4'-Amino-benzamido-taurocholic acid selectively solubilizes glycosyl-phosphatidylinositol-anchored membrane proteins and improves lipolytic cleavage of their membrane anchors by specific phospholipases.
Glycosyl-phosphatidylinositol-anchored membrane proteins (GPI-proteins) are normally identified either by cleavage of the lipid anchor using (glycosyl)phosphatidylinositol-specific phospholipases C or D (GPI-PLs) or by metabolic labeling of the lipid moiety with specific building blocks. Therefore, methods for discrimination between transmembrane proteins and GPI-proteins on the basis of their physicochemical properties are desirable. Here we are presenting a selective extraction method for typical well-characterized mammalian GPI-proteins, e.g., acetylcholine esterase, alkaline phosphatase, 5'-nucleotidase, and lipoprotein lipase, using a derivative of taurocholate. The results were compared to those obtained with well-characterized transmembrane proteins, e.g., insulin receptor and hydroxymethyl glutaryl coenzyme A-reductase, glucose transporters, or aminopeptidase M and several commercially available detergents. With regard to total membrane proteins, it was possible to selectively enrich GPI-proteins up to 8- to 14-fold by using concentrations between 0.1 and 0.3% of 4'-NH2-amino-7 beta-benzamido-taurocholic acid (BATC). In addition, the cleavage specificity and efficiency of (G)PI-PLs were increased in the presence of identical concentrations of BATC compared to commonly used detergents, e.g., Nonidet P-40. Therefore, the present study shows that the use of BATC facilitates the identification of glycosyl-phosphatidylinositol-anchored membrane proteins.
Müller
G
G
Hoechst AG Frankfurt a.M., Germany.
Korndörfer
A
A
Saar
K
K
Karbe-Thönges
B
B
Fasold
H
H
Müllner
S
S
D015720:5'-Nucleotidase / Q000032:analysis / Q000378:metabolism; D000110:Acetylcholinesterase / Q000032:analysis / Q000378:metabolism; D017667:Adipocytes / Q000737:chemistry; D000469:Alkaline Phosphatase / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D017261:Glycosylphosphatidylinositols / Q000032:analysis / Q000378:metabolism*; D007163:Immunosorbent Techniques; D008066:Lipolysis; D008071:Lipoprotein Lipase / Q000032:analysis / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000032:analysis / Q000378:metabolism; D009124:Muscle Proteins; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility; D013656:Taurocholic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology
C086340:4'-amino-7-benzamidotaurocholic acid; D003902:Detergents; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495694:Slc2a4 protein, rat; D013656:Taurocholic Acid; D008071:Lipoprotein Lipase; D000110:Acetylcholinesterase; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase