K+-stimulated phosphatase

The enzyme also displayes a K stimulated phosphatase activity, which is an inherent part of the Na-K ATPase system and which hydrolyses substrates containing phosphate bonds of an energy intermediate between that of an acid anhydride and of an ester bond. The hydrolysis of / -nitrophenylphosphate has been investigated most intensively. Its hydrolysis is normally stimulated by intracellular K via the transition E. K E2.K [15]. The for for the phosphatase... 

Drastic delipidation, which inhibits K -stimulated phosphatase activity nearly completely, apparently also interferes with the ,.K 2- transition. Although the capacity for binding of ADP was not impaired and the affinity for ADP was only reduced by 50%, the antagonism between binding of K and ADP was lost and was restored upon lipid reactivation [123]. This may indicate subunit interaction in the ATP-driven transition of 2- lo i.K. The latter hypothesis is supported by the... 

Another consequence of partial lipid depletion is that the residual K -stimulated phosphatase is not inhibited by Na nor undergoes stimulation by Na" " -I-ATP at low K concentrations [117,124]. This has led to the suggestion that the K" -stimulated phosphatase activity in preparations containing a full lipid complement is a monomeric enzyme function by virtue of a K -induced dissociation of the dimer into monomers. Na would inhibit the phosphatase by inducing dimerization. In lipid-depleted preparations, having only one of the monomers saturated with lipid as a prerequisite for phosphatase activity to occur, Na would be unable to cause dimerization. Hence, it does neither inhibit the phosphatase activity nor stimulate Na-K ATPase activity for which dimerization is essential. As a consequence, the Na -dependent reactions (ATP-phosphorylation and the ADP-ATP exchange) would be expressions of the dimer and the -dependent reactions (K -stimulated dephosphorylation and phosphatase) expressions of the monomer. However, thimerosal, which would inhibit monomer interaction, does not inhibit any of these partial reactions, but increases the affinities for K" " [125,126] and lipid depletion reduces the Na -dependent phosphorylation less than the overall Na-K ATPase activity [122]. It is clear from these studies that an unequivocal answer as to the monomeric and dimeric nature of the partial reactions in the overall Na-K ATPase reaction and their lipid dependence must await determination of their functional molecular weights. 

Forte et al. [52] found a K -stimulated phosphatase activity, which in contrast to such an activity in many other tissues could not be inhibited by ouabain, indicating that the activity was not due to the (Na +K )-ATPase system. Later studies of this group showed first that the appearance of the phosphatase activity ontogeneti-cally coincided with the development of acid secretion [53]. Later they found that a K -stimulated ATPase activity was closely associated with the -stimulated phosphatase activity [54]. The K -stimulated ATPase activity was shown to be stimulated by K -sensitive ionophores and by membrane disruption procedures [55], After membrane disruption the ionophore had no further effect, suggesting that the ATPase activity is present in the membranes of vesicles with low K -permeability. The K -sensitive site of the enzyme would then have to be located at the inside of the vesicular membrane. 

Upon purification, the K -stimulated phosphatase activity is always copurified with the (K )-ATPase activity [63-65]. Mitochondrial markers, such as cytochrome c oxidase, succinate dehydrogenase, monoamino-oxidase, and the ribo-somal marker RNA are largely removed by the purification procedure. The same is true for the anion-sensitive ATPase and 5 nucleotidase activities, but some (Na — K )-ATPase activity is still present in highly purified (K" -I-H )-ATPase preparations. Purification is also characterised by a lowering of the K -insensitive Mg ATPase activity, but even in the purest preparations some Mg -ATPase activity (4% of (K -I- H )-ATPase activity) is still present. This may represent an impurity or an inherent property of the enzyme. 

Ray [79] has presented evidence that in the soluble supernatant fraction of rabbit fundic mucosal cells there is a heat-labile, non-dialysible, and protease-sensitive factor, which is able to activate the (K -h H )-ATPase and K -stimulated phosphatase activity of frog, rabbit and pig mucosal microsomes by a factor 1.5-3.5. Ca in a concentration above 20 juM abolishes the activation by this factor. Since the activity of the preparation used in this study is only 10-15% of the highest activity reported, further investigations on this activator are warranted. 

