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.     

Increased Concentrations of 3-Hydroxykynurenine in Vitamin B6 Deficient Neonatal Rat Brain

Increased concentrations of the endogenous tryptophan metabolite 3-hydroxykynurenine (3-HK) were measured in the brains of vitamin B6 deficient neonatal rats. Mean concentrations of 3-HK in B6 deficient cerebellum, corpus striatum, frontal cortex, and pons/medulla ranged from 9.7 to 18.6 and 102 to 142 nmol/g of wet tissue at 14 and 18 days of age, respectively. 3-HK was not significantly increased in control neonatal or adult rat brain, vitamin B6 deficient rat brain at 7 days of age, or in brains from adult rats deprived of vitamin B6 for 58 days. The administration of daily intraperitoneal injections of vitamin B6 from the 14th to the 18th day of age decreased the concentration of 3-HK to control levels. 3-HK has been shown by other investigators to produce seizures when injected into the cerebral ventricles of adult rodents. Thus, our studies show the accumulation in brain of a putative endogenous convulsant as the result of a nutritional deficiency.     

Direct and Continuous Detection of ATP Secretion from Primary Monolayer Cultures of Bovine Adrenal Chromaffin Cells

A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin-luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KCl induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10(-7) M and a maximum at 10(-4) M. The dose-response curve for nicotine was bell shaped, with a threshold at 10(-7) M, a maximum at 10(-5) M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10(-4) M nicotine actually induced release of ATP at a faster rate than 10(-5) M nicotine. However, the rate of ATP release evoked by 10(-4) M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitization, whereas release induced by 10(-5) M nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5-1.5 min and a second phase of release beginning at 1.5-2 min, reaching a maximum by 2-3 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Integrated regulation of the photosynthetic gene family from Arabidopsis thaliana in transformed tobacco cells

Summary Expression of the three chlorophyll a/b binding protein (cab) genes of Arabidopsis thaliana was studied in transformed tobacco tissues. For each cab gene, approximately 1000 bp of the promoter region plus a portion of the structural gene was inserted into a promoter-expression vector such that a translational fusion between the cab gene and the promoter-less chloramphenicol acetyltransferase (cat) gene was formed. The constructed molecules were introduced into either cultured tobacco cells or tobacco leaves and the promoter activity was monitored as chloramphenicol acetyltransferase activity. The light-grown tissues exhibited 1.5- to 60-fold greater promoter activity than did dark-grown tissues. Expression of the cab promoters was tissue specific: activities were much stronger in green leaves than other tissues. The cab promoters were almost equally active in transformed calli or shoots derived from leaves. However, in cultured tobacco cells, one promoter was two to three times stronger than the other two. The chimeric gene fusion, cab-cat, segregated in the F1 generation as a dominant Mendelian trait.     

Characterization ofR. fredii QB1130, a strain effective on commercial soybean cultivars

Summary Two rhizobial strains (QB1130 and C3A) from northeast China were identified asRhizobium fredii on the basis of growth rate, media acidification and growth on a wide range of carbon substrates. The strains were shown to be distinct from USDA 191 on the basis of plasmid number and size. Bothnif and commonnod genes were located on the 295 kb plasmid of strains QB1130 and USDA 191, while onlynif genes were identified on this plasmid in C3A. When used to inoculate four commercial soybean (Glycine max) cultivars, one of the strains (C3A) was found to be ineffective, while the other (QB1130) was at least as effective as USDA 191, a strain ofR. fredii reported to be widely effective on North American cultivars of soybean. Further, QB1130 was capable of more effective nodulation of cowpea or the uncultivated soybean line, Peking, than either USDA 191 or the slow-growingBradyrhizobium japonicum USDA 16. Strain QB1130 should be useful for studies directed at improving symbiotic performance in soybean, or for studies of the comparative physiology and genetics of FG and SG strains on a single host.     

