Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membrane after treatment of intact tissues and cell free extracts with triacontanol

Ca²⁺- and Mg²⁺-dependent ATPase activity (EC 3.6.1.3) in a plasma membrane-enriched fraction increased rapidly after in vivo application of physiologically active concentrations of triacontanol (TRIA) to the roots of barley ( Hordeum vulgare L. cv. Conquest) seedlings. Ca²⁺- and Mg²⁺-dependent ATPase activity was 64 and 85% higher, respectively, in the roots of seedlings germinated in the presence of growth-promoting concentrations of TRIA compared to controls. The increase in vivo was concentration dependent, with the greatest increase obtained at 2.3 n M TRIA. Maximal stimulation of ATPase activity of excised tissue treated with TRIA coincided with the temperature at which the barley was grown. At this temperature the plasma membrane is primarily in a mixed gel/liquid crystalline state. Pretreatment of barley roots with cyclohexamide did not alter ATPase stimulation by TRIA. Two to three times more [¹⁴C]-TRIA (mg membrane protein)⁻¹ was found associated with plasma membrane-enriched vesicles treated with TRIA than with vesicles enriched for mitochondrial membranes or for vesicles enriched for tonoplast, Golgi and rough endoplasmic reticulum. Both Ca²⁺- and Mg²⁺-dependent ATPase activity increased by 40–60% within 30 min of the addition of 2.3 n M TRIA to cell-free extracts of barley roots. The addition of octacosanol, the C₂₈ analogue of TRIA, to cell-free extracts did not affect metal-dependent ATPase activity. Consistent with many studies in the green-house, simultaneous additions of equimolar amounts of TRIA and octacosanol to cell-free extracts resulted in inhibition of ATPase stimulation by TRIA. TRIA may directly affect plasma membrane function in barley roots.     

MORPHOLOGICAL CHANGES OF CULTURED MYOCARDIAL CELLS DUE TO CHANGE IN EXTRACELLULAR CALCIUM ION CONCENTRATION

Increase in the extracellular Ca²⁺ concentration from low (≤ 10⁻⁷ M) to normal (10⁻³ M) caused morphological changes of cultured myocardial cells obtained from fetal mouse heart. The extracellular Na⁺ and K⁺ concentrations of the normal medium (10⁻³ M Ca²⁺) did not significantly affect the genesis of these morphological changes. Like Ca²⁺, Ba²⁺ and Sr²⁺, but not Mg²⁺, Co²⁺ or Ni²⁺, could induce morphological changes. Increase in the extracellular Ca²⁺ concentration from 10⁻⁸ M to 10⁻³M also caused excess uptake of ⁴⁵Ca²⁺ by cultured myocardial cells. B–16CW 1 cells, which did not show these morphological changes, did not take up excess ⁴⁵Ca²⁺ on this treatment. Treatments, such as addition of verapamil or incubation at pH 6.3, which reduced the genesis of morphological changes, reduced the rate of ⁴⁵Ca²⁺ uptake by myocardial cells. These facts show that the morphological changes of myocardial cells induced by increasing the extracellular Ca²⁺ concentration from low to normal are due to excess uptake of Ca²⁺ by the myocardial cells.
The morphological changes of cultured myocardial cells induced by increasing the extracellular Ca²⁺ concentration from low to normal were reversed on further incubation of the cells in medium with or without Ca²⁺.     

STIMULATORY EFFECTS OF SUBSTANCE P ON NEURITE EXTENSION AND CYCLIC AMP LEVELS IN CULTURED NEUROBLASTOMA CELLS

Abstract— Synthetic substance P initially increased cyclic AMP levels and subsequently induced neurite extension in cultured neuroblastoma N 18 cells. The magnitude of these effects depended on the concentration of fetal calf serum (FCS) in the culture medium, being more evident in the presence of a lower (0.1%) concentration of FCS.
In Eagle's medium containing 0.1% FCS, low concentrations of substance P (10⁻⁷-10⁻⁵ M) increased cyclic AMP levels and stimulated neurite extension.
In Eagle's medium containing 5%FCS, both substance P at concentrations of 10⁻⁵-10⁻³M and dibutyryl cyclic AMP at concentrations of 10⁻⁴-10⁻²M increased cyclic AMP levels and stimulated neurite extension. The activities of acetylcholinesterase, (Na⁺+ K⁺)-, HCO₃⁻ and Mg²⁺ -stimulated-ATPase were also increased. Cell growth was inhibited.
Substance P at concentrations of 10-⁷-10⁻⁵M also stimulated the adenylate cyclase activity of a particulate fraction of N 18 in a concentration-dependent manner.     

