Partial purification and characterization of cytidine-5'-monophosphosialate synthase from rainbow trout liver.
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%).
Schmelter
T
T
Biochemisches Institut, Christian-Albrechts-Universität zu Kiel.
Ivanov
S
S
Wember
M
M
Stangier
P
P
Thiem
J
J
Schauer
R
R
D000818:Animals; D002846:Chromatography, Affinity; D002855:Chromatography, Thin Layer; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000201:enzymology*; D008970:Molecular Weight; D000216:N-Acylneuraminate Cytidylyltransferase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D007652:Oxo-Acid-Lyases / Q000378:metabolism; D014337:Trout / Q000378:metabolism*
D000216:N-Acylneuraminate Cytidylyltransferase; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase