It is proposed to study the physiological and biochemical mechanism of membrane transport using a comparative and evolutionary approach. We postulate that the earliest cell utilized a """"""""proton economy"""""""" (primary proton pumps generated a proton gradient which was used for proton-substrate cotransport and other processes). Later in evolution a """"""""sodium economy"""""""" developed and found its most sophisticated expression in animal cells. It is proposed to compare cotransport systems utilizing protons (lactose carrier of E. coli) with cotransport utilizing sodium ion (proline carrier of E. coli). In addition we plan to study a representative of the intermediate class of carriers that utilize either protons or sodium for cotransport (melibiose carrier of E. coli). Mutants will be isolated which show changes in sugar specificity or catiom recognition. We intend to clone the mutant gene for each of the transport proteins and determine the DNA sequence by the Sanger dideoxynucleotide method. A comparison between the altered amino acid sequence of the carrier protein and the altered physiology of transport will help to correlate three dimensional structure with function. A second project involves a study of the comparative physiology of cell volume regulation. As with animal cells microorganisms without cell walls (such as Mycoplasma gallisepticum) must continuously pump out the NaCl and water that diffuse into the cell as a result of colloid osmotic and Donnan forces. It is proposed to investigate the ionic mechanism for NaCl extrusion from M. gallisepticum and compare it with that used by animal cells.
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