Kamino K, Odo S, Maruyama T
Marine Biotechnology Institute, Shimizu Laboratory, Japan.
Biol Bull. 1996 Jun;190(3):403-9. doi: 10.2307/1543033.
Components of the proteinaceous cement secreted by barnacles have yet to be studied because of their insolubility. We solubilized and characterized the proteins of secondary cement, which is produced when the barnacle is detached from the substratum, in Megabalanus rosa. The cement was fractionated, according to its solubility in aqueous formic acid, into a soluble fraction, SF1 (21%); a fraction soluble after reduction, SF2 (37%); and a fraction insoluble after reduction, IF (42%). Analysis of the SF1 and SF2 by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that they contained three polypeptides (SF1-60 k, -57 k, -47 k) and one polypeptide (SF2-60 k), respectively. The amino acid compositions of these polypeptides were similar and their N-terminal amino acid sequences were identical. These polypeptides had an unusual amino acid composition, rich in Ser, Thr, Ala, and Gly, like the tube cement of a marine polychaete, Phragmatopoma californica. The IF, solubilized in aqueous formic acid after cleavage with cyanogen bromide, was shown by SDS-PAGE to contain eight fragment peptides (CB-peptides). N-terminal amino acid sequences of the CB-peptides were also determined. We conclude that the barnacle cement is composed of at least two types of protein: highly hydroxylated protein in the SF1 and SF2 and insoluble protein in the IF. The SDS-PAGE pattern of CB-peptides from the secondary cement was identical to that of the primary cement produced while the barnacle is attached to a substratum. In addition, immunoblot analysis, using a polyclonal antibody against one of the CB-peptides from the secondary cement, also cross-reacted with a CNBr-fragment peptide of the primary cement. These results indicate that the primary and secondary cements are similar in protein composition.
由于藤壶分泌的蛋白质类黏合剂不溶于水，其成分尚未得到研究。我们溶解并鉴定了玫瑰巨藤壶（Megabalanus rosa）中次级黏合剂的蛋白质，这种黏合剂是藤壶从基质上脱落时产生的。根据其在甲酸水溶液中的溶解性，将该黏合剂分为一个可溶部分SF1（21%）、一个还原后可溶部分SF2（37%）和一个还原后不溶部分IF（42%）。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）对SF1和SF2进行分析，结果显示它们分别包含三种多肽（SF1-60k、-57k、-47k）和一种多肽（SF2-60k）。这些多肽的氨基酸组成相似，其N端氨基酸序列相同。这些多肽具有不寻常的氨基酸组成，富含丝氨酸、苏氨酸、丙氨酸和甘氨酸，类似于海洋多毛类动物加州盘管虫（Phragmatopoma californica）的管黏合剂。用溴化氰裂解后在甲酸水溶液中溶解的IF，经SDS-PAGE显示含有八个片段肽（CB-肽）。还测定了CB-肽的N端氨基酸序列。我们得出结论，藤壶黏合剂至少由两种类型的蛋白质组成：SF1和SF2中的高度羟基化蛋白质以及IF中的不溶性蛋白质。次级黏合剂的CB-肽的SDS-PAGE图谱与藤壶附着在基质上时产生的初级黏合剂的图谱相同。此外，使用针对次级黏合剂的一种CB-肽的多克隆抗体进行的免疫印迹分析，也与初级黏合剂的溴化氰片段肽发生交叉反应。这些结果表明初级和次级黏合剂在蛋白质组成上相似。
