Phosphorylation of the Na,K-ATPase by Ca,phospholipid-dependent andcAMP-dependent protein kinases. Mapping of the region phosphorylated by Ca,phospholipid-dependent protein kinase

Ca,phospholipid-dependent (PKC) andcAMP-dependent (PKA) protein kinases phosphorylate the -subunit of the Na,K-ATPase from duck salt gland with the incorporation of 0.3 and 0.5 mol³²P/mol of -subunit, respectively. PKA (in contrast to PKC) phosphorylates the -subunit only in the presence of detergents. Limited tryptic digestion of the Na,K-ATPase phosphorylated by PKC demonstrates that³²P is incorporated into the N-terminal 41-kDa fragment of the -subunit. Selective chymotrypsin cleavage of phosphorylated enzyme yields a 35-kDa radioactive fragment derived from the central region of the -subunit molecule. These findings suggest that PKC phosphorylates the -subunit of the Na,K-ATPase within the region restricted by C₃ and T₁ cleavage sites.     

Study of Dimensional and Morphological Characteristics of Erythrocytes in Some Black Sea Fish of Different Evolution Position and Ecological Specialization

Journal of Ichthyology - A comparative analysis of the morphometric parameters of erythrocytes in two species of cartilage (Chondrichthyes) and five species of teleosts (Teleostei) Black Sea fish...     

Extracellular pH modulates kinetics of the Na(+),K(+)-ATPase

To investigate effects of pH on the Na(+),K(+)-ATPase, we used the Xenopus oocytes to measure transient charge movements in the absence of extracellular K(+), and steady-state currents mediated by the pump as well as ATPase activity. The activity of purified Na(+), K(+)-ATPase strongly depends on pH, which has been attributed to protonation of intracellular sites. The steady-state current reflects pump activity, the transient charge movement voltage-dependent interaction of external Na(+) ions with the pump molecule and/or conformational changes during Na(+)/Na(+) exchange. The steady-state current exhibits a characteristic voltage dependence with maximum at about 0 mV at low external K(+) (< or =2 mM) and with 50 Na(+). This dependency is not significantly affected by changes in external pH in the range from pH 9 to pH 6. Only below pH 6, the voltage dependence of pump current becomes less steep, and may be attributed to a pH-dependent inhibition of the forward pump cycle by external Na(+). External stimulation of the pump by K(+) in the absence of Na(+) can be described by a voltage-dependent K(m) value with an apparent valency z(K). At higher external pH the z(K) value is reduced. The transient current signal in the absence of external K(+) can be described by the sum of three exponentials with voltage-dependent time constants of about 50 ms, 700 micros and less than 100 micros during pulses to 0 mV. The charge distribution was calculated by integration of the transient current signals. The slowest component and the associated charge distributions do not significantly depend on external pH changes. The intermediate component of the transients is represented by a voltage-dependent rate constant which shows a minimum at about -120 mV and increases with decreasing pH. Nevertheless, the contribution to the charge movement is not altered by pH changes due to a simultaneous increase of the amplitude of this component. We conclude that reduction of external pH counteracts external K(+) and Na(+) binding.     

Functional role of the N-terminus of Na(+),K(+)-ATPase alpha-subunit as an inactivation gate of palytoxin-induced pump channel

The N-terminus of the Na(+),K(+)-ATPase alpha-subunit shows some homology to that of Shaker-B K(+) channels; the latter has been shown to mediate the N-type channel inactivation in a ball-and-chain mechanism. When the Torpedo Na(+),K(+)-ATPase is expressed in Xenopus oocytes and the pump is transformed into an ion channel with palytoxin (PTX), the channel exhibits a time-dependent inactivation gating at positive potentials. The inactivation gating is eliminated when the N-terminus is truncated by deleting the first 35 amino acids after the initial methionine. The inactivation gating is restored when a synthetic N-terminal peptide is applied to the truncated pumps at the intracellular surface. Truncated pumps generate no electrogenic current and exhibit an altered stoichiometry for active transport. Thus, the N-terminus of the alpha-subunit appears to act like an inactivation gate and performs a critical step in the Na(+),K(+)-ATPase pumping function.     

Voltage-dependent inhibition of the sodium pump by external sodium: Species differences and possible role of the N-terminus of the α-subunit