Characterization of an Initiating Cell-Free Protein Synthesis System Derived from Rabbit Brain

Abstract: Protein synthesis in the brain is known to be affected by a wide range of treatments. The detailed analysis of the mechanisms that are involved would be facilitated by the development of cell-free translation systems derived from brain tissue. To date, brain cell-free systems have not been fully characterized to demonstrate a capacity for initiation of translation. The following criteria were utilized to demonstrate that a cell-free protein synthesis system derived from rabbit brain was capable of initiation in vitro : (a) sensitivity of cell-free translation to the initiation inhibitor aurintricarboxylic acid (ATA); (b) binding of [³⁵S]Met-tRNA_f to 40S and 80S initiation complexes; (c) incorporation of labeled initiation methionine into high-molecular-weight proteins; and (d) the association of labeled exogenous mRNA with polysomes. The optimum conditions for amino acid incorporation in this system were 4 mM-Mg²⁺, 140 mM-K⁺, and pH 7.55. Incorporation was dependent on the addition of ATP, GTP, and an energy-generating system. Cell-free protein synthesis reflected the normal process, since a similar spectrum of proteins was synthesized in vitro and in vivo. This initiating cell-free translation system should have wide application in the analysis of the mechanisms whereby various treatments affect protein synthesis in the brain.     

Multisite Interactions Between Pb2+ and Protein Kinase C and Its Role in Norepinephrine Release from Bovine Adrenal Chromaffin Cells

Abstract: We investigated the interaction between Pb²⁺ and protein kinase C (PKC) in the Pb²⁺-induced release of norepinephrine (NE) from permeabilized adrenal chromaffin cells. Our analysis of endogenous PKC activity in permeabilized cells suggests that Pb²⁺ interacts with the adrenal enzyme at multiple sites. Pb²⁺ activates the enzyme through high-affinity ( K _A(Pb) = 2.4 × 10⁻¹² M ) interactions and inhibits the enzyme by competitive and noncompetitive interactions with nanomolar-( K _i = 7.1 × 10⁻⁹ M ) and micromolar- ( K '_i = 2.8 × 10⁻⁷ M ) affinity sites, respectively. Activation of PKC by 12- O -tetradecanoylphorbol 13-acetate (TPA) in Ca²⁺-deficient, Pb²⁺-containing medium, enhances the Pb²⁺-induced NE release from permeabilized chromaffin cells by lowering the concentration of Pb²⁺ required for half-maximal activation of the secretory response from 7.5 × 10⁻¹⁰ to 5.7 × 10⁻¹¹ M . The PKC inhibitors staurosporine and pseudosubstrate PKC (19–36) abolish the effect of TPA without affecting the Pb²⁺-induced secretion in the absence of TPA. These results indicate that (a) Pb²⁺ is a partial agonist of PKC, capable of both activating and inhibiting the enzyme and (b) synergistic activation of PKC by TPA and Pb²⁺ results in increased sensitivity of exocytosis to Pb²⁺ but is not obligatory for Pb²⁺-triggered secretion.     

Cell-Free Protein Synthesis by Rumen Protozoa

SYNOPSIS. The characteristics of protein synthesis by cell-free extracts of mixed rumen protozoa have been investigated. ATP,¹ GTP, and an energy supply system were necessary for amino acid incorporation which was partially inhibited by cycloheximide but not by chloramphenicol (100 μg/ml). The system was particularly sensitive to the cation concentration of the incubation mixture, maximal incorporation requiring 5 mM Mg⁺⁺ and 50 mM K⁺ Incorporation was further stimulated by the addition of 0.25 mM spermidine or 0.25 mM MnCl₂. Sucrose gradient centrifugation of the cell sap after amino add incorporation showed that most of the incorporated radioactivity was associated with free polysomes. These polysomes contained 82 S ribosomes which dissociated in high Tris concentrations to yield 40 S and 55 S ribosomes.     

