On the Assay of Acetyl-CoA Synthetase Activity in Chloroplasts and Leaf Extracts

• Published in: Analytical biochemistry 1994-01, Vol.216 (1), p.77-82
• Main Authors: Roughan, P.G., Ohlrogge, J.B.
• Format: Article
• Language: English
• Subjects: Acetate-CoA Ligase - metabolism; Acetyl Coenzyme A - biosynthesis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chloroplasts - enzymology; Enzymes and enzyme inhibitors; Filtration; Fundamental and applied biological sciences. Psychology; Ligases; Plant Extracts - analysis
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1994.1010

Acetyl-CoA synthetase activity in vitro is assayed quickly and conveniently by incubating whole chloroplast, 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.