共查询到20条相似文献,搜索用时 15 毫秒
1.
Baur R Tan KR Lüscher BP Gonthier A Goeldner M Sigel E 《Journal of neurochemistry》2008,106(6):2353-2363
Classical benzodiazepines, for example diazepam, interact with alpha(x)beta(2)gamma(2) GABA(A) receptors, x = 1, 2, 3, 5. Little is known about effects of alpha subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (NCS compound) with alpha(1)H101Cbeta(2)gamma(2) and with receptors containing the homologous mutation, alpha(2)H101Cbeta(2)gamma(2), alpha(3)H126Cbeta(2)gamma(2) and alpha(5)H105Cbeta(2)gamma(2). This comparison was extended to alpha(6)R100Cbeta(2)gamma(2) receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of alpha(6)R100Cbeta(2)gamma(2) enables best interaction with NCS compound, followed by alpha(3)H126Cbeta(2)gamma(2), alpha(1)H101Cbeta(2)gamma(2) and alpha(2)H101Cbeta(2)gamma(2), while alpha(5)H105Cbeta(2)gamma(2) receptors show little interaction. Our results allow conclusions about the relative apposition of alpha(1)H101 and homologous positions in alpha(2), alpha(3), alpha(5) and alpha(6) with the position occupied by -Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABA(A) receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the alpha/beta subunit interface pseudo-symmetrically to the site for classical benzodiazepines located at the alpha/gamma interface. 相似文献
2.
The beta sliding clamp encircles DNA and tethers DNA polymerase III holoenzyme to the template for high processivity. The clamp loader, gamma complex (gamma 3 delta delta'chi psi), assembles beta around DNA in an ATP-fueled reaction. The delta subunit of the clamp loader opens the beta ring and is referred to as the wrench; ATP modulates contact between beta and delta among other functions. Crystal structures of delta.beta and the gamma 3 delta delta' minimal clamp loader make predictions of the clamp loader mechanism, which are tested in this report by mutagenesis. The delta wrench contacts beta at two sites. One site is at the beta dimer interface, where delta appears to distort the interface by via a steric clash between a helix on delta and a loop near the beta interface. The energy for this steric clash is thought to derive from the other site of interaction, in which delta binds to a hydrophobic pocket in beta. The current study demonstrates that rather than a simple steric clash with beta, delta specifically contacts beta at this site, but not through amino acid side chains, and thus is presumably mediated by peptide backbone atoms. The results also imply that the interaction of delta at the hydrophobic site on beta contributes to destabilization of the beta dimer interface rather than acting solely as a grip of delta on beta. Within the gamma complex, delta' is proposed to prevent delta from binding to beta in the absence of ATP. This report demonstrates that one or more gamma subunits also contribute to this role. The results also indicate that delta' acts as a backboard upon which the gamma subunits push to attain the ATP induced change needed for the delta wrench to bind and open the beta ring. 相似文献
3.
The human T-cell leukemia/lymphotropic virus type 1 p12I proteins bind the interleukin-2 receptor beta and gammac chains and affects their expression on the cell surface. 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
p12I is a small hydrophobic protein encoded by the human T-cell leukemia/lymphotropic virus type 1 (HTLV-1) that interacts with the 16-kDa component of the H+ vacuolar ATPase and cooperates with bovine papillomavirus 1 E5 oncoprotein in cell transformation. Just as an important step in E5 action appears to be its binding to the platelet-derived growth factor receptor, it was found that p12I binds specifically to both the beta and gamma(c) chains of the interleukin-2 receptor (IL-2R). The IL-2R beta and gamma(c) chains associated with p12I are endoglycosidase-H sensitive, suggesting that their interaction occurs in a pre-Golgi compartment. p12I stabilizes the immature forms of the IL-2R beta and gamma(c) chains and decreases their cell surface expression. The interactions of p12I with IL-2R beta and gamma(c) may have important implications in the immunosuppressive effect of HTLV-1 in vivo as well as in the ligand-independent HTLV-1-mediated T-cell proliferation. 相似文献
4.
