The Titrimetric Assay of H+ Excreted by Ricinus communis Cultivated on NH4+-N Nutrient Media: The Effect of Ionic Strength and Nutrient Phosphate Concentration

Allen, C. R. and Allen, S. 1987. The titrimetric assay of H⁺excreted by Ricinus communis cultivated on -N nutrient media; the effect of ionic strengthand nutrient phosphate concentration.— J. exp. Bot. 38:597–606. The simple titrimetric assay of H⁺ excreted by -N plants into spent nutrient solution was criticallyassessed on the basis of equations for charge balance, conservationof matter and chemical equilibrium (including activity coefficients).For Ricinus communis, a simple volumetric calculation from back-titrationof the spent medium to 6–5 underestimated proton excretionby 3%, compared with the corresponding error of up to 50% fortitrimetric assay of base excreted into spent -N media. Key words: Ricinus, ammonium-N nutrient medium, proton excretion, ionic activity coefficients     

A Dynamic Growth Model of Vegetative Soya Bean Plants: Model Structure and Behaviour under Varying Root Temperature and Nitrogen Concentration

A differential equation model of vegetative growth of the soyabean plant (Glycine max (L.) Merrill cv. ‘Ransom’)was developed to account for plant growth in a phytotron systemunder variation of root temperature and nitrogen concentrationin nutrient solution. The model was tested by comparing modeloutputs with data from four different experiments. Model predictionsagreed fairly well with measured plant performance over a widerange of root temperatures and over a range of nitrogen concentrationsin nutrient solution between 0.5 and 10.0 mmol in the phytotron environment. Sensitivity analyses revealedthat the model was most sensitive to changes in parameters relatingto carbohydrate concentration in the plant and nitrogen uptakerate. Key words: Glycine max (L.) Merrill, dry matter, nitrogen uptake, partitioning, photosynthesis, respiration, sensitivity analysis     

Effects of Nutrient Limitation and {gamma}-Irradiation on Tracheary Element Differentiation and Cell Division in Single Mesophyll Cells of Zinnia elegans

The effects of nutrient limitation and -irradiation on trachearyelement differentiation and cell division were investigatedusing single cells isolated from the mesophyll of Zinnia elegans.When the phosphate concentration of the medium was reduced to10 µM (1/50 of Fukuda and Komamine's medium, 1980a), thefrequency of cell division during 4 days of culture decreased,while the frequency of tracheary element differentiation wasunaffected. -Irradiation with a dose of 92 Gy at 36 h of culturepreferentially and thoroughly suppressed cell division withoutreducing the number of tracheary elements formed. The appearanceof secondary cell wall thickenings was delayed by irradiation,but synchrony was maintained. Thus the Zinnia system previouslyreported [Fukuda and Komamine (1980a) Plant Physiol. 65: 57]was improved to give a more useful system for the study of cytodifferentiation,in which tracheary element formation occurred from single cellswithout cell division. ¹Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Miyagi 980, Japan. (Received November 28, 1985; Accepted February 22, 1986)     

Relative Uptake Rates of Inorganic Nutrients by NO3- and NH4+ Grown Ricinus communis and by two Plantago Species

Allen, S., Thomas, G. E. and Raven, J. A. 1986. Relative uptakerates of inorganic nutrients by and grown Ricinus communis and by two Plantago species.—J. exp. Bot. 37: 419–428. The relative rates of uptake and assimilation of C, N, P, S,Cl^–, K⁺ , Na⁺ Ca²⁺ and Mg²⁺ by and grown Ricinus conimunisand by NH₄NO₃- grown Plantago lanceolata and P. major were calculatedfrom data presented elsewhere. Results showed that for grown Ricinus the short term relativeuptake rates, for each nutrient X did not change significantly over the steady-state periodof exponential growth. The average gave , the mean relative uptake rate during exponential growth, for each nutrient. The amountof each nutrient taken up from a nutrient solution over a periodof time could, therefore, be calculated. For and -grown R. communis,the relative uptake rate of each nutrient was a constant fractionof the relative rate of carbon assimilation. It is suggestedthat this is typical of plants of cauline habit. For both Plantago spp., the relative rates of nitrogen uptakeand assimilation fell significantly during the exponential growthphase It is suggested that this could be a characteristic ofthe growth habit of the rosette plant. Key words: Relative uptake rates, Ricinus, Plantago, ammonium, nitrate, cauline, rosette     

A simplified strong ion model for acid-base equilibria: application to horse plasma

