The effects of several ligands on the potassium-vanadate interaction in the inhibition of the (Na+ + K+)-ATPase and the Na+, K+ pump

Inhibition by vanadate of the K+-dependent p-nitrophenylphosphatase activity catalyzed by the (Na+ + K+)-ATPase partially purified from pig kidney showed competitive behavior with the substrate, K+ and Mg2+ acted as cofactors in promoting that inhibition. Ligands which inhibited the K+-dependent p-nitrophenyl phosphate hydrolysis (Na+, nucleotide polyphosphates, inorganic phosphate) protected against inhibition by vanadate. The magnitude of that protection was proportional to the inhibition produced in the absence of vanadate. In the presence of only p-nitrophenyl phosphate and Mg2+, or when the protective ligands were tested alone, the activation of p-nitrophenyl phosphate hydrolysis by K+ followed a sigmoid curve in the presence as well in the absence of vanadate. However, the combination of 100 mM NaCl and 3 mM ATP resulted in a biphasic effect of K+ on the p-nitrophenyl phosphate hydrolysis in the presence of vanadate. After an initial rise at low K+ concentration, the p-nitrophenylphosphatase activity declined at high K+ concentrations; this decline became more pronounced as the vanadate concentration was increased. This biphasic response was not seen when a nonphosphorylating ATP analog was combined with Na+ (which favors the nucleotide binding) or with inorganic phosphate (a requirement for K+ - K+ exchange). Experiments with inside-out resealed vesicles from human red cells showed that in the absence of Na+ plus ATP, K+ promoted vanadate inhibition of p-nitrophenylphosphatase activity in a nonbiphasic manner, acting at cytoplasmic sites. On the other hand, in the presence of Na+ plus ATP, the biphasic response of p-nitrophenyl phosphate hydrolysis is due to K+ acting on extracellular sites. In vanadate-poisoned intact red blood cells, the biphasic response of the ouabain-sensitive Rb+ influx as a function of the external Rb+ concentration failed to develop when there was no Na+ in the extracellular media. In addition, in the absence of extracellular Na+, external Rb+ did not influence the magnitude of inhibition. The present findings indicate that external K+ favors vanadate inhibition by displacing Na+ from unspecified extracellular membrane sites.     

Duchenne muscular dystrophy

Summary In an extensive epidemiological survey of Duchenne muscular dystrophy carried out in Venetia (Italy) the incidence was found to be 28.2×10^-5 and female gamete mutation rate was estimated by the direct method between 61 and 35×10^-6. The percentage of isolated cases was 0.54. Indirect and direct estimates of this proportion suggest, however, that only a minor fraction arises from maternal mutation (from 0.11 to 0.18 of the total number of cases). Studies on pedigrees collected in the course of the survey indicate that there is a higher frequency of Duchenne carrier females than normal females in affected sibships. Additional evidence supporting the hypothesis of a reproductive heterozygote advantage and gametic selection is reported.This work was supported by an MDA grant and by funds from the Italian Muscular Dystrophy Assoc. (UILDM)     

Concanavalin A induced changes of lymphocyte p-nitrophenylphosphatase (author's transl)]

The effect of K+, Na+, Mg2+ and ATP on the p-nitrophenylphosphatase activity was investigated. As an enzyme preparation a microsomal fraction of sheep lymphocytes was used. Low concentrations of Mg2+, K+ and Na+ increased, whereas high concentrations decreased the enzyme activity. There was an inhibition of activity by ATP without Na+ in the incubation medium and an increase of enzyme activity at low K:Na-ratio. By concanavalin A in a concentration of 15 mug/ml the p-nitrophenylphosphatase activity was increased in intact cells and the microsomal fraction for 30-40%. The activation was not Na+, K+, Mg2+, p-nitrophenylphosphate or ATP dependent.     

