The project concerns the basic mechanism which governs the transport of water, urea and ethylene glycol across the human red cell membrane. The control of water and urea transport by the kidney is essential to life. The molecular details of these transport systems in the human red cell are not only important in themselves but may also provide clues to mechanisms used by the kidney and other tissues. A good deal of evidence supports the view that the anion transport protein, band 3, which comprises 25% of the red cell membrane proteins, provides the route for the passage of both water and urea; one major objective of the proposal is to show this definitively. Transport of both substances can be modulated by a specific extracellular SH group on band 3 which reacts with a mercurial SH reagent, pCMBS(p-chloromercuribenzene sulfonate). It is proposed to make a detailed study of the interaction of 14C-labelled pCMBS with external sites on band 3 and other membrane proteins to learn the total number of sites, where they are and how tightly pCMBS is bound. Another SH reagent, DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) binds to a different site on band 3 and modulates ethylene glycol transport; a detailed study of its binding to membrane proteins will be made. In order to determine whether water transport is mediated by band 3, purified band 3 will be prepared and reconstituted into lipid vesicles. Water flux will be measured either by 17O NMR or by light scattering methods to determine if pCMBS inhibitable water transport can be demonstrated in band 3 vesicles. Similar reconstitution techniques can be used to prepare band 4.5 and total integral membrane protein reconstituted vesicles for comparative purposes. Thiourea is a specific inhibitor of urea transport and betamercaptoethanol is a specific inhibitor of ethylene glycol transport. Studies will be made of urea and ethylene glycol permeation of reconstituted vesicles by the light scattering method. Addition of the specific inhibitors should make it possible to determine if band 3, or another membrane protein, mediates the transport of these nonelectrolytes.
