Salt activation and inhibition of membrane ATPase from roots of the halophyte Atriplex nummularia

Abstract Salt-stimulated ATPase activity in membrane preparations obtained from roots of Atriplex nummularia Lindl. at pH 5 was not suscep-tible to inhibition by KC1 or NaCl up to 450 mol m-3 but showed a broad peak of activity between 150 and 300 mol m^?3. At pH 8 stimulation occurred at 50 mol m^?3 but concentrations above 100 mol m^?3 depressed activity below the level of the MgATPase activity. By contrast, preparations from roots of Pisum sativum L. at pH 5 showed maximal stimulation at 25 to 50 mol m^?3 of NaCl or KC1; concentrations higher than 150 mol m^?3 depressed activity below that of MgATPase activity. At pH 8 maximal stimulation was observed at 5 to 10 mol m^?3 NaCl or KC1 while the threshold for inhibition was reduced to 15 mol m^?3. With increasing salt concentrations the pH profiles for NaCl stimulation of Atriplex ATPase activity (expressed as the difference between treatment and control) showed a progressive displacement of the apparent optimum towards lower pH. The shift was not apparent when stimulation was expressed as a percentage of MgATPase activity. This shift may be accounted for if NaCl stimulated the monovalent salt-activated ATPase activity but simultaneously inhibited MgATPase activity.     

Plasma membrane-bound NADH: Fe3+-EDTA reductase and iron deficiency in tomato (Lycopersicon esculentum). Is there a Turbo reductase?

The properties of NADH-dependent Fe³⁺-EDTA reductase in plasma membranes (PM) from roots of iron-deficient and -sufficient tomato plants [Lycopersicon esculentum L. (Mill.) cv. Abunda] were examined. Iron deficiency resulted in a 3-fold increase of in vivo root iron-chelate reductase activity with a K_m (Fe³⁺-EDTA) of 230 μM. In purified root PM, average specific activities of ferric chelate reductase of 410 and 254 nmol Fe (mg protein)^?1 min^?1 were obtained for iron-deficient and -sufficient plants, respectively. In both cases, the PM-bound activity showed a pH optimum at pH 6.8. Activity depended on NADH and not on NADPH and on the presence of detergent. The activity was inhibited 40-50% by superoxide dismutase (EC 1.15.1.1) and ca 30% by oxygen. Kinetic analysis of the membrane-bound enzyme revealed a K_m (Fe³⁺-EDTA) of ca 200 μM for both iron-stressed and -sufficient plants. For NADH, K_m values around 230 μM were obtained. The ferric chelate reductase could be solubilised from salt-washed PM with Triton X-100 at a protein:detergent ratio of 1:2.8 (w/w). The Triton-soluble fraction revealed one enzyme-stained band in native polyacrylamide electrophoresis. Although the membranes showed no nitrate reductase (NR; EC 1.6.6.1) activity, anti-spinach NR immunoglobulin G (IgG) recognized a 54 kDa band both in the PM and the Triton-soluble fraction, but not in the enzymatically active material obtained from the native gel. No evidence could be found for the synthesis of a new, biochemically distinct PM-bound ferric chelate reductase under iron deficiency, which might be identified as the so-called Turbo reductase. It is concluded that iron deficiency in tomato induces increased expression of a ferric chelate reductase in root PM, which is already present in iron-sufficient plants and probably also in plants, which do not contain the Turbo reductase, like the grasses. The iron reductase is not identical with the recently reported PM-associated nitrate reductase.     

Latent nitrate reductase activity is associated with the plasma membrane of corn roots

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U₂). Analysis with marker enzymes indicated that the U₂ fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.Abbreviations L₁ first lower phase - NR nitrate reductase - NRA nitrate-reductase activity - PM plasma membrane - T:p Triton X-100 (octylphenoxy polyethoxyethanol) to protein ratio - U₂ second upper phase     

Boric acid stimulates the plasma membrane H+-ATPase of ungerminated lily pollen grains