Muscle adenylate kinase in Duchenne muscular dystrophy

On the basis of electrophoretic and enzyme inhibition studies it was postulated that an aberrant adenylate kinase occurs in muscle and serum of patients with Duchenne muscular dystrophy (Schirmer, R.H. and Thuma, E. (1972) Biochim. Biophys. Acta 268, 92-97; Hamada, M. et al. (1981) Biochim. Biophys. Acta 660, 227-237; Hamada et al. (1985) J. Biol. Chem. 260, 11595-11602). On the basis of the following results we conclude that Duchenne muscular dystrophy patients do not possess an unusual adenylate kinase isoenzyme. In muscle biopsies from five Duchenne patients, the electrophoretic mobility of adenylate kinase and the inhibition of the enzyme by P1, P5-di(adenosine-5')pentaphosphate (Ap5A) was normal. Because of the high SH-group content of the extracts from Duchenne muscle, high concentrations of Ellman's reagent were needed to inhibit adenylate kinase activity in these samples. In Duchenne plasma the adenylate kinase activity was elevated. Like in muscle specimens, the DTNB inhibition curves were shifted to higher reagent concentrations; this was due to a high SH-group content of Duchenne plasma when compared with normal plasma. With respect to inhibition by Ap5A and electrophoretic mobility, Duchenne adenylate kinase in Duchenne plasma behaved like normal muscle adenylate kinase in normal plasma. It was noted that normal muscle adenylate kinase changes its electrophoretic behaviour when mixed with normal or Duchenne plasma. This finding had been considered previously as evidence for the presence of an aberrant adenylate kinase in Duchenne plasma.     

Plasma lipoproteins in Duchenne muscular dystrophy

Plasma lipoproteins of Duchenne muscular dystrophy patients and carriers of the disease, together with age- and sex-matched controls, were examined by density gradient ultracentrifugation and agarose gel electrophoresis. Analysis of density gradient profiles revealed a significant reduction in absorbance (435 nm) by low density and high density lipoproteins from Duchenne patients when compared with controls. Although no abnormalities were observed on electrophoresis of whole plasma samples, the isolated low density lipoprotein fractions from Duchenne patients and carriers displayed increased electrophoretic mobility compared with controls. The results obtained implicate the plasma lipoproteins, in particular the low density lipoproteins, as the primary site of the lesion in this disease.     

Progressive muscular dystrophy (Duchenne): Biochemical studies by flow-cytometry

Summary The peanut lectin (PNL) receptor density of the cell membrane and several metabolic parameters of cultured fibroblasts of normal human individuals and of patients with muscular dystrophy were measured by simultaneous two and three parameter flow cytometry. The PNL-receptor density was significantly decreased on muscular dystrophy fibroblasts (between 20.7 and 33.6%) as compared to normal fibroblasts. The cell volume, the esterase activity, the intracellular pH, and the percentage of proliferating cells of both types of fibroblasts were not significantly altered. The mean cell volume of different fibroblast cultures varied between 2500 and 6000m³. The concentration of the intracellular esterase activity of fibroblasts was low (0.169 relative units) as compared to lymphocytes and granulocytes of the peripheral blood (1.56 and 2.17 relative units). The fibroblasts had an acidic intracellular pH of 6.52 while lymphocytes and granulocytes had basic pH values of 7.30 and 7.17. Some of the fibroblasts were in the S+G2/M phase of the cell cycle (20%). The study shows that the measurement of biochemical parameters of vital and fixed single fibroblasts by flow-cytometry is of great interest for the recognition of differences between normal individuals and muscular dystrophy patients.     

Reversible inactivation of (Na+ + K+)-ATPase by use of a cleavable bifunctional reagent.

1. Purified (Na+ + K+)-ATPase, prepared from rabbit kidney outer medulla, is incubated with the bifunctional NH2-directed reagent dimethyl 3,3'-dithiobis-propionimidate. This results in a cross-link between the subunits of the enzyme and a simultaneous reduction of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 2. The most abundant cross-link product is a dimer of the two different subunits of the enzyme. 3. Reduction of the disulfide cross-link by dithioerythritol results in partial recovery of the original subunit structure of the enzyme and of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 4. These results suggest that a free mobility of the subunits of the (Na+ + K+)-ATPase system relative to each other is essential for proper functioning of both enzyme activities.     