THE RELEASE OF COPPER-COMPLEXING LIGANDS BY THE BROWN ALGA FUCUS VESICULOSUS (PHAEOPHYCEAE) IN RESPONSE TO INCREASING TOTAL COPPER LEVELS

The growth of Fucus vesiculosus L. germlings in chemically defined culture media containing a range of Cu concentrations (20–1000 nM) was monitored simultaneously with measurement of the Cu speciation in the media by competitive equilibrium-adsorptive cathodic stripping voltammetry. Fucus vesiculosus germlings were found to exude Cu-complexing ligands with conditional stability constants of the order of 1.6 × 10¹¹. Ligand concentrations increased with increasing total dissolved Cu concentrations (Cu_T) until a concentration of 500–800 neq Cu·L⁻¹ was reached. Concentrations of the ligand exceeded Cu_T in treatments containing 20 and 100 nM Cu, were similar to Cu_T in the 500-nM Cu treatment, but were less than Cu_T in the 1000-nM treatment. Therefore, [Cu²⁺] were calculated to be at concentrations of 10⁻¹¹− 10⁻¹⁰ M in the 20- and 100-nM treatments, 10⁻⁹ M in the 500-nM treatment, and 10⁻⁷ M in the 1000-nM treatment. Growth rates were lowest at Cu²⁺ concentration > 10⁻⁹. These results are discussed within the context of the potential roles for exuded copper-complexing ligands.     

Vitamin D3 affects growth and Ca2+ uptake by Phaseolus vulgaris roots cultured in vitro

Vitamin D₃ at low concentration (10⁻⁹ M) inhibited the growth of Phaseolus vulgaris L. (cv. Contrancha) roots in vitro as measured by elongation (14 h) and [³H]-leucine incorporation into protein (2 h), and increased their labelling with ⁴⁵Ca²⁺ (2 h). Cycloheximide and puromycin (50 u.M) blocked vitamin D₃ stimulation of root ⁴⁵Ca²⁺ labelling, indicating that it is mediated by de novo protein synthesis. The calcium ionophore X-537A (10⁻⁵JW) induced similar changes both in root elongation and ⁴⁵Ca²⁺ uptake (14 h). This may indicate that the inhibitory effects of the sterol on root growth are mediated by changes in Ca²⁺ fluxes. However, this interpretation should be further strengthened by additional studies as the ionophore may have acted on root growth, affecting physiological processes other than Ca²⁺ transport.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

STUDIES ON MEVALONATE KINASE, PHOSPHOMEVALONATE KINASE AND PYROPHOSPHOMEVALONATE DECARBOXYLASE IN DEVELOPING RAT BRAIN

Abstract— We have in the present study investigated the properties of mevalonate kinase, phosphomevalonate kinase and pyrophosphomevalonate decarboxylase in the 105,000 g supernatant fractions from rat brain, and determined whether the activities of these enzymes change during brain development. All three enzymes in brain showed a specific requirement for ATP for optimal activity. The presence of Mg²⁺ as divalent cation was also required for optimal activity of mevalonate kinase and phosphomevalonate kinase. Both Mg²⁺ and Mn²⁺ were equally effective divalent metal ions for pyrophosphomevalonate decarboxylase in brain. Mevalonate kinase as well as phosphomevalonate kinase were active in a broad pH range of 6.5–8 while the pH curve for pyrophosphomevalonate decarboxylase showed a peak activity at approx 6. No age-dependent change occurred in the activities of mevalonate kinase and phosphomevalonate kinase in developing brain, whereas pyrophosphomevalonate decarboxylase activity in brain increased during the 1st week after birth, reached a peak value at about the 8th day of age and declined slowly thereafter. The K_m for brain mevalonate kinase in 2, 13 and 52 day old rats were 312, 400 and 434 μM, respectively. The V _max for the kinase in 2, 13 and 52 day old rats were in the range of 45–52 nmol/h/mg protein, respectively. This suggests that, like in liver (R amachandran & S hah , 1976), pyrophosphomevalonate decarboxylase in brain may also be one regulatory step for cholesterol synthesis.     