Purification and Characterization of Cystathionine (gamma)-Lyase from Lactococcus lactis subsp. cremoris SK11: Possible Role in Flavor Compound Formation during Cheese Maturation
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A cystathionine (gamma)-lyase (EC 4.4.1.1) ((gamma)-CTL) was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris SK11 by a procedure including anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration chromatography. The activity of SK11 (gamma)-CTL is pyridoxal-5(prm1)-phosphate dependent, and the enzyme catalyzes the (alpha),(gamma)-elimination reaction of L-cystathionine to produce L-cysteine, (alpha)-ketobutyrate, and ammonia. The native enzyme has a molecular mass of approximately 120 to 200 kDa and apparently consists of at least six identical subunits of 20 kDa. In this respect, the SK11 enzyme clearly differs from other bacterial cystathionine lyases, which are all tetrameric proteins with identical subunits of approximately 40 kDa. In addition, the specific activity of purified SK11 (gamma)-CTL toward L-cystathionine is relatively low compared with those reported for other bacterial cystathionine lyases. The SK11 enzyme shows a broad substrate specificity. In the case of L-methionine, the action of SK11 (gamma)-CTL results in the formation of methanethiol, a volatile sulfur compound known to be required in flavor development in cheddar cheese. The (alpha),(beta)-elimination reaction of L-cysteine is also efficiently catalyzed by the enzyme, resulting in the formation of hydrogen sulfide. Although the conditions are far from optimal, cystathionine (gamma)-lyase is still active under cheddar cheese-ripening conditions, namely, pH 5.0 to 5.4 and 5% (wt/vol) NaCl. The possible role of the enzyme in cheese flavor development is discussed. 相似文献
5.
A series of 16 1-phenyl-1H-1,2,3-triazoles with substituents at both the 4- and 5-positions of the triazole ring were synthesized, and a total of 49 compounds, including previously reported 4- or 5-monosubstituted analogues, were examined for their ability to inhibit the specific binding of [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist, to human homo-oligomeric beta3 and hetero-oligomeric alpha1beta2gamma2 gamma-aminobutyric acid (GABA) receptors. Among all tested compounds, the 4-n-propyl-5-chloromethyl analogue of 1-(2,6-dichloro-4-trifluoromethylphenyl)-1H-1,2,3-triazole showed the highest level of affinity for both beta3 and alpha1beta2gamma2 receptors, with K(i) values of 659pM and 266nM, respectively. Most of the tested compounds showed selectivity for beta3 over alpha1beta2gamma2 receptors. Among all 1-phenyl-1H-1,2,3-triazoles, the 4-n-propyl-5-ethyl analogue exhibited the highest (>1133-fold) selectivity, followed by the 4-n-propyl-5-methyl analogue of 1-(2,6-dibromo-4-trifluoromethylphenyl)-1H-1,2,3-triazole with a >671-fold selectivity. The 2,6-dichloro plus 4-trifluoromethyl substitution pattern on the benzene ring was found to be important for the high affinity for both beta3 and alpha1beta2gamma2 receptors. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) provided similar contour maps, revealing that an electronegative substituent at the 4-position of the benzene ring, a compact, hydrophobic substituent at the 4-position of the triazole ring, and a small, electronegative substituent at the 5-position of the triazole ring play significant roles for the high potency in beta3 receptors. Molecular docking studies suggested that the putative binding sites for 1-phenyl-1H-1,2,3-triazole antagonists are located in the channel-lining 2'-6' region of the second transmembrane segment of beta3 and alpha1beta2gamma2 receptors. A difference in the hydrophobic environment at the 2' position might underlie the selectivity of 1-phenyl-1H-1,2,3-triazoles for beta3 over alpha1beta2gamma2 receptors. The compounds that had high affinity for beta3 receptors with homology to insect GABA receptors showed insecticidal activity against houseflies with LD(50) values in the pmol/fly range. The information obtained in the present study should prove helpful for the discovery of selective insect control chemicals. 相似文献
6.
