Further characterization of a myelin-associated neuraminidase: properties and substrate specificity.
A neuraminidase activity in myelin isolated from adult rat brains was examined. The enzyme activity in myelin was first compared with that in microsomes using N-acetylneuramin(alpha 2----3)lactitol (NL) as a substrate. In contrast to the microsomal neuraminidase which exhibited a sharp pH dependency for its activity, the myelin enzyme gave a very shallow pH activity curve over a range between 3.6 and 5.9. The myelin enzyme was more stable to heat denaturation (65 degrees C) than the microsomal enzyme. Inhibition studies with a competitive inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, showed the Ki value for the myelin neuraminidase to be about one-fifth of that for the microsomal enzyme (1.3 X 10(-6) M versus 6.3 X 10(-6) M). The apparent Km values for the myelin and the microsomal enzyme were 1.3 X 10(-4) M and 4.3 X 10(-4) M, respectively. An enzyme preparation that was practically devoid of myelin lipids was then prepared and its substrate specificity examined. The "delipidated enzyme" could hydrolyze fetuin, NL, and ganglioside substrates, including GM1 and GM2. When the delipidated enzyme was exposed to high temperature (55 degrees C) or low pH (pH 2.54), the neuraminidase activities toward NL and GM3 decreased at nearly the same rate. Both fetuin and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid inhibited NL and GM3 hydrolysis. With 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, inhibition of NL was greater than that of GM3; however, the Ki values for each substrate were almost identical. GM3 and GM1 also competitively inhibited the hydrolysis of NL and NL similarly inhibited GM3 hydrolysis by the enzyme. These results indicate that rat brain myelin has intrinsic neuraminidase activities toward nonganglioside as well as ganglioside substrates, and that these two enzyme activities are likely catalyzed by a single enzyme entity.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D005260:Female; D005732:Gangliosides / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008055:Lipids; D008861:Microsomes / Q000201:enzymology; D009186:Myelin Sheath / Q000201:enzymology*; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives*; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011489:Protein Denaturation; D051381:Rats; D011919:Rats, Inbred Strains; D012794:Sialic Acids / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D000509:alpha-Fetoproteins / Q000378:metabolism
D005732:Gangliosides; D008055:Lipids; D012794:Sialic Acids; D000509:alpha-Fetoproteins; C036137:2-deoxy-2,3-dehydro-N-acetylneuraminic acid; C029412:neuramin lactitol; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid