A radiometric assay for ganglioside sialidase applied to the determination of the enzyme subcellular location in cultured human fibroblasts.
A radiometric method for the assay of ganglioside sialidase in cultured human fibroblasts was set up. As substrate, highly radioactive (1.28 Ci/mmol) ganglioside GDla isotopically tritium-labeled at carbon C-3 of the long chain base was employed; the liberated, and TLC separated [3H]GM1 was determined by computer-assisted radiochromatoscanning. Under experimental conditions that provided a low and quite acceptable (4-5%) coefficient of variation, the detection limit of the method was 0.1 nmol of liberated GM1, using as low as 10 micrograms of fibroblast homogenate as protein. The detection limit could be lowered to 0.02-0.03 nmol, adopting conditions that, however, carried a higher analytical error (coefficient of variation over 10%). The content of ganglioside sialidase in human fibroblasts cultured in 75-cm2 plastic flasks was 5.8 +/- 2.5 (SD) nmol liberated GM1 h-1 mg protein-1. Subfractionation studies performed on fibroblast homogenate showed that the ganglioside sialidase was mainly associated with the light membrane subfraction that was rich in plasma and intracellular membranes. This subfraction displayed almost no sialidase activity on the artificial substrate 4-methylumbelliferyl-D-N-acetylneuraminic acid. A small but measurable ganglioside sialidase activity was also present in the lysosome-enriched subfraction, which contained a very high sialidase activity on the above artificial substrate. All this supports the hypothesis that human fibroblasts contain sialidases with different subcellular location and substrate specificity. Particularly, the sialidase acting on gangliosides seems to have two sites of subcellular location, a major one at the level of plasma membranes and/or intracellular organelles functionally related with the plasma membranes and a minor one in the lysosomes.
Chigorno
V
V
Cardace
G
G
Pitto
M
M
Sonnino
S
S
Ghidoni
R
R
Tettamanti
G
G
D002478:Cells, Cultured; D003201:Computers; D005347:Fibroblasts / Q000201:enzymology*; D005732:Gangliosides; D006801:Humans; D009439:Neuraminidase / Q000032:analysis*; D011854:Radiochemistry; D011874:Radiometry / Q000379:methods*; D012867:Skin / Q000201:enzymology; D013347:Subcellular Fractions / Q000201:enzymology
D005732:Gangliosides; C021298:ganglioside, GD1a; C036052:GM3 ganglioside sialidase; D009439:Neuraminidase