Kinetic properties and acceptor substrate preferences of two kinds of Gal beta 1,3GalNAc alpha 2,3-sialyltransferase from mouse brain.
The cDNAs encoding two kinds of Gal beta 1,3GalNAc alpha 2,3-sialytransferases (ST3GalA.1 and ST3GalA.2) have been cloned from mouse brain, both of which could synthesize the NeuAc alpha 2,3Gal beta 1,-3GalNAc sequence of gangliosides as well as O-glycosidically linked oligosaccharides of glycoproteins [Lee et al. (1993) Eur. J. Biochem. 216, 377-385; Lee et al. (1994) J. Biol. Chem. (in press)]. Kinetic analysis of the two sialyltransferases using Gal beta 1,3GalNAc, asialoGM1, or asialofetuin revealed that ST3GalA.1 exhibits the highest Km value for asialoGM1 (Km = 1.25 mM) and the lowest one for asialofetuin (Km = 0.10 mM), whereas the Km values of ST3GalA.2 for the substrates are very similar (Km approximately 0.5 mM). The synthesis of GM1b from asialoGM1 by ST3GalA.1 was clearly inhibited in the presence of Gal beta 1,3GalNAc or asialofetuin, but that by ST3GalA.2 was not at all. On the other hand, the activity of ST3GalA.2 toward Gal beta 1,3GalNAc or asialofetuin was inhibited by asialoGM1 or GM1. The results of acceptor competition experiments involving asialoGM1, Gal beta 1,3GalNAc, and asialofetuin indicated that ST3GalA.2 exhibits noncompetitive inhibition between asialoGM1 and Gal beta 1,3GalNAc or between asialoGM1 and asialofetuin, whereas ST3GalA.1 exhibits competitive inhibition between all kinds of acceptors. These results strongly indicate that acceptor preference of ST3GalA.1 is different from that of ST3GalA.2, although their acceptor substrate specificities are the same; i.e., gangliosides serve as predominant acceptors for the latter over O-glycosidically linked oligosaccharides of glycoproteins, which are much better acceptors for the former.
Kojima
N
N
Frontier Research Program, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Lee
Y C
YC
Hamamoto
T
T
Kurosawa
N
N
Tsuji
S
S
D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology*; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D005677:G(M1) Ganglioside / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D012799:Sialyltransferases / Q000378:metabolism*; D013379:Substrate Specificity; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D005677:G(M1) Ganglioside; C018835:asialo GM1 ganglioside; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase