In cystic fibrosis the symptoms of general obstruction of secretory organs and gastrointestinal tract, chronic progressive lung incapacitation and deranged transport of ions seem to be directly related to the modified glycoproteins and lipids in this disease. The projected studies are aimed to investigate the implications of the modified (overacylated with fatty acids) glycoproteins and the presence of ether lipid analogs in cystic fibrosis, to correlate the qualitative and quantitative results on lipids and glycoproteins with the pathological observations, to study the enzyme(s) involved in glycoprotein acylation (glycoprotein fatty acyl transferase(s)) and enzymes involved in regulation of the content of ether lipid analogs in the tissue (fatty acyl CoA reductase, fatty alcohol NAD+ oxidoreductase) and to correlate these findings with the health status of the patient. Further objectives of this proposal are to design a simple and reliable assay of fatty acyl transferase, or fatty acyl CoA reductase, or fatty alcohol NAD+ oxidoreductase using readily available specimens of the human tissue or fluids (skin biopsies, blood, amniotic fluid) which might be adopted for screening and for diagnostic purposes. The mucus glycoprotein will be isolated by combination of the organic solvent extraction, gel filtration and CsCl equilibrium density gradient centrifugation, and then analyzed for the content of covalently bound fatty acids. Using combination of chemical methods and proteases, the site(s) of fatty acid substitution will be determined. The glycoprotein, after release of fatty acids and deglycosylation, will be used as a substrate in the fatty acyl transferase(s) assay. The lipids isolated from samples prior to glycoprotein preparation will be separated into the individual components by means of thin layer chromatography, followed by analyses for the ether lipid analogs. Using palmitoyl (C14) CoA and (C14) hexadecanol the activity of fatty acyl transferase(s), fatty acyl CoA reductase, and fatty alcohol NAD+ oxidoreductase will be determined.
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