Changes in plasmalemma K+Mg2+ -ATPase dephosphorylating activity and H+ transport in relation to seasonal growth and freezing tolerance of Festuca pratensis Huds

Changes in plasmalemma K⁺Mg²⁺-ATPase dephosphorylating activity and H⁺ transport were examined in freezing-tolerant and non-tolerant genotypes of the perennial grass species Festuca pratensis Huds. Enzyme activity and ΔμH⁺ were measured in plasmalemma fractions isolated from basal nodes and roots. Three types of experiments were undertaken: (i) a field experiment, utilizing the seasonal growth and cessation cycle of a perennial plant; (ii) a cold acclimation experiment in hydroponics; and (iii) an instant freezing test. A specific fluctuation in K⁺Mg²⁺-ATPase activity was found throughout the seasonal growth of the plants (i). The K⁺Mg²⁺-ATPase activity peaks for both the basal node and the root plasmalemma were determined early in the spring before the renewal of growth. The lowest activity values in roots occurred at the time approaching flowering, and in basal nodes at the transition into the growth cessation. The K⁺Mg²⁺-ATPase activity was approximately 50% lower in the basal node plasmalemma of freezing-tolerant plants than of non-tolerant ones, when assessed at the optimal growth stage in hydroponics. In hydroponics (ii) and in the freezing test (iii), temperature stress was followed by a more pronounced change in the level of K⁺Mg²⁺-ATPase activity than in that of H⁺ transport, and this change was more clearly differentiated in the basal node plasmalemma of contrasting genotypes than in the roots. Stress response was manifested differently in freezing-tolerant and non-tolerant plants at cold acclimation (4–2 °C) and at freezing (−8 °C) temperatures. Proton transport regulation via coupled changes in the hydrolysed ATP/transported proton ratio, as an attribute of freezing-tolerant plants, is discussed.     

一氧化氮调节盐胁迫下小麦幼苗根部质膜H+-ATPase和焦磷酸酶活性提高耐盐性

采用外源一氧化氮(NO)供体硝普钠(SNP)研究了NO对盐胁迫下小麦(Triticum aestivum L.)幼苗耐盐性的影响.结果表明,0.1 mmol/L SNP处理显著缓解了1 50 mmol/L NaCl胁迫对小麦幼苗生长的抑制效应,包括水分丧失以及叶绿素降解,从而提高了小麦幼苗的耐盐性.进一步结合1 mg/mL血红蛋白处理则显著逆转了SNP诱导的上述效应;利用亚硝酸钠和铁氰化钾作为对照也证实了NO对小麦幼苗耐盐性的专一性调节作用,并可能与NO对小麦幼苗根部质膜H -ATPase和焦磷酸酶活性诱导有关.此外,尽管NO显著提高了盐胁迫下小麦幼苗根部细胞质膜H -ATPase和焦磷酸酶的ATP水解活性,但是对跨膜H 转运则没有明显影响.应用外源CaSO4和EGTA处理也证实,Ca2 可能在NO诱导的质膜H -ATPase和焦磷酸酶活性的提高过程中起信号作用.另外,分析盐胁迫下小麦幼苗根部Na 和K 含量的变化也发现,NO对Na 含量没有明显影响,但是却显著提高了K 水平和K /Na 比,这可能也是NO提高小麦幼苗耐盐性的原因之一.     

Dimeric structure of H-translocating pyrophosphatase from pumpkin vacuolar membranes

Vacuolar membrane H⁺-translocating pyrophosphatase (H⁺-PPase) was purified from pumpkin seedlings. Its enzymatic properties including molecular size of constituting polypeptide (75 kDa) were very similar to those of mung bean H⁺-PPase [(1989) J. Biol. Chem. 264, 20068–20073]. The native, functional molecular size of the pumpkin H⁺-PPase was estimated to be 135–139 kDa from gel permeation HPLC of the purified enzyme in the presence of detergent and from radiation inactivation of the enzyme in vacuolar membranes. It is concluded that native, functional pumpkin H⁺-PPase, and also probably H⁺-PPases from other plants, is a dimer of 75 kDa subunits.     