Spinach ferredoxin is a calcium-binding protein

Spinach-leaf ferredoxin was identified as a calcium-binding protein by ⁴⁵Ca autoradiography on nitrocellulose membranes and with the cationic carbocyanine dye 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl] naphtho[1,2-d]thiazolium bromide (stains-all). Binding of ⁴⁵Ca was observed at pH 6.8 and pH 7.8 and in the presence of 5 mM and 20 mM MgCl₂. At the higher MgCl₂ concentration the Ca²⁺-binding capacity is reduced. Only micromolar concentrations of LaCl₃, however, are required to achieve a similar effect. Both the oxidized and reduced forms of ferredoxin bind calcium.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - stains-all 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl] naptho[1,2-d]thiazolium bromide     

Analysis of labeling of plant protoplast surface by fluorophore-conjugated lectins

Summary Fluorescein or rhodamine conjugates of seventeen different lectins were tested for their ability to label the plasma membrane of live plant protoplasts. During the investigation, a strong effect of calcium was observed on the binding of several lectins to protoplasts derived from suspension cultured rose cells (Rosa sp. Paul's Scarlet). The binding of these lectins was increased by elevating the calcium concentration from 1 to 10 mM in the buffer. Other divalent cations had variable, but similar, effects on lectin binding. The mechanism of this effect appeared to involve the protoplast surface rather than the lectins. Although the cell wall-degrading enzymes used to isolate protoplasts had generally no effect on lectin binding, one clear exception was observed. Binding ofArachis hypogaea agglutinin was markedly reduced on protoplasts isolated with Driselase as compared to protoplasts isolated with a combination of Cellulysin and Pectolyase Y-23. Although most of the lectins that labeled protoplasts derived from cultured rose cells or from corn root cortex (Zea mays L. WF9 × Mo17) had specificities for galactose or N-acetylgalactosamine, some differences in protoplast labeling between lectins of the same saccharide specificity were observed. Two different analyses of the interaction betweenRicinus communis agglutinin and rose protoplasts showed that binding was cooperative with an apparent association constant of 7.2 × 10⁵M^–1 or 9.8 × 10⁵M^–1 with a maximum of approximately 10⁸ lectin molecules bound per protoplast. Treatment of protoplasts with glycosidases which hydrolyze either N- or O-glycosidic linkages of glycoproteins slightly enhanced labeling of protoplasts byRicinus communis agglutinin. Interpretation of these results are discussed.Abbreviations MPR medium, minimal organic medium (Nothnagel andLyon 1986) - APA Abrus precatorius agglutinin - CSA Cytisus sessilifolius agglutinin - ECA Erythrina cristagalli agglutinin - GS-I Griffonia simplicifolia agglutinin - LcH Lens culinarus agglutinin - PNA Arachis hypogaea agglutinin - SBA Glycine max agglutinin - VAA Viscum album agglutinin - VFA Vicia faba agglutinin - WGA Triticum vulgaris agglutinin - Con A Canavalia ensiformis agglutinin - HPA Helix pomatia agglutinin - TPA Tetragonolobus purpureas agglutinin - RCA Ricinus communis agglutinin - DBA Dolichos biflorus agglutinin - SJA Sophora japonica agglutinin - BPA Bauhinia purpurea agglutinin - FITC fluorescein isothiocyanate - Ga1NAc N-acetylgalactosamine - FDA fluorescein diacetate - 2-O-Me-D-Fuc 2-O-methyl-D-fucose Parts of the work presented here are also submitted in partial fulfillment of requirements for the Ph.D. degree.     

Effect of chemical modification on the binding of gossypol by gossypin (11S protein) and congossypin (7S protein) of cottonseed

Binding of gossypol by gossypin and congossypin and their succinylated and sulfhydryl group-blocked derivatives has been measured. The binding by gossypin and congossypin is characterized by weak interaction. Succinylation of gossypin decreases the binding affinity whereas that of congossypin increases it. Blocking of sulfhydryl groups of both the proteins does not significantly affect gossypol binding, Succinylation dissociates gossypin and causes conformational changes whereas it does not dissociate congossypin but causes conformational changes. Sulfhydryl group blocking does not dissociate gossypin or congossypin, nor does it cause any conformational changes.