Hemolytic action of staphylococcal α-hemolysin on human erythrocytes in a Na+- and K+-containing suspending fluid

The hemolytic action of staphylococcal α-hemolysin on human erythrocytes was studied. In a Na⁺- and K⁺-containing suspending fluid α-hemolysin caused a rapid potassium release from the cells, which probably preceded hemoglobin release, and an influx of sodium. After storage of the cells for 4 days the same dose of α-hemolysin lyzed more cells. Lysis increased with the potassium concentration in the suspending fluid, while no correlation could be demonstrated between lysis and intracellular potassium concentration nor between lysis and potassium leakage from the cells. α-Hemolysin stimulated Mg²⁺-activated ATPase activity but did not change Na⁺-K⁺-Mg²⁺-ATPase activity. α-Hemolysin may cause increased membrane permeability for sodium ions and still more so for potassium ions, which may lead to hemolysis through swelling of the cells.     

Plasma membrane H+-ATPase in sorghum roots as affected by potassium deficiency and nitrogen sources

We studied the influence of inorganic nitrogen sources (NO₃ ^? or NH₄ ⁺) and potassium deficiency on expression and activity of plasma membrane (PM) H⁺-ATPase in sorghum roots. After 15 d of cultivation at 0.2 mM K⁺, the plants were transferred to solutions lacking K⁺ for 2 d. Then, K⁺ depletion assays were performed in the presence or absence of vanadate. Further, PMs from K⁺-starved roots were extracted and used for the kinetic characterization of ATP hydrolytic activity and the immunodetection of PM H⁺-ATPase. Two major genes coding PM H⁺-ATPase (SBA1 and SBA2) were analyzed by real-time PCR. PM H⁺-ATPase exhibited a higher V_max and K_m in NH₄ ⁺-fed roots compared with NO₃ ^? -fed roots. The optimum pH of the enzyme was slightly lower in NO₃ ^? -fed roots than in NH₄ ⁺-fed roots. The vanadate sensitivity was similar. The expressions of SBA1 and SBA2 increased in roots grown under NH₄ ⁺. Concomitantly, an increased content of the enzyme in PM was observed. The initial rate of K⁺ uptake did not differ between plants grown with NO₃ ^? or NH₄ ⁺, but it was significantly reduced by vanadate in NH₄ ⁺-grown plants.     

ATPase activities in partially purified membranes of Acetabularia

Partly purified membranes (with plasmalemma material) of Acetabularia mediterranea were studied with respect to ATPase activity in alkali- and Ca⁺⁺-free media and its sensitivity to pH (5 – 9), oligomycin (200 ?g/mg protein), 100 ?M N-N′-dicyclohexylcarbodiimide (DCCD), and 50 ?M vanadate. Besides activities which may originate from mitochondrial H⁺ ATPase (oligomycin-sensitive, alkaline pH optimum) and tonoplast H⁺ ATPase (DCCD-sensitive, pH optimum 7.5), there is ATPase activity with a pH optimum around pH 6.5, sensitive to vanadate and insensitive to DCCD. These results strongly suggest that the electrogenic Cl^? pump in the plasmalemma of Acetabularia is an ATPase. Effects of Mg⁺⁺, Mg-ATP, ADP, GTP, UTP, CTP and HCO₃ ^? versus Cl^? on this ATPase activity are described.     

Mechanisms of Na+ uptake,ammonia excretion,and their potential linkage in native Rio Negro tetras (Paracheirodon axelrodi,Hemigrammus rhodostomus,and Moenkhausia diktyota)