Duchenne muscular dystrophy

Summary By a general survey in the hospitals of northeast Italy, Duchenne cases have been located and identified over a 20-year period.In a more restricted area screening for Duchenne carriers has been carried out in affected families. This procedure made possible an exact estimate of the incidence rate, prevalence rate, and mutation rate in a large sample of population. Prevalence rate was found to be 34x10^-6, incidence rate about 28x10^-5, while mutation rate was found lower than 50x10^-6 by the direct method.The discrepancy between the results obtained by the Haldane formula and those obtained by the direct method for the estimate of the mutation rate is discussed.     

Erythrocyte-ghost Ca2+-stimulated Mg2+-dependent adenosine triphosphatase in Duchenne muscular dystrophy.

The Ca2+-stimulated Mg2-dependent ATPase activities (Ca2+-ATPase) of erythrocyte-ghost membranes from patients with Duchenne muscular dystrophy (DMD) and carriers of DMD were compared with activities of normal controls. The Ca2+-ATPase activity of DMD-patient ghost preparations was found to follow the same pattern of activation by Ca2+ as the control membranes. However, the Ca2+-ATPase activity in DMD and some DMD-carrier preparations was substantially elevated compared with controls. To characterize further the elevated Ca2+-ATPase activity found in DMD-patient ghost membrane preparations, we estimated kinetic parameters using both fine adjustment and weighting methods to analyse our experimental data. It was established that in both DMD and DMD-carrier preparations the increase in Ca2+-ATPase activity was reflected by a significant increase in Vmax. rather than by any change in Km. The response of the membrane Ca2+-ATPase activity to changes in temperature was also investigated. In all preparations a break in the Arrhenius plot occurred at 20 degrees C, and in DMD and DMD-carrier preparations an elevated Ca2+-ATPase activity was detected at all temperatures. Above 20 degrees C the activation energy for all types of preparation was the same, whereas below this temperature there appeared to be an elevated activation in DMD and DMD-carrier preparations compared with normal controls. The concept that a generalized alteration in the physicochemical nature of the membrane lipid domain may be responsible for the many abnormal membrane properties reported in DMD is discussed.     

Duchenne muscular dystrophy

Summary A segregation analysis on 135 Duchenne families from Venetia (Italy) suggests that the proportion of sporadic cases might be less than expected. Support for this view is also given by an analysis of a pooled sample including 284 additional sibships from comparable studies published previously. Several hypotheses were tested: the maximum likelihood was obtained for a segregation frequency p=0.46 and for a proportion of sporadic cases x=0.227±0.048.Dedicated to Professor G. Montalenti in the occasion of his 80th birthday     

Specific modification of gastric K+-stimulated ATPase activity by thimerosal

Treatment of hog gastric microsomes with the sulfhydryl reagent, thimerosal (ethylmercurithiosalicylate), produced differential effects on the K+-ATPase and the K+-stimulated p-nitrophenylphosphatase activities. For example, exposure to 2 mM thimerosal for 3 min severely reduced the activity of K+-stimulated ATPase, while K+-p-nitrophenylphosphatase activity was enhanced 2- to 3-fold. Higher concentration of thimerosal, or longer incubation times, also led to inhibition of K+-p-nitrophenylphosphatase. The activated state of p-nitrophenylphosphatase could be sustained by a 20-fold, or greater, dilution of treated membranes, and could be reversed by reduction of membrane SH groups by exogenous thiols. Significant activation of K+-p-nitrophenylphosphatase was not produced by p-chloromercuribenzene sulfonate, p-chloromercuribenzoate or mersalyl; however, ethyl mercuric chloride had qualitatively similar activity effects as thimerosal. Kinetics of K+-p-nitrophenylphosphatase for thimerosal-treated membranes were altered as follows: V increased; Km for p-nitrophenylphosphate unchanged for Ka for K+ increased. ATP, which is a potent inhibitor of K+-p-nitrophenylphosphatase activity in native membranes (KI approximately 200 microM). These data suggest that there are multiple SH groups which differentially influence the gastric K+-stimulated ATPase activity. Defined treatments with thimerosal are interpreted as an uncoupling of the K+-stimulated phosphatase component of the enzyme (for which p-nitrophenylphosphatase is a presumed model reaction). Such differential modifications can be usefully applied to the study of partial reactions of the enzyme and their specific role in the related H+-transport reaction.     