ADP binding and ATP synthesis by reconstituted H-ATPase from chloroplasts

The H⁺-ATPase from chloroplasts, CF₀F₁, was isolated, purified and reconstituted into asolectin liposomes. The enzyme was brought either into the oxidized state or into the reduced state, and the rate of ATP synthesis was measured after energisation of the proteoliposomes with an acid—base transition ΔpH (pH_in = 5.0, pH_out = 8.5) and a K⁺/valinomycin diffusion potential, Δφ (K⁺_in = 0.6 mM, K⁺_out = 60 mM). A rate of 250 s⁻¹ was observed with the reduced enzyme (85 s⁻¹ in the absence of Δφ). A rate of 50 s⁻¹ was observed with the oxidized enzyme under the same conditions (15 s⁻¹ in the absence of Δφ). The reconstituted enzyme contained 2 ATP_bound per CF₀F₁ and 1 ADP_bound per CF₀F₁. Upon energisation the enzyme was activated and 0.9 ADP per CF₀F₁, was released. Binding of ADP to the active reduced enzyme was observed under different conditions. In the absence of phosphate the rate constant for ADP binding was 10⁵ M⁻¹·s⁻¹ under energized and de-energized conditions. In the presence of phosphate the rate of ADP binding drastically increased under energized conditions, and strongly decreased under de-energized conditions.     

Glutamate transporter GLAST/EAAT1 directs cell surface expression of FXYD2/gamma subunit of Na, K-ATPase in human fetal astrocytes

Na⁺-dependent uptake of excitatory neurotransmitter glutamate in astrocytes increases cell energy demands primarily due to the elevated ATP consumption by glutamine synthetase and Na⁺, K⁺-ATPase. The major pool of GLAST/EAAT1, the only glutamate transporter subtype expressed by human fetal astrocytes in undifferentiated cultures, was restricted to the cytoplasmic compartment. Elevated glutamate concentrations (up to 50 μM) stimulated both glutamate uptake and Na⁺, K⁺-ATPase activity and concomitantly increased cell surface expression of GLAST and FXYD2/γ subunit of Na⁺, K⁺-ATPase. Intracellular accumulation of glutamate or its metabolites per se was not responsible for these changes since metabolically inert transport substrate, d-aspartate, exerted the same effect. Nanomolar concentrations of TFB-TBOA, a novel nontransportable inhibitor of glutamate carriers, almost completely reversed the action of glutamate or d-aspartate. In the same conditions (i.e. block of glutamate transport) monensin, a potent Na⁺ ionophore, had no significant effect neither on the activation of Na⁺, K⁺-ATPase nor on the cell surface expression of γ subunit or GLAST. In order to elucidate the roles of γ subunit in the glutamate uptake-dependent trafficking events or the activation of the astroglial sodium pump, in some cultures γ subunit/FXYD2 was effectively knocked down using siRNA silencing. Unlike the blocking effect of TFB-TBOA, the down-regulation of γ subunit had no effect neither on the trafficking nor activity of GLAST. However, the loss of γ subunit effectively abolished the glutamate uptake-dependent activation of Na⁺, K⁺-ATPase. Following withdrawal of siRNA from cultures, the expression levels of γ subunit and the sensitivity of Na⁺, K⁺-ATPase to glutamate/aspartate uptake have been concurrently restored. Thus, the activity of GLAST directs FXYD2 protein/γ subunit to the cell surface, that, in turn, leads to the activation of the astroglial sodium pump, presumably due to the modulatory effect of γ subunit on the kinetic parameters of catalytic subunit(s) of Na⁺, K⁺-ATPase.     

Phosphorylation-dephosphorylation of membrane proteins controls the microsomal H-ATPase activity of corn roots

The Mg²⁺-dependent H⁺-ATPase activity of a sealed microsomal vesicle fraction isolated from corn (Zea mays L.) roots appears to be controlled by a phosphorylation-dephosphorylation cycle. Phosphorylation of the microsomal fraction is carried out by a Ca²⁺/calmodulin (CaM)-stimulated process. The H⁺-ATPase activity decreases with increasing phosphorylation of the membranes and becomes only slightly uncoupled by ionophores and less inhibited by dicyclohexylcarbodiimide (DCCD), diethylstilbestrol (DES), NO₃⁻ and vanadate. The inhibitory effect of phosphorylation is greater on the NO₃⁻-sensitive H⁺-ATPase activity than on the vanadate-sensitive activity. Restoration of H⁺-ATPase activity is achieved by allowing the phosphorylated membranes to dephosphorylate either in the absence or presence of exogenous alkaline phosphatase. Moreover, the presence of fluphenazine during the Ca²⁺/CaM-stimulated treatment inhibits membrane phosphorylation and protects the H⁺-ATPase activity from inhibition.     

