Isolation, purification, and characterization of glucosamine-6-phosphate-N-acetylase from pig liver.
The procedure of isolation, purification, and characterization of glucosamine-6-phosphate acetylase from the pig liver is described. The steps of purification were as follows: adsorption on hydroxylapatite, fractionation with ammonium sulfate, chromatography on cellulose phosphate, electrofocusing, and preparative gel electrophoresis. A highly purified (about 3000-fold) preparation of GlcN-6-P acetylase, with a yield of 23%, was obtained. It was found that GlcN-6-P acetylase from pig liver is heterogeneous and exists in two active forms. The characteristic features of the preparation were established: Mr, about 24 kDa; temperature optimum at 37 degrees; pH optimum at 7.45; and Km (GlcN-6-P) 3.7 x 10(-3) M and Km (AcCoA) 1.4 x 10(-3) M. The ions K+, Na+, NH4+, Mg2+, Mn2+, and CH3COO- do not stimulate the acetylase activity. The product of acetylase reaction (GlcNAc-6-P) inhibits this reaction according to the feedback process. The highly purified preparation of GlcN-6-P acetylase is unstable during storage and it is protected by ampholine or glycine from enzyme inactivation, but it is not protected by 2-mercaptoethanol.
Porowski
T S
TS
Department of General Chemistry and Organic Chemistry, Institute of Chemistry, Medical School, Białystok, Poland.
Porowska
H
H
Gałasiński
W
W
D000123:Acetyltransferases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D000327:Adsorption; D019747:Aldose-Ketose Isomerases; D000818:Animals; D002238:Carbohydrate Epimerases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D051081:Glucosamine 6-Phosphate N-Acetyltransferase; D006882:Hydroxyapatites; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis; D013552:Swine
D006882:Hydroxyapatites; D011506:Proteins; D000123:Acetyltransferases; D051081:Glucosamine 6-Phosphate N-Acetyltransferase; C020582:glucosamine-6-phosphate isomerase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases