Characterization of human placental neuraminidases. Stability, substrate specificity and molecular weight

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Abstract
1. At least two components of neuraminidase can be distinguished on the basis of thermolability and sedimentability by using the artificial fluorogenic substrate 4-methylumbelliferyl N-acetyl-alpha-D-neuraminate. 2. In crude homogenates, thermodenaturation at 25 degrees C showed a biphasic curve corresponding to component A (half-life, 21 min) and B (half-life, 85 min). The two components were partially resolved by centrifugation. A being soluble and B sedimentable. Both had similar pH-activity curves (pH optimum, 4.4), Km values (A, 0.10 mM; B, 0.06 mM) and molecular weight as determined by radiation inactivation (A, 67000; B, 63000). 3. The soluble A form was still aggregated or bound to membranous debris since almost all neuraminidase activity was eluted near or at the void volume of a Sephacryl S-300 column. 4. Both soluble and sedimentable fractions of placenta hydrolysed the GD1A ganglioside and N-acetyl-neuraminyl-D-lactose linearly for 12 h but no fetuin hydrolysis was detected. 5. The neuraminidase activity with the artificial fluorogenic substrate was inhibited by N-acetylneuraminyl-D-lactose but not by the GD1A ganglioside. These preliminary results suggest that there exist two closely related enzymes hydrolysing both the artificial substrate and N-acetylneuraminyl-D-lactose and a third one hydrolysing the GD1A ganglioside exclusively.