[A study of partial purification of ganglioside GD3 synthase and its enzymatic properties].
A simple and rapid procedure using anion exchange chromatography was established for determinations of the activity of ganglioside GD3 synthase (CMP-NeuAc: GM3, alpha 2----8 sialyltransferase) which catalyzes the conversion of ganglioside GM3 to GD3. The procedure was applicable for determination of activity of other ganglioside synthases. With the use of this procedure and GM3-acid affinity chromatography, the GD3 synthase was partially purified about 80 fold from a Lubrol PX extract of rat liver Golgi apparatus. The enzyme obtained had a pll optimum of 6.4. The preferred acceptors of the enzyme was GM3 containing N-acetylneuraminic acid (GM3 (NeuAc)) and GM3 containing N-glycolylneuraminic acid (GM3 (NeuGc)), with 2-fold higher V max in the latter than in the former. The Km values for GM3 (NeuAc), GM3 (NeuGc) and CMPNeuAc were 0.7 mM, 0.11 mM and 75 microM, respectively. The reaction product was identified as GD3 by thin layer chromatography. As to detergent, this enzyme showed maximum activity in 0.3% of Triton CF-54. The synthase did not require divalent cations for the activity, but rather Zn2+ and Cd2+ at 5 mM completely inhibited the enzyme activity. Cytidine nucleotides were strong inhibitors. The product, GD3, at 2 mM inhibited 30% the enzyme activity. The activity level of the GD3 synthase of rat liver was markedly different in rat strains, WKAH and TO strains being highest among eight strains examined. Male rat exhibited higher level than female. The synthase activity in rat liver was high at neonatal stage and decreased gradually thereafter.
Kawano
M
M
Department of Pediatric Dentistry, Hokkaido University, School of Dentistry, Sapporo, Japan.
D000818:Animals; D002292:Carcinoma, Renal Cell / Q000201:enzymology; D002846:Chromatography, Affinity; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007680:Kidney Neoplasms / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008545:Melanoma / Q000201:enzymology; D051381:Rats; D012799:Sialyltransferases / Q000302:isolation & purification* / Q000378:metabolism
D012799:Sialyltransferases; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase