Membrane (Na+ + K+)-ATPase of canine brain, heart and kidney. Tissue-dependent differences in kinetic properties and the influence of purification procedures (original) (raw)
Related papers
Biochemical Pharmacology, 1980
We have recently reported that the heart (Na+ + K+)ATPase occurred on high 7 specific binding and low affinity of r3H10uabain to human binding sites. The dissociation constant (KD) of i'H]ouabain low affinity binding sites was close to ouabain K i, an indication that these sites are involved in the inhibition of this enzyme by the glycoside. In guinea-pig heart microsomes, only one group of [3Hlouabain binding sites were identified and the KD of these sites was close to ouabain K i (1). In intact guinea-pig heart, it was previously shown that r3H]ouabain interacts withtwo groups of specific binding sites and that the proportion of the high affinity sites is increased by increasing extracellular K + (2,3).
FACTORS AND AGENTS THAT INFLUENCE CARDIAC GLYCOSIDE‐Na+, K+‐ATPase INTERACTION*
Annals of the New York Academy of Sciences, 1974
The optimal conditions for the binding of cardiac glycosides, such as ouabain, to Na+, K+-ATPase in vitro are the simultaneous presence of ATP, Na+ and Mg++ or the presence of inorganic phosphate (Pi) and Mg++ (Reference 1). It has been postulated that in vitro binding of ouabain in the presence of ATP, Na+ and Mg++ reflects the binding of cardiac glycosides t o this enzyme system in intact animals,2 and it is this interaction that ultimately results in the production of the positive inotropic and/or arrhythmic effects3 Thus, studies on the factors and agents that influence the cardiac glycoside-Na', K+-ATPase interaction in vitro appear important for an understanding of the factors and agents that modify the magnitude of response t o cardiac glycosides in patients when the plasma concentrations of cardiac glycosides are maintained at a fixed level Moreover, such studies may shed light on the mechanism of the Na' , K+-ATPase reaction itself. Monovalent Cations Na+, K+-ATPase has been shown t o be an allosteric enzyme and its configurations are determined by monovalent cation and phosphate ligand~.~-' Therefore, it is reasonable t o assume that these ligands affect the ouabain-enzyme interactions. The binding of cardiac glycosides t o Na+, K+-ATPase in vitro in the presence of ATP, Na+ and Mg++ is markedly inhibited by K+ (Reference 1). Since K+ has been shown t o reverse cardiac glycoside-induced arrhythmias,l2> it was once assumed that K+ reduces the level of cardiac glycoside bound t o the Na+, K+-ATPase, presumably by reducing the rate of binding, increasing the rate of dissociation, or both. Supporting this hypothesis, a number of investigators has reported that K+ antagonized the ouabain-inhibition of the Na+, K+-ATPase activity in vitro, although the evidence does not quite fit simple competitive inhibition for ouabain with respect t o KCl (see Reference 14). The steady-state levels of the enzyme-bond ouabain at a given drug concentration in the medium are determined by two independent variables, i.e., the rate of binding and the rate of release. These two parameters can be studied separately. FIGURE 1A shows the results of an experiment in which the rate of [ 3 H ] ouabain binding was monitored. Na+, K+-ATPase preparations obtained from rat brain microsomal fractions following deoxycholic acid and NaI treatments were incubated at 37OC with 0.01 pM [ 3 H ] ouabain (specific activity; 13.2 Ci/mmol) in the presence of 20 mM NaCl, 5 mM Tris-ATP, and 50 mM Tris-HC1 buffer (pH 7.5). Aliquots were taken at the indicated times and the bound
Archives of Biochemistry and Biophysics, 1999
In 40% dimethyl sulfoxide (Me2SO) high-affinity ouabain (O) binding to Na,K-ATPase (E) is promoted by Mg2+ in the absence of inorganic phosphate (Pi) (Fontes et al., Biochim. Biophys. Acta 1104, 215–225, 1995). Furthermore, in Me2SO the EO complex reacts very slowly with Pi and this ouabain binding can therefore be measured by the degree of inhibition of rapid phosphoenzyme formation. Here we found that, unexpectedly, the ouabain binding decreased with the enzyme concentration in the Me2SO assay medium. We extracted the enzyme preparation with Me2SO or chloroform/methanol and demonstrated that the extracted (depleted) enzyme bound ouabain poorly. Addition of such extracts to assays with low enzyme concentration or depleted enzyme fully restored the high-affinity ouabain binding. Dialysis experiments indicated that the active principle had a molecular mass between 3.5 and 12 kDa. It was highly resistant to proteolysis. It was suggested that the active principle could either be a low-molecular-weight, proteolysis-resistant-peptide (e.g., a proteolipid) or a factor with a nonproteinaceous nature. A polyclonal antibody raised against the C-terminal 10 amino acids of the rat kidney γ-subunit was able to recognize this low-molecular-weight peptide present in the extracts. The previously depleted enzyme displayed lower amounts of the γ-proteolipid in comparison to the native untreated enzyme, as demonstrated by immunoreaction with the antibody.