Currents generated by the Na⁺/K⁺ ATPase were measured under voltage clamp in oocytes of Xenopus laevis. The dependence of pump current on external [Na⁺] was investigated for the endogenous Xenopus pump as well as for wild-type and mutated pumps of electroplax of Torpedo californica expressed in the oocytes. The mutants had -subunits truncated before position Lys²⁸ (K28) or Thr²⁹ (T29) of the N-terminus. The currents generated by all variants of pump molecules in the presence of 5 mM K⁺ show voltage-dependent inhibition by external [Na⁺]. The apparent K₁ values increase with membrane depolarisation, and the potential dependence can be described by the movement of effective charges in the electrical potential gradient across the membrane. Taking into account Na⁺-K⁺ competition for external binding to the E₂P form, apparent K₁ values and effective charges for the interaction of the Na⁺ ions with the E₂P form can be estimated. For the Xenopus pump the effective charge amounts to 1.1 of an elementary charge and the K₁ value at 0 mV to 44 mM. For the wild-type Torpedo pump, the analysis yields values of 0.73 of an elementary charge and 133 mM, respectively. Truncation at the N-terminus removing a lysinerich cluster of the a-subunit of the Torpedo pump leads to an increase of the effective charge and decrease of the K₁ value. For K28, values of 0.83 of an elementary charge and 117 mM are obtained, respectively. If LyS²⁸ is included in the truncation (·T29), the effective charge increases to 1.5 of an elementary charge and the apparent K₁ value is reduced to 107 mM. The K, values for pump inhibition by external Na⁺, calculated by taking into account Na⁺-K⁺ competition, are smaller than the K_/12 values determined in the presence of 5 mM [K⁺]. The difference is more pronounced for those pump variants that have higher K_m, values. The variations of the parameters describing inhibition by external [Na⁺] are qualitatively similar to those described for the stimulation of the pumps by external [K⁺] in the absence of extracellular [Na⁺]. The observations may be explained by an acess channel within the membrane dielectric that has to be passed by the external Na⁺ and K⁺ ions to reach or leave their binding sites. The potential-dependent access and/or the interaction with the binding sites shows species differences and is affected by cytoplasmic lysine residues in the N-terminus.     

Etiological structure and clinical epidemiological characteristics of Leptospira infection in Rostov Province based on data from therapeutic institutions from 1960 to 1983

Changes in communal conditions, in economy, as well as in ecology and fauna, which took place in Rostov Province during the last decade (1973-1983) determined shifts in the etiological structure of Leptospira infection and in its course. The study revealed an increase in morbidity caused by L. icterohaemorrhagiae (up to 61%) and L. hebdomadis (up to 22%) with a simultaneous decrease in the isolation rate of L. grippotyphosa and L. pomona (up to 2-3%). In most cases (77%) the diseases caused by leptospires of different serogroups were found to take an icteric course accompanied by the development of hepatorenal insufficiency (46%). The similarity of clinical manifestations in different etiological forms of Leptospira infection was determined by common pathogenetic and pathophysiological features characteristic of the development of the leptospiral infectious process.     

Anti-arrhythmia action of the antioxidant SD-6 in the development of ischemic and reperfusion rhythm disorders of the isolated rat heart

An antiarrhythmic action of water-soluble antioxidant SD-6 from 3-hydroxypyridine class and its effect on the transmembrane potentials were studied using the isolated rat heart and papillary muscle. Ischemia was induced by the occlusion of the left anterior descending coronary artery. 10 minutes later the ligation was removed and reperfusion was achieved. In the control, ischemia induced premature ventricular complexes, tachycardia and, in some cases, fibrillation. During perfusion total fibrillation occurred in 100% of the experiments. SD-6 in the doses of 10(-6) g/ml and 5 X 10(-6) g/ml significantly reduced the incidence of fibrillation and tachycardia. In the experiments on the papillary muscle SD-6 during reperfusion completely normalized the action potential duration and removed depolarization developed in hypoxia, which suggests the ability of the antioxidant to block reperfusion-induced arrhythmias by normalization of the parameters of electrical heterogeneity. These data show that the origin of reperfusion-induced arrhythmias is connected with the activation of free radical metabolites and that their scavengers--synthetic antioxidants from 3-hydroxypyridine class--can be used as new antiarrhythmic agents.     

Stimulation of the Na+/K+ pump by external [K+] is regulated by voltage-dependent gating.

Wild-type and mutants with alpha-subunits truncated at the N terminus of Na+/K+ pumps of Torpedo electroplax were expressed in Xenopus oocytes by injection of cRNAs encoding for one of the alpha-subunits and for the beta-subunit. Currents generated by the pump were investigated under voltage clamp in Na(+)-free solution, a condition where stimulation by external [K+] is the only voltage-dependent and rate-determining step in the pump cycle (Rakowski, R. F., Vasilets, L. A., LaTona, J., and Schwarz, W. (1991) J. Membr. Biol. 121, 177-187). Voltage dependence of the apparent Km value for pump stimulation and of maximum transport activity was investigated. Truncation of the intracellular N-terminal end of the alpha-subunit at the trypsin-accessible site (alpha delta K37, leaving Lys37) leads to nearly complete inhibition of pump current at physiological potentials, whereas ouabain binding capacity is retained indicating an essential involvement of the N-terminal end in the process of ion translocation. Truncation at the N-terminal end leaving Lys28 (alpha delta K28) or Thr29 (alpha delta T29) leads to removal of 6 or 7 lysine residues, respectively, and has no effect on maximum transport activity. On the other hand, the mutated pumps with alpha delta K28 or alpha delta T29 exhibit more pronounced voltage dependences for stimulation of pump current by external [K+] compared with the wild-type Torpedo pump. In particular, a pronounced increase in voltage dependence of the apparent affinity of pump stimulation is obtained by the removal of the Lys28. The results support the view that the lysine-rich region in the N-terminal end affects the cation binding to the pump molecule and that Lys28 is important.