NH4+-stimulated and -inhibited components of K+ transport in rice (Oryza sativa L.)

The disruption of K(+) transport and accumulation is symptomatic of NH(4)(+) toxicity in plants. In this study, the influence of K(+) supply (0.02-40 mM) and nitrogen source (10 mM NH(4)(+) or NO(3)(-)) on root plasma membrane K(+) fluxes and cytosolic K(+) pools, plant growth, and whole-plant K(+) distribution in the NH(4)(+)-tolerant plant species rice (Oryza sativa L.) was examined. Using the radiotracer (42)K(+), tissue mineral analysis, and growth data, it is shown that rice is affected by NH(4)(+) toxicity under high-affinity K(+) transport conditions. Substantial recovery of growth was seen as [K(+)](ext) was increased from 0.02 mM to 0.1 mM, and, at 1.5 mM, growth was superior on NH(4)(+). Growth recovery at these concentrations was accompanied by greater influx of K(+) into root cells, translocation of K(+) to the shoot, and tissue K(+). Elevating the K(+) supply also resulted in a significant reduction of NH(4)(+) influx, as measured by (13)N radiotracing. In the low-affinity K(+) transport range, NH(4)(+) stimulated K(+) influx relative to NO(3)(-) controls. It is concluded that rice, despite its well-known tolerance to NH(4)(+), nevertheless displays considerable growth suppression and disruption of K(+) homeostasis under this N regime at low [K(+)](ext), but displays efficient recovery from NH(4)(+) inhibition, and indeed a stimulation of K(+) acquisition, when [K(+)](ext) is increased in the presence of NH(4)(+).     

K+-stimulated p-nitrophenyl phosphatase is not a partial reaction of the gastric (H+ + K+)-transporting ATPase. Evidence supporting a new model for the univalent-cation-transporting ATPase systems.

Studies with intact and lysed gastric microsomal vesicles demonstrate that there are two pNPP (p-nitrophenyl phosphate)-and one ATP-hydrolytic sites within the gastric H+, K+-ATPase [(H+ + K+)-transporting ATPase] complex. Whereas the ATPase site is located exclusively on the vesicle exterior, the pNPPase sites are distributed equally on both sides of the bilayer. Competition by ATP for the pNPPase reaction on the vesicle exterior suggests that both ATP and pNPP are hydrolysed at the same catalytic site present at the outside surface of the intact vesicles. However, a biphasic inhibition of the K+-pNPPase (K+-stimulated pNPPase) by ATP in the lysed vesicles suggest the pNPPase site of the vesicle interior to have very low affinity (Ki approximately equal to 1.2 mM) for ATP compared with the vesicle exterior (Ki approximately equal to 0.2 mM). Studies with spermine, which competes with K+ for the K+-pNPPase reaction without inhibiting the H+, K+-ATPase, suggest there are two separate K+ sites for the pNPPase reaction and another distinct K+ site for the ATPase reaction. In contrast with the K+ site for the ATPase, which is located opposite to the catalytic site across the bilayer, both the K+ and the catalytic site for the pNPPase are located on the same side. The data clearly demonstrate that the pNPPase is not a manifestation of the phosphatase step of the total H+, K+-ATPase reaction. The K+-pNPPase associated with the Na+, K+-ATPase also has properties strikingly similar to the gastric K+-pNPPase system, suggesting a resemblance in the basic operating principle of the two ion-transporting enzymes. A unified model has been proposed to explain the present data and many other observations reported in the literature for the ATPase-mediated transport of univalent cations.     

Gill (Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in the gilthead bream (Sparus auratus L.)

1. Gilthead gill 10(-3) M ouabain-inhibited (Na+ + K+)-ATPase and 10(-2) M ouabain-insensitive Na+-ATPase require the optimal conditions of pH 7.0, 160 mM Na+, 20 mM K+, 5 mM MgATP and pH 4.8-5.2, 75 mM Na+, 2.5 mM Mg2+, 1.0 mM ATP, respectively. 2. The main distinctive features between the two activities are confirmed to be optimal pH, the ouabain-sensitivity and the monovalent cation requirement, Na+ plus another cationic species (K+, Rb+, Cs+, NH4+) in the (Na+ + K+)-ATPase and only one species (Na+, K+, Li+, Rb+, Cs+, NH4+ or choline+) in the Na+-ATPase. 3. The aspecific Na+-ATPase activation by monovalent cations, as well as by nucleotide triphosphates, opposed to the (Na+ + K+)-ATPase specificity for ATP and Na+, relates gilthead gill ATPases to lower organism ATPases and differentiates them from mammalian ones. 4. The discrimination between the two activities by the sensitivity to ethacrynic acid, vanadate, furosemide and Ca2+ only partially agrees with the literature. 5. Present findings are viewed on the basis of the ATPase's presumptive physiological role(s) and mutual relationship.     

(Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.)

