Characterization of the plasmalemma ATPase activity from roots of Plantago major ssp. pleiosperma, purified by the two-phase partitioning method

A purified plasmalemma preparation from roots of Plantago major L. ssp. pleiosperma (Pilger) was obtained by the two-phase partitioning method, using 6.5% (w/w) of Dextran T-500 and polyethylene glycol 3350, respectively. The distribution of murker enzymes proved the purity of the plasmalemma fraction. The ATPase activity was characterized by determining its sensitivity to anions, cations and inhibitors. The Mg²⁺-dependent ATPase activity peaked at pH 7.25, K⁺-stimulation at pH 6.75, and the Cl⁻ -stimulation both at pH 6.75 and 7.5 (all in the presence of 3 m M MgSO₄). The plasmalemma preparations hydrolyzed preferentially ATP (in the presence of Mg²⁺), although they were less specific for ATP at pH 7.5 than at pH 6.75. The Cl⁻ - stimulated ATPase is probably associated with and located on the plasmalemma. The question if the Cl⁻ -stimulated activity is due to an ATPase distinct from the classical K⁺-stimulated ATPase is considered.     

Apoplastic pH and inorganic ion levels in tomato fruit: A potential means for regulation of cell wall metabolism during ripening

Apoplastic pH and ionic conditions exert strong influence on cell wall metabolism of many plant tissues; however, the nature of the apoplastic environment of ripening fruit has been the subject of relatively few studies. In this report, a pressure-bomb technique was used to extract apoplastic fluid from tomato fruit ( Lycopersicon esculentum Mill.) pericarp at several developmental stages. pH and the levels of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl⁻ and P were determined and compared with the values for the bulk pericarp and locule tissues. The pH of the apoplastic fluid from pericarp tissue decreased from 6.7 in immature and mature-green fruits to 4.4 in fully-ripe fruit. During the same period, the K⁺ concentration increased from 13 to 37 m M . The levels of Na⁺ and divalent cations did not change, whereas the anions P and Cl⁻ increased in ripe fruit. Ca²⁺ levels remained relatively constant during ripening at 4–5 m M , concentrations that effectively limit pectin solubilization. The electrical conductivity of the apoplastic liquid increased 3-fold during ripening, whereas osmotically active solutes increased 2-fold. Pressure-treated fruit retained the capacity to ripen. The decline in apoplastic pH and increase in ionic strength during tomato fruit ripening may regulate the activity of cell wall hydrolases. The potential role of apoplastic changes in fruit ripening and softening is discussed.     

Comparison of ion transportation before and after egg hatching in Amphinemura sp. (Plecoptera)

Abstract.  An increase in egg size with embryonic development in stoneflies is believed to result from the uptake of water by osmosis. The present study aims to investigate whether a selective ion transport through egg membranes exists before hatching, and whether ions are released after hatching. Viable and nonviable egg masses are incubated in Petri dishes filled with water, and the concentrations of the ions F⁻, Cl⁻, SO₄²⁻, NO₃⁻, Na⁺, K⁻, Mg²⁺ and Ca²⁺ in the water are determined. The ion transport of an egg mass before and after hatching and a nonviable egg mass is then calculated. Before hatching, Cl⁻, SO₄²⁻, NO₃⁻, Na⁺, Mg²⁺ and Ca²⁺ are taken up from the surrounding water into the inner egg. These ions are selectively taken into the egg. After hatching, Cl⁻, SO₄²⁻, Na⁺, Mg²⁺ and Ca²⁺ are released into the surrounding water. The amount of these ions released after hatching is lower than the amount taken up before hatching. Ions that are not released after hatching are considered to be used in embryonic development.     