Saccomani et al. [85] treated a vesicular (K + H )-ATPase preparation from pig gastric mucosa with phospholipase Aj, resulting in a breakdown of 50% of the phospholipids. This treatment also results in partial loss of ATPase activity, but the residual activity is still 25% of the original activity. The K -stimulated phosphatase activity is not affected by this treatment. Schrijen et al. [86] used two phospholipases with shght difference in substrate specificity, alone and in combination. With each of these phospholipases approx. 50% of the phospholipids could be hydrolysed resulting in a 50% loss in enzyme activity. When the two phospholipases were used successively 70% of the phospholipids were hydrolysed and the loss of activity was also 70%. This represents a striking parallelism between residual phospholipid content and ATPase activity. In this case the K -stimulated phosphatase activity... 

Figure 19. Time course of inactivation of Na+-K+-stimulated ATP hydrolysis ( ) or K+-phosphatase (o) activity of the Na+-K+-ATPase enzyme treated with trypsin or chymotrypsin under carefully controlled conditions in NaCI or KCI media. In NaCI, chymotrypsin (CHY) cleaves at Leu266 (3), while trypsin (TRY) cleaves at Lys30 (2) and Arg262 (3). In KCI, trypsin cleaves at Arg438 (1) and Lys30 (2) in sequence, while there is no cleavage site exposed to chymotrypsin. Data from Jorgensen and Andersen, 1988.

Two molecules of the active intermediate of omeprazole bind to one active site of gastric H /K -ATPase [63, 64], This binding is a disulphide linkage and can be prevented and reversed by the addition of mercaptan [65-67]. Detailed investigations of three reactions of H /K -ATPase enzyme cycle have shown that the K -stimulated ATPase-activity, / -nitrophenol-phosphatase(pNPPase)-activity and formation of phosphoenzyme are also inhibited [63, 68]... 

The molar ratio of Ser-P to Thr-P is only 1.3 in LC20 of actomyosin but as high as 11 in K+-stimulated arteries (Mougios and Barany, 1986), or 6 in oxytocin-stimulated uteri (Csabina et al., 1987). Several factors may influence LC20 phosphorylation (a) the conformation of the protein (which determines the accessibility of protein kinases and phosphatases to the phosphorylatable residues) is most likely different in the muscle from that in the test tube (b) the concentration of the cofactors, for example, calmodulin and Ca2+, is variable in vitro but regulated in the muscle and (c) MLCP is virtually absent in the isolated systems, but it is a major factor in the muscle. 

Lipids may well be involved in the subunit interactions in the Na-K ATPase complex, since this would explain why the monomeric K -activated phosphatase is less inhibited upon delipidation and is reactivated at lower lipid concentrations than the overall Na-K ATPase activity [117,120]. This requirement of subunit interaction is supported by the finding that in the presence of detergents covalent cross-linking of a-subunits by Cu or Cu -phenanthroline is inhibited and is replaced by cross-linking of an a- to a )8-subunit [121], despite the very low SH-group content of the j8-subunit (Section 3a). In a lipid-depleted enzyme preparation the ATP-dependent phosphorylation level is reduced less than the overall Na-K ATPase activity [122]. Addition of K to the phosphorylated lipid-depleted enzyme did not stimulate Pj production, whereas addition of K to the lipid-reactivated preparation increased hydrolysis. This implies that the P 2 — P conformational transition is blocked in the lipid-depleted preparations, and that this is one of the steps in which subunit interaction is involved. 

As previously discovered [52], the enzyme shows a cation-stimulated phosphatase activity, the cation specificity of which is the same as that of the ATPase and which is copurified with the enzyme [63]. The specific activity of the phosphatase reaction is 60-80% of that of the (K + H )-ATPase activity [63,77,78]. This is much higher than for (Na + K )-ATPase, where the activity of the phosphatase activity is only 10-20% of the ATPase activity [1]. 

