Identification of a putative Na(+)-H+ exchanger regulatory cofactor in rabbit renal BBM.
Previous in vitro studies with detergent-solubilized rabbit renal brush-border membrane (BBM) proteins have suggested that adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA)-mediated inhibition of the Na(+)-H+ exchanger requires the presence of 42-kDa cofactor that is distinct from the exchanger itself. We sought to determine whether there was a protein in native rabbit renal BBM vesicles that has characteristics similar to that of the 42-kDa cofactor. Incubation of native BBM vesicle proteins with a hypotonic phosphorylation solution containing purified catalytic subunit of PKA resulted in phosphorylation of a number of BBM proteins, including a protein with an apparent molecular weight that was similar but not identical to that of the 42-kDa cofactor obtained from anion-exchange column chromatography of n-octyl glucoside-extracted BBM proteins. The identity between the BBM vesicle protein and the 42-kDa cofactor was established by phosphopeptide maps and radioiodinated peptide maps. These results indicate that native BBM vesicles contain a number of proteins that are phosphorylated by PKA when the PKA and ATP are present inside the vesicle space. One of these proteins appears to be identical to the 42-kDa protein that, as previously suggested by in vitro studies, acts as a regulatory cofactor mediating the inhibitory effect of PKA on the renal Na(+)-H+ exchanger.
Morell
G
G
Department of Internal Medicine, University of Texas Medical School, Houston 77025.
Steplock
D
D
Shenolikar
S
S
Weinman
E J
EJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D002352:Carrier Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D008274:Magnesium