The tonoplast H+-pyrophosphatase of radish seedlings: biochemical characteristics

The activity of the H⁺-pyrophosphatase (H⁺-PPase) was characterized in microsomes from 24-h-old radish ( Raphanus sativus L., ev. Tondo Rosso Quarantino) seedlings, which are virtually devoid of the tonoplast H⁺-ATPase. The H⁺-PPase was localized to membranes which roughly comigrated with the plasma membrane in a sucrose density gradient, but clearly separated from plasma membrane when microsomes were partitioned in an aqueous dextran-polyethylene glycol two-phase system. The H⁺-PPase activity was strictly dependent on Mg²⁺ and on the presence of a monovalent cation (K⁺=Rb⁺=NH₃⁺Cs⁺≫Na⁺Li⁺) and was insensitive to anions such as Cl−, Br−, NO₃− and SO₄^2-. It was inhibited by F−, imidodiphosphate and Ca²⁺. It had a pH optimum between pH 7.5 and 8.5 and was saturated by low concentrations of pyrophosphate (half saturation at 30 μ M pyrophosphate). All of these characteristics are identical to those reported for the tonoplast H⁺-PPase from various plant materials. The functional molecular weight of the H⁺-PPase, measured with the radiation-inactivation technique was 96 kDa.     

Rb transport in Leishmania infantum promastigotes under various in vitro culture conditions

The survival of Leishmania, which encounter drastic changes of environment during their life-cycle, requires regulation and control of ionic concentrations within the cell. We analysed the influence of growth stage, ionic composition of the medium, heat and acidic stress on ⁸⁶Rb⁺ influx in L. infantum promastigetes. Proliferating promastigotes exibited faster and higher ⁸⁶Rb⁺ uptake than stationary cells. Cl⁻ anion did not have any effect, but in the presence of physiological concentration of HCO₃⁻, ⁸⁶Rb⁺ uptake was significantly increased. This enhancing effect was only partially inhibited by N,N′-dicyclohexylcarbodiimide (DCCD), a blocker of ion-translocating ATPases. ⁸⁶Rb⁺ influx was abolished by N-ethylmaleimide (NEM), indicating a major contribution of plasma membrane transporters. Heat shock and acidic shock notably decreased ⁸⁶Rb⁺ influx. Our data provide indirect evidence that an energy-dependent system which brings K⁺ in, such as K⁺/H⁺-ATPase evidenced by Jiang et al. (1994), is active in Leishmania in different environments. Mechanism(s) other than ion-translocating ATPase occur, at least in the presence of HCO₃⁻, and their contribution to K⁺ pathways varies in different environmental conditions.     

The sodium cycle. II. Na-coupled oxidative phosphorylation in Vibrio alginolyticus cells

The role of Na⁺ in Vibrio alginolyticus oxidative phosphorylation has been studied. It has been found that the addition of a respiratory substrate, lactate, to bacterial cells exhausted in endogenous pools of substrates and ATP has a strong stimulating effect on oxygen consumption and ATP synthesis. Phosphorylation is found to be sensitive to anaerobiosis as well as to HQNO, an agent inhibiting the Na⁺-motive respiratory chain of V. alginolyticus. Na⁺ loaded cells incubated in a K⁺ or Li⁺ medium fail to synthesize ATP in response to lactate addition. The addition of Na⁺ at a concentration comparable to that inside the cell is shown to abolish the inhibiting effect of the high intracellular Na⁺ level. Neither lactate oxidation nor Δω generation coupled with this oxidation is increased by external Na⁺ in the Na⁺-loaded cells. It is concluded that oxidative ATP synthesis in V. alginolyticus cells is inhibited by the artificially imposed reverse ΔPNa, i.e., [Na⁺]_in > [Na⁺]_out. Oxidative phosphorylation is resistant to a protonophorous uncoupler (0.1 mM CCCP) in the K⁺-loaded cells incubated in a high Na⁺ medium, i.e., when ΔpNa of the proper direction ([Na⁺]_in < [Na⁺]_out) is present. The addition of monensin in the presence of CCCP completely arrests the ATP synthesis. Monensin without CCCP is ineffective. Oxidative phosphorylation in the same cells incubated in a high K⁺ medium (ΔpNa is low) is decreased by CCCP even without monensin. Artificial formation of ΔpNa by adding 0.25 M NaCl to the K⁺-loaded cells (Na⁺ pulse) results in a temporary increase in the ATP level which spontaneously decreases again within a few minutes. Na⁺ pulse-induced ATP synthesis is completely abolished by monensin and is resistant to CCCP, valinomycin and HQNO. 0.05 M NaCl increases the ATP level only slightly. Thus, V. alginolyticus cells at alkaline pH represent the first example of an oxidative phosphorylation system which uses Na⁺ instead of H⁺ as the coupling ion.     

