For many years we have been interested in the synthesis and degradation of enterocyte proteins. This is a good system in which to study changes in these parameters, because this cell contains a number of specific proteins which have been purified to homogeneity and against which antibodies have been raised, and because the accumulation of many of these proteins respond to dietary or hormonal stimuli. Although we have successfully studied enterocyte protein degradation, we have been unable to investigate protein synthesis, due to the lack of a suitable cellular or organ culture system. For this reason we have chosen to look at synthesis at a more molecular level, concentrating on the translation of messenger RNA (mRNA) in the cell free system, and making the assumption that alterations in the content of mRNA will reflect synthetic capacity. We will use well characterized methods to determine mRNA content, primary structure of proteins, and processing of the primary translation product. Specific methods to be used will include identification of cell free translation products, microsequencing of primary translation products isolated by immune precipitation from cell free translation systems, protein sequencing of """"""""mature"""""""" proteins, co and post translational cleavage of polypeptides, and cDNA cloning using recombinant DNA techniques. Two classes of proteins will be studied: 1) cytosol proteins, especially fatty acid binding proteins in the enterocyte, and intrinsic factor in the stomach, because of their abundance, relatively small size, and availability of antibodcies. 2) Membrane proteins, especially alkaline phosphatase, because of its dual location on the membrane and in soluble form, and sucrase-isomaltase, because of its relative abundance, ease of isolation, and unusual structural properties. The special problems posed by the study of large proteins in cell free systems are recognized and wil be addressed.
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