Rat‐Liver‐Sialidase Activity Utilizing a Tritium‐Labeled Sialic‐Acid Derivative of Glycoprotein Substrates

Plasma glycoproteins were oxidized with periodic acid and reduced with tritiated KBH 4 , resulting in macromolecules specifically labeled with tritium on their sialic acid residues. The resulting tritiated seven‐carbon analog of N ‐acetylneuraminic acid, abbreviated [ 3 H]AcNeu, covalently attached as part of the fetuin molecule, [ 3 H‐AcNeu]fetuin, had a specific activity of 3.67 counts × min −1 × ng −1 . It served as a very suitable substrate for the measurement of bacterial and mammalian neuraminidase. Clostridium perfringens neuraminidase had a K m of 9 μM and a V of 50 counts × min −1 × h −1 × ng −1 . [ 3 H‐AcNeu]Fetuin was homogeneous on polyacrylamide gel electrophoresis and migrated exactly as native fetuin following treatment by sodium dodecyl‐sulfate, mercaptoethanol, and boiling. Mild acid hydrolysis removed 70% of the radioactivity, which chromatographed as a homogeneous product utilizing butylacetate, acetic acid and water (3:2:1, v/v/v) as the solvent system. Mammalian neuraminidase was located in a particulate fraction of rat liver. The enzyme had an acid pH optimum and may be lysosomal in origin. The use of [ 3 H‐AcNeu 7 ]glycoprotein substrates increased the sensitivity of measurement of the released sialic acid and provided a more reliable estimate of neuraminidase levels in mammalian tissue sources. Livers from rats fed a vitamin‐K‐deficient diet were also assayed for activity. The results indicated that vitamin‐K‐deficient animals had a slightly elevated amount of neuraminidase activity with [ 3 H‐AcNeu 7 ]prothrombin substrate. Prothrombin, unlabeled or labeled, was not extensively degraded by C l. perfringens neuraminidase. Therefore, either differences exist between bacterial enzyme and mammalian enzyme in the substrate specificities, or the mammalian enzyme source may contain more than one neuraminidase, i.e. one capable of the degradation of the sialic acid residues of fetuin and another capable of the degradation of the sialic acid residues of prothombin.