Renal excretion in channel catfish following injection of quinaldine sulphate or 3- trifluoromethyl-4-nitrophenol

Channel catfish, Ictalurus punctatus Rafinesque, injected intraperitoneally with 2-methyl-quinoline sulphate (QdSO₄) or 3-trifluoromethyl-4-nitrophenol (TFM) eliminate most of the dose of these compounds by extra-renal routes. Patterns of renal excretion of Na⁺, K⁺, Ca²⁺, Mg²⁺, and Cl⁻ (ρEq kg⁻¹ h⁻¹) appeared to be associated with the 'stress' of the urine collection technique rather than with the elimination of either compound. Concentrations of Na⁺, K⁺, Ca²⁺, Mg²⁺, and Cl⁻ (mEq/1) were determined in urine, plasma and gall bladder bile.     

Differences in osmoacclimation between sporophytes and gametophytes of the brown alga Ectocarpus siliculosus

The osmoacclimation of Ectocarpus siliculosus isolates known to have different salt tolerances was investigated. Included were isolates originating from 5 different locations in the northern hemisphere, and sporophyte and gametophyte phases of different ploidy from two of the locations were compared. The effect of salinity treatment (8–64%₀) on inorganic ions (K⁺, Na⁺, Mg²⁺, Cl⁻, SO²⁻₄, phosphate) and the low molecular weight carbohydrate mannitol was measured, together with complimentary measurements of cell viability. Very different responses between isolates were obtained, both between isolates of different geographic origin and between sporophytes and gametophytes from the same parent material. A similarity in response between haploid and diploid gametophytes, and diploid and triploid sporophytes indicates that physiological differences between gametophyte and sporophyte generations are not necessarily based on ploidy changes alone. There were no identifiable differences in the responses of male and female gametophytes. K⁺ is the major osmolyte within the species, and differences in the regulation of K⁺ largely account for the observed variation in osmoacclimation, both between life history phases and between isolates from different localities. Isolates with broader salt tolerances had the higher concentrations of mannitol. There were differences between isolates in the amounts and regulation of Cl⁻ and phosphate, the latter being present in unusually high concentrations. There were also isolate differences in the concentrations of Mg²⁺ and SO²⁻₄, although these divalent ions were present only in low concentrations.     

EFFECTS OF SULFHYDRYL REAGENTS ON BRAIN MICROTUBULE-ASSOCIATED ATPase ACTIVITY IN VITRO

Abstract— The effects of two sulfhydryl reagents, PCMBS ( p -chloromercuribenzene sulfonic acid) and NEM ( N -ethylmaleimide) on microtubule-associated Mg²⁺ -and Ca²⁺ -ATPase activity were studied in a MTP (microtubule proteins) preparation and in a MAP (microtubule-associated proteins) fraction. In the MTP preparation at pH 6.8, PCMBS stimulated the Mg²⁺ -ATPase activity at low concentrations and inhibited at higher, whereas the Ca²⁺ -ATPdse activity was only inhibited. NEM affected the activity in a similar way. At pH 8.0 PCMBS was only inhibitory. NEM showed stimulatory effects over a broader concentration range.
Preincubation in the presence of ATP counteracted the stimulatory effects of both PCMBS and NEM on Mg²⁺ -ATPase at pH 6.8.
In the MAP fraction at pH 6.8 PCMBS and NEM caused similar but less pronounced effects on the Mg²⁺ -and Ca²⁺ -ATPase.
The results show that brain microtubule-associated ATPase activity is similar to dynein and myosin ATPases with respect to biphasic alteration by sulfhydryl reagents.     

The effects of mineral nutrition and benzyladenine on the plasmalemma ATPase activity from roots of wheat and Plantago major ssp. pleiosperma