Constable, Peter D. A simplified strong ion model foracid-base equilibria: application to horse plasma. J. Appl. Physiol. 83(1): 297-311, 1997.TheHenderson-Hasselbalch equation and Stewart's strong ion model arecurrently used to describe mammalian acid-base equilibria. Anomaliesexist when the Henderson-Hasselbalch equation is applied to plasma,whereas the strong ion model does not provide a practical method fordetermining the total plasma concentration of nonvolatile weak acids([A_tot]) and theeffective dissociation constant for plasma weak acids(K_a). Asimplified strong ion model, which was developed from the assumptionthat plasma ions act as strong ions, volatile buffer ions(HCO₃), or nonvolatile buffer ions,indicates that plasma pH is determined by five independent variables:PCO₂, strong ion difference, concentration of individual nonvolatile plasma buffers (albumin, globulin, and phosphate), ionic strength, and temperature. The simplified strong ion model conveys on a fundamental level the mechanism for change in acid-base status, explains many of the anomalies when the Henderson-Hasselbalch equation is applied to plasma,is conceptually and algebraically simpler than Stewart's strong ionmodel, and provides a practical in vitro method for determining[A_tot] andK_a of plasma.Application of the simplified strong ion model toCO₂-tonometered horse plasmaproduced values for[A_tot] (15.0 ± 3.1 meq/l) and K_a(2.22 ± 0.32 × 10⁷ eq/l) that weresignificantly different from the values commonly assumed for humanplasma ([A_tot] = 20.0 meq/l, K_a = 3.0 × 10⁷ eq/l).Moreover, application of the experimentally determined values for[A_tot] andK_a to publisheddata for the horse (known PCO₂,strong ion difference, and plasma protein concentration) predictedplasma pH more accurately than the values for[A_tot] andK_a commonlyassumed for human plasma. Species-specific values for[A_tot] andK_a should beexperimentally determined when the simplified strong ion model (orstrong ion model) is used to describe acid-base equilibria.

     

Carbon Metabolism in Seagrasses: III. ACTIVITIES OF CARBON-FIXING ENZYMES IN RELATION TO INTERNAL SALT CONCENTRATIONS

The internal salt content and distribution in photosynthetictissues as well as the effect of NaCl on photosynthetic carbonfixation enzymes was investigated in two seagrass species fromthe Red Sea. Concentrations of both Na⁺ and Cl^– were lower in the chloroplast-richepidermis than in underlying cell layers in Halophila stipulacea.In Halodule uninervis, the concentration of Na⁺ was lower inthe epidermis than in the underlying cells, while K⁺ was evenlydistributed between cell layers. The epidermal concentrationsof Na+ were estimated to be 0.17 and 0.10 M for Halophila stipulaceaand Halodule uninervis, respectively, which were about to the average leaf concentrations. Epidermal Cl^– concentrationof Halophila stipulacea was estimated to be 0.08 M, a valueonly about of the overall leaf concentration. Phosphoenolpyruvate carboxylase (PEPcase) extracted from leavesof these seagrasses showed increased activity at 0.05–0.3M NaCl in vitro. Ribulose-l, 5-bisphosphate carboxylase (RuBPcase)activity, on the other hand, was inhibited by NaCl at all testedconcentrations. At epidermal NaCl concentrations, PEPcase activitywas thus stimulated while RuBPcase was inhibited. The reducedRuBPcase activity at such concentrations compared to salt-freeconditions was still sufficient to account for observed photosyntheticrates. We conclude that these seagrasses have adapted to a saline environmentboth by maintaining relatively low ion concentrations in theepidermis where photosynthesis occurs and by having carbon-fixingenzymes capable of functioning in the presence of salt.     