Mechanisms of Na⁺ uptake, ammonia excretion, and their potential linkage were investigated in three characids (cardinal, hemigrammus, moenkhausia tetras), using radiotracer flux techniques to study the unidirectional influx (J _in), efflux (J _out), and net flux rates (J _net) of Na⁺ and Cl^?, and the net excretion rate of ammonia (J _Amm). The fish were collected directly from the Rio Negro, and studied in their native “blackwater” which is acidic (pH 4.5), ion-poor (Na⁺, Cl^? ~20 µM), and rich in dissolved organic matter (DOM 11.5 mg C l^?1). J _in ^Na , J _in ^Cl , and J _Amm were higher than in previous reports on tetras obtained from the North America aquarium trade and/or studied in low DOM water. In all three species, J _in ^Na was unaffected by amiloride (10^?4 M, NHE and Na⁺ channel blocker), but both J _in ^Na and J _in ^Cl were virtually eliminated (85–99 % blockade) by AgNO₃ (10^?7 M). A time course study on cardinal tetras demonstrated that J _in ^Na blockade by AgNO₃ was very rapid (<5 min), suggesting inhibition of branchial carbonic anhydrase (CA), and exposure to the CA-blocker acetazolamide (10^?4 M) caused a 50 % reduction in J _in ^Na _.. Additionally, J _in ^Na was unaffected by phenamil (10^?5 M, Na⁺ channel blocker), bumetanide (10^?4 M, NKCC blocker), hydrochlorothiazide (5 × 10^?3 M, NCC blocker), and exposure to an acute 3 unit increase in water pH. None of these treatments, including partial or complete elimination of J _in ^Na (by acetazolamide and AgNO₃ respectively), had any inhibitory effect on J _Amm. Therefore, Na⁺ uptake in Rio Negro tetras depends on an internal supply of H⁺ from CA, but does not fit any of the currently accepted H⁺-dependent models (NHE, Na⁺ channel/V-type H⁺-ATPase), or co-transport schemes (NCC, NKCC), and ammonia excretion does not fit the current “Na⁺/NH₄ ⁺ exchange metabolon” paradigm. Na⁺, K⁺-ATPase and V-type H⁺-ATPase activities were present at similar levels in gill homogenates, Acute exposure to high environmental ammonia (NH₄Cl, 10^?3 M) significantly increased J _in ^Na , and NH₄ ⁺ was equally or more effective than K⁺ in activating branchial Na⁺,(K⁺) ATPase activity in vitro. We propose that ammonia excretion does not depend on Na⁺ uptake, but that Na⁺ uptake (by an as yet unknown H⁺-dependent apical mechanism) depends on ammonia excretion, driven by active NH₄ ⁺ entry via basolateral Na⁺,(K⁺)-ATPase.     

Arbuscular mycorrhizae improve low temperature tolerance in cucumber via alterations in H2O2 accumulation and ATPase activity

The combined effects of arbuscular mycorrhizal fungi (AMF) and low temperature (LT) on cucumber plants were investigated with respect to biomass production, H₂O₂ accumulation, NADPH oxidase, ATPase activity and related gene expression. Mycorrhizal colonization ratio was gradually increased after AMF-inoculation. However, LT significantly decreased mycorrhizal colonization ability and mycorrhizal dependency. Regardless of temperature, the total fresh and dry mass, and root activity of AMF-inoculated plants were significantly higher than that of the non-AMF control. The H₂O₂ accumulation in AMF-inoculated roots was decreased by 42.44 % compared with the control under LT. H₂O₂ predominantly accumulated on the cell walls of apoplast but was hardly detectable in the cytosol or organelles of roots. Again, NADPH oxidase activity involved in H₂O₂ production was significantly reduced by AMF inoculation under LT. AMF-inoculation remarkably increased the activities of P-type H⁺-ATPase, P-Ca²⁺-ATPase, V-type H⁺-ATPase, total ATPase activity, ATP concentration and plasma membrane protein content in the roots under LT. Additionally, ATP concentration and expression of plasma membrane ATPase genes were increased by AMF-inoculation. These results indicate that NADPH oxidase and ATPase might play an important role in AMF-mediated tolerance to chilling stress, thereby maintaining a lower H₂O₂ accumulation in the roots of cucumber.     

Evidence for an electrogenic adenosine-triphosphatase in Hevea tonoplast vesicles

The function of the Mg-dependent ATPase of Hevea tonoplast in active proton transport was investigated by using a purified tonoplast fraction containing tightly sealed vesicles. In the used experimental conditions, the uptake of [¹⁴C]triphenylmethyl-phosphonium ion ([¹⁴C]TPMP⁺) and [³H]tetraphenyl-phosphonium ion ([³H]TPP⁺) by the vesicles indicated a transmembrane potential difference, negative inside. In parallel, the uptake of [¹⁴C]methylamine into the vesicles monitored a transmembrane pH gradient, interior acid. The addition of 5 mM Mg-ATP markedly depolarized the membrane and increased the magnitude of trnasmembrane pH gradient. These ATP-driven events were substrate specific for Mg-ATP. They were strongly inhibited by ATPase inhibitors such as N, N′-dicyclohexylcar-bodiimide. They were completely eliminated by proton conductors such as carbonylcyanide-p-trifluoromethoxy-phenylhydrazone and 5-chloro-3-tert-butyl-2′-chloro-4-nitro-salicylanilide. They depended on the pH of the medium, the maximum being reached at about pH 7.0. These data provide in vitro evidence that the Mg-ATPase localized at tonoplast level is an electrogenic pump. They are consistent with the hypothesis that an electrogenic H⁺ pump is catalyzed by the tonoplast ATPase of higher plants.     