There is an increasing awareness of the possibilities of mineral nutrition as regulator of growth substance action and vice versa. The present paper focuses on the effects of mineral nutrition and benzyladenine at the level of the plasma membrane. Seedlings of wheat ( Triticum aestivum L. cv. Drabant) and juvenile plants of Plantago major L. ssp. pleiosperma (Pilger) were grown hydroponically at different mineral levels with or without benzyladenine. Purified plasmalemma preparations from roots of wheat and P. major ssp. pleiosperma were obtained by the two phase partitioning method, using 6.5% (w/w) of each of Dextran T-500 and polyethylene glycol 3350. The Mg²⁺ and (Mg²⁺+ K⁺) dependent ATPase activities of the root plasmalemma in both species and the (Mg²⁺+ Cl⁻) one in P. major ssp. pleiosperma increased with increasing mineral levels, but the ionic strength did not influence the substrate specificity, the sensitivity to inhibitors or the pH optima.
The addition of 10⁻⁸ M benzyladenine to a nutrient solution increased the ATPase activities. The pH optima and the sensitivity to several inhibitors were not affected by benzyladenine, but the substrate specificity for ATP decreased, except for the K⁺ stimulation. In conclusion, benzyladenine mimics the effects of a higher mineral level than actually applied. Data from this and previous experiments indicate that benzyladenine exerts its effects by increasing the endogenous cytokinin concentrations and by modulating membrane components.     

Excretion of ions (Cd2+, Li+, Na+ and Cl−) by Tamarix aphylla

Excretion of minerals by the NaCl-resistant and comparatively cadmium-resistant tree Tamarix aphylla (L.) Karst, was investigated. Cd²⁺ was excreted by plants exposed for 1–10 days to 9 or 45 μ M Cd²⁺ solutions. Excretion of this toxic ion increased considerably with time but was less than 5% of the quantities that had been accumulated in the shoots. Excretion of Na⁺ and Cl⁻ was positively correlated with NaCl concentration (1.5, 10, 50 m M ) of the medium. The Na⁺/Cl⁻ ratios of the excrete were positively correlated with the concentration of the treatment solution. Ca²⁺ excretion decreased with increasing NaCl concentrations of the solution. Excretion of K⁺ and Mg²⁺ was only little affected by NaCl. Excretion of Li⁺ occurred whenever this element was supplied in the uptake solution; daily excretion rates of Li⁺ increased with time. The ecological significance of excretion is discussed in relation to the low selectivity of the mechanism in T. aphylla .     

Element concentrations in mosses and surface waters of western Canadian mires relative to precipitation chemistry and hydrology

Concentrations of N, P, S, Na, K, Mg, Ca, Mn, Fe, Cu, Cd, Zn, Pb, Al, and AIA (acid insoluble ash) m mosses (three Sphagnum species and Tomenthypnum nitens, all hummock species) from a variety of mires, both ombrotrophic and minerotrophic, in the coastal western and central parts of Canada are considered in relation to surface water pH and concentrations of Na⁺, K⁺, Mg²⁺, Ca²⁺, Cl⁻, and SO₄^2- Distinct west-east concentration gradients were present for most elements in both mosses and water, but there were correlations between surface water and moss concentrations only for Ca and Mg
On ombrotrophic sites and sites characterized by poor fen vegetation, wet deposition is the main source of elements in the surface water On rich fen sites, additional Ca and Mg from surrounding soils change the elemental proportions We conclude that hydrochemically the limit between poor and rich fen sites is more decisive than between bog and fen The increase in Ca may give brown mosses a competitive advantage over Sphagnum
Moss concentrations of Na and Mg are the only ones decreasing inland The constancy or inland increase of moss elemental concentrations may depend on either an increasing atmospheric supply (e g Pb), differences in moss growth rates (especially N, P, and K) or site conditions related to the water regime (e g Fe and Al)     

Mg2+-free buffer elevates transformation efficiency of Vibrio parahaemolyticus by electroporation

Aims:  The ability to transform Vibrio spp. is limited by the extracellular nuclease that their cells secrete. The reported transformation efficiency of this organism is 10²–10⁵ transformants per microgram DNA. We tried different buffers and conditions, aiming to elevate its transformation efficiency.
Methods and Results:  MgCl₂ and sucrose are often included in the washing and/or electroporation buffers to stabilize the cell membrane. However, Mg²⁺ is required for production and activity of the extracellular nuclease. A simple electroporation buffer lacking Mg²⁺ was found to increase transformation efficiency dramatically, to levels 50-fold more than the buffers containing Mg²⁺. To maintain the stability of the cell membranes, Mg²⁺ was replaced with high concentrations of sucrose, from 272 to 408 mmol l⁻¹. With the new buffers, the transformation efficiency of Vibrio parahaemolyticus was increased to 2·2 × 10⁶ transformants per microgram DNA.
Conclusions:  Mg²⁺ in the buffer adversely affected transformation of V. parahaemolyticus by electroporation. The cell membranes of vibrio can be stabilized by high concentration of sucrose when Mg²⁺ is absent.
Significance and Impact of the Study:  A greater transformation efficiency can facilitate the genetic analysis of an organism and its pathogenicity. Buffers lacking Mg²⁺ can be used for other nuclease-producing organisms.     

