Kinetic properties of C 12E 8-solubilized (Na ++K +)-ATPase

Abstract

(1) The properties of the rectal gland (Na + + K +)-ATPase (ATP phosphohydrolase, EC 3.6.1.8) solubilized in octaethyleneglycol dodecylmonoether (C 12E 8) have been investigated. (2) The Kinetic properties of the solubilized enzyme resemble those of the membrane-bound enzyme to a large extent. The main difference is that K m for ATP for the (Na + + K +)-ATPase is about 30 μM for the solubilized enzyme and about 100 μM for the membrane-bound enzyme. (3) The Na +-form (E 1) and the K +-form (E 2) can also be distinguished in the solubilized enzyme, as seen from tryptic digestion, the intrinsic fluorescence and cosin fluorescence responses to Na + and K +. (4) 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 supernantant enzyme is about 3.8 nmol/mg, and is equal to the number of vanadate sites. (5) Inactivation of the enzyme by high concentrations of detergent can be shown to be related to the C 12E 8/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 C 12E 8 is required both for solubilization and inactivation. (6) It is observed that the commercially available detergent polyoxyethylene 10-lauryl ether is much less deleterious than C 12E 8, and its advantages in the assay of detergent-solubilized (Na + + K +)-ATPase are discussed. (7) The results show that (Na + + K +)-ATPase can be solubilized in C 12E 8 in an active form, and that most of the kinetic and conformational properties of the membrane-bound enzyme are conserved upon solubilization. C 12E 8-solubilized (Na + + K +)-ATPase is therefore a good model system for a solubilized membrane protein.