The stimulation of the plasma membrane (PM) H⁺-ATPase by boric acid was studied on a microsomal fraction (MF) obtained from ungerminated, boron-dependent pollen grains of Lilium longiflorum Thunb. which usually need boron for germination and tube growth. ATP hydrolysis and H+ transport activity increased by 14 and 18%, respectively, after addition of 2-4 mM boric acid. The optimum of boron stimulation was at pH 6.5-8.5 for ATP hydrolysis and at pH 6.5-7.5 for H⁺ transport. No boron stimulation was detected when vanadate was added to the MF, whereas an increase of 10-20% in ATP hydrolysis and H⁺ transport was still measured in the presence of inhibitors specific for V -type ATPase (nitrate and bafilomycin) and F-type ATPase (azide), respectively. A vanadate-sensitive increase in ATP hydrolysis activity was also observed in partially permeabilized vesicles (0.001%[w/v] Triton X-100) suggesting a direct interaction between borate and the PM H⁺-ATPase rather than a weak acid-induced stimulation. Additionally, we measured the effect of boron on membrane voltage (V_m) of ungerminated pollen grains and observed small hyperpolarizations in 48% of all experiments. Exposing pollen grains to a more acidic pH of 4 caused a depolarization, followed in some experiments by a repolarization (21%). In the presence of 2 mM boron such hyperpolarizations, perhaps caused by an enhanced activity of the H⁺-ATPase, were measured in 58% of all tested pollen grains. The effects of boron on V_m may be reduced by additional stimulation of a K⁺ inward current of opposite direction to the H⁺-ATPase. All experiments indicate that boron stimulates an electrogenic transport system in the plasma membrane which is sensitive to vanadate and has a pH optimum around 7, i.e. the plasma membrane H⁺-ATPase. A boron-increased PM H⁺-ATPase activity in turn may stimulate germination and growth of pollen tubes.     

Mg2+-ATPase activity in wheat root plasma membrane vesicles: Time-dependence and effect of sucrose and detergents

Purified plasma membrane vesicles were isolated in the presence of 250 mM sucrose from 7-day-old roots of Triticum aestivum L. cv. Drabant by aqueous polymer two-phase partitioning. When added to a low-salt medium containing 9-aminoacridine (9-AA), the vesicles caused a much larger total decrease in 9-AA fluorescence when sucrose was absent than when sucrose was present. A slow component of the decrease was also larger in the absence of sucrose. Triton X-100 reduced the decrease in 9-AA fluorescence upon vesicle addition and abolished completely the slow component of the decrease. There was no correlation between the time-dependence of 9-AA fluorescence and that of the Mg²⁺-ATPase described below. The time course of Mg²⁺-ATPase activity was followed by sampling at short intervals (down to 10 s) and analyzing for P, released. In the absence of detergent, the rates of P, release were linear from zero minutes, whether 250 mM sucrose was present or not, but the rate was 10^?50% higher in the absence of sucrose than in its presence. Sucrose (250 mM) added during a minus-sucrose assay lowered Mg²⁺-ATPase activity within 2 min to the level observed with 250 mM sucrose present from the start. The effect of 25-1 100 mM sucrose was tested and there was little or no effect below KM) mM. Above 100 mM sucrose the rate of P, release decreased drastically; at 1 100 mM sucrose the rate was ca 20% the rate at 25 mM sucrose. The inhibitory effect of sucrose was not alleviated by increased concentrations of Mg²⁺ and/or ATP. nor was it affected by the presence or absence of Triton X-100. We conclude that sucrose somehow inhibits the Mg²⁺-ATPase directly or affects the conformation of the plasma membrane in such a way as to inhibit the enzyme. The presence of detergents increased Mg²⁺-ATPase activity in the order Triton X-100 (4–5-fold) > Zwittergent 3–14 = Na-cholate = octylglucoside > digitonin (2-fold). In all cases optimal activity was observed at detergent concentrations at or below the critical micellar concentration. The detergent concentration curves could be simulated by the sum of a stimulatory and an inhibitory reaction. At the optimal concentration, digitonin gave a linear time-course of P, release, whereas all the other detergents showed a distinct lag of 1–3 min before maximal rates were attained. The problems of using detergents in polarity assays are discussed.     

Selective Solubilization of Membrane Proteins Differentially Labeled by p-Chloromercuribenzenesulfonic Acid in the Presence of Sucrose

Broadbean (Vicia faba L.) leaf discs have been incubated with the slowly permeant thiol reagent [²⁰³Hg]-para-chloromercuribenzenesulfonic acid (PCMBS) in the presence or in the absence of sucrose, and the release of PCMBS-labeled proteins has been monitored in media containing various concentrations of urea, ethyleneglycol-bis-(β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA), sodium cholate, sodium dodecyl sulfate, Triton X-100, octylglucoside or (3-[3-cholamidopropyl)-dimethylammonio] 1-propane-sulfonate) (CHAPS). The proteins differentially labeled by PCMBS in the presence of sucrose which, on the basis of previous results, are assumed to include the sucrose carrier, were preferentially solubilized by 1% CHAPS, 1% octylglucoside, or 1% Triton X-100. Other PCMBS-labeled proteins (`background' proteins) could be partially removed by EGTA, urea, or 0.1% cholate. Sequential treatment by 10 mm EGTA and 1% CHAPS was found to give a fraction highly enriched in the differentially labeled proteins. Analysis of the specific activity of microsomal pellets suggests that the results obtained with leaf discs give a good account of what is occurring at the plasma membrane level. These data, which suggest that the proteins differentially labeled by PCMBS in the presence of sucrose are intrinsic membrane proteins, can be used to solubilize these proteins from microsomal fractions.     

Duroquinone-stimulated NADH oxidase and b type cytochromes in the plasma membrane of cauliflower and mung beans

Microsomal membrane preparations of cauliflower inflorescences and mung bean hypocotyls possess duroquinone (DQ)-stimulated NADH oxidase activities at rates of 1–10 nmol NADH · min⁻ · mg⁻. These redox reaction are associated with the endoplasmic reticulum (ER) and the plasma membrane (PM) as shown by the distributions of marker enzymes in sucrose gradients. The NADH oxidase thus partially cosediments with a specific blue light (or ascorbate) reducible b type cytochrome of the PM.Cauliflower membranes are further purified by means of an aqueous polymer two phase method. The NADH oxidase in this presumptive PM fraction is to some extent stimulated by Triton X-100 and insensitive to KCN (1 mM) or quinacrine (0.4 mM). Kinetics for DQ stimulation showed a biphasic saturation curve. These membranes also have a high FeCN reduction capacity induced by NADH but insensitive to DQ.No evidence could be found in the present study for the involvement of the specific b type cytochrome in the NADH dehydrogenase system.     

Cytochromec oxidase fromParacoccus denitrificans in Triton X-100: Aggregation state and kinetics

Cytochromec oxidase fromParacoccus denitrificans was homogenously dispersed in Triton X-100. Using gel exclusion chromatography and sucrose gradient centrifugation analysis a molecular weight of the detergent-protein complex of 155,000 was determined. After subtraction of the bound detergent (111 mol/mol hemeaa ₃) a molecular weight of 85,000 resulted, which agreed well with the model of a monomer containing two subunits. This monomer showed high cytochromec oxidase activity when measured spectrophotometrically in the presence of Triton X-100 (V _max=85 s^–1). The molecular activity, plotted according to Eadie-Hofstee, was monophasic as a function of the cytochromec concentration. AK _m of 3.6×10^–6 M was evaluated, similar to theK _m observed in the presence of dodecyl maltoside [Naeczet al. (1985).Biochim. Biophys. Acta 808, 259–272].     

NADH-dependent Fe3+EDTA and oxygen reduction by plasma membrane vesicles from barley roots

Brüggemann, W. and Moog, P. R. 1989. NADH-dependent Fe³⁺EDTA and oxygen reduction by plasma membrane vesicles from barley roots. Biochemical properties of pyridine-dinucleotide-dependent Fe³⁺-EDTA reductase were analysed in purified plasma membranes (PM) from barley (Hordeum vulgare L. cv. Marinka) roots. The enzymatic activity preferred NADH over NADPH as electron donor and it was 3-fold increased in the presence of detergent. The reductase showed a pH optimum of 6.8 and saturable kinetics for NADH with K_m (NADH) of 125 μM and V_max of 143 nmol Fe (mg protein)^-1 min^-1 in the presence of 500 μM Fe³⁺EDTA. For the dependence of the reaction rate on the iron compound, K_m(Fe³⁺EDTA) of 120 μM and V_max of 184 nmol (mg protein)^-1 min^-1 were obtained. The activity was insensitive to superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6) and antimycin A, but stimulated by an oxygen-free reaction medium. It could be solubilized by 0.25% (w/v) Triton X-100. The solubilized enzyme revealed one band in native polyacrylamide gel electrophoresis (PAGE) and in isoelectric focussing (IEF) at pl 7.4 by enzyme staining. Major polypeptides with molecular weights of 94, 106, 120 and 205 kDa corresponded to the enzyme-stained band from native PAGE. Analysis of oxygen consumption by the membranes revealed the existence of NADH:CK oxidoreductase activity, which was stimulated by salicylhydroxamic acid (SHAM), chinhydron, Fe³⁺EDTA and Fe³⁺EDTA but not by K₃ [Fe(CN)₆] or K₄[Fe (CN)₆). The stimulating effect of the iron chelates on oxygen consumption was due to Fe²⁺ and could be suppressed by bathophenanthroline disulfonate (BPDS), SOD and p-chloromercurophenylsulfonic acid (PCMS). The results are discussed with respect to the nature of the stimulation.     

