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.     

Indole-3-ethanol oxidase in Phycomyces blakesleeanus. Is indole-3-ethanol a “storage pool” for IAA?

Indole-3-ethanol (IEt) was extracted from Phycomyces blakesleeanus Bgff. and purified by TLC and HPLC. Identification was performed by mass spectrum. The HPLC-purified compound showed an UV-spectrum typical for indoles, with absorption maxima at 220 and 281 nm. The IEt content varied between 1.5 nmol (g fresh weight)⁻¹ and 5.6 nmol (g fresh weight)⁻¹. The observed variations were strongly correlated with certain developmental stages of the fungus. Furthermore, the decrease of IEt between 60 and 84 h of fungal development coincides with a high IEt oxidase activity. The product of the enzyme reaction was indole-3-acetaldehyde, which was identified by co-chromatography with an authentic standard in several TLC and HPLC systems and by chemical conversion to indole-3-acetaldoxime.     

Periodicity of bud bursting in willow (Salix viminalis) as affected by growth regulators

Saunders, P. F. and Barros, R. S. 1987. Periodicity of bud bursting in willow ( Salix viminalis ) as affected by growth regulators.
Lateral vegetative buds of willow ( Salix viminalis L.) were only innately dormant for 3–5 weeks in October; during this time their apices were correlatively inhibited by the bud leaflets. Exogenous gibberellins stimulated the opening of cultured buds when the plants were dormant or entering dormancy. As dormancy was being released, however, cultured buds became more responsive to exogenous cytokinins. Thus the demand for gibberellins and cytokinins for bud opening seemed to be sequential rather than simultaneous. Dormant buds cultured in the presence of abscisic acid remained unopened, but they opened after a chilling treatment. Subsequent growth of such buds as measured by dry matter accumulation, was observed only if a cytokinin was added to the medium.     

Gas exchange studies in two Portuguese grapevine cultivars

Gas exchange characteristics of leaves of Vitis vinifera L. cvs Tinta Amarela and Periquita, two grapevine cultivars grown in distinct climatic regions of Portugal, were studied under natural and controlled conditions. Daily time courses of gas exchange were measured on both a hot, sunny day and a cooler, partly cloudy day. Responses of net photosynthesis to irradiance and internal partial pressure of CO₂, were also obtained. A strong correlation between net photosynthesis (P_N) and leaf conductance (g_s) was found during the diurnal time courses of gas exchange, as well as a relatively constant internal partial pressure of CO₂ (P_i), even under non-steady-state conditions. On the cloudless day, both P_N and g_s were lower in the afternoon than in the morning, despite similar conditions of leaf temperature, air to leaf water vapor deficit and irradiance. The response curves of net photosynthesis to internal CO₂ showed linearity up to p_i values of 50 Pa, possibly indicating a substantial excess of photosynthetic capacity. When measured at low partial pressures of O₂ (1 kPa), P_N became inhibited at high CO₂ levels. Inhibition of P_N at high CO₂ was absent under normal levels of O₂ (21 kPa). Significant differences in gas exchange characteristics were found between the two cultivars, with T. Amarela having higher rates under similar measurement conditions. In particular, the superior performance of T. Amarela at high temperatures may represent adaptation to the warmer conditions at its place of origin.     

Semiquantitative estimates of Al and other cations in the leaf tissues of some Al-accumulating species using electron probe microanalysis

Electron probe microanalysis of transverse sections was used to provide semiquantitative estimates of Al and other cations in the leaf tissues of some Al-accumulating plants of the cerrado vegetation of central Brazil. Concentrations of Al were generally higher than those of other cations in the phloem elements of these plants growing on dystrophic as well as fertile acid soils. When one of the Al-accumulating species,Vochysia thyrsoidea, was grown in a calcareous soil, the concentration of Al in the phloem elements of leaves was lower than that of Ca and K though the cotyledons showed higher concentrations of Al than those of other cations.     

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.     