ENaC proteins are required for NGF-induced neurite growth

Neurite growth is required for nervous system development and repair. Multiple signals, including neurotrophic factors and intact mechanosensing mechanisms, interact to regulate neurite growth. Degenerin/epithelial Na⁺ channel (DEG/ENaC) proteins have been identified as putative mechanosensors in sensory neurons. Recently, others have shown that the neurotrophic factor NGF stimulates expression of acid-sensing ion channel molecules, which are members of the DEG/ENaC family. However, it is unknown whether NGF regulates ENaC expression or whether ENaC expression is required for neurite formation. Therefore, the aims of the present study were to determine whether ENaC expression is 1) regulated by NGF and 2) required for NGF-induced neurite growth in pheochromocytoma PC-12 cells. We found NGF-induced expression of - and -subunits of ENaC, but not -ENaC. Tyrosine kinase A (TrkA) receptor blockade abolished NGF-induced - and -ENaC expression and neurite formation. NGF-induced neurite formation was inhibited by disruption of ENaC expression using 1) pharmacological blockade with benzamil, a specific ENaC inhibitor; 2) small interfering RNA; and 3) dominant-negative ENaC molecules. These data indicate NGF-TrkA regulation of ENaC expression may be required for neurite growth and may suggest a novel role for DEG/ENaC proteins in neuronal remodeling and differentiation. mechanosensation; degenerins; neurotrophins; tyrosine kinase A; pheochromocytoma cells     

Interaction of reactive oxygen species with ion transport mechanisms

The use ofelectrophysiological and molecular biology techniques has shed light onreactive oxygen species (ROS)-induced impairment of surface andinternal membranes that control cellular signaling. These deleteriouseffects of ROS are due to their interaction with various ion transportproteins underlying the transmembrane signal transduction, namely,1) ion channels, such asCa²⁺ channels (includingvoltage-sensitive L-type Ca²⁺currents, dihydropyridine receptor voltage sensors, ryanodine receptorCa²⁺-release channels, andD-myo-inositol1,4,5-trisphosphate receptor Ca²⁺-release channels),K⁺ channels (such asCa²⁺-activatedK⁺ channels, inward and outwardK⁺ currents, and ATP-sensitiveK⁺ channels),Na⁺ channels, andCl channels;2) ion pumps, such as sarcoplasmicreticulum and sarcolemmal Ca²⁺pumps,Na⁺-K⁺-ATPase(Na⁺ pump), andH⁺-ATPase(H⁺ pump);3) ion exchangers such as theNa⁺/Ca²⁺exchanger andNa⁺/H⁺exchanger; and 4) ion cotransporterssuch asK⁺-Cl,Na⁺-K⁺-Cl,andP_i-Na⁺cotransporters. The mechanism of ROS-induced modificationsin ion transport pathways involves1) oxidation of sulfhydryl groups located on the ion transport proteins,2) peroxidation of membrane phospholipids, and 3) inhibition ofmembrane-bound regulatory enzymes and modification of the oxidativephosphorylation and ATP levels. Alterations in the ion transportmechanisms lead to changes in a second messenger system, primarilyCa²⁺ homeostasis, which furtheraugment the abnormal electrical activity and distortion of signaltransduction, causing cell dysfunction, which underlies pathologicalconditions.

     

Measurement of Ammonium Ions in Flowing Solution Culture and Diurnal Variation Uptake by Lolium perenne L

An existing system of flowing solution culture, in which pHand the concentration of several nutrient ions are automaticallymonitored and controlled, has been extended to include at 10 mmol N m^–3. A brief account is givenof the use of the equipment with a simulated sward of perennialryegrass (Lolium perenne L.). Uptake of measured over three successive days in June, varied with dailysolar radiation and exceeded 1000 mg N m^–2 d^–1.The uptake of showed a pattern of diurnal variation similar to the variation in solar radiation, but witha lag period for uptake of 5 h. Hourly uptake rates ranged from32 to 67 mg N m^–2h^–1 and solar radiation from 0to 2.8 MJ m^–2 h^–1. During a 24 h period, additionalmeasurements were made of K⁺ uptake and net H⁺ efflux, bothof which showed patterns of diurnal variation with lag periodsof 6 h hand 7 h, respectively. The stoichiometric ratio of thesum of and K⁺ to the net efflux of H⁺ was1.02: 1. Key words: Ion uptake, diurnal variation, Lolium perenne L.     

Short-term Effects of Ferulic Acid on Ion Uptake and Water Relations in Cucumber Seedlings