Inhibition of Nitrate Transport by Anti-Nitrate Reductase IgG Fragments and the Identification of Plasma Membrane Associated Nitrate Reductase in Roots of Barley Seedlings

Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO₃⁻ uptake by more than 90% but had no effect on NO₂⁻ uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO₃⁻ uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO₃⁻ uptake. The results present the possibility that NO₃⁻ uptake and NO₃⁻ reduction in the PM of barley roots may be related.     

CHARACTERIZATION OF A CALCIUM-ACTIVATED CYTIDINE TRIPHOSPHATE PHOSPHOHYDROLASE PRESENT IN DORSAL SPINAL NERVE ROOTS

We report here the presence of a CTPase in rat dorsal spinal nerve roots. This is activated by Ca²⁺ (K_m= 2.1 mM) and to a lesser extent by Mg²⁺. It is active at 0°C and activity is increased 2-fold at 20°C. Substrate specificity is in the order CTP > UTP > TTP = ITP = GTP > ATP. The enzyme is totally membrane-bound and is inactivated by 0.1% Triton X-100. Its possible function is discussed     

Requirement for direct association of ammonia-excreting Anabaena variabilis mutant (SA-1) with roots for maximal growth and yield of wheat

Direct association between wheat roots and an ammonia-excreting mutant of the cyanobacterium Anabaena variabilis, strain SA-1, was required for maximal enhancement of growth of wheat plants in nitrogen (N)-free, hydroponic medium. Over 85% of the cyanobacterial mutant SA-1 inoculated to the roots were adsorbed under non-saturating conditions. The adsorption process of SA-1 to wheat roots was biphasic: an initial rapid adsorption was followed by a slow phase with about 10% of the initial adsorption rate. The maximal adsorption rate of filaments observed was 1.6 mg dry wt. SA-1 adsorbed·plant^–1·h^–1. Bypassing CO₂ fixation and sugar formation, the ¹⁴C label from [¹⁴C]sucrose was directly applied to leaf blades to study sugar translocation. The ¹⁴C label from this treatment appeared in the wheat culture medium within an hour. Nitrate-grown plants excreted about 30% of the ¹⁴C label into the medium, compared to only 10% excreted by wheat/Anabaena co-cultures. SA-1 assimilated 27% of all ¹⁴C translocated from [U-¹⁴C]sucrose applied to wheat leaves, and ¹⁴C label from this treatment was recovered from strain SA-1 after 30 min. Roots and cyanobacteria accounted for 51% of all radioactive label recovered in the plants co-cultured with SA-1 vs 20% for nitrate-grown plants. We studied the activity of -fructosidase (invertase) in wheat of variety Yecora rojo. Roots from nitrate-grown wheat plants produced high levels of invertase activity, which converted over 85% of 3 mm sucrose into glucose and fructose in 24 h. The rate of sucrose disappearance in the medium of co-cultures using A. variabilis SA-1, was 70% of that of nitrate-grown plants, but the levels of glucose and fructose in these cultures were always very low during sucrose conversion, suggesting hexose assimilation. To study the role of diffusible metabolites, a dialysis membrane was employed to separate the ammonia-excreting SA-1 from the wheat roots. Containing SA-1 in a dialysis bag away from direct root contact, severely limited leaf growth to less than one-third of the growth rate of nitrate control cultures. Ammonia produced by mutant SA-1 in dialysis bag cultures was excreted into the medium at 0.4 mm vs 1.2 mm in free-living cultures, but ammonia was not detectable in co-cultures with or without the dialysis bag containing the mutant. The nitrogenase activity derepressed in the mutant and responsible for ammonia excretion was always higher in the association co-cultures than in either free cells or in dialysis-bag cultures. The nitrogenase activity of strain SA-1 was highest (200 mol ethylene formed·mg^–1 Chl·h^–1) when the cyanobacterium was associated with the root tips. Dialysis membrane separation of plant and cyanobacterium severely inhibited growth of wheat during a complete growth cycle of 2 months. Total biomass and grain yield were very similar for control cultures without inorganic N or SA-1, and for diffusion cultures containing SA-1, kept in a dialysis bag around the roots. Total biomass of the association co-culture attained 75% of the biomass of the nitrate-grown control. It is proposed that wheat roots supplied fructose derived from sucrose for growth and nitrogen fixation of SA-1 in the light, and that ammonia excreted by SA-1 was utilized by the wheat plant for its own growth. Correspondence to: H. Spiller     

