Gangliosides modulate the activity of the plasma membrane Ca(2+)-ATPase from porcine brain synaptosomes.
We systematically examined the effects of gangliosides on the plasma membrane Ca(2+)-ATPase (PMCA) from porcine brain synaptosomes. Our results showed that GD1b (two sialic acid residues) stimulated the activity, GM1 (one sialic acid residue) slightly reduced the activity, while asialo-GM1 (no sialic acid residue) markedly inhibited it, suggesting that sialic acid residues of gangliosides are important in the modulation of the PMCA. We also examined the oligosaccharide effects by using GM1, GM2, and GM3 whose only difference was in the length of their oligosaccharide chain. GM1, GM2, and GM3 reduced the enzyme activities, whereas GM2 and GM3 were potent inhibitors. Gangliosides affect both affinity for Ca(2+) and the Vmax of enzyme. It was observed that GD1b and GM2 increased the affinity of the enzyme for Ca(2+). GD1b, GM2 affected the Vmax with an increase of GD1b, but decreases of GM2. The study of the affinity for ATP and the Vmax of enzyme in the presence of gangliosides showed that GD1b and GM2 had little effect on the ATP binding to the enzyme, but the Vmax was apparently changed. Moreover, the effects of gangliosides are additive to that of calmodulin, suggesting that the modulation of PMCA by gangliosides should be through a different mechanism. The conformational changes induced by gangliosides were probed by fluorescence quenching. We found that fluorescent quenchers (I(-) and Cs(+)) with opposite charges had different accessibility to the IAEDANS binding to the PMCA in the presence of gangliosides. An apparent red shift (25nm) with increased maximum of fluorescence spectrum was also observed in the presence of GD1b.
Zhao
Yongfang
Y
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Fan
Xiaoxuan
X
Yang
Fuyu
F
Zhang
Xujia
X
D000818:Animals; D001921:Brain / Q000201:enzymology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D002462:Cell Membrane / Q000201:enzymology*; D020536:Enzyme Activators / Q000737:chemistry; D005732:Gangliosides / Q000737:chemistry*; D013552:Swine; D013574:Synaptosomes / Q000201:enzymology*
D020536:Enzyme Activators; D005732:Gangliosides; D000252:Calcium-Transporting ATPases