PMID: 9187239May 23, 1997Paper

Characterization of the affinity of the G(M2) activator protein for glycolipids by a fluorescence dequenching assay

Biochimica Et Biophysica Acta
N Smiljanic-GeorgijevDon J Mahuran

Abstract

The G(M2) activator protein is a substrate specific cofactor for degradation of G(M2) ganglioside by lysosomal beta-hexosaminidase A. Mutations in the gene encoding the activator result in the AB-variant form of G(M2) gangliosidosis. The activator protein contains at least three functional elements; a hydrophobic binding pocket, an oligosaccharide binding site(s), and an area that interacts with hexosaminidase A. In this report a fluorescence dequenching assay specific for only the hydrophobic binding pocket is evaluated and optimized. It is shown that various glycolipids inhibit the transport between liposomes of a self-quenching fluorescent lipid probe, octadecylrhodamine, by the activator protein. The level of inhibition produced by each glycolipid is then used to characterize the oligosaccharide-binding specificity of the activator. The fluorescence dequenching assay is also used to evaluate the functionality of a truncated form of the activator protein. Our results indicate that this simple assay can be used to determine structure-function relationships within the normal or mutant forms of the activator. The data suggest that the C-terminus of the activator is required to produce a functional hydrophobic binding pocket.

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Citations

May 6, 1998·Protein Science : a Publication of the Protein Society·C G SchütteK Sandhoff
Oct 4, 2005·Acta Neuropathologica·Douglas R MartinHenry J Baker
Jan 24, 2007·The Journal of Biological Chemistry·Gustavo H B MaegawaDon J Mahuran
Jun 14, 2003·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·Konrad Sandhoff, Thomas Kolter
Jun 24, 2003·The American Journal of Pathology·Cecily P Vaughn, Kojo S J Elenitoba-Johnson
Jun 3, 2009·Biochemical and Biophysical Research Communications·Brigitte RigatDon Mahuran
Aug 13, 2008·Analytical Biochemistry·Yong Ran, Gail E Fanucci
Oct 16, 2004·Biochemical and Biophysical Research Communications·Maryam ZarghooniDon Mahuran
Nov 26, 1999·Biochimica Et Biophysica Acta·D J Mahuran
May 18, 1999·Biochemical and Biophysical Research Communications·B RigatD Mahuran
Aug 14, 2015·Journal of Lipid Research·Günter SchwarzmannKonrad Sandhoff
Feb 11, 2003·Journal of Lipid Research·Yoshimi ShimadaSu-Chen Li
Dec 29, 2006·Journal of Atherosclerosis and Thrombosis·Hidekatsu YanaiHitoshi Chiba
Nov 25, 2000·Journal of Molecular Biology·C S WrightF Rastinejad
Aug 12, 2003·Journal of Molecular Biology·Christine Schubert WrightFraydoon Rastinejad
Mar 30, 2006·Molecular and Biochemical Parasitology·Alexandra F BruceKleoniki Gounaris

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