Metal ion size selectivity in ligands with groups containing the neutral oxygen donor atom. A crystallographic and thermodynamic study

The coordinating properties of open-chain ligands containing alcoholic or ethereal oxygen donors are examined. Addition of oxygen donors usually leads to complex stabilisation for large metal ions (Pb²⁺, Cd²⁺) and to less favourable effects on complex stability for small metal ions (Cu²⁺, Ni²⁺). The formation constants of these metal ions with the set of ligands RN(CH₂CHOH·CH₃)₂ where R is ---H, ---CH₂CHOH·CH₃, ---CH₂CH₂OCH₃, ---CH₂CH₂OCH₂CH₂OH, and ---CH₂---CHOCH₂CH₂CH₂ are reported. The largest stabilisation for each case where R is an O-donor group relative to R = H occurs for Pb²⁺, the largest metal ion, while Cu²⁺, the smallest metal ion, shows the smallest stabilisation. The crystal structure of [Ni(HOCH₂CH₂NHCH₂CH₂NH₂)₂] (NO₃)₂ is reported. The space group is P , with cell constants a = 13.098(3), b = 8.737(4), and c = 7.746(3) Å, β = 112.66(3), β = 90.65(3), and γ = 85.03(2), and Z = 2. Disorder of the nitrate anions hindered refinement, with the result that a final conventional R factor of 0.0903 was achieved. The Ni---N bond lengths average 2.06(1) (secondary nitrogen) and 2.10(2) (primary nitrogen). The Ni---O bond lengths are rather long, averaging 2.15(1) Å, which is used to support the idea that the steric effects are responsible for destabilising the complexes of small metal ions such as Ni(11) when neutral oxygen donors are present.     

Populationsanteile und H?henverbreitung einj?hriger M?nnchen beim Hausrotschwanz (Phoenicurus ochruros) im Waldviertel, Nieder?sterreich

Zuammenfassung Im niederösterreichischen Waldviertel (200–700 m üNN) stieg der relative Anteil einjähriger von 17,9 auf 50% aller revierverteidigenden Hausrotschwanz- mit zunehmender Meereshöhe. Die Zunahme betrifft vor allem einjährige dercairei-Morphe, während dieparadoxus-Morphe in geringerer Anzahl und gleichförmiger als in allen Höhenbereichen auftrat.

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The portion of first-year males in breeding populations of Black Redstarts (Phoenicurus ochruros) and their altitudinal distribution in the Waldviertel, Lower Austria

Summary In 1986, between April 13 and July 5, breeding territories of male Black Redstarts were mapped in Lower Austria (200–700 m NN), with regard to their age (first-year or older) and the plumage types of first-year (femalelike or adult malelike). Due to a greater percentage of femalelike subadult (cairei-type) in higher altitudes, the proportion of first-year was found to increase from 17,9 per cent at 200–300 m to 50 per cent above 600 m. On the contrary adultlike first-year (paradoxus-type) are evenly distributed in smaller numbers over the whole altitudinal range.

     

Effects of Heavy Metal Cations and Other Sulfhydryl Reagents on Brain Dopamine D1 Receptors: Evidence for Involvement of a Thiol Group in the Conformation of the Active Site

To investigate aspects of the biochemical nature of membrane-bound dopamine D1 receptors, rat striatal homogenates were pretreated with heavy metal cations and some other chemical agents, and their effects on D1 receptors were subsequently determined using a standard [3H](R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1-N-3- benzazepine([3H]SCH 23390) binding assay. Incubation of striatal membranes with as little as 1 microM Hg2+, 10 microM Cu2+, and 10 microM Cd2+ completely prevented specific [3H]SCH 23390 binding. The effect of Cu2+, 1.5 microM, was noncompetitive in nature, whereas 3-5 microM Cu2+ afforded mixed-type inhibition. The inhibitory effect of Cu2+ was fully reversed by dithiothreitol (0.1-1 mM). Cu2+ (2 microM) did not affect the affinity of cis-flupenthixol or clozapine for remaining [3H]SCH 23390 sites. A second series of cations, Co2+ (30 microM), Ni2+ (30 microM), Mn2+ (1 mM), Ca2+ (25 mM), and Ba2+ (20 mM), inhibited specific [3H]SCH 23390 binding by 50% at the concentrations indicated. The thiol alkylating reagent N-ethylmaleimide (NEM) (0.2 mM) reduced specific binding by 70%. The effect of NEM was completely prevented by coincubation with a D1 receptor saturating concentration of SCH 23390 (20 nM) or dopamine (10 microM). The results indicated that the dopamine D1 receptor is a thiol protein and that a thiol group is essential for the ligand binding.