Methylumbelliferyl-N-acetylneuraminic acid sialidase in human liver.
Human liver sialidase was measured using methylumbelliferyl-N-acetylneuraminic acid as a substrate. The enzyme activity was linear for only 20 min and linearity was not improved by adding albumin, CaCl2, dithiothreitol, or Ep-459. The optimal pH was 4.5 and the apparent Km value, approximately 0.090 mM. Without substrate addition, the enzyme was unstable at temperatures between 0 and 37 degrees C, retaining only 35 and 5% of its activity, respectively, after 81/2 hr, but was protected by albumin at 5 mg/ml. The enzyme was more stable when either total liver or liver homogenate was kept frozen at -20 degrees C. Liver sialidase also retained about 70% of its activity after mechanical homogenization for 5 min. Potential inhibitors, notably, p- aminooxanilic acid, fetuin III, Triton X-100, mucin, sialyllactose, colominic acid, sodium taurocholate, N-acetylneuraminic acid, and methoxyphenyl-N-acetylneuraminic acid, were tested. Sialyllactose, methoxyphenyl-N-acetylneuraminic acid, fetuin, N-acetylneuraminic acid, and colominic acid were competitive inhibitors with Ki values of 1.12, 0.37, 0.20, 0.78, and 0.22 mM, respectively. The 0.11 M solutions of NaCl, LiCl, and KCl inhibited 20-30%, and CaCl2 about 60%, of the enzyme activity.
D000418:Albumins / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012794:Sialic Acids / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D014468:Umbelliferones / Q000378:metabolism*
D000418:Albumins; D002712:Chlorides; D012794:Sialic Acids; D014468:Umbelliferones; D006923:Hymecromone; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; D009439:Neuraminidase