Sialidase in cerebellar granule cells differentiating in culture.
The optimal conditions for the assay of sialidase in cerebellar granule cells cultivated in vitro, established using [3H]GD1a and 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUB-NeuNAc) as substrates, were the following: pH optimum for both substrates, 3.9; optimal molarity of sodium acetate/acetic acid buffer, 0.05 M with [3H]GD1a and 0.1 M for MUB-NeuNAc; substrate concentration for apparent maximal activity, 0.5 mM for MUB-NeuNAc and 0.1 mM for [3H]GD1a; enzyme activity linear with time up to 30 min with MUB-NeuNAc and up to 90 min with [3H]GD1a; and enzyme activity linear with enzyme protein content up to 80 micrograms with MUB-NeuNAc and up to 20 micrograms with [3H]GD1a. The assay with [3H]GD1a required the presence of Triton X-100 in a molar ratio to GD1a of 15:1. Poly-L-lysine, which was used for plating the cells, was capable of decreasing sialidase activity against [3H]GD1a/Triton X-100 when added to the incubation mixture. However, it had no effect on the enzyme working on MUB-NeuNAc. Using no more than 20 micrograms of cellular protein, the contamination, if any, by poly-L-lysine released from the dish was below the concentration limit exhibiting inhibition. Using the above optimal conditions, sialidase activity was measured during cerebellar granule cell differentiation in culture. From day 0 to day 7-8 in culture, the enzyme activity rose from 20 to 130 nmol of product released/h/mg of protein with MUB-NeuNAc and from 1 to 100 nmol of product released/h/mg of protein with [3H]GD1a.(ABSTRACT TRUNCATED AT 250 WORDS)
Pitto
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M
Department of Medical Chemistry and Biochemistry, Medical School, University of Milan, Italy.
Chigorno
V
V
Giglioni
A
A
Valsecchi
M
M
Tettamanti
G
G
D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000201:enzymology*; D005677:G(M1) Ganglioside / Q000096:biosynthesis; D005732:Gangliosides / Q000378:metabolism; D006098:Granulocytes / Q000166:cytology / Q000201:enzymology*; D007700:Kinetics; D009439:Neuraminidase / Q000378:metabolism*; D011107:Polylysine / Q000494:pharmacology; D013997:Time Factors
D005732:Gangliosides; C021298:ganglioside, GD1a; D011107:Polylysine; D005677:G(M1) Ganglioside; D009439:Neuraminidase