Ion transport in broad bean leaf mesophyll under saline conditions

Main conclusion

Salt stress reduces the ability of mesophyll tissue to respond to light. Potassium outward rectifying channels are responsible for 84 % of Na ⁺ induced potassium efflux from mesophyll cells. Modulation in ion transport of broad bean (Vicia faba L.) mesophyll to light under increased apoplastic salinity stress was investigated using vibrating ion-selective microelectrodes (the MIFE technique). Increased apoplastic Na⁺ significantly affected mesophyll cells ability to respond to light by modulating ion transport across their membranes. Elevated apoplastic Na⁺ also induced a significant K⁺ efflux from mesophyll tissue. This efflux was mediated predominately by potassium outward rectifying channels (84 %) and the remainder of the efflux was through non-selective cation channels. NaCl treatment resulted in a reduction in photosystem II efficiency in a dose- and time-dependent manner. In particular, reductions in Fv′/Fm′ were linked to K⁺ homeostasis in the mesophyll tissue. Increased apoplastic Na⁺ concentrations induced vanadate-sensitive net H⁺ efflux, presumably mediated by the plasma membrane H⁺-ATPase. It is concluded that the observed pump’s activation is essential for the maintenance of membrane potential and ion homeostasis in the cytoplasm of mesophyll under salt stress.     

Proton translation in submitochondrial vesicles

An investigation of proton translocation in submitochondrial vesicles from rat liver has been made under simple experimental conditions. Choline chloride was used both as the oxidizable substrate and the ionic medium for the measurement of activity during oxygen pulse experiments:

1. The passive permeability measured from the decay of proton efflux after an oxygen pulse could be described by a first-order equation. An H⁺/O ratio of 2·5 was obtained for choline oxidation in the presence of oligomycin and/or MgCl₂. Oligomycin decreased the passive proton permeability and respiration, concomitant with an increase in proton uptake. Respiratory control was directly related to the passive proton permeability and inversely related to the magnitude of the proton gradient. The decreased respiration and passive permeability reflecting respiratory control is most evident in the pH rang 5·8–7·5.
2. Preparation of submitochondrial vesicles in the presence of EDTA resulted in proton production during an oxygen pulse given at alkaline pH. Cytochromec enhanced proton uptake by approximately 1 H⁺/cytochromec, but only in the presence of Triton X-100. These results are indicative of the asymmetric behavior of the coupling membrane and provide direct evidence of the participation of electron transport components in proton translocation.

     

Enzymatic activity of basidiomycetes

In mycelial acetone powder of the basidiomyceteOudemansiella mucida, the presence of enzymatic systems of NADH₂-oxidase and-dehydrogenase type and of NAD⁺-dependent alcoholdehydrogenase (using ethanol as substrate) has been detected. NADH₂-oxidase has its optimal pH value in the region of 6,0 and is not too active. NADH₂-dehydrogenase which reduces 2,6-dichlorophenolindophenol is very active, may be eluted from the mycelial preparation with water and its adequate pH is in the region of 6,2. On the other hand, NADH₂-dehydrogenase reducing triphenyltetrazolium chloride is non-eluable with water, its activity depends on the presence of menadione and its adequate pH lies in the region of 7,4.     

Localization and phenotypic expression of a Tol marker in Citrobacter freundii

Among colicin-A-tolerant mutants of Citrobacter freundii we characterized some as Tol-2 mutants. The Tol-2 mutation results in insensitivity to bacteriocin S6 and an enhanced sensitivity to deoxycholate (DOC), ethylenediaminetetraacetic acid (EDTA) and to ampicillin. The Tol-2 mutation was mapped close near gal and the gene order pro-tol-gal was established in crosses between C. freundii Hfr tol ⁺ donor strains and C. freundii tol ^- acceptor strains. In these crosses a difference was observed in phenotypic expression of the pleiotropic properties of this tol ⁺ gene. Expression of resistance to DOC is substantially slower than the expression of EDTA-resistance. This phenomenon may play a disturbing role in those studies on cell envelope mutants, in which resistance to DOC is used as a selected marker. The differences in expression of DOC-and EDTA-resistance are discussed.     

Effect of long-term shock-avoidance training on certain biochemical constituents of rat brain

A few constituents of the brain of rats subjected to long-term shock avoidance training in the jump-box were investigated. The effects of training and that of the concomitant stress on these constituents were differentially examined. Brain DNA increased in both the trained rats and those subjected to stress. The total RNA showed a specific decrease in the trained rats. The total protein content remained unaffected, while the content of soluble protein showed a greater increase in the trained rats compared to that in animals subjected to stress. The Na⁺, K⁺-ATPase activity in the brain decreased in the trained rats, whereas the glial carbonic anhydrase activity increased in the stressed as well as the trained rats. No change was found in the activities of Mg⁺⁺-ATPase and pseudocholinesterase in the experimental groups.