Solubilisation of a Glutamate Binding Protein from Rat Brain

Rat brain synaptic plasma membranes were solubilised in either 1% Triton X-100 or potassium cholate and subjected to batch affinity adsorption on L-glutamate/bovine serum albumin reticulated glass fibre. The fibre was extensively washed, and bound proteins eluted with 0.1 mM L-glutamate in 0.1% detergent, followed by repeated dialysis to remove the glutamate from the eluted proteins. Aliquots of the dialysed extracts were assayed for L-[3H]glutamate binding activity in the presence or absence of 0.1 mM unlabelled L-glutamate (to define displaceable binding). Incubations were conducted at room temperature and terminated by rapid filtration through nitrocellulose membranes. Binding to solubilised fractions could be detected only following affinity chromatography. Binding was saturable and of relatively low affinity: KD = 1.0 and 1.8 microM for Triton X-100 and cholate extracts, respectively. The density of binding sites was remarkably high: approximately 18 nmol/mg protein for Triton X-100-solubilised preparations, and usually double this when cholate was employed. Analysis of structural requirements for inhibition of binding revealed that only a very restricted number of compounds were effective, i.e., L-glutamate, L-aspartate, and sulphur-containing amino acids. Binding was not inhibited significantly by any of the selective excitatory amino acid receptor agonists--quisqualate, N-methyl-D-aspartate, or kainate. The implication from this study is that the glutamate binding protein is similar if not identical to one previously isolated and probably is not related to the pharmacologically defined postsynaptic receptor subtypes, unless solubilisation of synaptic membranes resulted in major alterations to binding site characteristics. Since solubilisation with Triton X-100 is known to preserve synaptic junctional complexes, it seems likely that the origin of the glutamate binding protein may be extrajunctional, although its functional role is unknown.     

Ca2+/Calmodulin Distinguishes Between Guanyl-5''-yl-Imidodiphosphate-and Opiate-Mediated Inhibition of Rat Striatal Adenylate Cyclase

The inhibition of adenylate cyclase from rat striatal plasma membranes by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] and morphine was compared to determine whether Gpp(NH)p-mediated inhibition accurately reflected hormone-mediated inhibition in this system. Inhibition of adenylate cyclase activity by Gpp(NH)p and morphine was examined with respect to temperature, divalent cation concentration, and the presence of Ca2+/calmodulin (Ca2+/CaM). Gpp(NH)p-mediated inhibition was dependent on the presence of Ca2+/CaM at 24 degrees C; the inhibition was independent of Ca2+/CaM at 18 degrees C; and inhibition could not be detected in the presence, or absence, of Ca2+/CaM at 30 degrees C. In contrast, naloxone-reversible, morphine-induced inhibition of adenylate cyclase was independent of both temperature and the presence of Ca2+/CaM. Mg2+ dose-response curves also reinforced the differences in the Ca2+/CaM requirement for Gpp(NH)p- and morphine-induced inhibition. Because Gpp(NH)p-mediated inhibition was independent of Ca2+/CaM at low basal activities (i.e., 18 degrees C, or below 1 mM Mg2+) and dependent on the presence of Ca2+/CaM at higher basal activities (24 degrees C, or above 1 mM Mg2+), the inhibitory effects of Gpp(NH)p were examined at 1 mM Mg2+ in the presence of 100 nM forskolin. Under these conditions, both Gpp(NH)p- and morphine-induced inhibition of adenylate cyclase were independent of Ca2+/CaM. The results demonstrate that the requirement for Ca2+/CaM to observe Gpp(NH)p-mediated inhibition depends on the basal activity of adenylate cyclase, whereas hormone-mediated inhibition is Ca2+/CaM independent under all conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Implications for cytoplasmic pH,protonmotice force,and amino-acid transport across the plasmalemma of Riccia fluitans

