Alcoholism is known to produce clinically significant hematologic disorders including macrocytosis and sideroblastic anemia, but the molecular basis of these problems remains unclear. The major objective of this proposal is to employ recently developed membrane protein biochemical techniques in order to better understand the molecular pathology of red cell membrane damage in alcoholism. Potential long term clinical benefits from this study may include the development of laboratory tests to determine who among drinkers is developing specific forms of membrane pathology, the quantitation of which may guage the longitudinal severity an individual's alcohol consumption. Red cells from normal humans or lab rats will be compared to red cells from humans or lab rats suffering from excessive alcohol intake of a) acute or b) chronic duration or c) during recovery from a or b. Three basic lines of investigation will be followed: I. Evaluation of fatty acid acylations of the RH polypeptide and other major red cell acyl-proteins. Thioesterification and turnover of palmitic acid and other radiolabeled fatty acids onto free -SH groups will be assessed in an attempt to identify and quantitate specific functionally important red cell membrane protein sulfhydryl damage in alcoholism. The synthesis and stability of the Rh polypeptide itself will be assessed by immunological and physical analyses. II. A newly recognized Mr 28kDa red cell membrane channel protein will be evaluated in the different states of alcoholism. Synthesis and stability of 28kDa as well as possible alterations in the N-glycosylated subpopulation of 28kDa will be evaluated by immunological, chemical, physical and northern analyses. III. Potential defects in other red cell membrane proteins including spectrin, ankyrin, protein 4.1, and adducin will be assessed by high resolution gel electrophoresis, radioimmunoassays, protein binding assays, and membrane biophysical methods.
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