Uptake hydrogenase in Rhizobium and nodule leghemoglobin in cow pea miscellany hosts

Two effective strains of green gram rhizobia S24 (slow growing and Hup⁺) and M11 (fast growing and Hup^-) were tested for leghemoglobin production in nodules and effectivity on six species of cow pea miscellany hosts. Both strains nodulate green gram [Vigna radiata (L.) (Wilczek)], black gram [Vigna mungo (L.) (Hepper)], cow pea [Vigna unguiqulata (L.)], moth bean [Vigna aconitifolia (Jacq.) (Marechel)], Cluster bean [Cyamopsis tetragonoloba (L.) (Taub.)] and pigeon pea [Cajanus cajan (L.)]. In all these hosts, nodules formed by strain M11 contained 1.5 to 2 times more leghemoglobin than the nodules formed by strain S24. Gel electrophoresis of nodule contents of different host species showed a high concentration of a fast-moving ferricoxy leghemoglobin in the nodules of plants inoculated with strain M11 as compared to that of strain S24. Strain M11, however, was relatively less effective than strain S24 on black gram, cow pea and moth bean and was at par with the later on green gram, cluster bean and pigeon pea. Hydrogen recycling ability of the strain S24 was observed in nodules of all the host species. The effective functioning of strain S24 at low levels of leghemoglobin suggests an involvement of recycling hydrogenase in maintaining an appropriate oxidation-reduction potential in nodules.Abbreviations Lb Leghemoglobin - Cvr cultivar     

Rhizobium strains expressing uptake hydrogenase in different host species of cowpea miscellany

Uptake hydrogenase activity in nodules of green gram (Vigna radiata (L.) (Wilczek)), black gram (Vigna mungo (L.) (Hepper)), cowpea (Vigna unguiculata (L.) and cluster bean (Cyamopsis tetragonoloba (L.) (Taub.)), formed with two Hup⁺ (S24 and CT2014) and one Hup⁻ (M11)Rhizobium strains, was determined at different levels of external H2 in air atmosphere. Nodules of all the 4 host species formed by inoculation with strains S24 and CT2014, showed H2 uptake but not those formed with strain M11. H₂ uptake rates were higher in 1 and 2% H₂ in air atmosphere (v/v) than at 5 or 10% levels in all the host species. Variations in the relative rates of H₂ uptake were observed both, due to host species as well as due toRhizobium strains. However, no host dependent complete repression of the expression of H₂ uptake activity was observed in nodules of any of the host species formed with Hup⁺ strains.     

Hydrogen production and uptake by pea nodules as affected by strains of Rhizobium leguminosarum

Hydrogen evolution from root nodules has been reported to make N₂ fixation by some legume-Rhizobium symbiotic systems inefficient. We have surveyed the extent of H₂ evolution and estimated relative efficiencies of nodules of Austrian winter peas formed by 15 strains of R. leguminosarum. Their rates of H₂ evolution in air were about 30% of the rates of H₂ evolution under an atmosphere in which N₂ was replaced by Ar. Relative efficiency values based on C₂H₂ reduction rates ranged from 0.55 to 0.80. With some of the strains, hydrogenase activities were demonstrated in intact nodules and in bacteroids, but the levels of activity were insufficient to recycle all the H₂ evolved by the nitrogenase system. In both intact nodules and bacteroids the hydrogenase is less sensitive to O₂ damage than the nitrogenase system, so H₂ uptake capacity was observed in intact nodules by suppressing the nitrogenase-dependent H₂ evolution with an atmosphere containing a high O₂ concentration, and in bacteroids by using aerobically prepared bacteroid suspensions. The hydrogenase activity of both was dependent on O₂ consumption. A K _mfor H₂ of near 4 M was determined in suspension of bacteroids from nodules formed by strains 128C53 and 128C56.     