Quantitative aspects of the interaction between ouabain and (Na+ + K+)-activated ATPase in vitro
Biochimica Et Biophysica Acta - Biomembranes, 1971
The inhibitory effect of ouabain on (Na + + K+)-activated ATPase (Mg ~+dependent, (Na + + K+)-activated ATP phosphohydrolase, EC 3.6.1.3) obtained from rat brain microsomal fraction was reexamined using a modified method to estimate the inhibited reaction velocity. This method involves a preincubation of a ouabainenzyme mixture in the presence of Na ÷, Mg 2+ and ATP to bring the ouabain-enzyme reaction to near equilibrium. The (Na + + K+)-activated ATPase reaction was subsequently started by the addition of a KC1 solution. When the ouabain-enzyme reaction was brought to near equilibrium prior to the estimation of the ATPase activity, it was kinetically reversible, although overwhelmingly in favor of the ouabain-enzyme complex. This method resulted in a significant shift of the log concentration-response curve to the left. The concentration of ouabain to inhibit 50 % of the (Na + + K+)activated ATPase activity was o.12 #M, whereas it was 0.52 #M with the conventional method for the ATPase assay. Hence, the specific binding of ouabain to the (Na++ K+)-activated ATPase molecule was a slow process, This modified method was not suitable for the study of the effect of p-chloromercuribenzoate on (Na ÷ + K÷)-activated ATPase since the presence of ATP during the preincubation period protected the enzyme from the SH-blocking reagent. With the modified method, the effect of K + to antagonize ouabain inhibition of (Na + + K+)-activated ATPase was markedly reduced, indicating that the welldocumented effect of K ÷ was on the velocity of the ouabain-enzyme complex formation rather than on that of the ouabain-inhibited ATPase reaction. The ouabain-enzyme reaction was competitive with respect to K + at KC1 concentrations below 5 mM, although the competition by K ÷ was not remarkable. Above this concentration, the reaction was non-competitive with respect to K +. Ouabain released from the ouabain-enzyme complex was rebound to the enzyme during an incubation in the presence of Na ÷, Mg 2+ and ATP more easily than ouabain added to the incubation mixture. Abbreviations: PCMB, p-chloromercuribenzoate; S/M ratio, the ratio between the concentration of ouabain in the iooooo × g sediment and that in the medium.
Naunyn-schmiedebergs Archives of Pharmacology, 1973
The time course of the inotropic response to ouabain in Langendorff preparations was compared with that of the in vitro ATP-dependent (SH)-ouabain binding to cardiac (Na++K+)-activated ATPase preparations, and subsequent dissociation, to determine the temporal relationship between the inotropic response and (Na+ + K +)-activated ATPase inhibition. Species differences were minimal either in the onset of inotropic response or the (aIq)-ouabain binding. The rates of both loss of the inotropic response to ouabain during washout and the dissociation of the ouabain-enzyme complex, however, were rapid in guinea pig and rabbit (relatively ouabain-insensitive species) and slow in eat and dog (ouabain-sensitive species). The half-time of the loss of the inotropic response was similar to the half-time of the dissociation of the ouabain-enzyme complex in each species. Since ATP-dependent binding of cardiac glycosides has been related to enzyme inhibition, it was concluded that the time course of the inotropic response to ouabain parallels the time course of (Na++ K+)-activa~ed ATPase inhibition, and that the dissociation of ouabain from the enzyme may terminate the inotropic response.
