Roughan P G, Ohlrogge J B
Horticulture and Food Research Institute of New Zealand, Mt. Albert Research Centre, Auckland.
Anal Biochem. 1994 Jan;216(1):77-82. doi: 10.1006/abio.1994.1010.
Acetyl-CoA synthetase activity in vitro is assayed quickly and conveniently by incubating whole chloroplasts, chloroplast extracts, or leaf extracts with labeled acetate, CoA, ATP, and Mg and transferring aliquots of the reaction mixture to pieces of either Whatman No. 1 or DE81 filter paper. Unreacted acetate is quantitatively washed from the papers while the acetyl-CoA, which binds quantitatively, is determined by scintillation counting. Enzyme activity is absolutely dependent upon the presence of CoA, ATP, and Mg in reaction mixtures. The reaction has a broad pH optimum around pH 8.5. Potassium is required for maximum activity, and lithium strongly inhibits the reaction. The product retained on the papers was characterized as acetyl-CoA by several methods. On a chlorophyll basis, acetyl-CoA synthetase activities were about 25% higher in leaf homogenates than in intact chloroplasts isolated from similar leaves. Enzyme activities in the optimized assay were three- to fourfold greater than previously reported.
通过将完整叶绿体、叶绿体提取物或叶片提取物与标记的乙酸盐、辅酶A、三磷酸腺苷（ATP）和镁一起孵育，并将反应混合物的等分试样转移到沃特曼1号滤纸或DE81滤纸上，可快速方便地测定体外乙酰辅酶A合成酶的活性。未反应的乙酸盐从滤纸上被定量洗去，而定量结合的乙酰辅酶A则通过闪烁计数法测定。酶活性绝对依赖于反应混合物中辅酶A、ATP和镁的存在。该反应在pH 8.5左右有较宽的最适pH值。钾是最大活性所必需的，而锂则强烈抑制该反应。通过几种方法将保留在滤纸上的产物鉴定为乙酰辅酶A。以叶绿素为基础，叶片匀浆中的乙酰辅酶A合成酶活性比从相似叶片中分离出的完整叶绿体中的活性高约25%。优化测定中的酶活性比先前报道的高3至4倍。