Ferulic acid (FA) is commonly found in soils and is consideredan allelochemical. Studies have suggested that FA and otherphenolic acids decrease plant growth in part by decreasing theabsorption of mineral nutrients and water. However, no studieshave examined these parameters in a single experimental systemto investigate how FA affected both ion uptake and plant-waterrelations in whole plants. Using intact cucumber (Cucumis sativusL. cv. Early Green Cluster) seedlings, we examined short-termeffects of FA on ion uptake kinetics, transport promoters andinhibitors, and water relations as indicated by a pressure-volumeanalysis. We found that after 3 h of treatment, 200 µMFA inhibited net ion uptake, particularly NO₃, and promotednet K⁺ efflux from seedling roots. The addition of fusicoccin,a K⁺ transport promoter, counteracted the inhibitory effectof FA on net K⁺ uptake. Concurrent treatment of seedlings withFA and tetraethylammonium, a channel-blocking salt, reducedaverage K⁺ efflux by 66%. Treatment of seedlings with FA alsodecreased leaf water potential (₁ and turgor pressure (P_T).However, decreased ₁ and P_T were not caused by changes in theosmotic properties of the symplast or stomatal conductance.A decrease in water absorption is a likely explanation for theloss of P_T observed. The results of our experiments indicatethat both ion uptake and plant-water relations can be adverselyaffected by FA. Key words: Cucumis sativus, ferulic acid, allelopathy, ion uptake, water relations     

Properties of cytotoxic peptide-formed ion channels

Cytotoxic peptides are relatively small cationicmolecules such as those found 1) in venoms, e.g., melittin inbee, scorpion toxins in scorpion, pilosulin 1 in jumper ant, andlycotoxin I and II in wolf spider; 2) in skin secretions (e.g.,magainin I and II from Xenopus laevis, dermaseptin from frog,antimicrobials from carp) and cells of the immune system (e.g., insect,scorpion, and mammalian defensins and cryptdins); 3) asautocytotoxicity peptides, e.g., amylin cytotoxic to pancreatic-cells, prion peptide fragment 106-126[PrP-(106-126)], and amyloid -protein (AP)cytotoxic to neurons; and 4) as designed synthetic peptides based on the sequences and properties of naturally occurring cytotoxic peptides. The small cytotoxic peptides are composed of -sheets, e.g., mammalian defensins, AP, amylin, and PrP-(106-126),whereas the larger cytotoxic peptides have several domains composed of both -helices and -sheets stabilized by cysteine bonds, e.g., scorpion toxins, scorpion, and insect defensins. Electrophysiological and molecular biology techniques indicate that these structures modifycell membranes via 1) interaction with intrinsic ion transport proteins and/or 2) formation of ion channels. These twononexclusive mechanisms of action lead to changes in second messengersystems that further augment the abnormal electrical activity anddistortion of the signal transduction causing cell death.

     

Effects of Light on Transport by Azolla pinnata

Effects of light flux density (LFD) during growth and uptakeassay on induction of transport system and kinetics of transport were studied using the Azolla pinnata-Anabaena azollae association (Azolla). Theinduction and uptake kinetics of the transport system were determined using an automated system that measuredthe NO₃ concentration in the growth medium as a function oftime, using an on-line high performance liquid chromatograph(HPLC) with a UV-VIS detector. Full induction of the transport system required about 1.5 to 2.0 h and occurred without any apparent lag phase,regardless of the LFD provided. The level of induction of transport of Azolla grown at 600 µmol m^–2s^–1 LFD was higher than for that grown at 100 µmolm^–2 s^–1. Similarly, 600 µmol m^–1 s^–1LFD during the assay resulted in a higher level of inductionthan did 100 umol m^–2 s^–1. An increase in the LFDeither during the growth or the assay period increased the uptake rate; however, an increase in LFD duringthe latter period had greater effect. Azolla grown and assayedat 600 umol m^–2 s^–1 had the highest uptake rate. The uptake rate at 50 cm³ m^–3ambient CO₂ concentration was initially higher than at 305 cm³m^–3, but the uptake rate decreased rapidly with time andeventually dropped below that at 305 cm³ m^–3 CO₂. Thesedata suggest that the energy required for transport in Azolla may bypass the photosynthetic CO₂ fixationand carbon-cycling. Key words: carbon dioxide, concentration dependence, light flux density, uptake     

Dynamics of herbivorous grazing by the heterotrophic dinoflagellate oxyrrhis marina

