Photolysis of the lysosomal neuraminidase in cultured human skin fibroblasts in the presence of a photoreactive competitive inhibitor.
Photolysis of the lysosomal neuraminidase in crude homogenates of cultured human skin fibroblasts was carried out using the potent competitive enzyme inhibitor, 9-S-(4-azido-2-nitrophenyl)-5-acetamido-2,6-anhydro-2,3,5,9-tetradeoxy-9 -thio-D - glycero-D-galacto-non-2-enonic acid (9-PANP-2,3-D-NANA). Irradiation of the homogenate and the inhibitor (2 min, pH 4.3, 10 degrees C) with a medium pressure mercury lamp resulted in about a 24% reduction of enzyme activity compared to irradiated controls that did not contain additives. No significant loss of activity was observed with homogenate that contained a photoreactive thioglycoside of sialic acid that was not an inhibitor of the enzyme. Similarly, the enzyme activity was not affected when 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid was photolyzed with the homogenate. The latter is a potent competitive inhibitor but it is not photoreactive. Also, the products obtained by prephotolyzing 9-PANP-2,3-D-NANA gave similar enzyme levels under standard assay conditions when compared with the nonirradiated material. Together, these results demonstrate that the photoinactivation is highly specific and both the aryl azide and the unsaturated pyran portion of the molecule are required for inactivation. The title compound may be useful as a potential photolabeling reagent which may facilitate purification of the enzyme and permit further characterization of the mutation in sialidosis patients.
Warner
T G
TG
University of Tennessee, Pediatrics Department, Memphis.
Loftin
S K
SK
D001386:Azides / Q000494:pharmacology*; D001667:Binding, Competitive; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000528:radiation effects; D010782:Photolysis; D012867:Skin / Q000201:enzymology*; D013400:Sugar Acids / Q000494:pharmacology*
D001386:Azides; D013400:Sugar Acids; C061973:9-S-(4-azido-2-nitrophenyl)-5-acetamido-2,6-anhydro-2,3,5,9-tetradeoxy-9-thioglycerogalactonon-2-enonic acid; D009439:Neuraminidase