Regulators of G protein signaling (RGS) proteins that contain DEP (disheveled, EGL-10, pleckstrin) and GGL (G protein gamma subunit-like) domains form a subfamily that includes the mammalian RGS proteins RGS6, RGS7, RGS9, and RGS11. We describe the cloning of RGS6 cDNA, the specificity of interaction of RGS6 and RGS7 with G protein beta subunits, and certain biochemical properties of RGS6/beta5 and RGS7/beta5 complexes. After expression in Sf9 cells, complexes of both RGS6 and RGS7 with the Gbeta5 subunit (but not Gbetas 1-4) are found in the cytosol. When purified, these complexes are similar to RGS11/beta5 in that they act as GTPase-activating proteins specifically toward Galpha(o). Unlike conventional G(betagamma) complexes, RGS6/beta5 and RGS7/beta5 do not form heterotrimeric complexes with either Galpha(o)-GDP or Galpha(q)-GDP. Neither RGS6/beta5 nor RGS7/beta5 altered the activity of adenylyl cyclases types I, II, or V, nor were they able to activate either phospholipase C-beta1 or -beta2. However, the RGS/beta5 complexes inhibited beta(1)gamma(2)-mediated activation of phospholipase C-beta2. RGS/beta5 complexes may contribute to the selectivity of signal transduction initiated by receptors coupled to G(i) and G(o) by binding to phospholipase C and stimulating the GTPase activity of Galpha(o). 相似文献
7.
Human C8 gamma is a 22 kDa subunit of complement component C8, which is one of five components (C5b, C6, C7, C8, C9) that interact to form the cytolytic membrane attack complex (MAC) of complement. C8 contains three nonidentical subunits (alpha, beta, gamma) that are products of different genes. These subunits are arranged asymmetrically to form a disulfide-linked C8 alpha-gamma dimer that is noncovalently associated with C8 beta. C8 alpha and C8 beta are homologous to C6, C7 and C9 and together these proteins comprise what is referred to as the 'MAC protein family'. By comparison, C8 gamma is distinct in that it belongs to the lipocalin family of small, secreted proteins which have the common ability to bind small hydrophobic ligands. While specific roles have been identified for C8 alpha and C8 beta in the formation and function of the MAC, a function for C8 gamma and the identity of its ligand are unknown. This review summarizes the current status of C8 gamma structure and function and the progress made from efforts to determine its role in the complement system. 相似文献
8.
The purified gamma subunit of the eighth component of human complement (C8) was used to characterize its site of interaction within C8 and to probe the ultrastructure of membrane-bound C5b-8 and C5b-9 complexes. Purification of gamma was accomplished by separating the disulfide-linked alpha-gamma subunit from the noncovalently associated beta chain and subjecting the former to limited reduction, alkylation, and ion-exchange chromatography. Upon mixing, purified alpha and gamma exhibited a high affinity for each other, as evidenced by their ability to form a noncovalent, equimolar complex at dilute concentrations and in the presence of excess serum albumin. Purified gamma also exhibited an affinity for C8', a previously described derivative that is functionally similar to C8 although it is composed of only alpha and beta. These results indicate that alpha possesses a specific site for interaction with gamma and that this site is preserved in the isolated subunit. Furthermore, this site remains accessible when alpha is associated with beta. In related experiments, gamma was found to specifically associate with membrane-bound C5b-8' and C5b-(8')9 complexes. These results indicate that the site for gamma interaction remains accessible on alpha in C5b-8' and is not shielded by C9 within C5b-(8')9. It is concluded that the gamma subunit of C8 is located on the surface of membrane-bound C5b-8 and C5b-9. 相似文献
9.
