Abstract
Abstract Intracellular distribution of the sialidase of adult bovine brain gray matter was determined. The sialidase was found to be concentrated in the synaptosome fraction, and when the latter was disrupted and further fractionated, sialidase activity was found to be concentrated in the synaptosomal membrane. Sialidase in all cell fractions showed activity toward an intrinsic endogenous substrate, as well as toward added disialo- and trisialogangliosides, and sialyllactose. Km values for the sialidase in an acetone powder preparation from gross gray matter were 8 x 10-5 and 2 x 10-3 m for gangliosides and sialyllactose, respectively. There was an unequivocal lag phase of about 15 min between the time of addition of exogenous substrate and the time the sialidase measurably acted on it. The enzyme was found to act on the intrinsic endogenous substrate during the lag phase. Thin layer chromatography showed monosialo-, disialo-, and trisialogangliosides to be present in all cell fractions having sialidase activity. Analysis of the active fractions after incubation at 37° for 75 min without added exogenous substrate showed that monosialoganglioside, the end product of enzyme action on the intrinsic ganglioside substrate, remained associated with the membrane fragments.
Highlights
The enzyme was found to act on the intrinsic endogenoussubstrate during the lag phase
Thin layer chromatography showed monosialo, disialo, and trisialogangliosides to be present in all cell fractions having sialidase activity
Absorption spectra for the color obtained in the Warren thiobarbituric assay [11]. 1, Sialic acid; 2, the product obtained from sialidase of bovine brain acting on gangliosides; and 3, the product obtained from sialidase of bovine brain acting on gangliosides, after column chromatography on Dowex l-X10
Summary
The purpose of this work was to determine the intracellular distribution of sialidase in gray matter of bovine brain, and to compare the nature of enzymatic action on endogenous substrate (6) with that on added exogenous substrates
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