Uptake of metals by bacterial polysaccharides

J.L. GEDDIE AND I.W. SUTHERLAND. 1993. The binding of cations by a range of bacterial polysaccharides was examined. Comparison of native and deacetylated polymers indicated the influence of polysaccharide acetylation on ion uptake and selectivity. The effects of temperature and pH on ion uptake were also examined. Metal ion uptake was carried out by dialysis and samples were analysed using ion chromatography. The native acetylated polymers showed a selectivity for Ca²⁺ > Mg²⁺ > monovalent cations, whereas samples lacking acetyl groups showed a selectivity for monovalent cations > Mg²⁺ > Ca²⁺. Increased temperatures reduced the capacity for several of the polymers to bind the cations; The Zoogloea ramigera polymer appeared least affected. The pH value also affected uptake.     

Effects of NH+4-N and NO+3-N on growth and metabolism of Sphagnum magellanicum

Photosynthetic CO₂-fixation, chlorophyll content, growth rate and nitrate reductase activity were used to examine the influence of NH⁺₄-N and NO⁻₃-N on Sphagnum magellanicum cultivated under defined conditions in phytotrons. NO⁻₃-concentrations up to 322 μ M were found to be favourable. Increased NH⁺₄ concentrations, however, resulted in growth inhibition and decreased chlorophyll content at concentrations ≧ 255 μ M ; e.g. 600 μ M NH⁺₄ caused a 20% reduction of nitrate reductase activity and net photosynthesis. For raised bog Sphagna an improved standard nutrient solution is proposed with the following ion concentrations (μ M ): 55 Na⁺; 17 K⁺; 95 NH⁺₄; 22 Ca²⁺; 22 Mg²⁺; 2 Fe³⁺; 20 Cl⁻; 100 NO⁻₃; 57 SO^2-₄; 7.4 H₂PO⁻₄; trace elements: A-Z solution (Hoagland) 50 μl 1000 ml⁻¹; pH 5.8.     

Salinity adaptation in the snakehead, Ophiocephalus maculatus Lacépède: changes in oxygen consumption, branchial Na+-K+-ATPase and body composition

Snakeheads were adapted to fresh water (1 mOsm kg⁻¹), 25% sea water (230 mOsm kg⁻¹), 33% sea water (320 mOsm kg⁻¹) and 40% sea water (380 mOsm kg⁻¹) for 20 days. Exposure to salt water resulted in tissue dehydration, elevations of plasma osmolality, Na⁺, Mg²⁺, Cl⁻and protein concentrations and stimulation of branchial Na⁺-K⁺-ATPase activity. These changes were accompanied by concomitant decline of the hepatosomatic index and liver glycogen concentration. The routine rate of oxygen consumption was increased in snake-heads adapted to 33% sea water. These data were taken to indicate a stressful effect of salinity to the snakehead despite documentation of its ability to penetrate into brackish waters.     

Characteristics of the reverse reaction of NADP+-isocitrate dehydrogenase from castor bean seeds

The reductive carboxylation of α-ketoglutarate by purified NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) from maturing castor bean seeds ( Ricinus communis L. ) has been characterized. The optimum pH for the reaction was 6.5, whereas pH 8.5 was optimum for oxidation of isocitrate (forward reaction). The enzyme utilized NADH as well as NADPH as the reducing agent in the reverse reaction, but only NADP⁺ in the forward reaction. The Km values for NADPH and NADH were 0.044 and 2.8 m M respectively, and for α-ketoglutarate and HCO₃ 4.1 and 3.7 m M. The enzyme was activated by various cations including Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺ and Co²⁺. Km values for Mg²⁺ Mn²⁺, Co²⁺ and Zn²⁺ were 12, 34, 37 and 49μ M respectively.     

