AJP - Renal Physiology, Vol 259, Issue 6 867-F871, Copyright © 1990 by American Physiological Society
Identification of a putative Na(+)-H+ exchanger regulatory cofactor in rabbit renal BBM
G. Morell, D. Steplock, S. Shenolikar and E. J. Weinman
Department of Internal Medicine, University of Texas Medical School, Houston 77025.
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.
