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)-α-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 μg with MUB-NeuNAc and up to 20 μg 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 μg 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 proteins with [³H]GD1a. The values of enzyme activity in differentiated granule cells are the highest ever reported for mammalian sialidases in isolated cells or tissue homogenates. In fully differentiated cells, the sialidase activity against endogenous substrates was 4.2 nmol of liberated N-acetylneuraminic acid/h/mg of protein. The marked increase of sialidase activity in cerebellar granule cells during the process of differentiation with formation of functional synapses suggests that sialidase enrichment is a marker for the same process.