Picomolar concentrations of tentoxin affect Mg2+- and Ca2+-ATPase activities of plasma membrane vesicles from roots of winter wheat seedlings

Purified plasmalemma vesicles were isolated in the presence of 250 m M sucrose from roots of 14-day-old seedlings of winter wheat ( Triticum aestivum L. Martonvásári-8) by phase partitioning of salt-washed microsomal fractions in a Dextran-polyethylene glycol two-phase system, and both Mg²⁺- and Ca²⁺-ATPase activities were detected. Orthovanadate-sensitive Mg²⁺-ATPase activity associated with the inside of right side-out plasmalemma (PM) vesicles (latency 98%) was inhibited 76% by 0.3 m M Ca²⁺, Ca²⁺-dependent ATPase activity located partly on the inside and partly on the outside of plasmalemma vesicles (latency 47%) was not affected by Mg²⁺.
Mg²⁺-ATPase activity was inhibited by 68% and inhibition of Mg²⁺ activation by 0.3 m M Ca²⁺ partly disappeared in the presence of 10 p M tentoxin, a fungal phytotoxin. Mg²⁺-ATPase activity remained inhibited up to 10 n M tentoxin while at 1 μ M tentoxin Mg²⁺ activation was as high as without tentoxin. K⁺-stimulation and vanadate inhibition was increased and decreased, respectively, by 100 p M -10 n M tentoxin. Ca²⁺-dependent ATPase activity was continuously increased by 1 p M -10 n M tentoxin, but at 1 μ M tentoxin the stimulation disappeared. The effects of p M tentoxin on plasma-lemma Mg²⁺-ATPase are discussed in relation to its influence on K⁺ transport in wheat seedlings.     

Seed dimorphism in Salicornia europaea: Nutrient reserves

Median and lateral seeds of Salicornia europaea L. were separately analysed for their sizes and nutrient reserves. The mean air-dry weight of a single median and lateral seed was 0.31 and 0.25 mg, respectively. The composition as well as the concentration of the nutrient reserves were similar in both seed types. The bulk of the cations was derived from K⁺, followed by Mg²⁺, Na⁺ and Ca²⁺. The chloride content was somewhat higher than the sodium content, and phosphate was equalled by acid soluble Ca²⁺ and Mg²⁺. Starchy compounds and sucrose were present in equal amounts, each of them accounted for about 50% of the carbohydrates. Glucose and fructose were less than 1%. Protein-nitrogen (ethanol-insoluble N) was about 34 g (kg dry seeds)⁻¹. About 7 g (kg dry seeds)⁻¹ was ethanol-soluble nitrogen, of which 10% was derived from amino acids. The total lipid content was more than 290 g (kg dry seeds)⁻¹, 65% were calculated to be glycerides. More than 90% of the fatty acids consisted of linoleic and oleic acids, the majority (72%) of which was linoleic acid.     

The Effect of Divalent Cations on Aggregation of Dictyostelium discoideum

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20–40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl₂ accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 × 10⁻⁵ M progesterone considerably delays it These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg²⁺ or the ionophore and greatly decreased accumulation in the case of the steroid.
Treatment with EDTA and Mg²⁺ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn²⁺ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg²⁺ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca²⁺ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca²⁺.
These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

Homologous Mammalian Brain Cell Lysate System for the Initiation and Translation of Exogenous mRNAs

