Protonation-dependent inactivation of Na,K-ATPase by hydrostatic pressure developed at high-speed centrifugation.
Irreversible inactivation of membranous Na,K-ATPase by high-speed centrifugation in dilute aqueous solutions depends markedly on the protonation state of the protein. Pig kidney Na,K-ATPase is irreversibly inactivated at pH 5 but is fully protected at pH 7 and above. Shark rectal gland Na,K-ATPase is irreversibly inactivated at neutral or acidic pH and partially protected at an alkaline pH. The overall Na,K-ATPase activity and the K-dependent pNPPase activity were denatured in parallel. Cryoprotectants such as glycerol or sucrose at concentrations of 25-30% fully protect both enzymes against inactivation. The specific ligands NaCl and KCl protect the Na,K-ATPase activity partially and the pNPPase activity fully at concentrations of 0.2-0.3 M. Electron microscope analysis of the centrifuged Na,K-ATPase membranes revealed that the ultrastructure of the native membranes is preserved upon inactivation. It was also observed that the sarcoplasmic reticulum Ca-ATPase and hog gastric H, K-ATPase are susceptible to inactivation by high-speed centrifugation in a pH-dependent fashion. H,K-ATPase is protected at alkaline pH, whereas Ca-ATPase is protected only in the neutral pH range.
Esmann
M
M
Department of Biophysics, University of Aarhus, Ole Worms Alle 185, DK-8000 Aarhus C, Denmark. me@biophys.au.dk
Fedosova
N U
NU
Maunsbach
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AB
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry; D004284:Dogfish; D004789:Enzyme Activation; D004795:Enzyme Stability; D005990:Glycerol; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006874:Hydrostatic Pressure; D007668:Kidney / Q000201:enzymology; D011189:Potassium Chloride; D011487:Protein Conformation; D011522:Protons; D012491:Salt Gland / Q000201:enzymology; D012965:Sodium Chloride; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000302:isolation & purification; D013395:Sucrose; D013552:Swine; D013997:Time Factors; D014461:Ultracentrifugation
D011522:Protons; D012965:Sodium Chloride; D013395:Sucrose; D011189:Potassium Chloride; D017506:H(+)-K(+)-Exchanging ATPase; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D005990:Glycerol