PHYSIOLOGICAL SIGNIFICANCE OF NA, K-PUMP ALPHA ISOFORMS

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DESCRIPTION: The Na, K ATPase exchanges intracellular Na for extracellular K, hydrolyzing ATP to provide the energy for pumping these ions against their concentration gradients.The enzyme consists of alpha and beta subunits where the subunit contains the binding sites for ATP and cardiac glycosides. The goal of the proposal is to use site directed mutagenesis followed by expression of mutants in cos-1 or Ltk(- ) cell lines to examine the effect of the differential processing of the amino terminal sequences on pump function and of the isoform specific region near the ATP binding site on the kinetic properties of the isoforms. There are three specific aims. The first is to test whether isoform- specific processing is necessary for pump function. To accomplish this aim, antibodies will be generated against the first eight amino acids of each isoform and, along with polyclonal antibodies specific for nonprocessed regions of the three isotypes, will be used to examine the processing of four amino terminal chimeras. The second aim is to use deletion mutagenesis of the a1 amino terminal region (but maintaining the first eight amino acids) to isolate the site of proteolytic cleavage assaying functional expression and immunoreactivity (to measure cleavage). The final aim is to construct chimeras in which the eleven amino acid subtype specific regions located near the ATP binding site are interchanged. Analysis of the kinetic properties of chimeras and W.T. isoforms will include V(max), pump abundance by backdoor phosphorylation, apparent affinities for K, Na and ATP and cation concentration dependence for active transport. Correspondence of the kinetic parameters of the chimera's with respect to those of either of the two W.T. ATPases will be examined to detect nonspecific structural effects of a given mutation. If an assignment can be made from the chimeras, point mutants will be constructed to try to identify single amino acids responsible for the observed difference(s) in properties. If constructs are nonfunctional, possible defects in mutation, assembly and targeting will be examined using the immunological methods outlined above. If there appears to be no role for this region in catalysis, chimeras of the Na, K ATPase and homologous regions of the H, K or Ca pumps will be constructed to confirm this conclusion.