Kinetic properties of C12E8-solubilized (Na+ + K+)-ATPase.
The properties of the rectal gland (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.8) solubilized in octaethyleneglycol dodecylmonoether ( C12E8 ) have been investigated. The kinetic properties of the solubilized enzyme resemble those of the membrane-bound enzyme to a large extent. The main difference is that Km for ATP for the (Na+ + K+)-ATPase is about 30 microM for the solubilized enzyme and about 100 microM for the membrane-bound enzyme. The Na+-form (E1) and the K+-form (E2) can also be distinguished in the solubilized enzyme, as seen from tryptic digestion, the intrinsic fluorescence and eosin fluorescence responses to Na+ and K+. The number of vanadate-binding sites is unchanged upon solubilization, and it is shown that vanadate binding is much more resistant to detergent inactivation than the enzymatic activities. The number of phosphorylation sites on the 95-100% pure supernatant enzyme is about 3.8 nmol/mg, and is equal to the number of vanadate sites. Inactivation of the enzyme by high concentrations of detergent can be shown to be related to the C12E8 /protein ratio, with a weight ratio of about 4 being a threshold for the onset of inactivation at low ionic strength. At high ionic strength, more C12E8 is required both for solubilization and inactivation. It is observed that the commercially available detergent polyoxyethylene 10-lauryl ether is much less deleterious than C12E8 , and its advantages in the assay of detergent-solubilized (Na+ + K+)-ATPase are discussed. The results show that (Na+ + K+)-ATPase can be solubilized in C12E8 in an active form, and that most of the kinetic and conformational properties of the membrane-bound enzyme are conserved upon solubilization. C12E8 -solubilized (Na+ + K+)-ATPase is therefore a good model system for a solubilized membrane protein.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents; D004284:Dogfish; D007700:Kinetics; D009994:Osmolar Concentration; D011092:Polyethylene Glycols; D012491:Salt Gland / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013816:Thermodynamics; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D003902:Detergents; D014639:Vanadium; C017272:dodecyloctaethyleneglycol monoether; D014638:Vanadates; D011092:Polyethylene Glycols; D000254:Sodium-Potassium-Exchanging ATPase