Zeitschrift für Naturforschung C, 1979
Na+, K+-ATPase of the plasma membrane isolated from sheep kidney medulla exhibits functional asymmetry for the cardiac glycoside ouabain. In this vesicular membrane preparation the rate of binding of ouabain was slow (time constant > 60 min) when the vesicles were incubated in the presence of isotonic sucrose. Upon treatment of the preparation with hypoosmotic shock or phospholipase A the initial rate of ouabain binding was enhanced at least 3 fold. In equilibrium a concentration of the ouabain-enzyme-complex was obtained which was about twofold that of the untreated vesicles. This result suggests two types of ouabain binding sites with an approximate stoichiometry of 1 to 1. The stoichiometry seems to be maintained at high concentrations of ouabain where binding curves show a biphasic time course. Additional information about heterogeneity of binding sites comes through experiments in which the vesicles were treated with Mg2+ prior to the addition of ouabain. A minor fraction...
European Journal of Pharmacology, 1988
In intact heart muscle cells incubated in a physiological solution, i.e. under the condition in which the cardiac glycosides produce pharmacological and toxicological effects, receptors for these actions of the glycosides should be available to ouabain. In cell homogenates, [3H]ouabain binding observed in the presence of Mg 2+ and inorganic phosphate represents binding of the glycoside to Na+,K+-ATPase. Therefore, numbers of these two types of [3H]ouabain binding sites were compared using viable myocyte preparations obtained from ventricular muscle of guinea-pig heart. The number of ouabain binding sites observed in viable myocytes in the absence of Ca 2÷ and K + was not different from the number of ouabain binding sites on Na÷.K+-ATPase observed with sodium dodecylsulfate-treated homogenates prepared from isolated myocytes. These results do not support the hypothesis that there are receptors for the pharmacological or toxic actions of ouabain other than those that are associated with sarcolemmal Na+,K +-ATPase.
Turnover rates of the canine cardiac Na,K-ATPases
FEBS Letters, 1993
Two functional isoforms a (a,) and a' (a,) of the Na.K-ATPase catalytic subumt coexist m canine cardiac myocytes [J. Biol. Chem. (1987) 262, 8941-89431 The m vitro turnover rates of ATP hydrolysis have been determined in sarcolemma preparations by comparing ["Hlouabain-binding and Na,K-ATPase activity at various doses of ouabam (0.3-300 nM). The correlation between the occupancy of the ouabain-binding sites and the degree of Na.K-ATPase mhibition was not linear. The results showed that the form of low-affinity for ouabam (& = 300-700 nM) exhibited a lower turnover rate (88 + 10 vs. 147 ? 15 molecules of ATP hydrolyzed per second per ouabain-binding site) than the high affinity form (K, = I-8 nM). Thus our results indicate this specific isoform kmetic difference could contribute to differences in the cardiac cellular function.
Kinetics studies on the interaction between ouabain and (Na+,K+)-ATPase
Biochimica et biophysica acta, 1977
The association and dissociation rate constants for the interaction of [3H]-ouabain with partially purified rat brain (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in vitro were estimated from the time course of the [3H]-ouabain binding observed in the presence of Na+, Mg2+ and ATP by a polynomial approximation-curve-fitting technique. The reduction of the association rate constant by K+ was greater than its reduction of the dissociation rate constant. Thus, the affinity of Na+,K+)-ATPase for ouabain was reduced by K+. The binding-site concentration was unaffected by K+. Consistent with these findings, the addition of KCl to an incubation mixture at the time when [3H]-ouabain binding to (Na+,K+)ATPase is close to equilibrium, caused an immediate decrease in bound ouabain concentration, apparently shifting towards a new, lower equilibrium concentration. Dissociation rate constants which were estimated following the termination of the ouabain-binding reaction were different from ...
Detection of a highly ouabain sensitive isoform of rat brainstem Na,K-ATPase
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990
The present work provides evidence for the existence in rat brainstem of a form of the Na,K-ATPase catalytic subunit that displays a high affinity for ouabain (K,, about 10-9 M). Its kinetic identification was made out from studies on dose response curves of ouabain inhibition of Na,K-ATPase activity, ouabain inhibition of Na+-dependent phosphorylation from ATP and ouabain stabilized phosphoenzyme formation from inorganic phosphate (Pi). In all these studies this isoform comprises around 11 percent of the total Na,K-ATPase enzyme. The PAGE electrophoretic mobility of its phosphoprotein obtained from Pi in the presence of ouabain is lower than that of the alpha-l form but it cannot be distinguished from that of alpha-Z Whether this highly ouabain sensitive form corresponds to the alpha-3 isoenzyme or represents the translational product of one of the additional genes described for the large catalytic subunit remains at the moment an open question.