Purification, cloning, and sequence analysis of a M(r)=30,000 protein from sea urchin axonemes that is important for sperm motility - Relationship of the protein to a dynein light chain
Purification, cloning, and sequence analysis of a M(r)=30,000 protein from sea urchin axonemes that is important for sperm motility - Relationship of the protein to a dynein light chain
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DOI：
10.1074/jbc.271.22.12807
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发表时间：
1996-05-31
影响因子：
4.8
通讯作者：
Gagnon, C
中科院分区：
文献类型：
--
作者：
Gingras, D;White, D;Gagnon, C
我们从海胆精子中产生了一系列针对轴丝蛋白的单克隆抗体，以鉴定参与鞭毛运动调节的新蛋白。单克隆抗体 D405-14 在低浓度下抑制脱膜再激活精子模型的运动性，并在海胆轴丝蛋白的免疫印迹上识别 33 kDa (p33) 的单一多肽。用高盐溶液、加热和洗涤剂对轴丝进行分级分离，导致 p33 选择性提取成 0.6 M 氯化钠可溶性形式和 0.5% 月桂基肌氨酸钠 (Sarkosyl) 可溶性形式。通过单克隆抗体 D405-14-Sepharose 上的免疫亲和层析，将两种形式的 p33 纯化至明显同质。我们还分离并测序了编码 33 kDa 蛋白的全长 cDNA 克隆。该序列预测是一个由 260 个氨基酸组成的多肽，质量为 29,730 Da，等电点为 9.3。序列比较表明p33与莱茵衣藻轴丝内动力蛋白臂的p28轻链有66%相同（74%相似）。综上所述，这些结果表明我们已经在海胆精子轴丝中鉴定出 p28 轻链同源物，并且该蛋白质可能在鞭毛运动中发挥动态作用。
We have generated a series of monoclonal antibodies against axonemal proteins from sea urchin spermatozoa in order to identify novel proteins involved in the regulation of flagellar motility. The monoclonal antibody D405-14 inhibited the motility of demembranated-reactivated sperm models at low concentrations and recognized a single polypeptide of 33 kDa (p33) on immuno blots of sea urchin axonemal proteins. Fractionation of the axonemes with high salt solutions, heat, and detergent resulted in the selective extraction of p33 into a 0.6 M NaCl-soluble and a 0.5% sodium lauryl sarcosinate (Sarkosyl)-soluble form. Both forms of p33 were purified to apparent homogeneity by immunoaffinity chromatography on monoclonal antibody D405-14-Sepharose. We have also isolated and sequenced a full-length cDNA clone encoding the 33-kDa protein. The sequence predicts a polypeptide of 260 amino acids having a mass of 29,730 Da and an isoelectric point of 9.3. Sequence comparison indicates that p33 is 66% identical (74% similar) to the p28 light chain of axonemal inner dynein arm of Chlamydomonas reinhardtii. Taken together, these results suggest that we have identified a p28 light chain homolog in sea urchin sperm axoneme and that this protein may play a dynamic rob in flagellar motility.