Transduction mechanism Inhibition of adenylyl cyclase stimulation of tyrosine phosphatase activity stimulation of MAP kinase activity activation of ERK inhibition of Ca2+ channel activation stimulation of Na+/H+ exchanger stimulation of AM PA/kainate glutamate channels Inhibition of forskol in-stimulated adenylyl cyclase activation of phos-phoinositide metabolism stimulation of tyrosine phosphatase activity inhibition of Ca2+ channel activation activation of K+ channel inhibition of AM PA/ kainate glutamate channels inhibition of MAP kinase activity inhibition of ERK stimulation of SHP-1 and SHP-2 Inhibition of adenylyl cyclase stimulation of phosphoinositide metabolism stimulation of tyrosine phosphatase activation of K+ channel inhibi-tion/stimulation of MAP kinase activity induction of p53 and Bax Inhibition of adenylyl cyclase stimulation of MAP kinase stimulation of p38 activation of tyrosine phosphatase stimulation of K+ channels and phospholipase A2 Inhibition of adenylyl cyclase activation/ inhibition of phosphoinositide metabolism inhibition of Ca2+ influx activation of K+ channels inhibition of MAP kinase stimulation of tyrosine phosphatase... 

As noted earlier, studies with inhibitors have been of great value. One mole of ouabain binds per enzyme complex and inhibits all enzyme functions. It provides a convenient marker for the extracellular surface of the enzyme. Oligomycin inhibits the (Na+, K+)-ATPase but not the K+-phosphatase reaction. It stimulates the ADP/ATP exchange reaction and this led to the postulate for two phosphoenzymes in the reaction scheme. Anomalous kinetic behaviour for (Na+, K+)-ATPase, over some years, was eventually recognized57 to be due to a vanadate impurity in ATP, which binds with high affinity to the low affinity ATP site and with low affinity to the high affinity ATP site. In accord with this, vanadate effectively inhibits the K+-phosphatase... 

Ueki, K. (1979). Stimulation of phosphatase release from cultured tobacco cells by divalent cations. Plant and Cell Physiology 20, 789-96. 

Krasnoperov VG, Beavis R, Chepumy OG et al (1996) The calcium-independent receptor of a-latrotoxin is not a neurexin. Biochem Biophys Res Commun 227 868-75 Krasnoperov VG, Bittner MA, Beavis R et al (1997) a-Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor. Neuron 18 925-37 Krasnoperov VG, Bittner MA, Mo W et al (2002b) Protein tyrosine phosphatase-G is a novel member of the functional family of a-latrotoxin receptors. J Biol Chem 277 35887-95 Kreienkamp HJ, Zitzer H, Gundelfinger ED et al (2000) The calcium-independent receptor for a-latrotoxin from human and rodent brains interacts with members of the ProSAP/SSTRIP/Shank family of multidomain proteins. J Biol Chem 275 32387-90 Lajus S, Lang J (2006) Splice variant 3, but not 2 of receptor protein-tyrosine phosphatase a can mediate stimulation of insulin-secretion by a-latrotoxin. J Cell Biochem 98 1552-9 Lajus S, Vacher P, Huber D et al (2006) a-Latrotoxin induces exocytosis by inhibition of voltage-dependent K+ channels and by stimulation of L-type Ca2+ channels via latrophilin in [5-cells. J Biol Chem 281 5522-31... 

Lamer, J., Huang, L.C., Schwartz, C.EW., Oswald, A.S., Shen, T.-Y., Kinter, M., Tang, G., and Zeller, K., 1988, Rat liver insulin mediator which stimulates pyruvate dehydrogenase phosphatase contains galatosamine and D-chiroinositol. Biochem. Biophys. Res. Commun. 151 1416-1426. 

S.R. Lee, K.S. Kwon, S.R. Kim and S.G. Rhee, Reversible inactivation of protein-tyrosine phosphatase IB in A431 cells stimulated with epidermal growth factory. Biol. Chem. 17i (1998)... 

H9. Hertz, R., Westfall, B. B., Barrett, M. K., and Tullner, W. W., The effect of ectopic autologous grafts of androgen-stimulated prostate upon the serum acid phosphatase of the dog. J. Nat. Cancer Inst. 10, 61-66 (1949). 

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