The subunit combinations alpha1beta2gamma2, alpha6beta2gamma2, and alpha1alpha6beta2gamma2 of the GABA(A) receptor were functionally expressed in Xenopus oocytes. The properties of the resulting ion currents were characterized by using electrophysiological techniques. The concentration-response curve of the channel agonist GABA for alpha1alpha6beta2gamma2 showed a single apparent component characterized by an EC(50) of 107 +/- 26 microM (n = 4). It was different from the one for alpha1beta2gamma2, which had an EC(50) of 41 +/- 9 microM (n = 4), that for alpha6beta2gamma2, with an EC(50) of 6.7 +/- 1.9 microM (n = 5), and those for alpha1beta2 and alpha1alpha6beta2. There was no appreciable functional expression of alpha6beta2. Allosteric responses of alpha1alpha6beta2gamma2 to diazepam were intermediate to those of alpha1beta2gamma2 and alpha6beta2gamma2, and allosteric responses to flumazenil were comparable to the ones for alpha1beta2gamma2. The inhibition by furosemide of the currents elicited by GABA in alpha1alpha6beta2gamma2 [IC(50) = 298 +/- 116 microM (n = 7), assuming only one component] was not identical with inhibition of alpha6beta2gamma2 (IC(50) = 38 +/- 2 microM, n = 4), alpha1beta2gamma2 (IC(50) = 5,610 +/- 910 microM, n = 5), or a mixture of these components (assuming two components). These findings indicate unambiguously the formation of functional GABA(A) receptors containing two different alpha subunits, alpha1 and alpha6, with properties different from those of alpha1beta2gamma2 and alpha6beta2gamma2. Furthermore, we provide evidence for the facts that in the Xenopus oocyte (a) the formation of the different receptor types depends on the relative abundance of cRNAs coding for the different receptor subunits and (b) that functional dual subunit combinations alphabeta do not form in the presence of cRNA coding for the gamma subunit. 相似文献
10.
Effects of Mg2+ and the beta gamma-subunit complex on the interactions of guanine nucleotides with G proteins 总被引:16,自引:0,他引:16
T Higashijima K M Ferguson P C Sternweis M D Smigel A G Gilman 《The Journal of biological chemistry》1987,262(2):762-766
Mg2+ interacts with the alpha subunits of guanine nucleotide-binding regulatory proteins (G proteins) in the presence of guanosine-5'-[gamma-thio]triphosphate (GTP-gamma S) to form a highly fluorescent complex from which nucleotide dissociates very slowly. The apparent Kd for interaction of G alpha X GTP gamma S with Mg2+ is approximately 5 nM, similar to the Km for G protein GTPase activity X G beta gamma increases the rate of dissociation of GTP gamma S from G alpha X GTP gamma S or G alpha X GTP gamma S X Mg2+ at low concentrations of Mg2+. When the concentration of Mg2+ exceeds 1 mM, G beta gamma dissociates from G beta gamma X G alpha X GTP gamma S X Mg2+. Compared with the dramatic effect of Mg2+ on binding of GTP gamma S to G alpha, the metal has relatively little effect on the binding of GDP. However, G beta gamma increases the affinity of G alpha for GDP by more than 100-fold. High concentrations of Mg2+ promote the dissociation of GDP from G beta gamma X G alpha X GDP, apparently without causing subunit dissociation. The steady-state rate of GTP hydrolysis is strictly correlated with the rate of dissociation of GDP from G alpha under all conditions examined. Thus, there are at least two sites for interaction of Mg2+ with G protein-nucleotide complexes. Furthermore, binding of G beta gamma and GTP gamma S to G alpha is negatively cooperative, while the binding interaction between G beta gamma and GDP is strongly positive. 相似文献
11.