Phloem loading in Vicia faba leaves: Effect of N-ethylmaleimide and parachloromercuribenzenesulfonic acid on H+ extrusion,K+ and sucrose uptake

The effects of a penetrating (NEM) and a non-penetrating (PCMBS) sulfhydryl-specific reagent on proton extrusion, ⁸⁶Rb and [U-¹⁴C]sucrose uptake by Vicia faba leaves have been studied. Proton extrusion was strongly or completely inhibited by 0.1 mM NEM. ⁸⁶Rb and [U-¹⁴C]sucrose uptake were markedly reduced by NEM concentrations equal to or higher than 0.5 mM. Under our experimental conditions, PCMBS (1 mM) exerted a strong inhibition on [¹⁴C]sucrose uptake but did not inhibit proton extrusion and ⁸⁶Rb uptake. The sensitivity of phloem loading to PCMBS is thought to be a consequence of sugar-carrier blockage and not of inhibition of the proton pump.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - DES diethylstilbestrol - DCCD dicyclohexylcarbodiimide - FC Fusicoccin - NEM N-ethylmaleimide - PCMBS p-chloromercuribenzenesulfonic acid     

Close coupling between extrusion of H+ and uptake of K+ by barley roots

Extrusion of H⁺ by intact barley (Hordeum vulgare L.) roots was automatically titrated. Simultaneously, uptake of K⁺ into the roots, transport of K⁺ through the roots, and (as a residual term) accumulation of K⁺ within the root tissue were determined. When no monovalent cation was present in the medium the steady rate of H⁺ release was close to zero. Addition of K⁺ stimulated H⁺ extrusion within less than 1 min. The stimulation of H⁺ release was apparently limited only by the movement of K⁺ through the apoplast of the roots. The steady rate of H⁺ extrusion depended on the availability of external K⁺ and saturated at a K⁺ concentration of about 100 mol· dm^-3. Half-maximum rates of net K⁺ uptake and H⁺ extrusion were reached at a K⁺ concentration of about 10 mol·dm^-3. With (slowly absorbable) sulfate as the only anion present, the stoichoimetry between H⁺ release and net K⁺ uptake was one. In conclusion, the uptake of K⁺ across the plasmalemma of the cells of the root cortex is electrically coupled to H⁺ extrusion.     

The changing sensitivity to auxin of the plasma-membrane H+-ATPase: Relationship between plant development and ATPase content of membranes

The auxin sensitivity of the plasma-membrane H⁺-ATPase from tobacco leaves (Nicotiana tabacum L. cv. Xanthi) depends on the physiological state of the plant (Santoni et al., 1990, Plant Sci. 68, 33–38). Results based on the study of auxin sensitivity according to culture conditions which accelerate or delay tobacco development demonstrate that the highest auxin sensitivity is always associated with the end of the period of induction to flowering. Auxin stimulation of H⁺-translocation activity corresponds to an increase of the apparent ATPase affinity for ATP. The plasma-membrane H⁺-ATPase content, measured with an enzyme-linked immunosorbent assay using a specific anti-H⁺-ATPase antibody, varies according to plant development, and was found to increase by 100% during floral induction. The specific molecular ATPase activity also changes according to plant development; more particularly, the decrease in molecular ATPase activity upto and during the floral-induction period parallels the increase of sensitivity to indole-3-acetic acid.Abbreviations ELISA enzyme-linked immunosorbent assay - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate Authors are grateful to Mrs. Grosclaude (Lab. Virologie, INRA, Jouy-en-Josas, France) and Mrs. Boudon (Lab. Mycoplasmes, INRA, Dijon, France) for support and advice in the preparation of antibodies. This work was supported by grants No. 89/512/6 from the E.P.R of Bourgogne and No. 89 C 0662 from M.R.T.     

Low doses of vanadate and Trigonella synergistically regulate Na+/K+-ATPase activity and GLUT4 translocation in alloxan-diabetic rats