MAGNESIUM REQUIREMENT IN FERTILIZATION PROCESS OF SEA URCHINS

The Mg²⁺ requirement in fertilization was investigated in sea urchins. It was found that when sea urchin eggs were inseminated in sea water free of Mg²⁺, little fertilization took place. Even when spermatozoa pre-treated with dissolved egg-jelly to induce the acrosome reaction, which needs Ca²⁺, were used, the fertilization rate remained quite low in the absence of Mg²⁺. In Strongylo-centrotus intermedius , the lowest concentration of Mg²⁺ required for 50% fertilization was 0.05 mM in the presence of 10 mM Ca²⁺, whereas that of calcium was 3 mM in the presence of 49 mM Mg²⁺. These critical concentrations increased when the concentration of the other ion decreased. Removal of Mg²⁺ or Ca²⁺ or both from the suspending medium had little adverse effect on sperm motility. The elevation of the fertilization membrane was also induced by butyric acid independent of the presence or absence of Mg²⁺ and/or Ca²⁺. These results indicate that Mg²⁺ are required at least in some process(es) between acrosome reaction and fertilization membrane elevation, such as sperm penetration or membrane fusion.     

Isolation and characterization of cytoplasmic NADP+-malic enzyme from Lupinus luteus leaves

NADP⁺-dependent malic enzyme (L-malate : NADP⁺ oxidoreductase, decarboxylating, EC 1.1.1.40) was extracted from the leaves of yellow lupine. The purification procedure included fractionation with (NH₄)₂SO₄ and Sephadex G-25 chromatography, followed by purification on DEAE-cellulose and Sephadex G-200 columns. The enzyme was purified 122-fold. The enzyme affinity towards L-malate was found to be significantly higher with Mn²⁺ than with Mg²⁺. The Hill coefficient for Mg²⁺ depended on concentration and was 1.6 for the lower and 3.9 for the higher concentrations. The dependence of the enzyme activity on NADP⁺ followed a hyperbolic curve. K_m values and Hill coefficients for NADP⁺ were similar with both Mn²⁺ and Mg²⁺. The enzyme activity was strictly dependent on divalent cations and followed a sigmoidal curve at least for Mg²⁺. The enzyme had 4-fold higher affinity towards Mn²⁺ than towards Mg²⁺, the K_m values being 0.3 and 1.15 m M respectively. Of several tested organic acids, oxalate was the most effective inhibitor followed by oxaloacetate while succinate was the strongest activator.     

Effects of Microinjected Cations on the Early Event of Fertilization in the Medaka Egg

Effects of microinjected cations on the early events of fertilization were examined using eggs of Oryzias latipes . Microinjection of either Ca²⁺, Ba²⁺ or Sr²⁺ into the thin cortical cytoplasm induced breakdown of cortical alveoli (vesicles) (CABD) under Ca-Mg-free conditions, but microinjection of Mg²⁺, Mn²⁺ or Co²⁺ prevented CABD at the injected region when the eggs were inseminated in regular saline. Under Ca-Mg-free conditions, CABD could also be induced by microinjection of various solutions (NaCl, choline chloride, sucrose, pH buffer) without any divalent cations or ionophore A23187. Ca²⁺ microinjected into the cortical cytoplasm did not play a role in sperm penetration. Upon microinjection with either Ca²⁺, Mg²⁺ or K⁺, the resting membrane potential leakage was transiently observed. However, depolarization of the membrane followed by slow hyperpolarization was observed only upon microinjection of Ca²⁺. From these experiments, it was inferred that microinjected divalent cations such as Ca²⁺, Ba²⁺ or Sr²⁺ do not act directly upon the cortical alveolus membrane, but trigger the induction of CABD via depolarization of the membrne and increase in intracellular Ca²⁺.     

