Brawley S H, Roberts D M
Department of Biology, Vanderbilt University, Nashville, Tennessee 37235.
Dev Biol. 1989 Feb;131(2):313-20. doi: 10.1016/s0012-1606(89)80004-1.
Calcium-binding proteins and calmodulin-binding proteins were identified in gametes and zygotes of the marine brown algae Fucus vesiculosus, Fucus distichus, and Pelvetia fastigiata using gel (SDS-PAGE) overlay techniques. A calcium current appears to be important during cell polarization in fucoid zygotes (K.R. Robinson and L.F. Jaffe, 1975, Science 187, 70-72; K.R. Robinson and R. Cone, 1980, Science 207, 77-78), but there are no biochemical data on calcium-binding proteins in these algae. By using a sensitive 45Ca2+ overlay method designed to detect high-affinity calcium-binding proteins, at least 9-11 polypeptides were detected in extracts of fucoid gametes and zygotes. All samples had calcium-binding proteins with apparent molecular weights of about 17 and 30 kDa. A 17-kDa calcium-binding protein was purified by calcium-dependent hydrophobic chromatography and was identified as calmodulin by immunological and enzyme activator criteria. A 125I-calmodulin overlay assay was used to identify potential targets of calmodulin action. Sperm contained one major calmodulin-binding protein of about 45 kDa. Eggs lacked major calmodulin-binding activity. A 72-kDa calmodulin-binding protein was prominent in zygotes from 1-65 hr postfertilization. Both calmodulin-binding proteins showed calcium-dependent binding activity. Overall, the data suggest that the appearance and distribution of certain calcium-binding and calmodulin-binding proteins are under developmental regulation, and may reflect the different roles of calcium during fertilization and early embryogenesis.
利用凝胶(SDS-PAGE)覆盖技术,在海洋褐藻墨角藻、分歧墨角藻和鹿角菜的配子和受精卵中鉴定出了钙结合蛋白和钙调蛋白结合蛋白。在岩藻合子的细胞极化过程中,钙电流似乎很重要(K.R.罗宾逊和L.F.贾菲,1975年,《科学》187卷,70 - 72页;K.R.罗宾逊和R.科恩,1980年,《科学》207卷,77 - 78页),但关于这些藻类中钙结合蛋白的生化数据尚无报道。通过使用一种灵敏的45Ca2+覆盖方法来检测高亲和力钙结合蛋白,在岩藻配子和受精卵提取物中检测到了至少9 - 11种多肽。所有样品都含有表观分子量约为17 kDa和30 kDa的钙结合蛋白。一种17 kDa的钙结合蛋白通过钙依赖性疏水色谱法纯化,并通过免疫学和酶激活标准鉴定为钙调蛋白。使用125I-钙调蛋白覆盖测定法来鉴定钙调蛋白作用的潜在靶点。精子含有一种约45 kDa的主要钙调蛋白结合蛋白。卵子缺乏主要的钙调蛋白结合活性。一种72 kDa的钙调蛋白结合蛋白在受精后1 - 65小时的合子中很突出。这两种钙调蛋白结合蛋白都表现出钙依赖性结合活性。总体而言,数据表明某些钙结合蛋白和钙调蛋白结合蛋白的出现和分布受发育调控,可能反映了钙在受精和早期胚胎发生过程中的不同作用。