Oral administration of vanadate to diabetic animals have been shown to stabilize the glucose homeostasis and restore altered metabolic pathways. However, vanadate exerts these effects at relatively high doses with several toxic effects. Low doses of vanadate are relatively safe but unable to elicit any antidiabetic effects. The present study explored the prospect of using low doses of vanadate with Trigonella foenum graecum, seed powder (TSP), another antidiabetic agent, and to evaluate their antidiabetic effect in diabetic rats. Alloxan diabetic rats were treated with insulin, vanadate, TSP and low doses of vanadate with TSP for three weeks. The effect of these antidiabetic compounds was examined on general physiological parameters, Na⁺/K⁺ ATPase activity, membrane lipid peroxidation and membrane fluidity in liver, kidney and heart tissues. Expression of glucose transporter (GLUT4) protein was also examined by immunoblotting method in experimental rat heart after three weeks of diabetes induction. Diabetic rats showed high blood glucose levels. Activity of Na⁺/K⁺ ATPase decreased in diabetic liver and heart. However, kidney showed a significant increase in Na⁺/K⁺ ATPase activity. Diabetic rats exhibited an increased level of lipid peroxidation and decreased membrane fluidity. GLUT4 distribution was also significantly lowered in heart of alloxan diabetic rats. Treatment of diabetic rats with insulin, TSP, vanadate and a combined therapy of lower dose of vanadate with TSP revived normoglycemia and restored the altered level of Na⁺/K⁺ ATPase, lipid peroxidation and membrane fluidity and also induced the redistribution of GLUT4 transporter. TSP treatment alone is partially effective in restoring the above diabetes-induced alterations. Combined therapy of vanadate and TSP was the most effective in normalization of altered membrane linked functions and GLUT4 distribution without any harmful side effect.     

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na⁺,K⁺)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg⁻¹ with K_0.5=1.2±0.1 mmol l⁻¹; stimulation by potassium (V=121.0±6.1 U mg⁻¹; K_0.5=2.1±0.1 mmol l⁻¹) and magnesium ions (V=125.3±6.3 U mg⁻¹; K_0.5=1.0±0.1 mmol l⁻¹) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (n_H=2.7) to a rate of V=126.1±4.8 U mg⁻¹ with K_0.5=13.7±0.5 mmol l⁻¹. However, K⁺-phosphatase activity was not stimulated further by K⁺ plus NH₄⁺ ions. Sodium ions (K_I=36.7±1.7 mmol l⁻¹), ouabain (K_I=830.3±42.5 μmol l⁻¹) and orthovanadate (K_I=34.0±1.4 nmol l⁻¹) completely inhibited K⁺-phosphatase activity. The competitive inhibition by ATP (K_I=57.2±2.6 μmol l⁻¹) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K⁺-phosphatase activity corresponds strictly to a (Na⁺,K⁺)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K⁺-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.     

Tributylbenzylammonium (TBBA+)-dependent fusicoccin (FC)-induced H+ extrusion in maize roots: relationship between the stimulating effects of TBBA+ on H+ extrusion and on Cl- efflux

The mechanism of the stimulating effect of lipophilic cations on H⁺ extrusion in maize root segments (Zea mays L.) has been investigated. The measurement of the uptake of [³H]tributylbenzylammonium ([³H]TBBA⁺), the most active lipophilic cation on H⁺ extrusion, indicated that although a relevant fraction of TBBA⁺ taken up by the tissue is adsorbed to cell surfaces, a fraction of the cation enters the cells. However no correlation was observed between the rate of TBBA⁺ uptake and that of H⁺ extrusion. On the other hand, the lipophilic cations active on H⁺ extrusion (TBBA⁺ and dibenzyldimethylammonium (DDA⁺)), in the presence of fusicoccin (FC), induced under the same conditions an efflux of Cl^-, while tetramethylammonium (TMA⁺), inactive on H⁺ extrusion, did not. The stimulation of Cl^- efflux by TBBA⁺ was independent of the anion present in the medium and was inhibited by Na-orthovanadate, an inhibitor of plasma membrane ATPase and of TBBA⁺-induced H⁺ extrusion. These results suggest that the stimulation of H⁺ extrusion by TBBA⁺ depends on its effect on Cl^- efflux rather than on its penetration into the cells.Abbreviations DDA⁺ dibenzyldimethylammonium - FC fusicoccin - 3-O-MG 3-O-methyl glucose - PD transmembrane electric potential difference - TBBA⁺ tributylbenzylammonium - TCF tissue concentration factor - TMA⁺ tetramethylammonium - TPB^- tetraphenylboron     

Inositol 1,4,5-trisphosphate may mediate closure of K+ channels by light and darkness in Samanea saman motor cells