Abstract: The rate of protein synthesis in mammalian brain tissue is affected by a variety of physiological conditions, both natural and induced. The process of initiation may be involved in some of the observed changes, although as yet the actual rates of initiation of natural mRNAs have not been directly measured in these circumstances. One approach to studying the regulation of protein synthesis in brain tissue would be to utilize a homologous cell-free system to examine in vitro the translation of various added mRNAs. The present report describes a micrococcal nuclease-treated cell-free lysate system derived from fetal mouse brain tissue which is capable of actively initiating and translating exogenously added mRNA. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoretic analysis of the specific protein products of the reaction mixture allowed a qualitative and quantitative assessment of the translational process under a variety of experimental conditions. Optimal conditions for mRNA-dependent protein synthesis were the following: 30°C incubation temperature; 80–100 mM-KCl; 2.1 mM-Mg²⁺; 50 μM-spermhe; and 10 μg/ml poly A(+) mRNA. Incorporation of L-[³⁵S]methionine into proteins required ATP, GTP, and an energy regenerating system. The addition of saturating amounts of a homologous "initiation factors" fraction stimulated incorporation twofold during the first 20 min of incubation, while the patterns of inhibition observed upon the addition of 5 × 10^-5 M-aurin tricarboxylic acid at various periods during incubation demonstrated the occurrence of multiple rounds of initiation.     

Cross-Reaction of Anti-Rat B-50: Characterization and Isolation of a "B-50 Phosphoprotein" from Bovine Brain

Abstract: Antibodies to the phosphoprotein B-50 of rat brain were used to trace cross-reacting brain proteins of vertebrates. With the SDS-gel-immunoperoxidase method, a cross-reacting protein (CP) of apparent M_r 53,000 was demonstrated in the homogenate and the synaptic plasma membrane fraction of bovine brain. Sequence 1–24 of adrenocorticotropin (ACTH_1-24) (10⁻⁵ M and 10⁻⁴ M ) inhibited endogenous phosphorylation of CP in synaptic plasma membranes. The protein was partially characterized and purified to homogeneity from bovine brain by procedures previously described for rat B-50. CP was enriched in ammonium sulfate precipitated protein (ASP) fractions and phosphorylated by an endogenous protein kinase. Two-dimensional gel analysis of bovine and rat ASP showed that the cross-reacting protein had an isoelectric point less acidic than B-50. Limited proteolysis by Staphylococcus aureus protease yielded a "peptide map" analogous to B-50. Two major fragments of M_r 30,000 and 17,000 were produced. In addition, CP exhibited other similarities to rat B-50: phosphorylation by rat brain protein kinase C, microheterogeneity observed after isoelectric focusing, and possibly degradation by endogenous proteolysis. Cross-reaction of proteins in brain homogenates of other mammalian species and of chicken was demonstrated: the M_r of the proteins ranged from 47,000 to 53,000. We conclude that (1) the cross-reacting bovine protein is a "B-50 protein," and (2) the M _r of the "B-50 protein" varies from species to species.     

Chronic Ethanol Reduces Immunologically Detectable Gqα/11α in NG108–15 Cells

Abstract: The amyloid protein (βA₄) is found in the CNS of patients with Alzheimer's disease; however, the pathogenic role of this protein is not known. In the present study, a peptide fragment of βA₄βA₄ 25–35; Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-NH₂), which contains the conserved C-terminal sequence of substance P (X-Gly-Leu-Met-NH₂), and the neuropeptide substance P (SP) were examined for their ability to modulate nicotine-evoked secretion from cultured bovine adrenal chromaffin cells. Secretion of the released endogenous catecholamines was monitored by electrochemical detection after separation by HPLC. Secretion induced by 10⁻⁵ M nicotine was inhibited by SP and βA₄ 25–35. The IC₅₀ of SP and βA₄ 25–35 was 3 × 10⁻⁶ and 3 × 10⁻⁵ M , respectively. SP and βA₄ 25–35 both protected against nicotinic receptor desensitization. However, βA₄ 25–35 was ∼ 10-fold less effective than SP in its protective effect. The present work shows that βA₄ 25–35 can mimic the modulatory actions of SP on the nicotinic response of cultured bovine chromaffin cells, i.e., inhibition of the nicotinic response and protection against nicotinic desensitization. These modulatory actions may be associated with changes in nicotinic receptor levels reported to occur in Alzheimer's disease.     