Purification and Some Properties of an Isolate of Beet Yellows Virus from Ukraine

A purification procedure, which yielded up to 15–30 mg of beet yellows virus (BYV) per 100 g of infected Tetragonia expansa leaves, has been developed. The procedure included sap clarification with Triton X-100, and two cycles of ultracentrifugation through sucrose cushion, which contained PEG-6000 and NaCl. A specific antiserum was prepared, and BYV infection was successfully detected by the double-antibody sandwich (DAS) ELISA in infected sugar beet leaves and roots diluted up to 1 × 10⁵ and 1 × 10⁴, respectively. The virus concentration was demonstrated to decrease in infected sugar beet roots slowly during 7 months, thus allowing successful diagnosis of planting material in winter storage. BYV presence in Myzus persicae aphids was also reliably detectable using the DAS-ELISA. In a competitive DAS-ELISA test, the Ukraine and the British BYV isolates were found serologically indistinguishable.     

Scanning ion‐selective electrode technique and X‐ray microanalysis provide direct evidence of contrasting Na+ transport ability from root to shoot in salt‐sensitive cucumber and salt‐tolerant pumpkin under NaCl stress

Grafting onto salt‐tolerant pumpkin rootstock can increase cucumber salt tolerance. Previous studies have suggested that this can be attributed to pumpkin roots with higher capacity to limit the transport of Na⁺ to the shoot than cucumber roots. However, the mechanism remains unclear. This study investigated the transport of Na⁺ in salt‐tolerant pumpkin and salt‐sensitive cucumber plants under high (200 mM) or moderate (90 mM) NaCl stress. Scanning ion‐selective electrode technique showed that pumpkin roots exhibited a higher capacity to extrude Na⁺, and a correspondingly increased H⁺ influx under 200 or 90 mM NaCl stress. The 200 mM NaCl induced Na⁺/H⁺ exchange in the root was inhibited by amiloride (a Na⁺/H⁺ antiporter inhibitor) or vanadate [a plasma membrane (PM) H⁺‐ATPase inhibitor], indicating that Na⁺ exclusion in salt stressed pumpkin and cucumber roots was the result of an active Na⁺/H⁺ antiporter across the PM, and the Na⁺/H⁺ antiporter system in salt stressed pumpkin roots was sufficient to exclude Na⁺. X‐ray microanalysis showed higher Na⁺ in the cortex, but lower Na⁺ in the stele of pumpkin roots than that in cucumber roots under 90 mM NaCl stress, suggesting that the highly vacuolated root cortical cells of pumpkin roots could sequester more Na⁺, limit the radial transport of Na⁺ to the stele and thus restrict the transport of Na⁺ to the shoot. These results provide direct evidence for pumpkin roots with higher capacity to limit the transport of Na⁺ to the shoot than cucumber roots.     