Responses of apical ion fluxes to NaCl stress in Elaeagnus angustifolia seedlings

Aims Elaeagnus angustifolia is one of the most salt-tolerant species. The objective of this study was to understand the mechanisms of ion transporation in E. angustifolia exposed to different salt concentrations through manipulations of K⁺/Na⁺ homeostasis.
Methods Seedlings of two variants of the species, Yinchuan provenance (YC, salt-sensitive type) and the Alaer provenance (ALE, salt-tolerant type), were treated with three different NaCl application modes, and the ion fluxes in the apical regions were measured using non-invasive micro-test technology (NMT). In mode 1, Na⁺ and K⁺ fluxes were measured after 150 mmol·L^-1 NaCl stress lasted for 24 h. In mode 2, K⁺ and H⁺ fluxes were quantified with a transient stimulation of NaCl solution. In mode 3, Amiloride (Na⁺/H⁺ antiporters inhibitor) and tetraethylammonium (TEA, K⁺ channel inhibitor) was used to treat apical regions of E. angustifolia seedlings after NaCl stress for 24 h, respectively.
Important findings Under NaCl stress for 24 h, net effluxes of Na⁺ and K⁺ were increased significantly. The net Na⁺ effluxes of YC provenance seedlings (720 pmol·cm^-2•s^-1) were lower than that of ALE provenance (912 pmol·cm^-2·s^-1), but the net K⁺ efflux was higher in YC provenance. Under the instantaneous NaCl stimulation, net K⁺ efflux was remarkably increased, with the net K⁺ efflux of YC provenance always higher than that of ALE provenance. Interestingly, H⁺ at the apical regions was found from influx to efflux, with the net H⁺ efflux of ALE provenance greater than that of the YC provenance. Under the NaCl and NaCl + Amiloride treatment, the net Na⁺ efflux of ALE provenance seedlings was higher than that of YC provenance, while the net K⁺ efflux was less in ALE provenance seedlings. On the other hand, the differences in net Na⁺ and K⁺ effluxes were insignificant between the two provenances under the control group and NaCl + TEA treatment. In conclusion, NaCl stress caused Na⁺ accumulation and K⁺ outflows of E. angustifolia seedlings; The E. angustifolia seedlings utilize Na⁺/H⁺ antiporters to reduce Na⁺ accumulation by excretion; and the maintenance of K⁺/Na⁺ homeostasis in salt-tolerant E. angustifolia provenance seedlings roots accounted for a greater Na⁺ extrusion and a lower K⁺ efflux under NaCl stress. Results from this study provide a theoretical basis for further exploring salt-tolerant E. angustifolia germplasm resource.     

H2O2 and Src-dependent transactivation of the EGF receptor mediates the stimulatory effect of leptin on renal ERK and Na+, K+-ATPase

We examined the mechanism through which leptin increases Na⁺, K⁺-ATPase activity in the rat kidney. Leptin was infused under anaesthesia into the abdominal aorta proximally to the renal arteries and then Na⁺, K⁺-ATPase activity was measured in the renal cortex and medulla. Leptin (1 μg/kg min) increased Na⁺, K⁺-ATPase activity after 3 h of infusion, which was accompanied by the increase in urinary H₂O₂ excretion and phosphorylation level of extracellular signal regulated kinase (ERK). The effect of leptin on ERK and Na⁺, K⁺-ATPase was abolished by catalase, specific inhibitors of epidermal growth factor (EGF) receptor, AG1478 and PD158780, as well as by ERK inhibitor, PD98059, and was mimicked by both exogenous H₂O₂ and EGF. The effect of leptin was also prevented by the inhibitor of Src tyrosine kinase, PP2. Leptin and H₂O₂ increased Src phosphorylation at Tyr⁴¹⁸. We conclude that leptin-induced stimulation of renal Na⁺, K⁺-ATPase involves H₂O₂ generation, Src kinase, transactivation of the EGF receptor, and stimulation of ERK.     