Leaflet movements of Samanea saman (Jacq.) Merr. depend in part upon circadian-rhythmic, light-regulated K⁺ fluxes across the plasma membranes of extensor and flexor cells in opposing regions of the leaf-moving organ, the pulvinus. We previously showed that blue light appears to close open K⁺ channels in flexor protoplasts during the dark period (subjective night) (Kim et al., 1992, Plant Physiol 99: 1532–1539). In contrast, transfer to darkness apparently closes open K⁺ channels in extensor protoplasts during the light period (subjective day) (Kim et al., 1993, Science 260: 960–962). We now report that both these channel-closing stimuli increase inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] levels in the appropriate protoplasts. If extensor cells are given a pulse of red light followed by transfer to darkness, channels still apparently close (Kim et al. 1993) but changes in Ins(1,4,5)P₃ levels are complex with an initial decrease under red light followed by accumulation. Neomycin, an inhibitor of polyphosphoinositide hydrolysis, inhibits both blue-light-induced Ins(1,4,5)P₃ production and K⁺-channel closure in flexor protoplasts and both dark-induced Ins(1,4,5)P₃ production and K⁺ channel closure in extensor protoplasts. The G-protein activator, mastoparan, mimics blue light and darkness in that it both increases Ins(1,4,5)P₃ levels and closes K⁺ channels in the appropriate cell type at the appropriate time. These results indicate that phospholipase C-catalyzed hydrolysis of phosphoinositides, possibly activated by a G protein, is an early step in the signal-transduction pathway by which blue light and darkness close K⁺ channels in S. saman pulvinar cells.Abbreviations DiS-C₃-(5) 3,3-dipropylthiadicarbocyanine iodide - F measure change in Dis-C₃-(5) fluorescence - F_o initial Dis-C₃-(5) fluorescence - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PtdIns(4,5)P₂ phosphatidylinositol 4,5-bisphosphate - rbc red blood cell Supported by grants from NSF (IBN 9206179 and MCB 9305154) and U.S.-Israel Binational Agricultural Research and Development Fund (IS-1670-90RC) to R.C.C. We thank the University of Connecticut Biotechnology Center for the use of a fluorescent spectrophotometer.     

Fluorescent labelling of Na+,K+-ATPase in intact cells by use of a fluorescent derivative of ouabain: Salinity and teleost chloride cells

Summary Anthroylouabain, a fluorescent derivative of ouabain, was used to localize Na⁺,K⁺-ATPase in transport epithelia of two species of teleosts. Exposure of the opercular membrane of seawater-adapted tilapia (Oreochromis mossambicus) and the jaw skin of the long-jawed mudsucker (Gillichthys mirabilis) to a 2 M anthroylouabain solution resulted in the appearance of cells stained bright blue. These were deemed to be chloride cells by their large size, distinct morphology and co-localization of DASPEI fluorescence, a mitochondrial stain. Addition of ouabain (1 mM final concentration) greatly decreased anthroylouabain fluorescent staining of chloride cells of seawater-adapted fish. Exposure of opercular membranes from freshwater tilapia to 2 M anthroylouabain did not result in significant staining. Anthroylouabain is therefore a useful vital stain for localizing Na⁺,K⁺-ATPase in chloride cells of seawater-adapted teleosts, and may be useful for fluorescent labelling of ouabain-sensitive Na⁺,K⁺-ATPase in other tissues and species.     

Nitrite reduction in barley-root plastids: Dependence on NADPH coupled with glucose-6-phosphate and 6-phosphogluconate dehydrogenases,and possible involvement of an electron carrier and a diaphorase

