analysis of six tryptic peptides of PRP 1 followed by homology search in a protein sequence data base revealed 100% identity of all six peptides with the deduced amino acid sequence of human calpactin I heavy chain. The activities of PRP and calpactin I coelute on gel filtration columns, and a high correlation of PRP and calpactin I activities was seen at different stages of purification. A rabbit polyclonal anti-chicken calpactin I antibody was shown to cross-react with PRP 1 polypeptide at various stages of PRP purification, and the homogeneous preparation of PRP exhibits 3-phosphoglycerate kinase (PRP 2) and calpactin I (PRP 1) activities. PRP activity is neutralized by a mouse monoclonal anti-calpactin II antibody although having no effect on the polymerase alpha activity itself. Calpactin II has a 50% amino acid sequence homology with calpactin I. However, PRP 1 is not calpactin II as shown by lack of cross-reaction to a monoclonal anti-calpactin II antibody on Western blots. Calpactin I and 3-phosphoglycerate kinase, purified independently, cannot be efficiently reconstituted into the PRP complex, indicating that their association in the PRP complex involves specific protein-protein interactions that remain to be elucidated. The biochemical and immunological data presented here revealing the identity of PRP 1 as calpactin I provide evidence for one physiological role of calpactin I in the cell.
引物识别蛋白（PRP）可在含有少量引物的长单链模板的DNA底物上刺激DNA聚合酶α的活性。从HeLa细胞和人胎盘中纯化得到的PRP由两个亚基组成，分别为36,000道尔顿（PRP 1）和41,000道尔顿（PRP 2）。通过氨基酸序列同源性分析，我们已确定PRP 2为糖酵解酶3 - 磷酸甘油酸激酶。在此，我们提供的数据表明PRP 1是蛋白酪氨酸激酶底物钙结合蛋白I重链。对PRP 1的六个胰蛋白酶肽段进行氨基酸序列分析，随后在蛋白质序列数据库中进行同源性搜索，结果显示所有六个肽段与人类钙结合蛋白I重链的推导氨基酸序列具有100%的同一性。PRP和钙结合蛋白I的活性在凝胶过滤柱上共同洗脱，并且在纯化的不同阶段，PRP和钙结合蛋白I的活性呈现出高度相关性。在PRP纯化的各个阶段，兔抗鸡钙结合蛋白I多克隆抗体均显示与PRP 1多肽发生交叉反应，并且PRP的纯化物表现出3 - 磷酸甘油酸激酶（PRP 2）和钙结合蛋白I（PRP 1）的活性。尽管小鼠单克隆抗钙结合蛋白II抗体对DNA聚合酶α活性本身没有影响，但它可中和PRP活性。钙结合蛋白II与钙结合蛋白I具有50%的氨基酸序列同源性。然而，Western印迹显示PRP 1与单克隆抗钙结合蛋白II抗体无交叉反应，表明PRP 1不是钙结合蛋白II。单独纯化的钙结合蛋白I和3 - 磷酸甘油酸激酶不能有效地重组为PRP复合物，这表明它们在PRP复合物中的结合涉及特定的蛋白质 - 蛋白质相互作用，有待进一步阐明。此处呈现的生化和免疫学数据揭示了PRP 1与钙结合蛋白I的同一性，为钙结合蛋白I在细胞中的一种生理作用提供了证据。