Solubilization of active (H+ + K+)-ATPase from gastric membrane

(H+ + K+)-ATPase-enriched membranes were prepared from hog gastric mucosa by sucrose gradient centrifugation. These membranes contained Mg2+-ATPase and p-nitrophenylphosphatase activities (68 +/- 9 mumol Pi and 2.9 +/- 0.6 mumol p-nitrophenol/mg protein per h) which were insensitive to ouabain and markedly stimulated by 20 mM KCl (respectively, 2.2- and 14.8-fold). Furthermore, the membranes autophosphorylated in the absence of K+ (up to 0.69 +/- 0.09 nmol Pi incorporated/mg protein) and dephosphorylated by 85% in the presence of this ion. Membrane proteins were extracted by 1-2% (w/v) n-octylglucoside into a soluble form, i.e., which did not sediment in a 100 000 X g X 1 h centrifugation. This soluble form precipitated upon further dilution in detergent-free buffer. Extracted ATPase represented 32% (soluble form) and 68% (precipitated) of native enzyme and it displayed the same characteristic properties in terms of K+-stimulated ATPase and p-nitrophenylphosphatase activities and K+-sensitive phosphorylation: Mg2+-ATPase (mumol Pi/mg protein per h) 32 +/- 9 (basal) and 86 +/- 20 (K+-stimulated); Mg2+-p-nitrophenylphosphatase (mumol p-nitrophenol/mg protein per h) 2.6 +/- 0.5 (basal) and 22.2 +/- 3.2 (K+-stimulated); Mg2+-phosphorylation (nmol Pi/mg protein) 0.214 +/- 0.041 (basal) and 0.057 +/- 0.004 (in the presence of K+). In glycerol gradient centrifugation, extracted enzyme equilibrated as a single peak corresponding to an apparent 390 000 molecular weight. These findings provide the first evidence for the solubilization of (H+ + K+)-ATPase in a still active structure.     

Diphenoloxidases in X-linked recessive (Duchenne) muscular dystrophy

Summary In extracts derived from whole blood, a high molecular weight fraction of the diphenoloxidase enzymes has a significantly diminished specific activity in patients and definite carriers (heterozygotes) of the X-linked, recessive (Duchenne) form of muscular dystrophy. This anomaly was studied using spots of blood which had been collected on absorbent paper and stored at 4°C for variable periods of time. Fractions enriched in the enzymes were obtained by subjecting aqueous extracts of the spots to treatment with an anion exchange resin (DEAE Sephadex A 50) followed by gel filtration on Sephadex G-25. It is of interest that this anomaly was observed in some definite carriers of the mutant gene who had had on several occasions a serum creatine kinase level in the normal range. The significance of these observations is discussed.     

A monoclonal antibody against horse kidney (Na+ + K+)-ATPase inhibits sodium pump and E2K to E1 conversion of (Na+ + K+)-ATPase from outside of the cell membrane

