title: A calnaktin-like inhibitor of Na,K-ATPase in rat brain: regulation of alpha 1 and alpha 2 isozymes.
abstract: This study was designed to determine if a Ca(2+)-dependent, calnaktin-like inhibitor of Na,K-ATPase existed in rat brain and to compare the inhibition of different Na,K-ATPase isozymes in brain, heart and kidney. Based on the size and characteristics of human red blood cell calnaktin, a soluble protein fraction was obtained from rat brain and subjected to ultrafiltration and gel filtration to restrict the proteins to an appropriate molecular range of 6-50 kDa (6/50 fraction) for a crude calnaktin preparation. The 6/50 fraction was reconstituted with semipurified rat brain Na,K-ATPase and resulted in Ca(2+)-dependent inhibition of Na,K-ATPase activity. A 6/50 fraction was also prepared from rat heart ventricles, and, in its presence, Ca(2+)-dependent inhibition of cardiac Na,K-ATPase activity was observed. With brain preparations, the threshold for inhibition was approximately 100 nM free Ca2+, and inhibition was half maximal at 3-10 microM free Ca2+. Different isozymes of Na,K-ATPase were examined using differential sensitivity to ouabain and differential tissue distribution in brain, heart and kidney. The alpha 1 activity was inhibited in all three tissues. The alpha 2 activity of heart and the alpha 2 and/or alpha 3 activity of brain were also inhibited by the brain 6/50 fraction. In synaptosomal preparations from rat forebrain, resting intracellular (intrasynaptosomal) free Ca2+ was close to the threshold for calnaktin-like inhibition. The results are consistent with the presence of a calnaktin-like inhibitor of Na,K-ATPase in rat brain and indicate that calnaktin could be a widespread regulator of the alpha 1 isozyme. In addition, this study provides the first evidence that calnaktin also inhibits the alpha 2 activity of heart and the alpha 2 and/or alpha 3 isozymes of brain.
authors.affiliation: Department of Biochemistry, Microbiology and Molecular Genetics, University of Rhode Island, Kingston, USA.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: M
authors.initials: M
authors.lastname: Adachi
authors.forename: D E
authors.initials: DE
authors.lastname: Rhoads
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002540:Cerebral Cortex / Q000201:enzymology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000032:analysis*; D006801:Humans; D007527:Isoenzymes / Q000032:analysis*; D007668:Kidney / Q000201:enzymology; D008297:Male; D009206:Myocardium / Q000201:enzymology; D009928:Organ Specificity; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D013574:Synaptosomes / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium