Leucine 332 at the Boundary Between the Fourth Transmembrane Segment and the Cytoplasmic Domain of Na+,K+-ATPase Plays a Pivotal Role in the Ion Translocating Conformational Chan
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- 作者：Bente Vilsen
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：October 28, 1997
- 年：1997
- 卷：36
- 期：43
- 页码：13312 - 13324
- 全文大小：223K
- 年卷期：v.36,no.43(October 28, 1997)
- ISSN：1520-4995
文摘
Mutants Gly330
Ala, Leu332Ala, Leu332Pro, andPro780Ala of the 
1-isoform ofrat kidney Na+,K+-ATPase were expressed inCOS-1 cells to active site concentrations between 20 and70 pmol per mg of membrane protein. The functional properties ofthe mutants were characterized bytitrations of Na+-, K+-, andATP-dependencies of Na+,K+-ATPase activityas well as by a series of assaysmeasuring the K+-dependence of the steady-statephosphoenzyme level, the kinetics of dephosphorylationunder a variety of conditions, and the ADP-ATP exchange activity.In mutants Gly330Ala,Leu332Ala, and Leu332Pro, the molecular turnover number wasreduced relative to that of the wild-type Na+,K+-ATPase, and the steady-statephosphoenzyme level was high even in the presence ofseveralmillimolar K+. At a low Na+concentration in the absence of K+, mutants Leu332Proand Gly330Aladisplayed high ADP-ATP exchange activity and formed a high level ofthe ADP-sensitive phosphoenzyme(E1P). The phosphoenzyme decayed slowly following a jump in saltconcentration and chase with ATPand K+. Hence, the conversion of E1P to theK+-sensitive phosphoenzyme (E2P) was inhibited inmutantsLeu332Pro and Gly330Ala. In the Leu332Ala mutant, a highlevel of E2P was accumulated in theabsence of K+, and the ADP-ATP exchange activity waslow at low Na+ concentration in the absenceof K+, but rose sharply on addition of K+.Dephosphorylation experiments indicated that in theLeu332Ala mutant K+ induced reversal of thephosphoenzyme interconversion, forming E1P from E2P.Leu332 is therefore a pivotal residue in the conformationalchange. Mutants Gly330Ala and Pro780Aladisplayed reduced K+ affinities relative to the wildtype, determined in three independent assays.
Ala, Leu332Ala, Leu332Pro, andPro780Ala of the 1-isoform ofrat kidney Na+,K+-ATPase were expressed inCOS-1 cells to active site concentrations between 20 and70 pmol per mg of membrane protein. The functional properties ofthe mutants were characterized bytitrations of Na+-, K+-, andATP-dependencies of Na+,K+-ATPase activityas well as by a series of assaysmeasuring the K+-dependence of the steady-statephosphoenzyme level, the kinetics of dephosphorylationunder a variety of conditions, and the ADP-ATP exchange activity.In mutants Gly330Ala,Leu332Ala, and Leu332Pro, the molecular turnover number wasreduced relative to that of the wild-type Na+,K+-ATPase, and the steady-statephosphoenzyme level was high even in the presence ofseveralmillimolar K+. At a low Na+concentration in the absence of K+, mutants Leu332Proand Gly330Aladisplayed high ADP-ATP exchange activity and formed a high level ofthe ADP-sensitive phosphoenzyme(E1P). The phosphoenzyme decayed slowly following a jump in saltconcentration and chase with ATPand K+. Hence, the conversion of E1P to theK+-sensitive phosphoenzyme (E2P) was inhibited inmutantsLeu332Pro and Gly330Ala. In the Leu332Ala mutant, a highlevel of E2P was accumulated in theabsence of K+, and the ADP-ATP exchange activity waslow at low Na+ concentration in the absenceof K+, but rose sharply on addition of K+.Dephosphorylation experiments indicated that in theLeu332Ala mutant K+ induced reversal of thephosphoenzyme interconversion, forming E1P from E2P.Leu332 is therefore a pivotal residue in the conformationalchange. Mutants Gly330Ala and Pro780Aladisplayed reduced K+ affinities relative to the wildtype, determined in three independent assays.