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

✍ Scribed by P.G. Roughan; J.B. Ohlrogge

• Publisher: Elsevier Science
• Year: 1994
• Tongue: English
• Weight: 488 KB
• Volume: 216
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1994.1010

✦ Synopsis

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 (\mathrm{Mg}) in reaction mixtures. The reaction has a broad (\mathrm{pH}) optimum around (\mathrm{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. ic 1994 Academic Press, Inc.