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The fluorescent ATP derivative 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) adenosine 5'-triphosphate (TNP-ATP) binds specifically with enhanced fluorescence to the ATP site of purified eel electroplax sodium-potassium adenosine triphosphatase, (Na,K)-ATPase. A single homogeneous high affinity TNP-ATP binding site with a KD of 0.04 to 0.09 microM at 3 degrees C and 0.2 to 0.7 microM at 21 degrees-25 degrees C was observed in the absence of ligands when binding was measured by fluorescence titration or with [3H]TNP-ATP. ATP and other nucleotides competed with TNP-ATP for binding with KD values similar to those previously determined for binding to the ATP site. Binding stoichiometries determined from Scatchard plot intercepts gave one TNP-ATP site/175,000 g of protein (range: 1.64 X 10(5) to 1.92 X 10(5) when (Na,K)-ATPase protein was determined by quantitative amino acid analysis. The ratio of [3H]ouabain sites to TNP-ATP sites was 0.91. These results are inconsistent with "half-of-sites" binding and suggest that there is one ATP and one ouabain site/alpha beta protomer. (Na,K)-ATPase maintained a high affinity for TNP-ATP regardless of the ligands present. K+ increased the KD for TNP-ATP about 5-fold and Na+ reversed the effect of K+. The effects of Na+, K+, and mg2+ on ATP binding at 3 degrees C were studied fluorimetrically by displacement of TNP-ATP by ATP. The results are consistent with competition between ATP and TNP-ATP for binding at a single site regardless of the metallic ions present. The derived KD values for ATP were : no ligands, 1 microM; 20 mM NaCl, 3-4 microM; 20 mM KCl, 15-19 microM; 20 mM Kcl + 4 mM MgCl2, 70-120 microM. These results suggests that a single ATP site exhibits a high or low affinity for ATP depending on the ligands present, so that high and low affinity ATP sites observed kinetically are interconvertible and do not co-exist independently. We propose that during turnover the affinity for ATP changes more than 100-fold owing to the conformational changes associated with ion binding, translocation, and release.
The responses of the cytosolic pH of hepatocytes in suspension to agents affecting the activity of vacuolar adenosine triphosphatase (V-ATPase) and Na/H exchange have been studied. Changes of cytosolic pH were determined both with dual-wavelength excitation (500/440 nm) of the fluorescence of 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein and from the distribution of 14C-dimethyloxazolidinedione; both methods gave very similar results. Changes of vesicular pH were determined by comparing the fluorescence of fluorescein isothiocyanate-dextran and rhodamine B isothiocyanate-dextran taken up by endocytosis. Nitrate, which inhibits V-ATPase in isolated organelles, induced a concentration-dependent acidification of the cytosol and alkalinization of vesicles, with maximal effects at 25-37.5 mM in each case, indicating that V-ATPase contributes to removal of cytosolic protons. On continued exposure to nitrate, the acidification underwent an amiloride-inhibitable reversal. At the higher concentrations of NO3-, both cytosolic acidification and vesicular alkalinization were reduced or absent. Bafilomycin A1 caused alkalinization of vesicular pH; cytosolic acidification was not observed, possibly because of other ionic exchanges. Recovery of cytosolic pH from an acid load (2 min exposure to 5% CO2) was sensitive to both 25 mM NO3- and to ouabain. The pH dependence of the nitrate effect was tested with media of different pH; the activity was negligible at cytosolic pH 6.2 and rose to a maximum at cytosolic pH 7.3. Treatment of hepatocytes with 0.5-1.0 mM ouabain resulted in an initial alkalinization (0.5-2 min duration) of the cytosol, followed by a spontaneous reversal and, on occasion, further acidification. The alkalinization was blocked by 25 mM NO3-, but not by 25 mM gluconate. The results suggest that the cytosolic alkalinization is caused by a stimulation of H+ uptake by V-ATPase activity. We conclude that V-ATPase make an important contribution to the regulation of the cytosolic pH of hepatocytes.
