Abstract
Concanavalin A (Con A) affinity chromatography is the standard procedure to separate cholesterol nucleating biliary proteins from lipids and pigment. We observed that even after extensive washing following application of bile, lipid contaminants remain. We have determined the contribution of lipid contamination to cholesterol nucleation and assessed a modified procedure to remove lipids from the column. Human gallbladder bile was spiked with [3H]cholesterol and [14C]phospholipid and applied to two sets of Con A-Sepharose columns. One column was washed in the usual manner with Tris-HCl buffer and the other with buffer containing 10 mM taurocholate prior to eluting bound glycoproteins with alpha-D-methylmannopyranoside. Eluted proteins were added to heated abnormal bile at a final concentration of 250 micrograms/ml to study the effect on cholesterol nucleation. A separate aliquot (20 microliter) of the protein solutions was counted for radioactivity. Cholesterol nucleating activity was less in samples from columns washed with 10 mM taurocholate than in samples from columns not washed with the bile salt. Lipid radioactivity was associated with Con A-binding proteins prepared without taurocholate, but not in those prepared with taurocholate wash. Light microscopy revealed the presence of cholesterol microcrystals and vesicles in some Con A-binding protein preparations prepared without a taurocholate wash. However, pellets from ultracentrifuged Con A preparations prepared without a bile salt wash revealed cholesterol crystals in all samples (n = 6). Washing with taurocholate did not affect recovery of protein mass and appearance of bands on SDS-PAGE gel showed an identical pattern in the two groups. This modified procedure to prepare potential nucleating proteins from gallbladder bile should eliminate erroneous results that may arise due to lipid contamination.
Highlights
Concanavalin A (Con A) affinity chromatography is the standard procedure to separate cholesterol nucleating biliary proteins from lipids and pigment
We studied the effect of ultracentrifugation of Con A preparations prepared by standard technique on cholesterol nucleation as, our hypothesis was that cholesterol microcrystals contaminate Con A glycoproteins prepared by the standard procedure
Eluates were examined by light microscopy using polarizing filters to determine whether they contained particulate lipids such as vesicles or cholesterol microcrystals
Summary
Concanavalin A (Con A) affinity chromatography is the standard procedure to separate cholesterol nucleating biliary proteins from lipids and pigment. Washing with taurocholate did not affect recovery of protein mass and appearance of bands on SDS-PAGEgel showed an identical pattern in the two groups This modified procedure to prepare potential nucleating proteins from gallbladder bile should eliminate erroneous results that may arise due to lipid contamination.-Harvey, P. Nucleating proteins appear to be responsible for the rapid nucleation of cholesterol from bile of gallstone patients Many of these potential pronucleating proteins have been isolated using concanavalin A lectin affinity chromatography that separates biliary glycoproteins from biliary lipids and pigments. We observed that even after extensive washing following the binding of biliary proteins to concanavalin A, lipid contaminants still remain These contaminants could affect the nucleating activity of putative pronucleating proteins. The purpose of this study was to investigate the contribution of lipid contamination to cholesterol nucleation and to compare the standard isolation method to a modified one that completely removes all the lipids from the isolated biliary proteins
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Published Version
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