Divalent cations enhance ammonia excretion in Lahontan cutthroat trout in highly alkaline water

The Lahontan cutthroat trout lives under highly alkaline and saline conditions in Pyramid Lake, Nevada (pH 9.4; 0.2 mmol 1⁻¹ Ca⁺⁺; 7.3 mmol 1⁻¹ Mg⁺⁺). These experiments were conducted to study the possible roles of water Ca⁺⁺ and Mg⁺⁺ concentrations on ammonia excretion in the Lahontan cutthroat trout under highly alkaline conditions. The basic protocol of the experiments was to determine ammonia excretion rates during the following three exposure periods (each of 3-h duration) in sequence: (a) in normal lake water; (b) in soft lake water with the divalent cation concentrations reduced; and (c) in the soft lake water with either Ca⁺⁺ or Mg⁺⁺ (or no divalent cations added) added back at the appropriate lake water concentration. The soft-water exposure caused a significant reduction in ammonia excretion to about half of the control (original lake water) levels. When either Ca⁺⁺ or Mg⁺⁺ was added to the soft water in the third exposure period, the ammonia excretion rates were increased more than twofold back to lake water levels.     

ATPASE AND PHOSPHATIDYLINOSITOL KINASE ACTIVITIES OF ADRENAL CHROMAFFIN VESICLES

Abstract— The hypothesis that the ATPase and phosphatidyhnositol (PI) kinase activities of chromaffin vesicle membranes are catalysed by same enzyme was investigated. The two activities exhibited entirely different responses to variations in Mg²⁺ or Mn²⁺ concentrations. In the presence of 1 mM ATP, maximal ATPase activity occurred with 1 mM Mg²⁺ while maximal PI kinase activity required 100 mM Mg²⁺ Similar differences were observed with Mn²⁺ with the exception that maximal ATPase activity occurred with 0.5 mM Mn²⁺ and maximal PI kinase activity occurred with 5 mM Mn²⁺ Mn²⁺ was more effective than Mg²⁺ in stimulating PI kinase activity at low concentrations, but at optimal concentrations of each, the maximal activity obtained with Mg²⁺ was 5-fold greater than the maximal activity obtained with Mn²⁺ The heat stabilities of the two enzymes are vastly different. At 50°C the ATPase activity of the intact membranes was stable for up to 20 min while the t _l/2 of PI kinase was less than 2 min. After solubilization in Lubrol PX or at higher temperatures both enzymes were less heat stable, but PI kinase was still inactivated at a much greater rate than the ATPase. The evidence suggests that the ATPase and the PI kinase are different proteins.
The major phosphorylated product was diphosphatidylinositol and once formed, it was stable. Phosphorylation of membrane protein accounted for less than 10% of the total ³²P-incorporated into chromaffin vesicles. SDS gel electrophoresis of the solubilized membranes showed the presence of at least 2 major phosphorylated high molecular weight components.     

A study of the helminth parasites of Anarhichas lupus (Perciformes: Anarhichadidae) in the North Atlantic

The North Atlantic wolffish, Anarhichas lupus , has a broad, relatively short gastro-intestinal tract (0.55–1.05 of body length). The stomach-like region has a high pH, suggesting a functionally stomachless condition. The 'stomach' is rarely parasitized by helminths. The gut pH tends to rise along its length as does the concentration of the ion Mg²⁺, while osmolarity and the ions Na⁺, Cl⁻ and K⁺ tend to decrease and Ca²⁺ stays practically the same. The intestinal wall is deeply folded, particularly in the anterior region. The intestine commonly harbours several helminth species. The gall bladder and urinary bladder tend to be voluminous and both are commonly parasitized by digeneans. A. lupus has been recorded as harbouring over 30 species of parasitic helminth of which 10 are commonly found, often in large numbers. There is little overlap of parasite sites and there appear to be several empty niches. The parasites tend to fall into three categories, those stenoxenic to this genus of fish, those stenoxenic to other groups and accidental in A. lupus , and those forms which are euryxenic.