Hack N, Carey F, Crawford N
Biochem J. 1984 Oct 1;223(1):105-11. doi: 10.1042/bj2230105.
Previous studies by Roth & Majerus [J. Clin. Invest. (1975) 56, 624-632] showed that exposure of platelets to [acetyl-14C]aspirin resulted in the radioactive labelling of three polypeptides, two of which were in the cytosol and not saturable, whilst the third was located in particulate material, and was saturated at 30 microM-aspirin. By using high voltage free flow electrophoresis to separate a platelet mixed membrane fraction into highly purified surface and intracellular membrane subfractions, we have confirmed that the major polypeptide acetylated after exposing whole platelets to [acetyl-14C]aspirin is almost exclusively associated with intracellular membrane structures. We have shown previously that these intracellular membranes are the major site for prostanoid biosynthesis [Carey, Menashi & Crawford (1982) Biochem. J. 204, 847-851] and in the present study the extent of the radioactive labelling correlated well with inhibition of the cyclo-oxygenase activity in these intracellular membranes. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the [14C]acetylated component, which appears to be a dimer, migrates with a mobility corresponding to 72kDa. Although cyclo-oxygenase is inhibited, there is no discernible radioactive labelling when the platelets are exposed to aromatic-ring-labelled [14C]aspirin. We suggest that the site or sites for aspirin acetylation and cyclo-oxygenase activity are structurally associated in the platelet's intracellular membranes referred to by electron microscopists as the dense tubular membrane system.
罗斯和马耶鲁斯此前的研究[《临床研究杂志》(1975年)56卷，624 - 632页]表明，将血小板暴露于[乙酰基 - 14C]阿司匹林会导致三种多肽被放射性标记，其中两种位于胞质溶胶中且不饱和，而第三种位于颗粒物质中，在30微摩尔阿司匹林浓度下达到饱和。通过使用高压自由流动电泳将血小板混合膜部分分离为高度纯化的表面膜和细胞内膜亚组分，我们证实了将完整血小板暴露于[乙酰基 - 14C]阿司匹林后被乙酰化的主要多肽几乎完全与细胞内膜结构相关。我们之前已经表明，这些细胞内膜是类前列腺素生物合成的主要部位[凯里、梅纳希和克劳福德(1982年)《生物化学杂志》204卷，847 - 85页]，在本研究中，放射性标记的程度与这些细胞内膜中环氧化酶活性的抑制密切相关。在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳中，[14C]乙酰化成分似乎是一种二聚体，迁移率对应于72 kDa。尽管环氧化酶受到抑制，但当血小板暴露于芳环标记的[14C]阿司匹林时，没有可察觉的放射性标记。我们认为，阿司匹林乙酰化位点和环氧化酶活性位点在血小板的细胞内膜中在结构上相互关联，电子显微镜学家将其称为致密管状膜系统。
