Binding and elution of EGTA to anion exchange columns: implications for study of (Ca+Mg)-ATPase inhibitors.
EGTA bound to DEAE-Sephadex and DEAE-cellulose at low ionic strength in the presence and absence of applied protein. It remained bound when the column was washed at low ionic strength, but was eluted as the ionic strength was increased. The amount of EGTA recovered at high ionic strength was 60 to 90% of that applied to the column. At the peak of its elution, the concentration of EGTA in the eluted fractions was 25 mM, over 10-fold higher than the concentration of EGTA applied to the column. Eluted fractions containing EGTA inhibited the (Ca+Mg)-ATPase by two mechanisms: (1) by chelating the Ca and (2) by affecting activity even when the free Ca was held constant. We suggest that at least some of the inhibitory effects previously attributed to a cytoplasmic inhibitor of the (Ca+Mg)-ATPase may in fact be due to contaminating amounts of EGTA.
Yingst
D R
DR
Department of Physiology, School of Medicine, Wayne State University, Detroit, MI 48201.
Barrett
V E
VE
D001798:Blood Proteins / Q000737:chemistry* / Q000302:isolation & purification; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors*; D002118:Calcium / Q000494:pharmacology; D002852:Chromatography, Ion Exchange / Q000379:methods; D004533:Egtazic Acid / Q000737:chemistry*; D004910:Erythrocyte Membrane / Q000201:enzymology; D006801:Humans
D001798:Blood Proteins; D004533:Egtazic Acid; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium