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31.
Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) serve to transduce information from agonist-bound receptors to effector enzymes or ion channels. Current models of G protein activation-deactivation indicate that the oligomeric GDP-bound form must undergo release of GDP, bind GTP and undergo subunit dissociation, in order to be in active form (GTP bound subunits and free dimers) and to regulate effectors. The effect of receptor occupation by an agonist is generally accepted to be promotion of guanine nucleotide exchange thus allowing activation of the G protein. Recent studies indicate that transphosphorylation leading to the formation of GTP from GDP and ATP in the close vicinity, or even at the G protein, catalysed by membrane-associated nucleoside diphosphate kinase, may further activate G proteins. This activation is demonstrated by a decreased affinity of G protein-coupled receptors for agonists and an increased response of G protein coupled effectors. In addition, a phosphorylation of G protein subunits and consequent phosphate transfer reaction resulting in G protein activation has also been demonstrated. Finally, endogenously formed GTP was preferentially effective in activating some G proteins compared to exogenous GTR The aim of this report is to present an overview of the evidence to date for a transphosphorylation as a means of G protein activation (see also refs [1 and 2] for reviews). (Mol Cell Biochem 157: 593, 1996)Recipient of Servier Investigator Award 相似文献
32.
Jan O. Gordeladze Trine Haugen Eivind J. Paulssen Ruth H. Paulssen 《Bioscience reports》1996,16(1):65-74
The presence of the pertussis toxin (PTX) insensitive GTP-binding proteins (G-proteins) Gq and/or G11 has been demonstrated in three different prolactin (PRL) and growth hormone (GH) producing pituitary adenoma cell lines. Immunoblocking of their coupling to hormone receptors indicates that Gq and/or G11 confer throliberin (TRH) responsive phospholipase C (PL-C) activity in these cells. The contention was substantiated by immunoprecipitation analyses snowing that anti Gq/11-sera coprecipitated PL-C activity. In essence, only Gq/11 (but neither Gi2, Gi3 nor Go) seems to mediate the TRH-sensitive PL-C activity, while Go may be coupled to a basal or constitutive PL-C activity. Immunoblocking studies imply that the B-complex also, to some extent, may stimulate GH3 pituitary cell line PL-C activity. Finally, the steady state levels of Gq/11 mRNA and protein were downregulated upon long term exposure of the GH3 cells to TRH (but not to vasoactive intestinal peptide = VIP). 相似文献
33.
Roots of ten-days-old seedlings obtained from a maize hybrid grown in complete or in sulphate-deprived medium were used to extract Poly(A)+RNA. The response to sulphate deprivation, which is known to increase the uptake capacity up to ten times, was manifested also by the expression of three mRNA species, as shown by the in vitro translation of the mRNA population. One hour after transfer to complete nutrient medium all three mRNAs were still present.Abbreviations BSA
Bovine Serum Albumine
- DTT
1,4-Dithio-DL-Threitol
- EDTA
Ethylenediaminetetraacetic-acid
- MES
2-(N-Morpholino) ethane sulfonic acid
- SDS
Sodium Dodecyl Sulfate
- TCA
Trichloracetic acid
- TRIS
Tris(hydroxy-methyl)-aminomethane 相似文献
34.
The topological disposition of Wolfgram proteins (WP) and their relationship with 2, 3-cyclic nucleotide 3-phosphodiesterase (CNPase) in human, rat, sheep, bovine, guinea pig and chicken CNS myelin was investigated. Controlled digestion of myelin with trypsin gave a 35KDa protein band (WP-t) when electrophoresed on dodecyl sulfate-polyacrylamide gel in all species. Western blot analysis showed that the WP-t was derived from WP. WP-t was also formed when rat myelin was treated with other proteases such as kallikrein, thermolysin and leucine aminopeptidase. Staining for CNPase activity on nitrocellulose blots showed that WP-t is enzymatically active. Much of the CNPase activity remained with the membrane fraction even after treatment with high concentrations of trypsin when WP were completely hydrolysed and no protein bands with M.W>14KDa were detected on the gels. Therefore protein fragments of WP with M.W<14KDa may contain CNPase activity. From these results, it is suggested that the topological disposition of all the various WP is such that a 35KDa fragment is embedded in the lipid bilayer and the remaining fragment exposed at the intraperiod line in the myelin structure which may play a role in the initiation of myelinogenesis. 相似文献
35.
Anthony P. Fordham-Skelton F. Safadi M. Golovkin A. S. N. Reddy 《Plant Molecular Biology Reporter》1994,12(4):358-366
Calmodulin labeled with125I or34S has been used to screen expression libraries to isolate cDNAs encoding calmodulin-binding proteins (CBPs) from several eukaryotic
systems. The use of radiolabeled calmodulin has, however, several disadvantages. We have developed a nonradiactive method
to isolate cDNAs for CBPs using biotinylated calmodulin. Screening of a cDNA library in an expression vector with biotinylated
calmodulin resulted in the isolation of cDNAs encoding CBPs. Avidin and biotin blocking steps, prior to incubation of the
filters with biotinylated calmodulin, are found to be essential to eliminate the cDNAs that code for biotin-containing polypeptides.
The cDNA clones isolated using this nonradioactive method bound calmodulin in a calcium-dependent manner. The binding of biotinylated
calmodulin to these clones was completely abolished by ethylene glycolbis(\-aminoethylether)-N,N′-tetraacetic acid (EGTA),
a calcium chelator. Furthermore, the isolated cDNAs were confirmed by probing the clones with35S-labeled calmodulin. All the isolated clones bound to radiolabeled calmodulin in the presence of calcium but not in the presence
of EGTA. The method described here is simple, fast, and does not involve preparation and handing of radiolabeled calmodulin.
All the materials used in this method are commercially available; hence, this procedure should be widely applicable to isolate
cDNAs encoding CBPs from any eukaryotic organism. 相似文献
36.
Shohab Youssefian Michimi Nakamura Hiroshi Sano 《Molecular & general genetics : MGG》1993,237(1-2):187-192
Summary We previously reported the isolation of rgp1, a gene from rice, which encodes a ras-related GTP-binding protein, and subsequently showed that the gene induces specific morphological changes in transgenic tobacco plants. Here, we report the isolation and characterization of an rgp1 homologue, rgp2, from rice. The deduced rgp2 protein sequence shows 53% identity with the rice rgp1 protein, but 63% identity with both the marine ray ora3 protein, which is closely associated with synaptic vesicles of neuronal tissue, and the mammalian rab11 protein. Conservation of particular amino acid sequence motifs places rgp2 in the rab/ypt subfamily, which has been implicated in vesicular transport. Northern blot analysis of rgp1 and rgp2 suggests that both genes show relatively high, but differential, levels of expression in leaves, stems and panicles, but low levels in roots. In addition, whereas rgp1 shows maximal expression at a particular stage of plantlet growth, rgp2 is constitutively expressed during the same period. Southern blot analysis suggests that, in addition to rgp1 and rgp2, several other homologues exist in rice and these may constitute a small multigene family. 相似文献
37.
Arthur M. Brown 《The Journal of membrane biology》1993,131(2):93-104
Summary Ion channels are signaling molecules and by them-selves perform no work. In this regard they are un like the usual membrane
enzyme effectors for G proteins. The pathways of G protein receptor, G protein and ion channels are, therefore, purely infor
mational in function. Because a single G protein may have several ion channels as effectors, the effects should be coordinated
and this seems to be the case. Inhibition of Ca2+ current and stimulation of K+ currents would have a greater impact than either alone. Additional flexibility is provided by spontane ous noise in the complexes
of G protein receptor, G protein, and ion channel. By having a non-zero setpoint, the range of control is extended and the
responses become bi-directional. 相似文献
38.
Recent progress in plant transformation for insect resistance has increased the interest in the potential toxicity of proteins
towards insect pests. While studies have been targeted to a large array of insect species, phloem-feeding Homoptera have not
been investigated yet. The paper describes a routine test for screening toxicity and growth inhibition of purified proteins
in artificial diets onAcyrthosiphon pisum (Harris). Twenty-five commercially available proteins of different classes were tested and compared to some non-protein chemicals
(an insecticide, an antibiotic …).A. pisum proved to be very sensitive to all proteases tested and to some venoms with general cytolytic properties. A plant lectin,
concanavalin A, displayed significant toxicity and growth inhibition, while various proteins such as a soybean proteinase
inhibitor, a chitinase, and bovine serum albumin showed measurable impairments of growth only at higher dose (≥250 μg.ml−1). Some proteins were without short-term effect onA. pisum physiology. The influence of these results on aphid-plant interactions are discussed.
Résumé L'effet de protéines alimentaires sur les insectes phloémophages, dont les pucerons, n'a jamais été étudié. Nous proposons ici un test biologique standardisé sur milieu artificiel permettant d'analyser les effets de différentes classes de protéines sur la physiologie d'A. pisum. La validité de ce test est éprouvée (protocole, reproductibilité) et les différentes données récoltées (mortalité et inhibition de croissance) permettent de définir des paramètres toxicologiques tels que concentration létale 50 ou concentration inhibitrice 50. Cette caractérisation toxicologique a été réalisée sur 25 protéines appartenant à des classes différentes, ainsi que plusieurs substances non protéiques utilisées comme témoin de toxicité (insecticide, antibiotique, inhibiteur de synthèse protéique et glucoside phénolique). Les regroupements de protéines par proximités de profils toxicologiques ont été corrélés aux activités biochimiques des différentes protéines. Les implications de ces résultats sur les interactions plante-puceron sont discutées, ainsi que le potentiel d'une stratégie de création de variétés transgéniques résistantes aux pucerons.相似文献
39.
S. Butz R. Benz T. Wacker W. Welte A. Lustig R. Plapp J. Weckesser 《Archives of microbiology》1993,159(4):301-307
Oligomeric porin of the phototrophic bacterium Rhodopseudomonas blastica DSM 2131 was obtained from cell envelopes by differential temperature extraction in the presence of detergent and salt. The isolated porin exhibited strong porin activity after reconstitution into lipid bilayer membranes. The effective channel diameter for the trimer was estimated as 1.5 nm from single channel conductance measurements in the presence of 1 M KCl. Moderate cation-selectivity was observed. Oligomeric porin migrated as a single band (apparent molecular weight 81 kDa) on sodium dodecyl sulfate polyacrylamide gelelectrophoresis when solubilized below 70 °C. The oligomers were converted into monomers on heating to 70 °C or above forming two bands with apparent molecular weight of 36 kDa and 35 kDa. The oligomer was not sensitive to EDTA. Its molecular weight was determined to be 119.3 kDa by analytical ultracentrifugation. The isoelectric point was 5.7. Circular dichroism data indicated a high content of -sheet structure. Gasphase sequencing of the N-terminal residues revealed the sequence: NH2-Glu-Ile-Ser-Leu-Asn-Gly-Tyr-Gly-Arg-Phe. Crystals with a maximal side length of 300 m and diffracting to 0.32 nm resolution were obtained with the porin oligomer in the presence of C8E4 and 1,2,3-heptanetriol by using the vapor phase equilibration technique.Abbreviations C8E4
n-octyl tetraoxyethylene
- Mr
apparent molecular weight
- Octyl-POE
n-octyl polyoxyethylene
- LDAO
N,N-dimethyl dodecyl aminoxide
- LPS
lipopolysaccharide
- PAGE
polyacrylamide gel-electrophoresis
- PEG
polyethylene glycol 相似文献
40.
Stuart B. Moss Brenda L. Burnham Anthony R. Bellv 《Molecular reproduction and development》1993,34(2):164-174
The presence of lamin proteins in mouse spermatogenic cells has been examined by using an anti-lamin AC and an anti-lamin B antisera which recognize somatic lamins A and C, and somatic lamin B, respectively. Anti-lamin B binds to the nuclear periphery of all cell types examined, including Sertoli cells, primitive type A spermatogonia, preleptotene, leptotene, zygotene and pachytene spermatocytes, and round spermatids. In sperm nuclei, the antigenic determinants are localized to a narrow domain of the nucleus. However, after removing the perinuclear theca, anti-lamin B localizes to the entire nuclear periphery in a punctate pattern, suggesting that it is binding to determinants previously covered by the theca constituents. On immunoblots anti-lamin B reacts with a ~ 68 kD polypeptide in all germ cells and, to a lesser extent, with four additional polypeptides present only in meiotic and post-meiotic nuclear matrices. Anti-lamin AC also reacts with the perinuclear region of the somatic cells in the testes, in particular, those of the interstitium and also the Sertoli cells of the seminiferous epithelium. In contrast to anti-lamin B, anti-lamin AC does not bind to the germ cells at any stage of spermatogenesis. In addition, nuclear matrix proteins from isolated spermatogenic cells do not bind anti-lamin AC on immunoblots, suggesting the lack of reactivity is not due to the masking of any antigenic sites. These data demonstrate that germ cells contain lamin B throughout spermatogenesis, even during meiosis and spermiogenesis when the nuclear periphery lacks a distinct fibrous lamina. © 1993 Wiley-Liss, Inc. 相似文献