By means of pH-sensitive microelectrodes, cytoplasmic pH has been monitored continuously during amino-acid transport across the plasmalemma of Riccia fluitans rhizoid cells under various experimental conditions. (i) Contrary to the general assumption that import of amino acids (or hexoses) together with protons should lead to cytoplasmic acidification, an alkalinization of 0.1–0.3 pH_c units was found for all amino acids tested. Similar alkalinizations were recorded in the presence of hexoses and methylamine. No alkalinization occurred when the substrates were added in the depolarized state or in the presence of cyanide, where the electrogenic H⁺-pump is inhibited. (ii) After acidification of the cytoplasm by means of various concentrations of acetic acid, amino-acid transport is massively altered, although the protonmotive force remained essentially constant. It is suggested that H⁺-cotransport is energetically interconnected with the proton-export pump which is stimulated by the amino-acid-induced depolarization, thus causing proton depletion of the cytoplasm. It is concluded that, in order to investigate H⁺-dependent cotransport processes, the cytoplasmic pH must be measured and be under continuous experimental control; secondly, neither pH nor the protonmotive force across a membrane are reliable quantities for analysing a proton-dependent process.Abbreviations 3-OMG 3-oxymethylglucose - pH_c cytoplasmic pH - _m electrical potential difference across the respective membrane, i.e. membrane potential - _H+/F (=pmf) electrochemical proton gradient     

Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: effects on the components of transmembrane transport of indol-3yl-acetic acid

Phenylacetic acid (PAA), a naturally-occurring acidic plant growth substance, was readily taken up by pea (Pisum sativum L. cv. Alderman) stem segments from buffered external solutions by a pH-dependent, non-mediated diffusion. Net uptake from a 0.2 M solution at pH 4.5 proceeded at a constant rate for at least 60 min and, up to approx. 100 M, the rate of uptake was directly proportional to the external concentration of the compound. The net rate of uptake of PAA was not affected by the inclusion of indol-3yl-acetic acid (IAA) in the uptake medium (up to approx. 30 M) and, unlike the net uptake of IAA, was not stimulated by N-1-naphthylphthalamic acid (NPA) or 2,3,5-triiodobenzoic acid. At an external concentration of 0.2 M and pH 4.5, the net rate of uptake of PAA was about twice that of IAA. It was concluded that the uptake of PAA did not involve the participation of carriers and that PAA was not a transported substrate for the carriers involved in the uptake and polar transport of IAA. Nevertheless, the inclusion of 3–100 M unlabelled PAA in the external medium greatly stimulated the uptake by pea stem segments of [1-¹⁴C]IAA (external concentration 0.2 M). It was concluded that whilst PAA was not a transported substrate for the NPA-sensitive IAA efflux carrier, it interacted with this carrier to inhibit IAA efflux from cells. Over the concentration range 3–100 M, PAA progressively reduced the stimulatory effect of NPA on IAA uptake, indicating that PAA also inhibited carrier-mediated uptake of IAA. The consequences of these observations for the regulation of polar auxin transport are discussed.Abbreviations IAA indol-3yl-acetic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - NPA N-1-naphthylphthalamic acid - PAA phenylacetic acid - TIBA 2,3,5-triiodobenzoic acid     

OmpR and EnvZ are pleiotropic regulatory proteins: positive regulation of the tripeptide permease (tppB) of Salmonella typhimurium

Summary The tppB locus of Salmonella typhimurium encodes the anaerobically-induced tripeptide permease. We have demonstrated that expression of tppB requires the function of the ompR and envZ gene products, originally identified as positive regulatory proteins required for the osmotic regulation of porin expression. Significantly, tppB expression is not osmotically regulated. We have also identified three additional genes whose expression depends on OmpR. Thus OmpR and EnvZ serve a more general regulatory role than has previously been supposed. This study provides the first detailed genetic analysis of the ompB locus of S. typhimurium.     

Ionic balance,proton efflux,nitrate reductase activity and growth ofHippophaë rhamnoides L. ssp.rhamnoides as influenced by combined-N nutrition or N2 fixation

Growth of 2-month-old nonnodulatedHippophaë rhamnoides seedlings supplied with combined N was compared with that of nodulated seedlings grown on zero N. Plant growth was significantly better with combined N than with N₂ fixation and, although not statistically significant for individual harvests, tended to be highest in the presence of NH ₄ ⁺ , a mixture of NH ₄ ⁺ and NO ₃ ^? producing the highest yields. Growth was severely reduced when solely dependent on N₂ fixation and, unlike the combined-N plants, shoot to root ratios had only slightly increased after an initial decrease. An apparently insufficient nodule mass (nodule weight ratio <5 per cent) during the greater part of the experimental period is suggested as the main cause of the growth reduction in N₂-fixing plants. Thein vivo nitrate reductase activity (NRA) of NO ₃ ^? dependent plants was almost entirely located in the roots. However, when grown with a combination of NO ₃ ^? and NH ₄ ⁺ , root NRA was decreased by approximately 85 per cent.H. rhamnoides demonstrated in the mixed supply a strong preference for uptake of N as NH ₄ ⁺ , NO ₃ ^? contributing only for approximately 20 per cent to the total N assimilation. Specific rates of N acquisition and ion uptake were generally highest in NO ₃ ^? +NH ₄ ⁺ plants. The generation of organic anions per unit total plant dry weight was approximately 40 per cent less in the NH ₄ ⁺ plants than in the NO ₃ ^? plants. Measured extrusions of H⁺ or OH^? (HCO ₃ ^? ) were generally in good agreement with calculated values on the basis of plant composition, and the acidity generated with N₂ fixation amounted to 0.45–0.55 meq H⁺. (mmol N_org)^?1. Without acidity control and in the presence of NH ₄ ⁺ , specific rates of ion uptake and carboxylate generation were strongly depressed and growth was reduced by 30–35 per cent. Growth of nonnodulatedH. rhamnoides plants ceased at the lower pH limit of 3.1–3.2 and deterioration set in; in the case of N₂-fixing plants the nutrient solution pH stabilized at a value of 3.8–3.9 without any apparent adverse effects upon plant performance. The chemical composition of experimental and field-growing plants is being compared and some comments are made on the nitrogen supply characteristics of their natural sites.     

Calculation of leaf photosynthetic parameters from light-response curves for ecophysiological applications

Measurement of the light response of photosynthetic CO₂ uptake is often used as an implement in ecophysiological studies. A method is described to calculate photosynthetic parameters, such as the maximum rate of whole electron transport and dissimilative respiration in the light, from the light response of CO₂ uptake. Examples of the light-response curves of flag leaves and ears of wheat (Triticum aestivum cv. ARKAS) are shown.Abbreviations and symbols A net photosynthesis rate - D ¹ rate of dissimilative respiration occurring in the light - f loss factor - I incident PPFD - I effective absorbed PPFD - J rate of whole electron transport - J _m maximum rate of whole electron transport - p ^c intercellular CO₂ partial pressure - PPFD photosynthetic photon flux density - q effectivity factor for the use of light (electrons/quanta) - absorption coefficient - I ^* CO₂ compensation point in the absence of dissimilative respiration (bar) - II conversion factor for calculation of CO₂ uptake from the rate of whole electron transport - convexity factor Gas-exchange rates relate to the projective area and are given in mol·m^-2·s^-1. Electron-transport rates are given in mol electrons·m^-2·s^-1; PPFD is given in mol quanta·m^-2·s^-1.     

Calcium-activated protease in the giant algaChara australis

Summary There is a protease, which is activated by Ca²⁺ (about 100 M), works at neutral pH and exists in the cytoplasm inChara australis. This protease may correspond to calpain, the calcium-activated neutral protease, which has been studied in animal cells. This is the first report showing the existence of a calcium-activated protease in plant cells.     

Cytoplasmic streaming does not drive intercellular passage in staminal hairs ofSetcreasea purpurea

Summary The effect of inhibition of cytoplasmic streaming on intercellular passage of carboxyfluorescein (CF) in staminal hairs ofS. purpurea was examined. Tip cells of staminal hairs were microinjected with buffered-CF. Cytoplasmic streaming was then inhibited by addition of KCN or NaN₃ to the external bathing solution. In separate experiments, cytoplasmic streaming was inhibited by microinjection of cytochalasin D along with the buffered-CF. CF passage over a 5 minutes treatment period was monitored by video fluorescence microscopy and video intensity analysis. Cytoplasmic streaming ceased within 1 minute of inhibitor agent treatment, however, little change in the kinetics of intercellular passage was noted over the 5 minute experimental period. Th us, cytoplasmic streaming plays no major role in the regulation of intercellular passage of the hydrophilic, negatively charged molecule CF.The work is dedicated to professor Saal Zalik, Department of Plant Science, University of Alberta, on his 65th birthday.