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DEMONSTRATION AND PURIFICATION OF A MYRISTOYL-COA BINDING PROTEIN FROM BOVINE CARDIAC MUSCLE

机译:牛心肌中肉豆蔻酰-COA结合蛋白的展示和纯化

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Protein myristoylation refers to the co-translational addition of myristoyl group to an amino-terminal glycine residue of a protein by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). The myristoylation reaction depends on the availability of the cellular pools of coenzyme A and myristate and their subsequent formation of myristoyl-CoA the substrate of NMT. In the present study a myristoyl-CoA binding protein (MCBP) was purified using various column chromatographies: hydroxylapatite, DEAE Sepharose CL-6B and Sephacryl S-300 gel filtration. The purified protein exhibited an apparent molecular mass of 50 kDa on SDS-polyacrylamide gel electrophoresis. Incubation of protein with [C1-14]myristoyl-CoA, followed by denaturing gel electrophoresis, fluorography and treatment with hydroxylamine yielded results that are highly suggestive of a covalent ester-linked acyl-protein complex. This complex formation was not observed in the crude cytosolic fractions. The addition of cytosolic fraction to a progressing acyl-protein complex, resulted in deacylation suggesting a role for thioesterase or/proteinases in the regulation of the acylation reaction in bovine cardiac muscle. The acyl-protein complex formation was not inhibited by NLP71, a 71 kDa NMT inhibitory protein from bovine brain. [References: 21]
机译:蛋白质肉豆蔻酰基化是指通过肉豆蔻酰基-CoA:蛋白N-肉豆蔻酰基转移酶(NMT)将肉豆蔻酰基共翻译到蛋白质的氨基末端甘氨酸残基上。肉豆蔻酰化反应取决于辅酶A和肉豆蔻酸酯的细胞池的可用性及其在NMT底物上形成的肉豆蔻酰-CoA的能力。在本研究中,肉豆蔻酰-CoA结合蛋白(MCBP)使用各种柱色谱进行了纯化:羟磷灰石,DEAE Sepharose CL-6B和Sephacryl S-300凝胶过滤。纯化的蛋白质在SDS-聚丙烯酰胺凝胶电泳上表现出50 kDa的表观分子量。用[C1-14]肉豆蔻酰基-CoA孵育蛋白质,然后进行变性凝胶电泳,荧光照相和羟胺处理,得到的结果高度暗示了共价酯连接的酰基-蛋白质复合物。在粗胞质级分中未观察到这种复合物的形成。向进展的酰基-蛋白质复合物中添加胞质部分会导致脱酰作用,表明硫酯酶或/蛋白酶在调节牛心肌中的酰化反应中起作用。 NLP71(一种来自牛脑的71 kDa NMT抑制蛋白)不会抑制酰基-蛋白质复合物的形成。 [参考:21]

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