Plastids from roots of barley (Hordeum vulgare L.) seedlings were isolated by discontinuous Percoll-gradient centrifugation. Coinciding with the peak of nitrite reductase (NiR; EC 1.7.7.1, a marker enzyme for plastids) in the gradients was a peak of a glucose-6-phosphate (Glc6P) and NADP⁺-linked nitrite-reductase system. High activities of phosphohexose isomerase (EC 5.3.1.9) and phosphoglucomutase (EC 2.7.5.1) as well as glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) were also present in the isolated plastids. Thus, the plastids contained an overall electron-transport system from NADPH coupled with Glc6PDH and 6PGDH to nitrite, from which ammonium is formed stoichiometrically. However, NADPH alone did not serve as an electron donor for nitrite reduction, although NADPH with Glc6P added was effective. Benzyl and methyl viologens were enzymatically reduced by plastid extract in the presence of Glc6P+ NADP⁺. When the plastids were incubated with dithionite, nitrite reduction took place, and ammonium was formed stoichiometrically. The results indicate that both an electron carrier and a diaphorase having ferredoxin-NADP⁺ reductase activity are involved in the electron-transport system of root plastids from NADPH, coupled with Glc6PDH and 6PGDH, to nitrite.Abbreviations Cyt cytochrome - Glc6P glucose-6-phosphate - Glc6PDH glucose-6-phosphate dehydrogenase - MVH reduced methyl viologen - NiR nitrite reductase - 6PG 6-phosphogluconate - 6PGDH 6-phosphogluconate dehydrogenase     

Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: Effects on gill (Na,K)-ATPase α-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‰ 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 K_0.5 = 1.31 ± 0.05 mmol L^− 1. Stimulation of K⁺-phosphatase activity by magnesium (V_max = 125.3 ± 7.5 U mg^− 1; K_0.5 = 2.09 ± 0.06 mmol L^− 1), potassium (V_max = 134.2 ± 6.7 U mg^− 1; K_0.5 = 1.33 ± 0.06 mmol L^− 1) and ammonium ions (V_max = 130.1 ± 5.9 U mg^− 1; K_0.5 = 11.4 ± 0.5 mmol L^− 1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (K_I = 304.9 ± 18.3 μmol L^− 1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the ≈2-fold reduction in K⁺-phosphatase specific activity, Western blotting analysis revealed similar α-subunit expression in gill tissue from shrimps acclimated to 21‰ salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na⁺,K⁺)-ATPase was stimulated by high salinity acclimation.     

Growth inhibition of unicellular and colonial Microcystis strains (Cyanophyceae) by compounds isolated from rice (Oryza sativa) hulls

The algicidal effects of crude and pure rice hull extracts on the growth of Microcystis aeruginosa were investigated using cultured unicellular and colonial strains. Upon treatment with rice hull crude extract (RHE), growth inhibition of unicellular M. aeruginosa was much higher than that of colonial M. aeruginosa. However, purified compounds from the crude extract, β-sitosterol-β-d-glucoside and dicyclohexanyl orizane, powerfully inhibited the growth of colonial M. aeruginosa cells. At the same concentrations, the two compounds were almost equipotent (66% and 80% growth inhibition for colonial M. aeruginosa, respectively; P < 0.05). As rice hulls are readily obtainable, and as extracts show high algicidal activity (targeting colonial algae rather than unicellular organisms) at low concentrations, the results suggest that some pure compounds extracted from rice hulls, such as β-sitosterol-β-d-glucoside and dicyclohexanyl orizane, may serve as environmentally friendly agents for controlling the growth of toxic colonial M. aeruginosa in eutrophic waters.     

B2 receptor-mediated dual effect of bradykinin on proximal tubule Na-ATPase: Sequential activation of the phosphoinositide-specific phospholipase Cβ/protein kinase C and Ca-independent phospholipase A2 pathways

In a previous paper we showed that bradykinin (BK), interacting with its B₂ receptor, inhibits proximal tubule Na⁺-ATPase activity but does not change (Na⁺ + K⁺)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B₂-mediated modulation of proximal tubule Na⁺-ATPase by BK. To abolish B₁ receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg⁹-[Leu⁸]-BK (DALBK), a specific antagonist of B₁ receptor. A dual effect on the Na⁺-ATPase activity through the B₂ receptor was found: short incubation times (1-10 min) stimulate the enzyme activity; long incubation times (10-60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C β (PI-PLCβ)/protein kinase C (PKC); its inhibitory action is mediated by Ca²⁺-independent phospholipase A₂ (iPLA₂). Prior activation of the PI-PLCβ/PKC pathway is required to activate the iPLA₂-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B₂ receptor and activation of membrane-associated iPLA₂.