Monoclonal antibodies against horse kidney outer medulla (Na+ + K+)-ATPase were prepared. One of these antibodies (M45-80), was identified as an IgM, recognized the alpha subunit of the enzyme. M45-80 had the following effects on horse kidney (Na+ + K+)-ATPase: (1) it inhibited the enzyme activity by 50% in 140 mM Na+ and by 80% in 8.3 mM Na+; (2) it increased the Na+ concentration necessary for half-maximal activation (K0.5 for Na+) from 12.0 to 57.6 mM, but did not affect K0.5 for K+; (3) it slightly increased the K+-dependent p-nitrophenylphosphatase (K-pNPPase) activity; (4) it inhibited phosphorylation of the enzyme with ATP by 30%, but did not affect the step of dephosphorylation; and (5) it enhanced the ouabain binding rate. These data are compatible with a stabilizing effect on the E2 form of (Na+ + K+)-ATPase. M45-80 was concluded to bind to the extracellular surface of the plasmamembrane, based on the following evidence: (1) M45-80 inhibited by 50% the ouabain-sensitive 86Rb+ uptake in human intact erythrocytes from outside of the cells; (2) the inhibition of (Na+ + K+)-ATPase activity in right-side-out vesicles of human erythrocytes was greater than that in inside-out vesicles; and (3) the fluorescence intensity due to FITC-labeled rabbit anti-mouse IgM that reacted with M45-80 bound to the right-side-out vesicles was much greater than that in the case of the inside-out vesicles.     

Effects of an ATP site affinity analog on some conformational and enzymatic properties of the canine kidney (Na+ + K+)-ATPase

We have shown previously that the canine kidney Na+,K+ pump [Na+ + K+)-ATPase) reacts with the ATP affinity analog p-fluorosulfonylbenzoyladenosine (FSBA). At 20 degrees C, we find the time-course of this reaction to be that predicted for a first-order reaction accompanied by competing solvolysis of the reagent. The FSBA-inactivated (Na+ + K+)-ATPase retains the ability to move between the E1 and E2 conformations that predominate in Na+ and K+ medium, respectively. Therefore, FSBA reaction with the enzyme does not interfere significantly with either its alkali metal cation binding or its conformational freedom. The ability of ATP to influence the enzyme's conformation by binding to the high-affinity nucleotide site is decreased, however, in proportion to the degree of inhibition of enzyme activity by FSBA. In addition, the ability of the enzyme to shift from the E1 to the E2 conformation through the (ATP + Na+)-dependent phosphorylation cycle is inhibited by FSBA treatment, as shown by the decreased ability of these substrates to stimulate the K+-dependent p-nitrophenylphosphatase activity. Both of these effects are consistent with specific reaction of FSBA with the ATP binding site of the enzyme. An additional effect of FSBA treatment is that it causes loss of p-nitrophenylphosphatase activity, but to a lesser extent than (Na+ + K+)-ATPase or Na+-ATPase activity. Binding of p-nitrophenylphosphate to the enzyme is apparently unaffected by FSBA treatment, since the Km for p-nitrophenylphosphate is not changed.     

Thallous ion activation of p-nitrophenylphosphatase of rat myometrium plasma membrane

Addition of thallous ion (Tl+) inhibited the spontaneous mechanical activity of rat myometrium in K-free Krebs solutions with an I50 value of 30 microM. The corresponding value for I50 for similar inhibition by addition of K+ was 150 microM. Tl+ as well as K+-activated p-nitrophenylphosphatase (PNPPase) of isolated rat myometrium plasma membrane vesicles. Half maximal activation was caused by 0.47 mM Tl+ or 1.6 mM K+. Maximal enzyme activities obtained using Tl+ and K+ were comparable. The Km values for the substance p-nitrophenylphosphatase using Tl+ (1.24 mM) and K+ (1.46 mM) were also similar. Activation by either ion was inhibited by ouabain, Na+, inorganic phosphate, and vanadate (V +5). The results suggest that Tl+ can substitute for K+ for activation of the Na-K pump of rat myometrium plasma membrane.     

Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: effects on gill (Na+,K+)-ATPase alpha-subunit expression and K+-phosphatase activity

The kinetic properties of a microsomal gill (Na+,K+)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21 per thousand salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6+/-4.9 U mg-1 and K0.5=1.31+/-0.05 mmol L-1. Stimulation of K+-phosphatase activity by magnesium (Vmax=125.3+/-7.5 U mg-1; K0.5=2.09+/-0.06 mmol L-1), potassium (Vmax=134.2+/-6.7 U mg-1; K0.5=1.33+/-0.06 mmol L-1) and ammonium ions (Vmax=130.1+/-5.9 U mg-1; K0.5=11.4+/-0.5 mmol L-1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (KI=304.9+/-18.3 micromol L-1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the approximately 2-fold reduction in K+-phosphatase specific activity, Western blotting analysis revealed similar alpha-subunit expression in gill tissue from shrimps acclimated to 21 per thousand salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na+,K+)-ATPase was stimulated by high salinity acclimation.     

