Binding of sodium ions and cardiotonic steroids to native and selectively trypsinized Na,K pump, detected by charge movements
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Binding of sodium ions and cardiotonic steroids to native and selectively trypsinized Na,K pump, detected by charge movements. / Schwappach, B; Stürmer, W; Apell, H J; Karlish, S J.
In: J BIOL CHEM, Vol. 269, No. 34, 26.08.1994, p. 21620-6.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Binding of sodium ions and cardiotonic steroids to native and selectively trypsinized Na,K pump, detected by charge movements
AU - Schwappach, B
AU - Stürmer, W
AU - Apell, H J
AU - Karlish, S J
PY - 1994/8/26
Y1 - 1994/8/26
N2 - A fluorescent dye, RH421, has been used to characterize charge movements associated with cation and cardiotonic steroid binding to Na,K-ATPase and to a specifically trypsinized preparation, so-called "19-kDa membranes." A fluorescence decrease induced by Na+ is attributed to electrogenic binding of one Na+ ion from the cytoplasm. The apparent affinity for Na+ is the same in both preparations. (ATP + Na + Mg) or (P(i) + Mg)-induced fluorescence signals observed with native enzyme are not observed in 19-kDa membranes, consistent with loss of ATP binding and phosphorylation. Cardiotonic steroids (CS) bind to native enzyme and 19-kDa membranes as judged by RH421 signals, fluorescence of anthroyl ouabain, and inhibition of Rb+ occlusion. Binding affinities to both preparations are in the micromolar range, and binding is prevented by the presence of Na+ or K+. The kinetics of glycone binding and dissociation are identical in both preparations, but aglycones bind and dissociate about 6-fold faster to 19-kDa membranes. Binding of Na+ and cardiotonic steroids is inactivated upon heating or extensive Pronase digestion of 19-kDa membranes. This suggests that cation and CS binding depend on the structural integrity of a complex of the proteolytic fragments, and that sites for both cations or CS consist of ligating groups located on more than one fragments of 19-kDa membranes.
AB - A fluorescent dye, RH421, has been used to characterize charge movements associated with cation and cardiotonic steroid binding to Na,K-ATPase and to a specifically trypsinized preparation, so-called "19-kDa membranes." A fluorescence decrease induced by Na+ is attributed to electrogenic binding of one Na+ ion from the cytoplasm. The apparent affinity for Na+ is the same in both preparations. (ATP + Na + Mg) or (P(i) + Mg)-induced fluorescence signals observed with native enzyme are not observed in 19-kDa membranes, consistent with loss of ATP binding and phosphorylation. Cardiotonic steroids (CS) bind to native enzyme and 19-kDa membranes as judged by RH421 signals, fluorescence of anthroyl ouabain, and inhibition of Rb+ occlusion. Binding affinities to both preparations are in the micromolar range, and binding is prevented by the presence of Na+ or K+. The kinetics of glycone binding and dissociation are identical in both preparations, but aglycones bind and dissociate about 6-fold faster to 19-kDa membranes. Binding of Na+ and cardiotonic steroids is inactivated upon heating or extensive Pronase digestion of 19-kDa membranes. This suggests that cation and CS binding depend on the structural integrity of a complex of the proteolytic fragments, and that sites for both cations or CS consist of ligating groups located on more than one fragments of 19-kDa membranes.
KW - Animals
KW - Cardiac Glycosides/metabolism
KW - Cations, Monovalent/metabolism
KW - Cell Membrane/metabolism
KW - Electricity
KW - Fluorescent Dyes/metabolism
KW - Kidney Medulla/metabolism
KW - Models, Biological
KW - Motion
KW - Peptide Fragments/metabolism
KW - Pyridinium Compounds/metabolism
KW - Rabbits
KW - Rubidium/metabolism
KW - Sodium/metabolism
KW - Sodium-Potassium-Exchanging ATPase/drug effects
KW - Spectrometry, Fluorescence
KW - Styrenes/metabolism
KW - Trypsin/pharmacology
M3 - SCORING: Journal article
C2 - 8063803
VL - 269
SP - 21620
EP - 21626
JO - J BIOL CHEM
JF - J BIOL CHEM
SN - 0021-9258
IS - 34
ER -