TY - JOUR
T1 - [30] Acetyl coenzyme A
T2 - Amine acetyltransferase from the soluble fraction of Hansenula ciferri
AU - Barenholz, Yechezkel
AU - Gatt, Shimon
PY - 1975/1/1
Y1 - 1975/1/1
N2 - This chapter discusses the determination of amine acetyltransferase from the soluble fraction of Hansenula ciferri. This enzyme transfers the acetyl group of acetyl coenzyme A to an amine acceptor. The acetyltransferase from the soluble fraction of the yeast H. ciferri utilizes water-soluble amines but also acetylates normal amines up to hexadecylamine. Three assay methods are used. In two (one for the water-soluble and the second for the long-chain amines) radioactively labeled acetyl-CoA is used and the radioactivity of the acetylated amino is determined. In the third, the reaction is terminated, DTNB [5,5-dithiobis(2-nitrobenzoic acid) ] is added and the color resulting from the coenzyme A released is read. For the purification procedure, Hansenula ciferii can be obtained from the American type collection. The lyophilized culture is transferred to 100 ml erlenmeyers containing 40 ml of a yeast maintenance medium consisting of 0.3% malt extract, 0.3% yeast extract, 0.5% peptone, and 1% glucose. The purified enzyme is directly proportional to enzyme concentration up to at least 12 μg of protein and incubation time of 20–30 min. The linearity with time is improved by increasing the concentration of glycerol. Using the high-speed supernatant, the curves describing the rate as a function of enzyme concentration are not linear, and the specific activities are not constant, but increase on dilution of the enzyme with PG.
AB - This chapter discusses the determination of amine acetyltransferase from the soluble fraction of Hansenula ciferri. This enzyme transfers the acetyl group of acetyl coenzyme A to an amine acceptor. The acetyltransferase from the soluble fraction of the yeast H. ciferri utilizes water-soluble amines but also acetylates normal amines up to hexadecylamine. Three assay methods are used. In two (one for the water-soluble and the second for the long-chain amines) radioactively labeled acetyl-CoA is used and the radioactivity of the acetylated amino is determined. In the third, the reaction is terminated, DTNB [5,5-dithiobis(2-nitrobenzoic acid) ] is added and the color resulting from the coenzyme A released is read. For the purification procedure, Hansenula ciferii can be obtained from the American type collection. The lyophilized culture is transferred to 100 ml erlenmeyers containing 40 ml of a yeast maintenance medium consisting of 0.3% malt extract, 0.3% yeast extract, 0.5% peptone, and 1% glucose. The purified enzyme is directly proportional to enzyme concentration up to at least 12 μg of protein and incubation time of 20–30 min. The linearity with time is improved by increasing the concentration of glycerol. Using the high-speed supernatant, the curves describing the rate as a function of enzyme concentration are not linear, and the specific activities are not constant, but increase on dilution of the enzyme with PG.
UR - http://www.scopus.com/inward/record.url?scp=0016415610&partnerID=8YFLogxK
U2 - 10.1016/0076-6879(75)35161-6
DO - 10.1016/0076-6879(75)35161-6
M3 - Article
C2 - 235701
AN - SCOPUS:0016415610
SN - 0076-6879
VL - 35
SP - 247
EP - 253
JO - Methods in Enzymology
JF - Methods in Enzymology
IS - C
ER -