Effects of Na+ and K+ on Artemia salina (Na,K)-ATPase solubilized with a zwitterionic detergent, CHAPS.
The membrane bound (Na,K)-ATPase prepared from Artemia salina nauplii was solubilized with a zwitterionic detergent, 3[3(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and then purified on a Bio-Gel A-1.5 m column in the presence of the detergent. 1) Upon solubilization, both NaCl and KCl protected the enzyme against loss of activity, KCl being more effective than NaCl. 2) Gel filtration of the solubilized enzyme on a Bio-Gel A-1.5 m column in the presence of 5 mM CHAPS resulted in loss of the enzyme activity even when one of the cations was added. Most of the phospholipids in the solubilized enzyme preparation were removed during the gel filtration (delipidation) and 10-25 phospholipids were left on a protomer (alpha beta) of the enzyme irrespective of the cation present during the gel filtration. With the addition of exogenous phospholipids, the activity was restored. The activity of the enzyme delipidated in the presence of KCl was restored to 3-4 times higher than in the case of that delipidated in the presence of NaCl. 3) Relipidation experiments with a fluorescent phospholipid, dansyl phosphatidylethanolamine (Dans-PE), suggested that the enzyme delipidated in the presence of KCl reassociated with phospholipids more firmly than the enzyme delipidated in the presence of NaCl. From these results we concluded that K+ stabilized the (Na,K)-ATPase more effectively than Na+, even when the enzyme was delipidated.
Morohashi
M
M
Department of Biology, University of Occupational and Environmental Health, Japan.
Konishi
K
K
Kawamura
M
M
D000818:Animals; D001156:Artemia / Q000201:enzymology*; D002793:Cholic Acids / Q000494:pharmacology*; D002850:Chromatography, Gel; D004793:Enzyme Reactivators / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology*; D012964:Sodium / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D012995:Solubility; D013050:Spectrometry, Fluorescence
D002793:Cholic Acids; D004793:Enzyme Reactivators; D010743:Phospholipids; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D011188:Potassium