Extracellular Na removal enhances granule secretion in platelets 3-evidence that Na/H exchange is inhibitory to secretion induced by some agonists

The effect of extracellular Na⁺ ([Na⁺]_e) removal on agonist-induced granule secretion in platelets in relation to [ph]_i and [Ca²⁺]_i changes was investigated. Substitution of [Na⁺]_e with choline⁺ of K⁺ resulted in a significant enhancement of 5HT secretion induced by thrombin, collagen, U46619 and the protein kinase C activators, PMA and diC₈. Increases in [Ca²⁺]_i induced by thrombin and U46619 were slightly inhibited or unaffected in these buffers, but [pH]_i increases induced by thrombin, U46619, PMA and diC₈ were abolished and a drop in [pH]_i (0.05–0.1 units below resting) was observed. Although preincubation with potassium acetate produced a big drop in [pH]_i and greatly increased secretion with all the agonists, particularly in the absence of [Na⁺]_e, clear evidence that [pH]_i rises due to Na⁺/H⁺ exchange are inhibitory to secretion was obtained only with thrombin. Thus, (i) NH₄Cl, which restored the increase in [pH]_i in the absence of [Na⁺]_e reduced the potentiated secretory response to thrombin, (ii) no increase in thrombin-induced secretion was observed when Na⁺ was replaced with Li⁺, which allowed a normal increase in [pH]_i and (iii) ethyl isopropyl amiloride (EIPA) abolished the [pH]_i rise and potentiated thrombin-induced secretion. With collagen and U46619, the results suggest that removal of [Na⁺]_e per se rather than inhibition of Na⁺/H⁺ exchange results in enhanced secretion. It is concluded that [Na⁺]_e per se and [pH]_i elevations via Na⁺/H⁺ exchange both have important inhibitory roles in the control of platelet granule secretion.     

Hyperbaric oxygen treatment: the influence on the hippocampal superoxide dismutase and Na+,K+-ATPase activities in global cerebral ischemia-exposed rats

The influence of hyperbaric oxygen (HBO) treatment on the activities of superoxide dismutase (SOD) and Na⁺,K⁺-ATPase was determined during different time periods of reperfusion in rats exposed to global cerebral ischemia. Ischemic animals were either sacrificed or exposed to the first HBO treatment 2, 24, 48 or 168 h after ischemic insult (for SOD activities measurement) or immediately, 0.5, 1, 2, 6, 24, 48, 72 or 168 h after ischemic procedure (for Na⁺,K⁺-ATPase activities measurement). Hyperbaric oxygenation procedure was repeated for seven consecutive days. The results of presented experiments demonstrated the statistically significant increase in the hippocampal SOD activity 24 and 48 h after global cerebral ischemia followed by a decrease in the enzymatic activity 168 h after ischemic insult. In the ischemic rats treated with HBO the level of hippocampal SOD activity was significantly higher after 168 h of reperfusion in comparison to the ischemic, non HBO-treated animals. In addition, it was found that global cerebral ischemia induced a statistically significant decrease of the hippocampal Na⁺,K⁺-ATPase activity starting from 1 to 168 h of reperfusion. Maximal enzymatic inhibition was obtained 24 h after the ischemic damage. Decline in Na⁺,K⁺-ATPase activity was prevented in the animals exposed to HBO treatment within the first 24 h of reperfusion. Our results suggest that global cerebral ischemia induces significant alterations in the hippocampal SOD and Na⁺,K⁺-ATPase activities during different periods of reperfusion. Enhanced SOD activity and preserved Na⁺,K⁺-ATPase activity within particular periods of reperfusion, could be indicators of a possible benefitial role of HBO treatment in severe brain ischemia.     

