Abstract
A sialyltransferase activity which catalyzes the synthesis of the trisialoganglioside GT1a from added disialoganglioside TD1a and CMP-N-acetylneuraminic acid has been demonstrated using a particulate fraction of 9-day-old embryonic chick brains. The enzyme exhibited optimum activity with the detergent Triton CF-54 and showed a broad pH optimum of 6.0 to 7.2. Ca2+ inhibited the reaction, whereas Mn2+, Mg2+, and EDTA had no effect. Slight elevations in activity were seen in the presence of Hg2+ or histone. The apparent Km for GD1a leading to GT1a was estimated to be 10(-3) M. When the monosialoganglioside, GM1, was used as the glycolipid substrate under conditions optimum for the synthesis of GR1a from GD1a, approximately 65% of the radioactive label was found in GD1a. However, about 50% of the remaining radioactivity was found in GT1a. The results suggest that the synthesis of GR1a could proceed via the sequence GM1 yields GD1a yields GT1a.
Highlights
Inhibited the reaction,whereas Mn2+,M&+,and EDTA had no effect.Slight elevations in activity were seen in reagent grade and solvents were redistilled prior to use
MO; detergents Tween 20, Tween 80, and MYRJ59 were from Atlas Chemical Co., Wilmington, DE, and detergents Triton X-100 and Triton CF-54 were from Sigma.DEAE-Sephadex A-25and Sephadex G-50 wereobtained from Pharmacia Fine Chemicals, Piscataway, NJ
In one experiment,the final supernatant (70,000 X g for 90 min) was aliquot taken for total radioactivity determination, was taken to dryness by lyophilization which removed the acetic acid
Summary
Subcellular distribution ofprotein a n d Gn,-synthesizing activity relative to total homogenate. Relative specific activity is defined as counts permin in G.l-l./mg of protein/2 h/cpm in G,rl./mg of total homogenate protein/2 h. The individual ganglioside bands were located on the thin layer chromatogram by water spray. Areas corresponding to GI, and Grlh were scraped and the gangliosides eluted from the silica gel via sintered glass funnels using aliquots (6 X 1 rnl) of chloroform/methanol (1:4). The initial volume of buffer was determined to give a total incubation volume of 50 pl. The sample was taken up in methanol,and two-fifths of the sample was analyzed after thin layer chromatography as described above. Due to slight distortion by salt of the ganglioside chromatographic pattern, theremaining portion of the sample was desalted and analyzed again by thin layer chromatography. T h e desalting step separated free sialic acid from the remaining gangliosides
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