Wright L S, Siegel F L
Department of Pediatrics, University of Wisconsin Medical School, Madison 53705.
J Neurochem. 1993 Apr;60(4):1475-82. doi: 10.1111/j.1471-4159.1993.tb03310.x.
Synaptosomes from five regions of adult rat brain were isolated, analyzed for methyl acceptor proteins, and probed for methyltransferases by photoaffinity labeling. Methylated proteins of 17 and 35 kDa were observed in all regions, but cerebellar synaptosomes were enriched in a 21-26-kDa family of methyl acceptor proteins and contained a unique major methylated protein of 52 kDa and a protein of 50 kDa, which was methylated only in the presence of EGTA. When cerebellar and liver subcellular fractions were compared, the cytosolic fractions of each tissue contained methylated proteins of 17 and 35 kDa; liver membrane fractions contained few methylated proteins, whereas cerebellar microsomes had robust methylation of the 21-26-kDa group. Differential centrifugation of lysed cerebellar synaptosomes localized the 17- and 35-kDa methyl acceptor proteins to the synaptoplasm, the 21-26-kDa family to the synaptic membranes, and the 52-kDa to synaptic vesicles. The 21-26-kDa family was identified as GTP-binding proteins by [alpha-32P]GTP overlay assay; these proteins contained a putative methylated carboxyl cysteine, based on the presence of volatile methyl esters and the inhibition of methylation by acetylfarnesylcysteine. The 52-kDa methylated protein also contained volatile methyl esters, but did not bind [alpha-32P]GTP. When synaptosomes were screened for putative methyltransferases by S-adenosyl-L-[methyl-3H]methionine photoaffinity labeling, a protein of 24 kDa was detected only in cerebellum, and this labeled protein was localized to synaptic membranes.
从成年大鼠大脑的五个区域分离出突触体，分析其甲基受体蛋白，并通过光亲和标记检测甲基转移酶。在所有区域均观察到17 kDa和35 kDa的甲基化蛋白，但小脑突触体富含21 - 26 kDa的甲基受体蛋白家族，并且含有一种独特的主要甲基化蛋白，分子量为52 kDa，还有一种50 kDa的蛋白，该蛋白仅在存在乙二醇双乙醚四乙酸（EGTA）时才被甲基化。当比较小脑和肝脏的亚细胞组分时，每个组织的胞质组分都含有17 kDa和35 kDa的甲基化蛋白；肝膜组分含有的甲基化蛋白很少，而小脑微粒体中21 - 26 kDa组的甲基化很明显。对裂解的小脑突触体进行差速离心，将17 kDa和35 kDa的甲基受体蛋白定位到突触浆，21 - 26 kDa家族定位到突触膜，52 kDa的蛋白定位到突触小泡。通过[α - 32P]GTP覆盖试验将21 - 26 kDa家族鉴定为GTP结合蛋白；基于挥发性甲酯的存在以及法尼基乙酰半胱氨酸对甲基化的抑制作用，这些蛋白含有一个推定的甲基化羧基半胱氨酸。52 kDa的甲基化蛋白也含有挥发性甲酯，但不结合[α - 32P]GTP。当通过S - 腺苷 - L - [甲基 - 3H]甲硫氨酸光亲和标记筛选突触体中的推定甲基转移酶时，仅在小脑中检测到一种24 kDa的蛋白，并且这种标记蛋白定位在突触膜上。
