RESUMEN
Trout liver is a rich source of sialate cytidylyltransferase activity. Three procedures are described by which the enzyme was enriched between 67- and 647-fold with high specific activities varying between 0.67 and 1.88 U/mg protein. In the simplest procedure studied, 100,000 x g supernatant of liver homogenate was chromatographed on Q-Sepharose and beta-[3-(2-aminoethylthio)propyl]-N- acetylneuraminic acid as affinity matrix, leading to an enzyme preparation (0.67 U/mg protein) well suited for the synthesis of CMP-N-acetylneuraminic acid. The synthase has a molecular mass of 160 kDa, a temperature optimum of 28 degrees C, a pH-optimum of 9.3 and exhibits Km-values for CTP, N-acetylneuraminic acid and N-glycoloylneuraminic acid of 1.7 mM, 2.1 mM and 2.9 mM, respectively. It is inactive with N-acetyl-9-O-acetylneuraminic acid. The enzyme is inhibited by CMP, CDP and 2'-deoxy-CTP. The sialic acid fraction of trout liver after hydrolysis is composed by N-acetylneuraminic acid (86%), N-acetyl-9-O-acetylneuraminic acid (12%) and N-acetyl-9-O-lactoylneuraminic acid (2%).
Asunto(s)
Hígado/enzimología , N-Acilneuraminato Citidililtransferasa/aislamiento & purificación , Trucha/metabolismo , Animales , Cromatografía de Afinidad , Cromatografía en Capa Delgada , Concentración de Iones de Hidrógeno , Cinética , Hígado/química , Peso Molecular , N-Acilneuraminato Citidililtransferasa/análisis , N-Acilneuraminato Citidililtransferasa/antagonistas & inhibidores , Oxo-Ácido-Liasas/metabolismoRESUMEN
As a precursor for the chemical synthesis of sialylated oligosaccharides, the trisaccharide glycoside Neu5Ac alpha (2-8)Gal beta (1-4)GlcNAc beta (1-O)-pent-4-ene was synthesized starting from GlcNAc beta (1-O)-pent-4-ene, UDP-glucose and N-acetylneuraminic acid in a one pot reaction employing galactosyltransferase and alpha (2-6)sialyltransferase in a complete cofactor regeneration system.