1. Sea bass kidney microsomal preparations contain two Mg2+ dependent ATPase activities: the ouabain-sensitive (Na+ + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na+ + K+)-ATPase under the optimal conditions of pH 7.0, 100 mM Na+, 25 mM K+, 10 mM Mg2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 mumol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH 6.0, 40 mM Na+, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 mumol Pi/mg protein per hr. 2. The (Na+ + K+)-ATPase is specifically inhibited by ouabain and vanadate; the Na+-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca2+. 3. The (Na+ + K+)-ATPase is specific for ATP and Na+, whereas the Na+-ATPase hydrolyzes other substrates in the efficiency order ATP greater than GTP greater than CTP greater than UTP and can be activated also by K+, NH4+ or Li+. 4. Minor differences between the two activities lie in the affinity for Na+, Mg2+, ATP and in the thermosensitivity. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na+ transport and, on the other, a distinct modulation in vivo in different tissues.     

(Ca + Mg)-stimulated ATPase activity of a rat brain microsomal preparation.

ATPase activity of freshly prepared brain microsomes was stimulated 20% when 0.1 mm CaCl₂ was added in the presence of a “saturating” concentration of MgCl₂ (4 mm). This (Ca + Mg)-stimulated activity declined rapidly on storage. Treatment of the microsomes with 0.12% deoxycholate in 0.15 m KCl, followed by centrifugation and resuspension in sucrose, produced a preparation both stable on storage at ?15 °C and with an increased stimulation in the presence of CaCl₂. SrCl₂ was more effective than CaCl₂, but BaCl₂ was a poor activator. KCl and NaCl stimulated the (Ca + Mg)-ATPase activity by reducing substrate (ATP) inhibition. The K_m for ATP was 0.1 mm, a third that of the Mg-ATPase. CTP, ITP, and GTP could not substitute for ATP, although they were fair substrates for the Mg-ATPase. The energy of activation of the (Ca + Mg)-ATPase was 21 kcal, nearly twice that of the Mg-ATPase. After sucrose density-gradient centrifugation of the microsomal preparation, the (Ca + Mg)-ATPase activity was distributed with the (Na + K)-ATPase and not with the mitochondrial marker succinic dehydrogenase. Studies with ouabain, oligomycin, and azide distinguished the (Ca + Mg)-stimulated ATPase from (Na + K)- and mitochondrial ATPases. Sensitivity to ruthenium red suggested a link to Ca transport, although the microsomal ⁴⁵Ca accumulating system was much more sensitive to the inhibitor than was this ATPase activity.     

Two molecular forms of (Na+ + K+)-stimulated ATPase in brain. Separation, and difference in affinity for strophanthidin.

The brain contains two distinct molecular forms of the (Na,K)-ATPase (sodium and potassium ion-stimulated adenosine triphosphatase). They can be resolved by gel electrophoresis in sodium dodecyl sulfate, and can be identified by sodium-dependent, potassium-sensitive phosphorylation by [gamma-32P]ATP. They are present in the brain of every animal species examined, while only one molecular form is detected in the other organs examined. They are located in different kinds of cells within the brain, and can be physically separated while fully active by gentle tissue fractionation procedures. One is the only (Na,K)-ATPase of brain non-neuronal cells (astrocytes), while the other is the only (Na,K)-ATPase of axolemma (plasma membrane of myelinated axons). They differ in at least one kinetic parameter: the affinity for the specific inhibitor strophanthidin. They have similar one-dimensional peptide maps, but differ in their sensitivity to digestion by trypsin and in the number or reactivity of sulfhydryl groups. It is anticipated that they will be found to play functionally different roles in the complex ion transport mechanisms of the brain.     

Osmoregulation in Lilium pollen grains occurs via modulation of the plasma membrane H+ ATPase activity by 14-3-3 proteins

To allow successful germination and growth of a pollen tube, mature and dehydrated pollen grains (PGs) take up water and have to adjust their turgor pressure according to the water potential of the surrounding stigma surface. The turgor pressure of PGs of lily (Lilium longiflorum) was measured with a modified pressure probe for simultaneous recordings of turgor pressure and membrane potential to investigate the relation between water and electrogenic ion transport in osmoregulation. Upon hyperosmolar shock, the turgor pressure decreased, and the plasma membrane (PM) hyperpolarizes in parallel, whereas depolarization of the PM was observed with hypoosmolar treatment. An acidification and alkalinization of the external medium was monitored after hyper- and hypoosmotic treatments, respectively, and pH changes were blocked by vanadate, indicating a putative role of the PM H(+) ATPase. Indeed, an increase in PM-associated 14-3-3 proteins and an increase in PM H(+) ATPase activity were detected in PGs challenged by hyperosmolar medium. We therefore suggest that in PGs the PM H(+) ATPase via modulation of its activity by 14-3-3 proteins is involved in the regulation of turgor pressure.     

