SEPARATION OF ACETYL TRANSFER ENZYMES IN PIGEON LIVER EXTRACT

Abstract

Pigeon liver extract has proved to be a store-house of biosynthetic and, particularly, acetylation reactions.Acetylation of arylamine (I), histamine (Z), and glucosamine (3) and the synthesis of acetoacetate (4) and of citrate (5) were found in this extract.In all these systems, acetate, coenzyme A (CoA), and adenosinetriphosphate (ATP) react with the various acetyl acceptors.From early data, it had become quite likely that acetate, CoA, and ATP reacted enzymatically to yield the common acetyl donor, which carried the acetyl to the various acceptors by way of more or less specific transfer enzymes (4, 6).A rigorous proof for this interpretation could be obtained only through a separation of the specific enzyme systems involved.Some preliminary progress had been made by fractionation with ammonium sulfate (7), but fractionation with acetone in the cold proved to be most successful for this purpose.A preliminary report of the results of these experiments was published previously (8). Analytical MethodsAcetylation of aromatic amines was determined by the method of Bratton and Marshall (9).Citrate was determined by the method of Krebs and Eggleston (10) as modified by Novelli and Lipmann (11).Determination of acet.oacetate was carried out by the salicylaldehyde method of Behre (12), by applying a microdiffusion technique similar to that described by Stadtman et al. ( 13).As a diffusion chamber, a 25 ml.Pyrex Erlenmeyer flask was used, into which a glass cup containing bisulfite was inserted.The glass cup was prepared by sealing 1.5 to 2 cm. of the lower end of a 13 X 100 mm.Pyrex test-tube to a Pyrex glass rod, 8 cm.long and 4 mm. in diameter.The glass rod was fitted tightly into a No. 1 rubber stopper and adjusted to leave about a 1 cm.space between the bottom of the cup and the fluid level. 1 to 2 ml. of the sample was placed in the bottom of the Erlenmeyer flask and 0.2 ml. of 15 N sulfuric acid was added.Immediately, the cup containing 0.4 ml. of M sodium bisulfite solution was inserted.The tightly stoppered flask was incubated for 12 to 16 hours at