Effect of auxin concentration and growth phase on the plasma membrane H-ATPase of tobacco calli

The activity of plasma membrane H⁺-ATPase increases up to 3 fold during the growth of tobacco calli for 6 weeks. In medium with optimal auxin this activity is always greater than in medium with suboptimal hormone. The amount of H⁺-ATPase measured with specific antibody only experiences small changes under the same conditions. Therefore, auxin level and growth phase mostly influence the molecular activity of the enzyme and not its amount. Despite the increase in H⁺-ATPase activity, there is a decrease in K⁺ content and ths decrease is greater in medium with suboptimal auxin. Apparently, there is a progressive deenergization of the plasma membrane which is only partially compensated by an auxin-dependent increase in activity of plasma membrane H⁺-ATPase.     

The effect of hydroxylamine on microsomal ATPase

The (Na⁺ + K⁺)-stimulated Mg²⁺-ATPase, but not the Mg²⁺-ATPase, is irreversibly inhibited when turtle bladder microsomes were incubated with hydroxylamine.

The Mg²⁺-dependent or the (Mg²⁺ + Na⁺)-dependent phosphorylation of ADP by the phospho-protein (the exchange reaction) is reversibly inhibited when the microsomes are incubated with hydroxylamine.

The Na⁺-induced increment of ³²P-labelling of microsomes previously incubated with [λ-³²P]ATP is completely eliminated by hydroxylamine, but the Mg²⁺-dependent ³²P-labelling of such microsomes is unaffected by hydroxylamine.

It is concluded that the phospho-enzyme formed during the Mg²⁺-dependent hydrolysis does not contribute to the Mg²⁺-dependent exchange reaction. Instead, the phosphoenzyme formed during the (Na⁺ + K⁺)-stimulated hydrolysis is apparently the only substance which phosphorylates ADP in the exchange reaction, even in the absence of Na⁺ and/or K⁺.

The hydroxylamine-sensitive nature of the sodium form of the phospho-enzyme in the (Na⁺ + K⁺)-stimulated ATPase sequence is consistent with the existence of an enzyme-acyl-phosphate bond of high internal energy with respect to that of ADP.

On the other hand, the hydroxylamine-resistant nature of the phospho-enzyme in the Mg²⁺-ATPase sequence suggests the existence of a non-acyl type of enzyme phosphate bond with low internal energy relative to that of ADP.     

Functional regulation of reconstituted Na, K-ATPase by protein kinase A phosphorylation

Reconstituted Na⁺,K⁺-ATPase from either pig kidney or shark rectal glands was phosphorylated by cAMP dependent protein kinase, PKA. The stoichiometry was 0.9 mole P_i/mole -subunit in the pig kidney enzyme and 0.2 mol P_i/mol -subunit in the shark enzyme. In shark Na⁺,K⁺-ATPase PKA phosphorylation increased the maximum hydrolytic activity for cytoplasmic Na⁺ activation and extracellular K⁺ activation without affecting the apparent K_m values. In contrast, no significant functional effect after PKA phosphorylation was observed in pig kidney Na⁺,K⁺-ATPase.     

盐地碱蓬叶片液泡膜H+-ATPase B亚基的克隆及盐胁迫下表达分析

植物液泡膜H -ATPase在建立跨液泡膜质子梯度、促进液泡Na 区域化、提高植物耐盐性方面发挥着重要作用.本实验从盐生植物盐地碱蓬(Suaeda salsa L.)cDNA文库分离到碱蓬叶片液泡膜H -ATPase B亚基cDNA克隆.测序表明该基因长达1 974 bp,开放阅读框有1 470 bp编码489个氨基酸,含有一个保守的ATP结合位点,其蛋白分子量约为54.29 kD.Northem及Western印迹表明盐地碱蓬液泡膜H -ATPase B亚基表达明显受NaCl胁迫诱导,并且在NaCl胁迫下,B亚基在转录及翻译水平上与液泡膜H -ATPase c亚基存在协同作用.盐胁迫下,盐地碱蓬液泡H -ATPase B亚基与c亚基的协同表达增加了液泡H -ATPase的数量,从而提高了液泡H -ATPase活性,为碱蓬叶片液泡Na 区域化提供了动力,最终提高了碱蓬植株的耐盐性.     