The role of sucrose and nitrogen in adventitious root formation on cultured rose shoots

Cultured shoots ofRosa ‘Improved Blaze’ were used to determine the effects of sucrose and inorganic nitrogen on adventitious root formation. Shoots grown in media containing high sucrose concentrations (146.07–262.93 mM) produced more and longer roots than those grown in media containing 0–87.64 mM sucrose. This response to sucrose was related to the metabolism of sucrose rather than its osmotic properties since the use of mannitol and 3-O-methyl-α-D-glucopyranoside as osmotic substitutes did not reproduce the effect on rooting. The number and length of roots increased when the shoots were grown in media with the nitrogen concentration of the Murashige-Skoog (MS) salt formulation reduced from 60 to 7.5 mM. Neither nitrate (NO ₃ ⁻ ) nor ammonium (NH ₄ ⁺ ) alone at any of the concentrations tested had the effect on rooting that both had together in the ratio of the MS salt formulation. When the sucrose and nitrogen concentrations were both varied, the greatest rate of root initiation occurred on shoots grown in media with a high sucrose to nitrogen concentration ratio.     

A re‐assessment of sucrose signaling involved in cluster‐root formation and function in phosphate‐deficient white lupin (Lupinus albus)

Apart from substrate functions, a signaling role of sucrose in root growth regulation is well established. This raised the question whether sucrose signals might also be involved in formation of cluster‐roots (CRs) under phosphate (Pi) limitation, mediating exudation of phosphorus (P)‐mobilizing root exudates, e.g. in Lupinus albus and members of the Proteaceae. Earlier studies demonstrated that CR formation in L. albus was mimicked to some extent by external application of high sucrose concentrations (25 mM) in the presence of extremely high P supply (1–10 mM), usually suppressing CR formation. In this study, we re‐addressed this question using an axenic hydroponic culture system with normal P supply (0.1 mM) and a range of sucrose applications (0.25–25 mM). The 2.5 mM sucrose concentration was comparable with internal sucrose levels in the zone of CR initiation in first‐order laterals of P‐deficient plants (3.4 mM) and induced the same CR morphology. Similar to earlier studies, high sucrose concentrations (25 mM) resulted in root thickening and inhibition of root elongation, associated with a 10‐fold increase of the internal sucrose level. The sucrose analog palatinose and a combination of glucose/fructose failed to stimulate CR formation under P‐sufficient conditions, demonstrating a signal function of sucrose and excluding osmotic or carbon source effects. In contrast to earlier findings, sucrose was able to induce CR formation but had no effect on CR functioning with respect to citrate exudation, in vitro activity and expression of genes encoding phosphoenolpyruvate carboxylase, secretory acid phosphatase and MATE transporters, mediating P‐mobilizing functions of CRs.     

Isolation of plasma membrane and binding of the Ca2+ antagonist nimodipine in Chlamydomonas reinhardtii

Chlorophyll-free plasma membranes of the unicellular green alga Chlamydomonas reinhardtii Dangeard were purified from a microsomal fraction using an aqueous polymer two-phase system of 6.5% (w/w) dextran T500, 6·5% (w/w) polyethylene glycol 3350, 60 mM NaCI, 0 33 M sucrose and 5 mM potassium phosphate (pH 7·8). The plasma membrane fraction contained only 2·4% of the microsomal membrane protein. Specific activity of the plasma membrane marker enzyme, K*, Mg²⁺-ATPase (EC 3.6.1.3). was enriched 9-fold over the microsomal fraction, and 22% of total activity was recovered in the upper, polyethylene glycol-rich phase. Contamination from intracellular membranes was minimal. K*, Mg²⁺-ATPase showed a pH optimum at about 6·5, and addition of 0·05% (w/v) Triton X-100 stimulated the activity 3-fold. [³H]-Nimodipinc was employed to characterize 1,4-dihydropyridine-specific membrane receptors. Two apparent binding sites with different affinities to nimodipine were found in the crude microsomal fraction. The separation of plasma membranes from intracellular membranes revealed that one binding site with higher affinity (K_D= 9 nM) was located on the plasma membrane and a second binding site with lower affinity (K_D= 36 nM) on an intracellular membrane The apparent dissociation constants determined from the association and dissociation rate constants in kinetic experiments were comparable to those determined by equilibrium experiments. The maximum number of binding sites of the plasma membrane fraction and the intracellular membrane fraction was B_max= 440 and 470 fmol (mg protein)^-1, respectively. [³H]-Nimodipinc binding was inhibited by (±) verapamil and stimulated by D-cis-diltiazem in both fractions. Moreover, ethyle-neglycol-bis(2-aminoethylcther)-N, N'-tetraacctic acid (EGTA) inhibited [³H]-nimo-dipinc binding in the plasma membrane fraction but not in the intracellular membrane fraction This effect was cancelled by the addition of CaCl₂.     

