Active transport of amino acids by liver tissue is an important site for regulation of hepatic amino acid and carbohydrate metabolism. Transport System A is unique because of the complex nature of its regulation. In addition to stimulation by hormones and growth factors, the hepatic System A activity is enhanced when the hepatocytes are starved for amino acids. This process, referred to as """"""""adaptive regulation"""""""", is thought to result from a derepression of a gene coding for a System A-associated glycoprotein. Most of the previous research concerned with the regulation of System A has been descriptive, involving whole-cell studies. The purpose of the research is to provide a sub-cellular approach to the study of adaptive regulation. The primary components of the proposal are as follows. 1) Definition of the substrate specificity of a possible apo-repressor molecule: Comparison of the substrate specificity for amino acid-dependent repression with that for transport will provide information about the binding sites on the molecules that mediate these two processes. 2) Characterization of the System A activity in plasma membrane vesicles prepared from hepatocytes incubated in amino acid-rich or amino acid-free medium: To investigate the nature of the membrane-bound protein complex that represents System A, the research will establish whether the starvation-induced activity is retained in isolated plasma membrane vesicles. 3) Reconstitution of the System A activity following detergent-solubilization of plasma membrane proteins: To study the proteins that are responsible for System A activity effectively, they must be purified. At the present time, reconstitution into proteoliposomes appears to be the best method for assay of the activity during purification. 4) Identification of the System A-associated protein(s): Purification of the System a complex could be aided by the prior identification of the individual protein(s). The research will use various labelling methods to identify the protein that contains the amino acid binding site and the putative glycoprotein that is induced by hormones and adaptive regulation. Whether these two """"""""activities"""""""" actually represent one protein is unknown.
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