The plasma membrane enzyme (Ca2+ + Mg2+)-adenosine triphosphatase (ATPase) is hormonally regulated and may participate in Ca2+ signaling by removing excess Ca2+ from the cell. Therefore, observations of a hormone-specific loss of insulin stimulation of ATPase in kidney membranes from non-insulin-dependent diabetic (NIDDM) rats may reflect their insulin-resistant state. Consequently, to evaluate whether additional insulin-resistant conditions are associated with impaired function of ATPase and with loss of regulation of the enzyme by insulin, studies were extended to investigate (Ca2+ + Mg2+)-ATPase activities and hormonal regulation of the enzyme in kidney basolateral membranes from obese and lean Zucker rats. (Ca2+ + Mg2+)-ATPase activity was lower in membranes from obese rats compared with lean rats. Maximal velocity (Vmax) of the enzyme activity was 29.2 +/- 2.6 nmol Pi/mg/min in obese rats versus 57.2 +/- 6.5 in lean rats (P < .05). However, the affinity of the enzyme for Ca2+ was similar in obese and lean rats (Km Ca2+, 0.23 +/- 0.025 v 0.23 +/- 0.032 mumol/L Ca2+). Also, the Km for ATP of the enzyme was similar in membranes from obese and lean rats. Insulin, parathyroid hormone (PTH), and cyclic adenosine monophosphate (cAMP) stimulated the ATPase activity in membranes from lean rats in a dose-dependent manner (15% to 28%). Also, the protein kinase C (PKC) stimulator 12-O-tetradecanoyl phorbol-13-acetate (TPA) increased the ATPase activity in membranes from lean rats.(ABSTRACT TRUNCATED AT 250 WORDS)
To determine a possible role of sodium-potassium adenosine triphosphatase (Na+, K+-ATPase) in the regulation of membrane functions in hypertension, we investigated the effects of ouabain on the membrane fluidity of erythrocytes in spontaneously hypertensive rats (SHR) by means of an electron paramagnetic resonance (EPR) and spin labeling method. Erythrocytes obtained from SHR were examined compared with age matched Wistar-Kyoto (WKY) rats, and the EPR spectra for 5-nitroxide stearate incorporated into the erythrocyte membranes were studied. The value of order parameter (S) of the EPR spectra was significantly higher in the erythrocytes of SHR than in the erythrocytes of WKY rats (S value: SHR, 0.721+/-0.009, n = 10; WKY, 0.652+/-0.008, n = 10; P < .05). The finding shows that the membrane fluidity of erythrocytes was lower in SHR than in WKY rats. Ouabain loading to erythrocytes significantly decreased the membrane fluidity (S value was increased) in both SHR and WKY rats. The ouabain induced change was significantly greater in SHR than in WKY rats. These results demonstrate that the membrane fluidity of erythrocytes might be highly dependent on the Na+, K+-ATPase activity in SHR, which would suggest an abnormality in Na+ related cellular functions in hypertension.
The interactions of gadolinium ion, lithium, and two substrate analogues, beta,gamma-imido-ATP (AMP-PNP) and tridentate CrATP, with the calcium ion transport adenosine triphosphatase (Ca2+-ATPase) of rabbit muscle sarcoplasmic reticulum have been examined by using 7Li+ NMR, water proton NMR, and Gd3+ EPR studies. Steady-state phosphorylation studies indicate that Gd3+ binds to the Ca2+ activator sites on the enzyme with an affinity which is approximately 10 times greater than that of Ca2+. 7Li+, which activates the Ca2+-ATPase in place of K+, has been found to be a suitable nucleus for probing the active sites of monovalent cation-requiring enzymes. 7Li+ nuclear relaxation studies demonstrate that the binding of Gd3+ ion to the two Ca2+ sites on Ca2+-ATPase increases the longitudinal relaxation rate (1/T1) of enzyme-bound Li+. The increase in 1/T1 was not observed in the absence of enzyme, indicating that the ATPase enhances the parmagnetic effect of Gd3+ on 1/T1 of 7Li+. Water proton relaxation studies also show that the ATPase binds Gd3+ at two tight-binding sites. Titrations of Gd3+ solutions with Ca2+-ATPase indicate that the tighter of the two Gd3+-binding sites (site 1) provides a ghigher enhancement of water relaxation than the other, weaker Gd3+ site (site 2) and also indicate that the average of the enhancements at the two sites is 7.4. These data, together with a titration of the ATPase with Gd3+ ion, yield enhancements, epsilonB, of 9.4 at site 1 and 5.4 at site 2. Analysis of the frequency dependence of 1/T1 of water indicates that the electron spin relaxation taus of Gd3+ is unusually long (2 X 10(-9) s) and suggests that the Ca2+-binding sites on the ATPase experience a reduced accessiblity of solvent water. This may indicate that the Ca2+ sites on the Ca2+-ATPase are buried or occluded within a cleft or channel in the enzyme. The analysis of the frequency dependence is also consistent with three exchangeable water protons on Gd3+ at site 1 and two fast exchanging water protons at site 2. Addition of the nonhydrolyzing substrate analogues, AMP-PNP and tridenate CrATP, to the enzyme-Gd3+ complex results in a decrease in the observed enhancement, with little change in the dipolar correlation time for Gd3+, consistent with a substrate-induced decrease in the number of fast-exchanging water protons on enzyme-bound Gd3+. From the effect of Gd3+ on 1/T1 of enzyme-bound Li+, Gd3+-Li+ separations of 7.0 and 9.1 A are calculated. On the assumption of a single Li+ site on the enzyme, these distances set an upper limit on the separation between Ca2+ sites on the enzyme of 16.1 A.