On the concentration of acetic acid in straw and soil

Summary Freshly harvested wheat straw contained 0.096 g water g^–1 dry straw and 180 mM acetic acid. The straw absorbed water more rapidly from wet soil. The concentration of acetic acid fell to about 10 mM within 6 h of incorporation of straw in the soil and then remained relatively constant for a period of 12 days, irrespective of soil moisture content. In soil at its maximum water holding capacity after gravitational drainage, the decline in acetic acid concentration (c) with distance (d) from wheat or barley straw was exponential, with c=c_o e^–nd where c_o is the concentration of acetic acid at the straw surface and n is a constant (0.46 for barley and 0.42 for wheat straw). The presence of acetic acid seems to be a major cause of poor establishment and growth when seeds and seedling roots come into contact with straw.     

Lectin activity in pollen from plants representative of thirty orders of spermatophyta

Summary Pollen extracts from a variety of species representative of thirty orders of spermatophyta, including gymnosperms, dicotyledons and monocotyledons, were examined for the presence of lectin activity by means of a hemagglutination assay. Hemagglutinating activity (HA) was detected in the pollen extracts of all the species examined, indicating that lectins are generally present in the pollen of spermatophyta. The response of this pollen hemagglutinating activity to the sugars and glycoproteins tested as potential inhibitors was identical in all species examined. Moreover, the hemagglutinating activity of pollen extracts from eight species which had been selected as representative of the gymnosperms and both subclasses of angiosperms exhibited similar properties (e.g. distribution by differential centrifugation, stability to heat, response to bivalent ions). The bulk of the hemagglutinating activity was always recovered in the pellet after centrifugation at 1000 g for 5 min. Although sequential treatments with 1% Triton X-100 and 1 M KCl were ineffective, subsequent incubation of the pellet with saline phosphate buffer released hemagglutinating activity. The solubilized hemagglutinating activity was destroyed by protease treatment, indicating that the substance(s) responsible for the activity is (are) protein in nature and, consequently, might be considered to be a lectin. The sugar specifity of the pollen lectin activity from wheat, potato and bean was compared with that of wheat germ agglutinin (WGA), potato agglutinin and bean agglutinin — the lectins present in sporophytic tissues of these plants. For all three plants, the response of the pollen lectin activity to sugars and glycoproteins was different from that shown by the lectin from sporophytic tissues.Abbreviations HA Hemagglutinating activity - PBS 150 mM Na-phosphate buffer (pH 7.2) containing 0.9% NaCl - PHA Phaseolus vulgaris agglutinin - STA Solanum tuberosum agglutinin - WGA wheat germ agglutinin     

Chlorophyllase and Photsystem

• 1 Preliminary experiments showed that Triton X-100 at low concentrations (appr. 0.01%) induces reversible changes in some photosynthetic reactions in Phaeodactylum cells without influencing their absorption spectrum; presumably membrane permeability is effects. Although DC PIP can pass through the cell membrane in the presence of 0.01% Triton X-100, only a very low PS I activity, measured as MV photoreduction by DCPIP-ascorbate, is observed with whole cells.
• 2 MV photoreduction sensitized by membrane fragments isolated from Phaetdactylum, Euglena, Porphyridium and Synechococcus after French Press treatment is much higher with Phaeodactylum than with the other organisms. Spinach shroma membrane fragments show higher photosensitizing activity than grana fragments.
• 3 MV photoreduction in the above mentioned experiments is not influenced by DCMU (2 × 10^?6M) or sodium azide (0.01–0.05 M); KCN (4 × 10^?3M) has an inhibiting effect only with the blue-green alga Synechococcus. The reaction mechanism of chlorophyll-sensitized MV photoreduction is discussed.
• 4 Chlorophyll in an aqueous medium containing Triton X-100 (JsO.01%) sensitizes the photoreduction of MV by DCPIP ascorbate. A similar reaction is observed with chlorophyll combined with solubilized Phaeocdactylum chlorophyllase: in the latter case the presence of both MgCl₂ and DTT is required.
• 5 MV-photoreduction is concluded to be a very unreliable procedure for determining PS I activity in membranes if these membranes have been prepared in (the presence of detergents.
• 6 The results support the hypothesis that (prochlorophyllase is a PS I protein.
• 7 The capacity of PS I to reduce MV in vivo is discussed.