The calmodulin-binding domain as an endogenous inhibitor of the p-nitrophenylphosphatase activity of the Ca2+ pump from human red cells.

Digestion of red cell membranes with chymotrypsin elicited p-nitrophenylphosphatase activity. During digestion, the p-nitrophenylphosphatase appeared in parallel with the activation of the Ca(2+)-ATPase (in the absence of calmodulin). The chymotrypsin-activated p-nitrophenylphosphatase was inhibited by C20W, a 20 amino acid peptide modelled after the sequence of the calmodulin-binding site of the red cell Ca2+ pump (Vorherr et al. (1990) Biochemistry 29, 355-365). On the contrary, the (ATP + Ca(2+)-dependent p-nitrophenylphosphatase activity of intact red cell membranes was not affected by C20W. Ca2+ inhibited the chymotrypsin-induced p-nitrophenylphosphatase (Ki for Ca2+ = 2 microM). In the absence of ATP, C20W and Ca2+ did not interact in apparent affinity as inhibitors of this activity. On the other hand, in the presence of 2 mM ATP, Ca2+ antagonized the inhibition produced by C20W. The results are consistent with the idea that the calmodulin-binding site is an 'autoinhibitory domain' of the Ca2+ pump, and that removal of this domain by proteolysis, or its modification by calmodulin binding is the reason for the activation of both the ATPase and the p-nitrophenylphosphatase activity of the pump. The results presented in this paper give new information about the mechanism of the two kinds of p-nitrophenylphosphatase and about the nature of the apparent competition between C20W and Ca2+.     

Mechanisms of inhibition of (Na,K)-ATPase by hydrostatic pressure studied with fluorescent probes

To investigate the mechanisms by which hydrostatic pressure inhibits (Na,K)-ATPase, we measured enzyme activity, as a function of pressure and temperature, of purified (Na,K)-ATPase from dog kidney and eel electroplax, and we monitored protein conformation, possible subunit interactions, and the fluidity of the membrane with fluorescent probes. The (Na,K)-ATPase and p-nitrophenylphosphatase activities were inhibited reversibly by pressures below 1.5 kilobars (eel enzyme) and 2.5 kilobars (dog kidney enzyme). Above these pressures, the enzymes were inactivated irreversibly. The plots of 1n(activity) versus pressure were curvilinear; this indicates that the reversible inhibition by pressure involves two or more rate-limiting steps. The calculated activation volumes varied with temperature and pressure and were larger for the (Na,K)-ATPase activity compared to the p-nitrophenylphosphatase activity. The fluorescence polarization of three hydrophobic probes decreased with increasing temperature (10-36 degrees C) and increased with increasing pressure (10(-3)-1.5 kilobars), reversibly, without any evidence of a lipid phase transition. Plots of enzyme activity versus fluorescence polarization of the lipid probes showed an inverse relationship; this indicates that enzyme activity was directly related to the fluidity of the membrane as measured by the lipid probes. Pressure had no effect on the fluorescence polarization of two cardiac glycoside probes nor on the efficiency of resonance energy transfer between either donor and acceptor cardiac glycosides specifically bound to the ouabain sites of different alpha-subunits, or tryptophan and the bound cardiac glycoside probe. These results suggest that dissociation of dimeric alpha-subunits is not related to the inhibition by pressure, and that the cardiac glycoside-complexed enzyme conformation is stabilized by pressure. It is concluded that increased pressure decreases the membrane fluidity which hinders conformational transitions associated with rate-limiting steps of the (Na,K)-ATPase reaction. It is proposed that ion-bound or -occluded forms of (Na,K)-ATPase are stabilized by pressure because they occupy a smaller volume.