Swain L D, Renthal R D, Boyan B D
Department of Orthopaedics, University of Texas Health Science Center,San Antonio 78284-7774.
J Dent Res. 1989 Jun;68(6):1094-7. doi: 10.1177/00220345890680060701.
Formation of calcium hydroxyapatite occurs on membrane surfaces via interaction of calcium, inorganic phosphate, phospholipids, calcifiable proteolipids, and ion flux to and from the nucleating site. Recently, this laboratory reported that proteolipids from the calcifying bacterium, Bacterionema matruchotti, act as an ionophore when reconstituted into bacteriorhodopsin-proteoliposomes. This ionophoric activity is blocked by [14C]dicyclohexylcarbodiimide ([14C]DCCD). SDS-PAGE shows that [14C]DCCD binds to a single band of Mr 8500. To determine whether proteins other than the [14C]DCCD-binding protein are involved, we examined the function of proteolipid species extracted by solvents of differing polarity. Proteolipids were isolated independently from chloroform:methanol (2:1) and chloroform:methanol:HCl (200:100:1) extracts of the bacteria by Sephadex LH-20 chromatography and were electrophoresed on 12.5% acrylamide gels. The chloroform:methanol extract contained a major hand at Mr 10,000 that was not present in gels of proteolipid isolated by acidified solvent. Proteolipids extracted in chloroform:methanol:HCl included a broad band at Mr 8500, which co-migrated with the [14C] DCCD-binding protein. The rate and extent of proton translocation were not altered when either proteolipid extract was added individually to bacteriorhodopsin proteoliposomes. However, when proteolipids isolated from the chloroform:methanol and chloroform:methanol:HCl extracts were combined, the rate and extent of translocation were increased. These data demonstrate that at least two proteolipid proteins are necessary for ionophoric activity, the Mr 10,000 protein isolated by chloroform:methanol 2:1 and the [14C]DCCD-binding protein requiring acidified solvent for extraction.
通过钙、无机磷酸盐、磷脂、可钙化蛋白脂质以及进出成核位点的离子流之间的相互作用，羟基磷酸钙在膜表面形成。最近，本实验室报道，钙化细菌马氏杆菌的蛋白脂质在重构到细菌视紫红质 - 蛋白脂质体中时可作为离子载体。这种离子载体活性被[14C]二环己基碳二亚胺（[14C]DCCD）阻断。SDS - 聚丙烯酰胺凝胶电泳显示[14C]DCCD与一条分子量为8500的单带结合。为了确定除[14C]DCCD结合蛋白之外的其他蛋白质是否参与其中，我们研究了用不同极性溶剂提取的蛋白脂质种类的功能。通过Sephadex LH - 20柱色谱法从细菌的氯仿：甲醇（2:1）和氯仿：甲醇：盐酸（200:100:1）提取物中分别分离出蛋白脂质，并在12.5%丙烯酰胺凝胶上进行电泳。氯仿：甲醇提取物在分子量为10,000处有一条主要条带，而在酸化溶剂分离的蛋白脂质凝胶中不存在。在氯仿：甲醇：盐酸中提取的蛋白脂质包括一条分子量为8500的宽带，它与[14C]DCCD结合蛋白共迁移。当将任何一种蛋白脂质提取物单独添加到细菌视紫红质蛋白脂质体中时，质子转运的速率和程度没有改变。然而，当将从氯仿：甲醇和氯仿：甲醇：盐酸提取物中分离出的蛋白脂质组合时，转运的速率和程度增加。这些数据表明，离子载体活性至少需要两种蛋白脂质蛋白，即通过氯仿：甲醇2:1分离的分子量为10,000的蛋白和需要酸化溶剂提取的[14C]DCCD结合蛋白。
