[29] Acetyl coenzyme A: Long-chain base acetyltransferase from the microsomes of Hansenula ciferri

Yechezkel Barenholz, Shimon Gatt

Research output: Contribution to journalArticlepeer-review

Abstract

This chapter discusses the determination of long-chain base acetyltransferase from the microsomes of Hansenula ciferri. This enzyme transfers the acetyl portion of acetyl coenzyme A to the amino groups of primary amines of 6–18 carbon atoms and to the amino and hydroxyl groups of sphingosine base. The enzyme differs from the arylamine acetyltransferase of the soluble fraction of H. ciferri or of mammalian and avian liver. The transfer reaction is directly proportional to enzyme concentration (up to at least 1 mg of microsomal protein) and to the reaction time (for at least 2 hours). The optimal pH of the reaction depends on the type of buffer used; this is related to the charge imparted to the micelles of the base. The curves that describe the rate of acetyl transfer as a function of the concentration of acetyl-CoA (while maintaining the base at a fixed concentration) are hyperbolic. Similar curves, in which aeetyl-CoA is the fixed and dihydrosphingosine or hexadecylamine are the variable substrates, have a nonsymmetric sigmoidal shape. Serum albumin increases the reaction rates, and at an optimal molar ratio of albumin to base of about 0.5 the V/S curves are hyperbolic. Two assay methods are used for the determination of long-chain base acetyltransferase. In one, radioactively labeled acetyl-CoA is used and the radioactivity of the acetylated base is determined. In the second, the reaction is run in the presence of DTNB [5,5-dithiobis-(2-nitrobenzoic acid)] and the color resulting from the coenzyme A released is read.

Original languageEnglish
Pages (from-to)242-247
Number of pages6
JournalMethods in Enzymology
Volume35
Issue numberC
DOIs
StatePublished - 1 Jan 1975

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