Mechanisms of Saccharomyces Cerevisiae PMA1 H+-ATPase inactivation by Fe2+, H2O2 and Fenton reagents

Although considerably more oxidation-resistant than other P-type ATPases, the yeast PMA1 H⁺-ATPase of Saccharomyces cerevisiae SY4 secretory vesicles was inactivated by H₂O₂, Fe²⁺, Fe- and Cu-Fenton reagents. Inactivation by Fe²⁺ required the presence of oxygen and hence involved auto-oxidation of Fe²⁺ to Fe³⁺. The highest Fe^2- (100 μM) and H₂O₂ (100 mM) concentrations used produced about the same effect. Inactivation by the Fenton reagent depended more on Fe²⁺ content than on H₂O₂ concentration, occurred only when Fe²⁺ was added to the vesicles first and was only slightly reduced by scavengers (mannitol, Tris, NaN₃, DMSO) and by chelators (EDTA, EGTA, DTPA, BPDs, bipyridine, 1, 10-phenanthroline). Inactivation by Fe- and Cu- Fenton reagent was the same; the identical inactivation pattern found for both reagents under anaerobic conditions showed that both reagents act via OH^·. The lipid peroxidation blocker BHT prevented Fenton-induced rise in lipid peroxidation in both whole cells and in isolated membrane lipids but did not protect the H⁺-ATPase in secretory vesicles against inactivation. ATP partially protected the enzyme against peroxide and the Fenton reagent in a way resembling the protection it afforded against SH-specific agents. The results indicate that Fe²⁺ and the Fenton reagent act via metal-catalyzed oxidation at specific metal-binding sites, very probably SH-containing amino acid residues. Deferrioxamine, which prevents the redox cycling of Fe²⁺, blocked H⁺-ATPase inactivation by Fe²⁺ and the Fenton reagent but not that caused by H₂O₂, which therefore seems to involve a direct non-radical attack. Fe-Fenton reagent caused fragmentation of the H⁺-ATPase molecule, which, in Western blots, did not give rise to defined fragments bands but merely to smears.     

不同家系凡纳滨对虾对低盐度的适应能力及其适应机理

为研究不同遗传背景的凡纳滨对虾(Litopenaeus vannamei)在对盐度的适应能力上具有明显的差异的机理, 比较了30个凡纳滨对虾家系在3个不同盐度水体(5‰、20‰和30‰)中饲养30d后的生长性状。研究结果证实了不同家系对虾在不同盐度条件下的生长性能和适应能力存在显著差异。研究进一步对比分析了各盐度条件下不同家系间生理代谢、ATP含量及ATP合成关键酶酶活力的差异, 并检测了不同家系凡纳滨对虾鳃Na+/K+-ATPase、Ca2+-ATPase酶活水平。结果发现盐度适应力差的对虾家系的Na+/K+-ATPase、Ca2+-ATPase酶活力较弱, 这可能是由于其机体能量供给不足所导致。此外, 研究以血浆中皮质醇浓度为指标, 对比了不同盐度下不同对虾家系的机体应激水平, 结果显示盐度适应力差的对虾家系在经30d饲养后仍处于应激状态。综合研究结果得出, 不同遗传背景的对虾对盐度的适应能力不同, 可能是由其机体代谢、离子转运及能量合成能力所决定。     

Effects of Tetraethylammoniumchloride (TEA), Vanadate, and Alkali Ions on the Lateral Leaflet Movement Rhythm of Desmodium motorium (Houtt.) Merr

The period (∼3-5 min) of the ultradian rhythm of the lateral leaflet movement of Desmodium motorium is strongly lengthened (≤30-40%) by the K⁺ channel blocker tetraethylammoniumchloride (20, 30, and 40 mM) and vanadate (0.5 and 1 mM), which is an effective inhibitor of the plasma membrane-bound H⁺ pump. The alkali ions K⁺, Na⁺, Rb⁺, and Cs⁺ (10-40 mM) shorten the period only slightly (≤ 10-15%). Li⁺ (5-30 mM), however, increases the period of the leaflet rhythm drastically (≤80%). We concluded that the plasmalemma-H⁺-ATP-ase-driven K⁺ transport through K⁺ channels is an essential component of the ultradian oscillator of Desmodium, as has been proposed for the circadian oscillator.