Gel chromatographic characterization of the hydrophobic interaction of glycosylphosphatidylinositol-alkaline phosphatase with detergents.
The interaction of a glycosylphosphatidylinositol (GPI) protein with different detergents was studied for the first time with a purified protein. Four differently hydrophobic fractions of GPI-alkaline phosphatase (GPI-AP) from calf intestine were used as model proteins. The mode of interaction was determined by investigating (i) the self-aggregation behaviour of the GPI-AP fractions, (ii) the interference of detergents with GPI-AP binding to octyl-Sepharose, and (iii) the elution of GPI-AP bound to octyl-Sepharose. It was shown that polyoxyethylene-type detergents surprisingly interact much stronger than n-octylglucoside with GPI-AP, which is in contrast to the known behaviour of GPI-proteins in natural membranes. Gel filtration chromatography of Triton X-100 at concentrations above the critical micellar concentration yields three different micelle species with apparent molecular weights of about 166, 54, and 16 kDa. GPI-AP fraction II, which is shown to bear only one anchor per dimer, does not bind to any of these micelles. We demonstrate that a complex is formed containing about 150 Triton X-100 molecules and about 4700 molecules of water per molecule of GPI-AP dimer. The experimental findings are in accordance with a simple geometrical model based on the physical data of fatty acids and the arrangement, mean size, and shape of Triton X-100 molecules.
Gehrhardt
S
S
Institute of Biochemistry, Medical Faculty, Friedrich Schiller University, Jena, Germany.
Blume
E
E
Cumme
G A
GA
Bublitz
R
R
Rhode
H
H
Horn
A
A
D000433:1-Propanol / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002850:Chromatography, Gel; D003902:Detergents / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000378:metabolism*; D007422:Intestines / Q000201:enzymology; D008024:Ligands; D008823:Micelles; D008956:Models, Chemical; D008970:Molecular Weight; D017830:Octoxynol / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014867:Water / Q000378:metabolism
D003902:Detergents; D005960:Glucosides; D017261:Glycosylphosphatidylinositols; D008024:Ligands; D008823:Micelles; D014867:Water; C018619:octyl-beta-D-glucoside; C027176:octyl-sepharose CL-4B; D017830:Octoxynol; D012685:Sepharose; D000433:1-Propanol; D000469:Alkaline Phosphatase