Determination of turgor pressure in Bacillus subtilis: a possible role for K+ in turgor regulation

The effects of hypersaline treatment (osmotic upshock) on cell water relations were examined in the Gram-positive bacterium Bacillus subtilis using particle size analysis. Application of the Boyle-van't Hoff relationship (cell volume versus reciprocal of external osmolality) permitted direct determination of turgor pressure, which was approximately 0.75 osmol kg-1 (1.9 MPa) in exponentially growing bacteria in a defined medium. The abolition of turgor pressure immediately after upshock and the subsequent recovery of turgor were investigated. Recovery of turgor was K+ dependent. Calculation of turgor by an alternative method involving spectrophotometric analysis of shrinkage gave somewhat lower estimates of turgor pressure.     

Frosthardiness development in Pinus sylvestris: The involvement of a K+-stimulated Mg2+-dependent ATPase from purified plasma membranes of pine

Purified plasma membranes were prepared from needles of Scots pine (Pinus sylvestris L., Provenance Sodra Ydre), and the behavior of a K+ stimulated Mg2+-dependent ATPase was studied. The microsomal fraction (1000-30 000 g) was used for partition in a dextranpolyethylene glycol two-phase system. Both upper and lower phases were washed twice with new upper and lower phase, respectively, yielding the fractions U3 and L3. After staining with STA (silicotungstic acid) and examining with an electron microscope, an enrichment of the plasma membranes in the U, phase was seen. This fraction was also enriched in K+ stimulated Mg2+-dependent ATPase. The ATPase was rather specific towards ATP compared to other nucleosides, and had a pH optimum between 6 and 7. The activity of this ATPase was followed during a frost hardening-dehardening cycle and showed an increased activity towards the end of the hardening period, whereas the activity decreased to normal during the dehardening period.     

(Na+-K+)-stimulated ATPase in Leaves of C4 Plants: Possible Involvement in Active Transport of C4 Acids

Leaves of three C₄ plants, Setaria italica, Pennisetum typhoides,and Amaranthus paniculatus possessed five- to ten-fold higheractivities of a (Na⁺-K⁺)-dependent ATPase than those of twoC₃ plants, Oryza sativa and Rumex vesicarius. Na⁺-K⁺ ATPasefrom leaves of Amarathus exhibited an optimal pH of 7?5 andan optimal temperature of 35 ?C. It required 40 mM K⁺ and 80mM Na⁺ for maximal activity. Ouabain partially inhibited (Na⁺-K⁺)-dependentATPase activity in leaves of C₄ plants. Ouabain also blockedthe movement of label from initially formed C₄ acids into endproducts in leaves of only C₄ plants, Setaria and Amaranthusbut not in a C₃ plant, Rumex. We propose that Na⁺-K⁺ ATPasemay mediate transfer of energy during active transport of C₄acids from mesophyll into the bundle sheath.     

Intracellular serine protease-4, a new intracellular serine protease activity from Bacillus subtilis

A previously undiscovered intracellular serine protease activity, which we have called intracellular serine protease-4, was identified in extracts of stationary Bacillus subtilis cells, purified 260 fold from the cytoplasmic fraction, and characterized. The new protease was stable and active in the absence of Ca²⁺ ions and hydrolyzed azocasein and the chromogenic substrate carbobenzoxy-carbonyl-alanyl-alanyl-leucyl-p-nitroanilide, but not azocollagen or a variety of other chromogenic substrates. The protease was strongly inhibited by phenylmethylsulfonylfluoride, chymostatin and antipain, but not by chelators, sulfhydryl-reactive agents or trypsin inhibitors. Its activity was stimulated by Ca²⁺ ions and gramicidin S; its pH and temperature optima were 9.0 and 37°C, respectively. Although intracellular serine protease-4 was immunochemically distinct from intracellular serine protease-1, it was absent from a mutant in which the gene encoding the latter was disrupted.     

Expression of a P-type Ca(2+)-transport ATPase in Bacillus subtilis during sporulation

The open reading frame designated yloB in the genomic sequence of Bacillus subtilis encodes a putative protein that is most similar to the typically eukaryotic type IIA family of P-type ion-motive ATPases, including the endo(sarco)plasmic reticulum (SERCA) and PMR1 Ca(2+)-transporters, located respectively in the SERCA and the Golgi apparatus. The overall amino acid sequence is more similar to that of the Pmr1s than to the SERCAs, whereas the inverse is seen for the 10 amino acids that form the two Ca(2+)-binding sites in SERCA. Sporulating but not vegetative B. subtilis cells express the predicted protein, as shown by Western blotting and by the formation of a Ca(2+)-dependent phosphorylated intermediate. Half-maximal activation of phosphointermediate formation occurred at 2.5 microM Ca(2+). Insertion mutation of the yloB gene did not affect the growth of vegetative cells, did not prevent the formation of viable spores, and did not significantly affect 45Ca accumulation during sporulation. However, spores from knockouts were less resistant to heat and showed a slower rate of germination. It is concluded that the P-type Ca(2+)-transport ATPase from B. subtilis is not essential for survival, but assists in the formation of resistant spores. The evolutionary relationship of the transporter to the eukaryotic P-type Ca(2+)-transport ATPases is discussed.