The role of Mg2+ in proton transport by the tonoplast pyrophosphatase in Riccia fluitans vacuoles

The Mg²⁺-dependent activity of the tonoplast pyrophosphatase (PPase) was investigated by measuring proton transport and by using the acridine orange technique on intact vacuoles of the aquatic liverwort Riccia fluitans L. In solutions with both Mg²⁺ and pyrophosphate present, a number of complexes are formed, which could all influence the enzymatic and hence the transport activity of the PPase. Therefore, the individual concentrations of these complexes were calculated and their contributions to proton transport across the tonoplast were tested. From these experiments we conclude that Mg²⁺ has three different roles: (i) Mg²⁺ stimulates transport activity of the PPase. (ii) Mg₂PP_i inhibits PPase-mediated H⁺ transport, (iii) MgPP_i* (= MgPP_i^2-+ MgHPP_i^-) is the substrate with an apparent K_1/2= 5–10 μM, with no discrimination between MgPP_i^2- and MgHPP_i^-.     

Characterization of ATPase activities in rice root plasmalemma vesicles isolated by two-phase partitioning

Plasma membrane vesicles were isolated from the roots of 7-day-old rice plants ( Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two-phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate-sensitive K⁺, Mg²⁺-ATPase activity that was 18 times higher in the upper (PEG-rich) phase than in the lower (Dextran-rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 m M KCl and 3 m M ATP with apparent K_m (ATP) and V_max of 0.75 m M and 79 μmol (mg protein)⁻¹ h⁻¹, respectively. With 50 m M KCl and 7 m M ATP a pH optimum of 6.5, an apparent K_m (ATP) of 6.3 m M and V_max of 159 μmol (mg protein)⁻¹ h⁻¹ were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca²⁺ but insensitive to azide and nitrate.     

Development of pH sensitivity of sucrose uptake during ageing of Vicia faba leaf discs

Uptake of [U-¹⁴C]-sucrose (40 m M ) by fresh and aged peeled leaf discs of broad bean ( Vicia faba L. cv. Aguadulce) has been studied. In fresh discs, uptake was nearly insensitive to external pH, whereas the pH response of absorption in discs aged for 12 h was bell-shaped, with an optimum between pH 5 and 6. At this pH, uptake was nearly twice that in fresh tissue. The passive (insensitive to carbonyl cyanide m -chlorophenylhydrazone and to cold treatment) uptake was the same in fresh or aged discs. The development of pH sensitivity of absorption did not appear when ageing was performed in the presence of 10^−H M cycloheximide or 5.7 × 10⁻⁵ M actinomycin D. Similarly, when the tissues were treated with 10⁻³ M spermidine for 2 h after excision and then aged for 10 h, the development of the pH-sensitive uptake system was inhibited. Ca²⁺ (10⁻² M ) supplied together with spermidine prevented the inhibiting effect of spermidine. The appearance of the pH-sensitive system was also markedly reduced if ageing took place in the presence of 10⁻³ M aminoethoxyvinylglycine. Autoradiographs from fresh discs and from discs aged with or without the inhibitors suggest that pH sensitivity developed more intensively in the parenchyma than in the veins.
The results suggest some caution when using excised leaf discs for studies on sucrose uptake and phloem loading. Development of pH sensitivity of uptake may require the synthesis of both DNA-dependent RNA and protein and could be related to ethylene metabolism.     

Regulation of the First Step of the Initiation of Brain Protein Synthesis by Guanosine Diphosphate

Abstract: Initiation factor elF-2-like activity has been measured in the 0.5 M-KCl wash of rat brain microsomes. Ternary complex formation (elF-2 GTP Met-tRNA_r), one of the early steps in protein synthesis initiation, is optimal in a high-[K⁺], low-[Na⁺] environment. Mg²⁺, Ca²⁺, Li⁺, spermine and spermidine reduce and the antibiotic aurin tricarboxylic acid can effectively eliminate ternary complex formation. The formation of ternary complex requires GTP or its nonhydrolyzable analog, GMP-P(NH)P. Ternary complex formation is particularly sensitive to the ratio of GDP to GTP. When the ratio of GDP to GTP added is 1: 10, ternary complex formation is inhibited between 40 and 50% over a 30-fold concentration range of GTP. Other nucleotides exert little inhibition. These results suggest that the regulation of brain protein synthesis initiation may be tightly linked to the ratios of guanosine nucleotide concentrations in brain tissue.