Determination of methylumbelliferyl-N-acetylneuraminic acid sialidase for clinical purposes.
In order to determine optimal conditions for the clinical application of leukocyte acid sialidase determination, we have studied some characteristics of the enzyme giving attention more specifically to the enzyme's thermolability and the influence of potential effectors. The enzyme has a pH optimum around 4 and a Km value of 0.25 mmol . 1(-1). A decrease of activity is observed when the enzyme is measured in leukocytes isolated from total blood or leukocytes containing plasma kept for several hours at room temperature. On the other hand, in a leukocyte homogenate, the enzyme is stable at 0 degree C for 8 hours although pronounced thermolability is observed at 23 and 37 degrees C and total inactivation occurs after 10 minutes at 50 degrees C. The addition of albumin to the homogenate partially protects the enzyme. Residual activities of 40% and 50% were measured in leukocytes and leukocyte homogenates frozen during 2 months at -20 degrees C. A marked inhibition was obtained by Triton X-100, sodium taurocholate, and methoxyphenyl-N-acetylneuraminic acid. Immediate isolation of the leukocytes after the blood drawing and subsequent homogenization at 0 degree C of a dense pellet make it possible to achieve enzyme linearity and reproducible results.
D003124:Colorimetry / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives; D007962:Leukocytes / Q000201:enzymology*; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000097:blood*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature; D013997:Time Factors
D006923:Hymecromone; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; D009439:Neuraminidase