Radiometric assay for ganglioside sialidase applied to the determination of the enzyme subcellular location in culture human fibroblasts

Journal Article · Sat Mar 01 00:00:00 EST 1986 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(86)90094-1· OSTI ID:5690699

Chigorno, V; Cardace, G; Pitto, M; Sonnino, S; Ghidoni, R; Tettamanti, G

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 GD/sub 1a/ isotopically tritium-labeled at carbon C-3 of the long chain base was employed; the liberated, and TLC separated (/sup 3/H)GM/sub 1/ 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 GM/sub 1/, using as low as 10 ..mu..g 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-cm/sup 2/ plastic flasks was 5.8 -/+ 2.5 (SD) nmol liberated GM/sub 1/ h/sup -1/ mg protein/sup -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.

• Research Organization: Univ. of Milan, Italy
• OSTI ID: 5690699
• Journal Information: Anal. Biochem.; (United States), Vol. 153:2
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
GANGLIOSIDES
GLYCOSYL HYDROLASES
RADIOMETRIC ANALYSIS
CELL CULTURES
CELL MEMBRANES
ENZYME ACTIVITY
EXPERIMENTAL DATA
FIBROBLASTS
HOMOGENATES
LYSOSOMES
QUANTITATIVE CHEMICAL ANALYSIS
RADIOCHROMATOGRAPHY
THIN-LAYER CHROMATOGRAPHY
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ANIMAL CELLS
CARBOHYDRATES
CELL CONSTITUENTS
CHEMICAL ANALYSIS
CHROMATOGRAPHY
CONNECTIVE TISSUE CELLS
DATA
ENZYMES
HYDROLASES
INFORMATION
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIPIDS
MEMBRANES
NUMERICAL DATA
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
SACCHARIDES
SEPARATION PROCESSES
SOMATIC CELLS
550201* - Biochemistry- Tracer Techniques