TOPOLOGY AND MECHANISMS BY SITE-DIRECTED IMMUNOCHEMISTRY

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DESCRIPTION: The covalent modification of particular amino acids in selected proteins is being used to examine structural and mechanistic questions. The structures of three proteins that span the bilayer and transport ions across the plasma membrane of animal cells, anion exchanger, Na/K-ATPase and acetylcholine receptor, are being probed in sealed systems with impermeant chemical reagents that react with particular types of animo acids on their surfaces. By assigning specific amino acids in the native protein to its cytoplasmic surface or its extracytoplasmic surface, the hydrophobic segments of the amino acid sequence of each protein that form the pathways along which the ions are transported across the membrane can be identified. Cysteines within the active site of the enzyme ribonucleotide reductase are also being examined. It has been proposed that during the chemical reaction catalyzed by this enzyme, these cysteines either become free radicals or combine with each other to form cystines. The rates at which and the sequence in which these transformations occur are being examined by rapidly mixing the enzyme with a ribonucleotide and quenching the reaction at short times. In all of these experiments, specific peptides are being isolated from the digested proteins by using antibodies that recognize characteristic sequences of amino acids in these peptides. These antibodies are able to pick out just the desired peptide containing the amino acid of interest from the complex mixture of peptides resulting from the digestion of each of these large proteins. In this way, efficient and rapid purifications are achieved. Each of the proteins being studied is responsible for an important biological function. Anion exchanger is responsible for transport of bicarbonate across the plasma membrane of the erythrocyte that permits the efficient transport of carbon dioxide from the peripheral tissues to the lungs. Na/K-ATPase creates the imbalances in ion concentration across the plasma membrane of the cell that provide the energy for nutrient uptake and the electrical activity of nervous tissue. Acetylcholine receptor creates the electrical signals that transform nerve impulses into the contractions of a muscle. Ribonucleotide reductase produces all of the deoxyribonucleotides needed for the duplication and repair of DNA in the cell.