The rate of cholesterol nucleation and crystal growth from metastably supersaturate model and native human gallbladder biles has been shown to importantly affected by factors present in biliary proteins. These kinetic factors are both inhibiting and promoting in activity. Our recent studies show that they are both glycoproteins, a well-defined 63 kDa inhibitor and most likely @ 140 kDa promotor. These glycoproteins have been found in different lectin chromatography-bound fractions with differing sugar specificities. We have developed a sensitive and reproducible new spectrophotometric assay system that facilitates not only their detection but permits estimates of their relative potency. Using this assay, we have found that both glycoproteins are present in normal biles. This suggests that in Health a balance of the kinetic factors is present and that an undefined imbalance may play a significant role in cholesterol gallstone pathogenesis. Given our present findings, with progress in coming months we expect to complete the Isolation and partial characterization of the two glycoproteins with optimal """"""""scaling-up"""""""" of production. The following logical objectives have therefore been established. Firstly, we wish to accomplish quantitation of the two effector glycoproteins. Radioimmunoassay (RIA) (and related immunoblotting for final purity assessment) seems the only reasonable methodologic approach. Secondly, a biophysicochemical characterization will be undertaken. This will consist of an assessment of the magnitude and functional importance of the sugar- moieties, followed by amino acid analysis of both effector glycoproteins. Thirdly,, their individual mechanism(s) of action will be examined. This will be done by Quasi-elastic light-scattering (QELS) analysis of the crystal growth process to assess the normal pattern as altered by the glycoproteins, especially the inhibitor. Analysis of the spectrophotometric crystal growth curve will also be undertaken to define quantifiable parameters. For study of mechanistic aspects of the promotor glycoprotein,k we propose to use Differential Scanning Calorimetry, QELS and related techniques to assess fusogenic vesicle protein-lipid interactions. Comparative surface activity measurements will also be made. Lastly, we propose to use the previously-developed quantitation methods (RIA) to determine the source(s) and natural distribution of the effector glycoprotein.
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