Sambasivam H, Murray R K
Department of Biochemistry, University of Toronto, Ont., Canada.
Biochem Cell Biol. 1988 Oct;66(10):1152-61. doi: 10.1139/o88-132.
The activity of acetyltransferase was detected in the microsomal fraction of rat liver by incubation with [3H]acetyl-CoA and by analyses using sodium dodecyl sulfate - polyacrylamide gel electrophoresis. Endogenous membrane proteins of relatively high molecular weight were found to serve as substrates. Optimal conditions for assay of the enzyme were defined. A deacetylase activity was also detected, which was inhibited by 2 mM ethylenediaminetetraacetic acid. Further subfractionation disclosed that the acetyltransferase activity was most enriched in the Golgi fraction, in which its specific activity was some ninefold greater than in the total homogenate. The radioactive labelling of Golgi-associated proteins observed was relatively intense, exceeding that of histone and ribosomal proteins in the homogenate. Analysis of the acetylated Golgi fraction by two-dimensional electrophoresis revealed approximately 90 radioactive polypeptides. Various treatments demonstrated that a minimum of 80% of the incorporated radioactivity was present as derivatives of N-acetylneuraminic acid, principally N-acetyl-9-mono-O-acetylneuraminic acid (Neu5,9Ac2). The sialic acid O-acetyltransferase activity detected is thus probably identical to that reported by Varki and Diaz; the intense labelling of proteins reflects the ability of Golgi apparatus fractions to take up and concentrate acetyl-CoA. Protein-bound radioactive Neu5,9Ac2 was also detected in the medium of hepatocytes incubated with N-[3H]acetylmannosamine, demonstrating that these cells synthesize certain proteins containing acetylated sialic acids, some of which may be secreted. The data confirm that the Golgi apparatus is a major site of acetylation of protein-bound sialic acids in rat liver in vitro and provide new information showing that many glycoproteins undergo this particular type of modification.
通过与[3H]乙酰辅酶A孵育并使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳进行分析，在大鼠肝脏微粒体部分检测到乙酰转移酶的活性。发现相对高分子量的内源性膜蛋白可作为底物。确定了该酶测定的最佳条件。还检测到一种脱乙酰酶活性，其受到2 mM乙二胺四乙酸的抑制。进一步的亚分级分离表明，乙酰转移酶活性在高尔基体部分中最为富集，其比活性比总匀浆中的高约九倍。观察到的与高尔基体相关蛋白的放射性标记相对较强，超过了匀浆中组蛋白和核糖体蛋白的标记。通过二维电泳对乙酰化高尔基体部分进行分析，发现了大约90种放射性多肽。各种处理表明，至少80%掺入的放射性以N-乙酰神经氨酸的衍生物形式存在，主要是N-乙酰-9-单-O-乙酰神经氨酸（Neu5,9Ac2）。因此，检测到的唾液酸O-乙酰转移酶活性可能与Varki和Diaz报道的相同；蛋白质的强烈标记反映了高尔基体部分摄取和浓缩乙酰辅酶A的能力。在用N-[3H]乙酰甘露糖胺孵育的肝细胞培养基中也检测到了与蛋白质结合的放射性Neu5,9Ac2，表明这些细胞合成了某些含有乙酰化唾液酸的蛋白质，其中一些可能会被分泌。这些数据证实，高尔基体是大鼠肝脏体外蛋白质结合唾液酸乙酰化的主要部位，并提供了新的信息，表明许多糖蛋白会经历这种特殊类型的修饰。
