Gołebiowski Filip, Szulc Aneta, Sakowicz Monika, Szutowicz Andrzej, Pawełczyk Tadeusz
Department of Molecular Medicine, Medical University of Gdańsk, 80-211 Gdańsk, Poland.
Acta Biochim Pol. 2003;50(4):1065-73.
Ubc9 is a homologue of the E2 ubiquitin conjugating enzyme and participates in the covalent linking of SUMO-1 molecule to the target protein. In this report we describe a simple and efficient method for obtaining pure human recombinant Ubc9 protein. The purified Ubc9 retained its native structure and was fully active in an in vitro sumoylation assay with the promyelocytic leukaemia (PML) peptide as a substrate. In order to better understand the physiology of Ubc9 protein we examined its levels in several rat tissues. Immunoblot analyses performed on tissue extracts revealed quantitative and qualitative differences in the expression pattern of Ubc9. The Ubc9 protein was present at a high level in spleen and lung. Moderate level of Ubc9 was detected in kidney and liver. Low amount of Ubc9 was observed in brain, whereas the 18 kDa band of Ubc9 was barely visible or absent in heart and skeletal muscle. In heart and muscle extracts the Ubc9 antibodies recognized a 38 kDa protein band. This band was not visible in extracts of other rat tissues. A comparison of the relative levels of Ubc9 mRNA and protein indicated that the overall expression level of Ubc9 was the highest in spleen and lung. In spleen, lung, kidney, brain, liver and heart there was a good correlation between the 18 kDa protein and Ubc9 mRNA levels. In skeletal muscle the Ubc9 mRNA level was unproportionally high comparing to the level of the 18 kDa protein. The presented data indicate that in the rat the expression of the Ubc9 protein appears to have some degree of tissue specificity.
Ubc9是E2泛素缀合酶的同源物，参与SUMO-1分子与靶蛋白的共价连接。在本报告中，我们描述了一种获得纯人重组Ubc9蛋白的简单有效方法。纯化后的Ubc9保留了其天然结构，并且在以早幼粒细胞白血病（PML）肽为底物的体外SUMO化分析中具有完全活性。为了更好地理解Ubc9蛋白的生理学特性，我们检测了它在几种大鼠组织中的水平。对组织提取物进行的免疫印迹分析揭示了Ubc9表达模式在数量和质量上的差异。Ubc9蛋白在脾脏和肺中高水平存在。在肾脏和肝脏中检测到中等水平的Ubc9。在脑中观察到Ubc9含量较低，而在心脏和骨骼肌中几乎看不到或不存在18 kDa的Ubc9条带。在心脏和肌肉提取物中，Ubc9抗体识别出一条38 kDa的蛋白条带。这条带在其他大鼠组织的提取物中不可见。Ubc9 mRNA和蛋白相对水平的比较表明，Ubc9的整体表达水平在脾脏和肺中最高。在脾脏、肺、肾脏、脑、肝脏和心脏中，18 kDa蛋白与Ubc9 mRNA水平之间存在良好的相关性。在骨骼肌中，Ubc9 mRNA水平与18 kDa蛋白水平相比异常高。所呈现的数据表明，在大鼠中，Ubc9蛋白的表达似乎具有一定程度的组织特异性。
