Plasma membrane calcium-ATPase in cultured human retinal pigment epithelium.
The present work demonstrates the presence of plasma membrane (Ca(2+) + Mg2+)-ATPase (PMCA) activity in cultured human retinal pigment epithelium (HRPE). Whole-cell HRPE homogenates exhibited Ca(2+)-stimulated ATPase activity that was inhibited with high affinity (IC50 = 60 nM) by eosin, a potent inhibitor of the erythrocyte PMCA. This activity was not inhibited by thapsigargin, a selective inhibitor of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA). A PMCA-specific monoclonal antibody exhibited staining in attached HRPE monolayers. By Western blot analysis of SDS-PAGE separations of whole cell lysates of HRPE, this antibody identified a single band at approximately 145 kD. Finally, a Ca(2+)-dependent, La(3+)-augmented phosphoprotein, which comigrated with anti-PMCA immunoreactivity, was also detected. Taken together, these results show that cultured HRPE express PMCA activity.
Kennedy
B G
BG
Northwest Center for Medical Education, Indiana University School of Medicine, Gary 46408, USA.
Mangini
N J
NJ
D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000201:enzymology; D004801:Eosine Yellowish-(YS) / Q000494:pharmacology; D006801:Humans; D007124:Immunoenzyme Techniques; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation; D010857:Pigment Epithelium of Eye / Q000201:enzymology*; D019284:Thapsigargin / Q000494:pharmacology
D010042:Ouabain; D019284:Thapsigargin; D017301:Ca(2+) Mg(2+)-ATPase; D004801:Eosine Yellowish-(YS)