Protein Na+/K+ ATPase

Much of current interest in vanadium stems from the discovery that vanadate (HV042 at pH 7) is a powerful inhibitor of ATPases such as the sodium pump protein (Na+ + K+)ATPase (Chapter 8), of phosphatases,623 and of kinases.624 This can be readily understood from comparison of the structure of phosphate and vanadate ions. 

Staining Applications Atria cardiac tissues cardiac myocytes neurons ° proteins Na" /K -ATPase ... 

FIGURE 10.8 A schematic diagram of the Na, K -ATPase in mammalian plasma membrane. ATP hydrolysis occurs on the cytoplasmic side of the membrane, Na ions are transported out of the cell, and ions are transported in. The transport stoichiometry is 3 Na out and 2 in per ATP hydrolyzed. The specific inhibitor ouabain (Figure 7.12) and other cardiac glycosides inhibit Na, K -ATPase by binding on the extracellular surface of the pump protein. 

FIGURE 10.13 Some of the sequence homologies in the nucleotide binding and phosphorylation domains of Na, K -ATPase, Ca -ATPase, and gastric H, K -ATPase. (Adapted from j0rgensm, P. L., and Andersen, J. R, 1988. Structnral basis for Ei - E2 confoyinational transitions in Na, K -pnmp and Cc -pnmp proteins. Journal of Membrane Biology 103 95-120)... 

ATP certainly fulfils the criteria for a NT. It is mostly synthesised by mitochondrial oxidative phosphorylation using glucose taken up by the nerve terminal. Much of that ATP is, of course, required to help maintain Na+/K+ ATPase activity and the resting membrane potential as well as a Ca +ATPase, protein kinases and the vesicular binding and release of various NTs. But that leaves some for release as a NT. This has been shown in many peripheral tissues and organs with sympathetic and parasympathetic innervation as well as in brain slices, synaptosomes and from in vivo studies with microdialysis and the cortical cup. There is also evidence that in sympathetically innervated tissue some extracellular ATP originates from the activated postsynaptic cell. While most of the released ATP comes from vesicles containing other NTs, some... 

The procedure for purification of Na,K-ATPase in membrane-bound form from the outer renal medulla of mammalian kidney offers the opportunity of exploring the structure of the Na,K-pump proteins in their native membrane environment. The protein remains embedded in the membrane bilayer throughout the purification procedure thus maintaining the asymmetric orientation of the protein in the baso-lateral membrane of the kidney cell in the purified preparation. This preparation has been particularly useful in studies of ultrastructure, protein conformation and for... 

In an ideal pure preparation of Na,K-ATPase from outer renal medulla, the al subunit forms 65 70% of the total protein and the molar ratio of a to is 1 1, corresponding to a mass ratio of about 3 1 [1,5]. Functionally the preparation should be fully active in the sense that each a/ unit binds ATP, Pj, cations and the inhibitors vanadate and ouabain. The molecular activity should be close to a maximum value of 7 000-8 000 Pj/min. The highest reported binding capacities for ATP and phosphate are in the range 5-6 nmol/mg protein and close to one ligand per otjS unit [29], when fractions with maximum specific activities of Na,K-ATPase [40 50 pmo Pj/min mg protein) are selected for assay. 

Low resolution models (20-30 A) based on diffraction analysis of membrane crystals of Na,K-ATPase [34,35,39] and Ca-ATPase [40,41] show that the cytoplasmic protrusions of the proteins are remarkably similar. A notable difference is a 10-20 A... 

Proteolytic cleavage has proven to be an efficient tool for exploring the structure and function of the Na,K-ATPase. Exposure and protection of bonds on the surface of the cytoplasmic protrusion provides unequivocal evidence for structural changes in the a subunit accompanying E1-E2 transition in Na,K-ATPase [52]. Localization of the proteolytic splits provided a shortcut to identification of residues involved in E1-E2 transition [33,53,54] and to detection of structure-function correlations [33]. Further proteolysis identifies segments at the surface of the protein and as the cytoplasmic protrusion is shaved off all ATP-dependent reactions are abolished. 

Labelling Na,K-ATPase with ATP analogues provides evidence for contribution from charged residues that are widely separated in the sequence of a subunit of Na,K-ATPase. The first indication came from ATP sensitive covalent insertion of fluorescein-isothiocyanate (FITC) into Lys ° in the a subunit [90], The strong fluorescence signal provides a convenient probe for monitoring conformational transitions in the proteins. Site-directed mutagenesis of Lys reduces the activity of... 

Transition from the high-energy phosphoform E]P[3Na] to the K-sensitive E2P[2Na] of Na,K-ATPase are accompanied by conformational transitions in protein structure and changes of the capacity and orientation of cation sites. In the Ej form of Na,K-ATPase, the exposure of Chys (Leu ) and Trys (Arg ) to cleavage reflects that the cation sites of the phosphoprotein are in a conformation oriented towards the cytoplasm with a capacity for occlusion of three Na ions. The E2 form... 

Definition of Ej and E2 eonformations of the a subunit of Na,K-ATPase involves identification of cleavage points in the protein as well as association of cleavage with different rates of inactivation of Na,K-ATPase and K-phosphatase activities [104,105]. In the Ei form of Na,K-ATPase the cleavage patterns of the two serine proteases are clearly distinct. Chymotrypsin cleaves at Leu (C3), Fig. 3A, and both Na,K-ATPase and K-phosphatase are inactivated in a monoexponential pattern [33,106]. Trypsin cleaves the E form rapidly at Lys ° (T2) and more slowly at Arg (T3) to produce the characteristie biphasic pattern of inactivation. Localization of these splits was determined by sequencing N-termini of fragments after isolation on high resolution gel filtration columns [107]. 

Hall et al. [62] identified in a separate study the same glycoprotein in H,K-ATPase vesicles isolated from porcine gastric mucosa. A stoichiometric ratio of 1.2 1.0 was found for the deglycosylated protein (35 kDa)/catalytic 94-kDa protein. Furthermore, compelling evidence that this glycoprotein is the H,K-ATPase p subunit was provided by N-terminal sequence analysis of three protease V8-obtained peptides of the 35-kDa core protein. These peptides showed 30% and 45% homology with the Na,K-ATPase pi and pi subunit, respectively. 

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