Coupled-enzyme system for measuring viral neuraminidase activity.
A coupled-enzyme assay for determining viral neuraminidase activity is described. All reactants-viral neuraminidase, the initial substrate (fetuin), N-acetylneuraminic acid aldolase, lactic acid dehydrogenase, and reduced nicotinamide adenine dinucleotide-are combined in a single cuvette. Thus, in a single coupled system neuraminidase releases N-acetylneuraminic acid, which is cleaved to N-acetyl-D-mannosamine and pyruvic acid; finally, pyruvate is reduced to lactate as reduced nicotinamide adenine dinucleotide is oxidized. The rate of change of absorbance at 340 nm, as reduced nicotinamide adenine dinucleotide is oxidized, is a measure of the rate of reaction of the coupled system. This procedure, which measures the rate of release of N-acetylneuraminic acid by neuraminidase, is an alternate method for those procedures which require multistep, colorimetric determinations.
D000510:Alpha-Globulins / Q000378:metabolism; D002474:Cell-Free System; D006595:Hexosamines / Q000096:biosynthesis; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007773:Lactates / Q000096:biosynthesis; D008190:Lyases / Q000378:metabolism; D008722:Methods; D009243:NAD / Q000378:metabolism; D009438:Neuraminic Acids / Q000096:biosynthesis / Q000378:metabolism; D009439:Neuraminidase / Q000378:metabolism*; D009975:Orthomyxoviridae / Q000201:enzymology*; D010084:Oxidation-Reduction; D011773:Pyruvates / Q000096:biosynthesis / Q000378:metabolism; D013053:Spectrophotometry; D013997:Time Factors; D014776:Virus Cultivation
D000510:Alpha-Globulins; D006595:Hexosamines; D007773:Lactates; D009438:Neuraminic Acids; D011773:Pyruvates; D009243:NAD; D007770:L-Lactate Dehydrogenase; D009439:Neuraminidase; D008190:Lyases