Enzymatic 4-O-acetylation of N-acetylneuraminic acid in guinea-pig liver.
Sialic acids from the liver and serum of guinea-pig are composed of N-acetylneuraminic acid (Neu5Ac; 85% and 61%, respectively), N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2; 10% and 32%, respectively) and N-glycolylneuraminic acid (Neu5Gc; 5% and 7%, respectively), besides traces of N-glycolyl-4-O-acetylneuraminic acid in serum. The analysis was carried out using thin-layer chromatography, high-performance liquid chromatography, electron impact ionization mass spectrometry, and different enzymes (sialidase, sialate esterase, and sialate-pyruvate lyase after hydrolysis and purification of the sialic acids by ion-exchange chromatography). We showed that this O-acetylation of sialic acids is due to the activity of an acetyl-coenzyme A:sialate-4-O-acetyltransferase (EC 2.3.1.44), which occurs together with sialyltransferase activity in Golgi-enriched membrane fractions of guinea-pig liver. The enzyme operates optimally at 30 degrees C in 70 mM potassium phosphate buffer at pH 6.7 and in the presence of 90 mM KCI with an apparent KM for AcCoA of 0.6 1microM and a Vmax of 20 pmol/mg protein x min. The enzyme is inhibited by coenzyme A in a mixed-competitive manner (Ki = 4.2 microM), as well as by parachloromercuribenzoate, MnCl2, saponin and Triton X-100.
Biochemisches Institut, Christian-Albrechts-Universität, Kiel, Germany.
M
M
Iwersen
V
V
Vandamme-Feldhaus
R
R
Schauer
D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism*; D000818:Animals; D003065:Coenzyme A / Q000494:pharmacology; D005260:Female; D006056:Golgi Apparatus / Q000201:enzymology; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D009438:Neuraminic Acids / Q000378:metabolism; D012794:Sialic Acids / Q000032:analysis / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D009438:Neuraminic Acids; D012794:Sialic Acids; C032592:N-glycolylneuraminic acid; C041901:4-O-acetyl-N-acetylneuraminic acid; D000123:Acetyltransferases; C118584:N-acetylneuraminate 4-O-acetyltransferase; D019158:N-Acetylneuraminic Acid; D003065:Coenzyme A