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41.
Solubilisation of a Glutamate Binding Protein from Rat Brain 总被引:2,自引:2,他引:0
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. 相似文献
42.
Luisa Castagnoli 《Molecular & general genetics : MGG》1987,206(1):178-180
Summary A point mutation in the -35 region of the promoter of the primer for initiation of DNA replication in the plasmid pMB1 was characterized. This base change causes a promoter up phenotype. The analysis of a second mutant obtained by site-directed mutagenesis allowed the exclusion of a role in the phenotype for the potential intrastrand secondary structure as well as for the methylation state of the DNA in the promoter region. The promoter up phenotype is concluded to be due to a change in the primary structure of the — 35 element with the consequent production of a better cluster of hydrogen bond donors and acceptors for the RNA polymerase. 相似文献
43.
A. Tyagi J. Hermans J. Steppuhn Ch. Jansson F. Vater R. G. Herrmann 《Molecular & general genetics : MGG》1987,207(2-3):288-293
Summary Several cDNA clones encoding the 33 kDa protein associated with the photosynthetic water oxidation activity of spinach were sequenced. A 1208 bp insert of one of the clones encodes the entire 331 amino acid residues of the precursor protein including 84 amino acids (8.5 kDa) of the amino-terminal transit peptide, 49 bp of the 5 and 111 bp of the 3 untranslated segment of the mRNA. The 3 poly(A) tail starts 19 bp downstream from a putative polyadenylation signal, TATAAA. The hydrophilic mature protein consists of 247 amino acid residues corresponding to an Mr of 26.5 kDa, which is 6.5 kDa smaller than the value determined by SDS-polyacrylamide gel electrophoresis (33–34 kDa), and shows a certain degree of conservation with the putative Mn-complexing active sites of bacterial Mn-dependent superoxide dismutases. The anatomy of the unusually long transit sequence is discussed with regard to current concepts of protein import into and protein routein within the organelle. 相似文献
44.
Gynheung An 《Molecular & general genetics : MGG》1987,207(2-3):210-216
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. 相似文献
45.
Nicolas Mermod Juan L. Ramos Amos Bairoch Kenneth N. Timmis 《Molecular & general genetics : MGG》1987,207(2-3):349-354
46.
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 相似文献
47.
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. 相似文献
48.
Effect of chemical modification on the binding of gossypol by gossypin (11S protein) and congossypin (7S protein) of cottonseed 总被引:1,自引:0,他引:1
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. 相似文献
49.
On binding toVicia faba lectin, the fluorescence of 4-methylumbelliferyl-α-D-glucoPyranoside was quantitatively quenched showing that the interaction
of 4-methylumbelliferyl-α-D-glucoPyranoside took Place in a binding environment. The binding of the fluorescent sugar was
saccharide sPecific as evidenced by the reversal of 4-methylumbelliferyl-α-D-glucoPyranoside fluorescence quenching by D-fructose.
The association constant,K
a, values for the 4-methylumbelliferyl-α-D-glucoPyranoside was determined by comPetition study emPloying reversal of fluorescence
quenching of 4-methylumbelliferyl-α-D-glucoPyranoside by D-fructose. TheK
a value obtained for D-fructose was 1.07 ±0.03 X 104 M-1 and for 4-methylumbelliferyl-α-D-glucoPyranoside was 1.60 ±0.05 X 104 M-1 at 15°C. TheK
a values of 2.51 ±0.06 X 104M-1, l.26 ±0.02 X 104 M-1 and 0.56 ±0.01 X 104M-1, resPectively at 10°, 20° and 30°C were obtained from the ChiPman equation. The relative fluorescence quenching, ΔF
a, at infinite concentration of the free saccharide sites ofVicia faba lectin [P′] was 93.5% at 30°C and the binding constant for 4-methylumbelliferyl-α-D-glucoPyranoside lectin interaction as derived by
Yank and Hanaguchi equation was 0.63 ±0.01 X 104M-1. 相似文献
50.
Summary Phloridzin-insensitive, Na+-independentd-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll® gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed aK
t of 48mm and aV
max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK
t of 23mm and aV
max of 0.20 nmol glucose/mg protein/sec.d-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. TheK
t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK
D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na+-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and theK
t for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream. 相似文献