In a series of batch experiments in the dark the heterotrophicdinoflagellate Oxyrrhis marina grazed three phytoplankton prey(Phaeodactylun tricornutum, Isochrysis galbana and Dunaliellateriolecta) with equal efficiency. Growth rates of the dinoflagellateranged between 0.8 and 1.3 day–1 Maximum observed ingestionrates on a cell basis varied according to the size of the preyfrom about 50 cells flagellate^–1 day^–1 when D.tertiolectawas the prey to 250–350 cells fiagellate^–1 day^–1when the other species were eaten. However, when compared ona nitrogen basis, ingestion rates were independent of prey type.Both ingestion and growth ceased when prey cell concentrationsfell below a threshold concentration of about 10⁵ cells ml^–1.Maximum specific clearance rates were 0.8x10⁴0ndash;5.7x10⁴it day which is considerably lower than that found for heterotrophicdinoflagellates in oceanic waters and may explain why O.marinagenerally thrives only in productive waters. The timing of NHregeneration was linked to the C:N ratio of the prey at thestart of grazing. Regeneration efficiencies for NH₄. never exceeded7%; during the exponential phase and were 45% well into thestationary phase. These results are comparable to those obtainedwith heterotrophic flagellates and demonstrate that the bioenergeticpatterns of grazing and nutrient cycling by different protozoaare very similar. Moreover, they support the notion that toachieve 90+% nutrient regeneration in the open ocean, as iscurrently believed, the microbial food loop must consist ofmultiple feeding steps. Alternatively, nutrient regenerationefficiencies may be considerably lower than 90%.     

Mechanisms of Inhibition of Water Movement in Anaerobically Treated Roots of Zea mays L.

Changes in water flux (Jv) across detopped, 7-d-old, maize rootswere characterized during the initial 24 h of being made anoxicby exposure to an anaerobic nutrient solution. Suction (50 kPa)was applied to the xylem and samples of the xylem sap were collectedat intervals and the osmolality and ionic content were measured. Values of Jv through anoxic roots fell below those of aerobiccontrols 1 h after the equilibrium oxygen partial pressure inthe bathing medium dropped below 20 kPa (air = 20.6 kPa). Thereduction in Jv was due primarily to a nullification of thediurnal rhythm in hydraulic conductivity (Lp) that was measuredin aerobic roots. However, about one-quarter of the reductionin Jv could be accounted for by a smaller osmotic componentof the driving force () on water movement. The significance of changes in Jv in anoxic roots is discussedin terms of the reliability of estimates of Lp, the reflectioncoefficient () and . Key words: Anaerobiosis, hydraulic conductivity, osmotic potential, water     

The Titrimetric Assay of OH- and HCO3- Excreted by Ricinus Cultivated on NO3--containing Nutrient Media: The Influence of Ionic Strength, End Point pH and CO2 Supersaturation

Allen, S. and Allen, C. R. 1987. The titrimetric assay of OH^–and excreted by Ricinus cultivatedon -containing nutrient media: the influence of ionic strength, end point pH and CO² supersaturation.–J. exp. Bot. 38: 607–617. When spent -containing nutrient media were titrated to the starting pH of 6.5 the titre wasequivalent to 50%orless of the base (i.e. ) excreted. Calculation of the total amount of baseexcreted could only be made from data obtained by titrationto pH 4–5. An accurate calculation of the amount of baserequired the inclusion of activity coefficients, estimated fromionic strength, in the calculations. Spent nutrient media contained from four to ten times the concentrationof CO₂ predicted from equilibrium values. It is probable that is very slow. Key words: Ricinus, nitrate-N nutrient medium, base excretion, ionic activity coefficients, carbon dioxide supersaturation     

The Rate of Photosynthesis in Isolated Chloroplasts

Chloroplasts were isolated using aqueous and nonaqueous procedures.Aqueous chloroplasts lost approximately 50 per cent, of theirsoluble proteins during isolation. Nonaqueous chloroplasts retainedall their soluble enzymes, but lost their ability to performthe light reactions of photosynthesis. It was possible to reconstitutea chloroplast system of higher activity by adding soluble enzymesfrom nonaqueous chloroplasts to protein-deficient aqueous chloroplasts.The properties of the reconstituted chloroplast system wereas follows: 1. The CO₂ fixation rate of the reconstituted chloroplast system( 4 µM./. chlorophyll/hr.) was 3–4 times that ofthe aqueous chloroplasts ( I µM./. chlorophyll/hr.). Thefixation of aqueous chloroplasts isapparently limited in partby lack of soluble enzymes. 2. During light-fixation, the reconstituted chloroplast systemaccumulated PGA. This indicates that the reduction of PGA totriosephosphate is a rate-limiting step in this system. 3. It was possible to increase the CO₂ fixation to 12 µM.CO₂/mg. chlorophyll/ hr. by addition of ATP and TPNH to thesystem, but the reduction of PGA was still rate-limiting. 4. Further increase in the fixation rate was obtained by concentratingthe reaction mixture. Part of the striking differences of theCO₂-fixing capabilities of chloroplasts in vivo and in vitrois caused by dilution effects. Extrapolation of the dilutioneffect to the protein concentration which exists in chloroplastsyields a CO₂ fixation rate of approximately 30 µM./mg.chlorophyll/hr. 5. Inhibitors which are located in vivo outside the chloroplastsaffect the CO₂ fixation in vitro. 6. Under consideration of the examined factors which influencethe CO₂ fixation of isolated chloroplasts, it is possible toraise the fixation from approximately 1 per cent, to at least15 per cent, of the fixation in vivo.     