BACKGROUND: Helicobacter pylori is a major cause of gastritis, and the parietal cell H,K-adenosine triphosphatase (ATPase) is a major autoantigen in autoimmune atrophic corpus gastritis, which may eventually lead to pernicious anemia and/or neuropathy. Whether the bacterium induces the autoimmune response is unknown. METHODS: By means of enzyme-linked immunosorbent assay the occurrence of antibodies against porcine H,K-ATPase and H. pylori was determined in sera from 30 patients with pernicious anemia. RESULTS: All sera scored positive against H,K-ATPase, and 25 (83%) scored positive against H. pylori. The titers of antibodies against both antigen preparations inversely correlated with the duration of disease. A possible common epitope in the antigen preparations was tested with a competition assay. There was no indication of a common epitope in either human or porcine H,K-ATPase and H. pylori. CONCLUSIONS: There was a positive correlation and a high incidence of antibodies against H,K-ATPase and H. pylori in sera from patients with pernicious anemia. These antibodies recognized different epitopes.
These in vitro studies of golden hamster sperm were undertaken to determine whether: Na+, K+-adenosine triphosphatase (ATPase) activity is required for capacitation; Na+, K+-ATPase activity is altered during capacitation; and cyclic nucleotides can control this enzyme activity. Hamster sperm were incubated in a medium in which capacitation occurred in an asynchronous manner and in which acrosome reactions began to occur after approximately 3.5 h of incubation. Inhibition of the hamster sperm acrosome reaction by the Na+, K+-ATPase inhibitor ouabain (1 microM) added at Time (T) = 2 or T = 3 h could be fully reversed by the addition of the ionophore nigericin (0.1 microM) at T = 3.5 h. However, when ouabain was added at T = 0 or T = 1 h, similar nigericin addition could not completely reverse the inhibition. Na+, K+-ATPase activity of hamster sperm increased by 2 h of incubation (compared to that measured initially after 15 min) and this activity remained elevated at 3.5 h. Addition of either monobutyryl cyclic adenosine 3':5'-monophosphate ( BtcAMP ) (12.9 microM) or monobutyryl cyclic guanosine monophosphate ( BtcGMP ) (10.5 microM), or the phosphodiesterase inhibitor SQ20009 (10 microM) at 2 h produced a stimulation of acrosome reactions at 4 and 5 h. However, while BtcGMP and SQ 20009 also induced a further increase in Na+, K+-ATPase activity measured at 3.5 h, BtcAMP had no effect. Intracellular cAMP and cGMP levels measured showed cAMP increased by 2 h and remained elevated when measured at 3.5 h, while cGMP could not be consistently detected at 15 min, 2 h or 3.5 h. However, assays of high numbers of uncapacitated sperm did detect a low level of cGMP. These results suggest that Na+, K+-ATPase activity increases in and is essential for early capacitation [and thereby eventually for the acrosome reaction (AR)] of hamster sperm and that the increase in Na+, K+-ATPase activity occurring during capacitation is probably mediated by intracellular cGMP but not cAMP, although both cyclic nucleotides stimulate the hamster sperm AR.
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