Pirner M A, Linck R W
Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis 55455.
J Biol Chem. 1994 Dec 16;269(50):31800-6.
Tektins are proteins that copartition with tubulin in a stable ribbon of three protofilaments from ciliary and flagellar microtubules. After purification, tektins A, B, and C from sea urchin sperm flagellar microtubules appear as extended relatively insoluble filaments, < 5 nm in diameter. We used cross-linking reagents to investigate the associations and structural organization of subunits within tektin polymers isolated from stable protofilament ribbons of Strongylocentrotus purpuratus. We show by SDS-polyacrylamide gel electrophoresis, immunoblots, and transmission electron microscopy that tektins are continuous heteropolymers in the stable protofilament ribbons, and thus flagellar microtubules. Our results also provide evidence for the arrangement of different tektin polypeptides within "core" filaments containing equimolar tektins A and B. Treatment of these core filaments with bis(sulfosuccinimidyl)suberate and with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide yielded a predominant cross-linked approximately 106-kDa heterodimer of tektins A and B; similar results were obtained by glutaraldehyde cross-linking of tektins solubilized under mild conditions. Finally, cross-linking with 3,3'-dithiobis(sulfosuccinimidylpropionate) revealed a 16 nm periodicity in isolated tektin AB filaments that can be related to the 8 nm tubulin dimer lattice and to periodically associated microtubule components.
轴丝蛋白是一类能与微管蛋白共同分配到来自纤毛和鞭毛微管的由三条原纤维组成的稳定带中的蛋白质。纯化后，来自海胆精子鞭毛微管的轴丝蛋白A、B和C呈现为直径小于5纳米的延伸的相对不溶性细丝。我们使用交联剂来研究从紫海胆稳定原纤维带中分离出的轴丝蛋白聚合物内亚基的关联和结构组织。我们通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-聚丙烯酰胺凝胶电泳）、免疫印迹和透射电子显微镜表明，轴丝蛋白在稳定的原纤维带中，因而也在鞭毛微管中是连续的杂聚物。我们的结果还为不同的轴丝蛋白多肽在含有等摩尔轴丝蛋白A和B的“核心”细丝中的排列提供了证据。用双（磺基琥珀酰亚胺基）辛二酸酯和1-乙基-3-（3-二甲基氨基丙基）碳二亚胺处理这些核心细丝，产生了一种主要的交联的大约106 kDa的轴丝蛋白A和B的异二聚体；通过在温和条件下溶解的轴丝蛋白的戊二醛交联也得到了类似结果。最后，用3,3'-二硫代双（磺基琥珀酰亚胺基丙酸酯）交联揭示了分离的轴丝蛋白AB细丝中有16纳米的周期性，这可能与8纳米的微管蛋白二聚体晶格以及与周期性相关的微管成分有关。
