The long-term goal of my research is to understand the molecular mechanism of protein-catalyzed ion transport across biological membranes. One of the systems we have chosen for our study of iron transport is the band 3 protein of human red blood cell membranes. This protein catalyzes inorganic anion transport, and it is one of the few transport proteins in nature for which it is possible to study both function and molecular structure. The approach that we have taken is 1) to perform irreversible chemical or enzymatic modification of band 3 in the native membrane, 2) to make a detailed statement on the effects of that modification on substrate anion binding and translocation, and 3) to isolate band 3 or band 3 peptides under denaturing conditions to determine the nature and location of the modification in the primary structure. One segment of band 3 which we will study in detail is the 5,000 - dalton peptide which is produced by extracellular papain cleavage of band 3. This peptide is of functional significance because the papain cleavage inhibits outward anion translocation. We will also try to localize the site of covalent H2DIDS (4,4 feet-diisothiocyanodihydrostilbene-2,2 feet-disulfonate) binding in the primary structure of the C-terminal third of the band 3 molecule. Other studies will investigate sites of proteolytic cleavage at the cytoplasmic surface of the membrane-associated portion of band 3. Another erythrocyte anion transport protein which we will study in detail is that which transports lactate and pyruvate. We have recently provided evidence that a polypeptide of Mr 43,000 is associated with lactate and pyruvate transport, and we plan to isolate and reconstitute this protein in an artificial lipid membrane in order to establish conclusively whether or not the protein is in fact the catalyst of lactate transport. We will also raise antibodies to the Mr 43,000 polypeptide and determine whether or nor similar proteins exist in the plasma membranes of hepatocytes, skeletal muscle, and Ehrlich ascites tumor cells.

K04AM001137

1983-07-01

1988-06-30