Etiolated Growth as Measure of Non-structural Biomass in Lucerne Taproots

Enzymatic hydrolysis of starch in lucerne (Medicago sativa L.)taproots is the conventional method used to determine the quantityof carbohydrates allocated to regrowth. Etiolated growth froma taproot could be used to quantify total root biomass allocatedto regrowth. This study compared concentrations of non-structuralcarbohydrates, as measured by -amylase hydrolysis of starchto glucose, to concentrations of non-structural biomass, asmeasured by etiolated growth from lucerne taproots placed inan incubator and plants in situ. The concentration of starchfrom enzymatically assayed taproots was 325 g kg^-1 expressedas glucose equivalents. Etiolated growth and weight loss byrespiration from plants grown in the incubator accounted for524 g of actual biomass per kg of root. There was 46·2g kg^-1 of N, 3·1 of P, and 33·1 of K in the etiolatedgrowth. An 88% increase in etiolated growth dry weight was observedfrom plants in situ compared to taproots placed in the incubator.Accurate quantification on non-structural biomass should notbe limited to sampling just the taproot, but must included theentire root system. Compared to determining non-structural carbohydratesby enzymatic hydrolysis of starch, the procedure used in determiningnon-structural biomass by etiolate growth gave results in unitsrelative to the plant. The use of etiolate growth also providedinformation on mineral nutrient partitioning from root to shoots,was less technically demanding, and could be applied to theentire root system.Copyright 1993, 1999 Academic Press Medicago sativa, root carbohydrates, etiolated growth, taproot     

Unique temperature-activated neurons from pit viper thermosensors

Rattlesnakes, copperheads, and other pit vipers have highly sensitive heat detectors known as pit organs, which are used to sense and strike at prey. However, it is not currently known how temperature change triggers cellular and molecular events that activate neurons supplying the pit organ. We dissociated and cultured neurons from the trigeminal ganglia (TG) innervating the pit organs of the Western Diamondback rattlesnake (Crotalus atrox) and the copperhead (Agkistrodon contortix) to investigate electrophysiological responses to thermal stimuli. Whole cell voltage-clamp recordings indicated that 75% of the TG neurons from C. atrox and 74% of the TG neurons from A. contortix showed a unique temperature-activated inward current (I_T). We also found an I_T-like current in 15% of TG neurons from the common garter snake, a species that does not have a specialized heat-sensing organ. A steep rise in the current-temperature relationship of I_T started just below 18°C, and cooling temperature-responsive TG neurons from 20°C resulted in an outward current, suggesting that I_T is on at relatively low temperatures. Ion substitution and Ca²⁺ imaging experiments indicated that I_T is primarily a monovalent cation current. I_T was not sensitive to capsaicin or amiloride, suggesting that the current did not show similar pharmacology to other mammalian heat-sensitive membrane proteins. Our findings indicate that a novel temperature-sensitive conductance with unique ion permeability and low-temperature threshold is expressed in TG neurons and may be involved in highly sensitive heat detection in snakes. snake; thermosensory; trigeminal; ion conductance     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A     

Purification and properties of {delta}-aminolevulinic acid dehydratase from radish cotyledons

-Aminolevulinic acid dehydratase (5-aminolevulinate hydro-lyase,EC 4.2.1.24 [EC] ) was purified from greening radish cotyledons. Thefinal product was homogeneous on polyacrylamide disc gel electrophoresisand had a molecular weight, estimated by gel filtration, of282,000 daltons. The enzyme seems to require magnesium ion aswell as sulfhydryl compounds for maximum activity. EDTA anda low concentration of zinc ion markedly inhibited the activity.The optimum pH was 8.0; the Km value for -aminolevulinic acidwas 3.85?10^–4M. Levulinic acid was a competitive inhibitorof the enzyme, with a Ki of 2.14?10^–4M. These propertieswere compared with those of microorganism and animal -aminolevulinicacid dehydratases. (Received November 22, 1976; )