Tuzi S Uekama N Okada M Yamaguchi S Saito H Yagisawa H 《The Journal of biological chemistry》2003,278(30):28019-28025
Despite the importance of signal transduction pathways at membrane surfaces, there have been few means of investigating their molecular mechanisms based on the structural information of membrane-bound proteins. We applied solid state NMR as a novel method to obtain structural information about the phospholipase C-delta1 (PLC-delta1) pleckstrin homology (PH) domain at the lipid bilayer surface. NMR spectra of the alanine residues in the vicinity of the beta5/beta6 loop in the PH domain revealed changes in local conformations due to the membrane localization of the protein. We propose that these conformational changes originate from a hydrophobic interaction between the amphipathic alpha-helix located in the beta5/beta6 loop and the hydrophobic layer of the membrane and contribute to the membrane binding affinity, interdomain interactions and intermolecular interactions of PLC-delta1. 相似文献
12.
Mitochondrial F1 from the yeast Schizosaccharomyces pombe, in contrast to the mammalian enzyme, exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 291 nm and a maximal emission at 332 nm. Low values of Stern-Volmer quenching constants, 4.0 M-1 or 1.8 M-1, respectively, in the presence of either acrylamide or iodide, indicate that tryptophans are mainly buried inside the native enzyme. Upon subunit dissociation and unfolding by 6 M guanidine hydrochloride (Gdn.HCl), the maximal emission is shifted to 354 nm, a value very similar to that obtained with N-acetyltryptophanamide, a solute-tryptophan model compound. The tryptophan content of each isolated subunit has been estimated by fluorescence titration in the presence of Gdn.HCl with free tryptophan as a standard. Two tryptophans and one tryptophan are found respectively in the alpha and epsilon subunits, whereas none is detected in the beta, gamma, and delta subunits. These subunit contents are consistent with the total of seven tryptophans estimated for native F1 with alpha 3 beta 3 gamma 1 delta 1 epsilon 1 stoichiometry. The maximal emission of the isolated epsilon subunit is markedly blue-shifted to 310-312 nm by interaction with the isolated delta subunit, which suggests that the epsilon subunit tryptophan might be a very minor contributor to the native F1 fluorescence measured at 332 nm. This fluorescence is very sensitive to phosphate, which produces a marked blue shift indicative of tryptophans in a more hydrophobic environment. On the other hand, ADP and ATP quench the maximal emission at 332 nm, lower tryptophan accessibility to acrylamide, and reveal tryptophan heterogeneity. 相似文献
13.
Bovine lens alpha A- and alpha B-crystallin polypeptides show extensive sequence homology with each other, but apparently none with beta Bp- and gamma 2-crystallin. Despite only 30% sequence homology, the latter two proteins are assumed to have a strong correspondence in tertiary structure, consisting of four structurally similar folding units of antiparallel beta-sheet. We have tested for internal structural repeats in all crystallins, and structural homology between crystallins, by comparing various physical properties of the amino acid residues, such as bulkiness and propensity to form beta-sheet and beta-turn structure. Two procedures used a combination of five physical parameters to calculate correlation coefficients. The 4-fold structural repeat in gamma 2-crystallin and the internal duplication in beta Bp-crystallin were readily detectable, as was also the strong structural homology between corresponding folding units in beta Bp- and gamma 2-crystallin. However, for alpha-crystallin polypeptides, no conclusive support was obtained for either a four-unit or a six-unit folding, the two models previously considered by us. The third procedure compared smoothened hydropathy plots, representing hydrophilic and hydrophobic regions along the polypeptide sequences. Hydropathy profiles were found to show strong correspondence, particularly between alpha B-crystallin and beta Bp-crystallin. These observations support a similar 4-fold folding pattern for all bovine crystallins. A possible role in subunit interactions of the N-terminal folding unit, which has hydrophobic surface characteristics in both alpha- and beta-crystallin polypeptides, is proposed. 相似文献
14.
Smith KJ Baillie GS Hyde EI Li X Houslay TM McCahill A Dunlop AJ Bolger GB Klussmann E Adams DR Houslay MD 《Cellular signalling》2007,19(12):2612-2624
The unique 88 amino acid N-terminal region of cAMP-specific phosphodiesterase-4D5 (PDE4D5) contains overlapping binding sites conferring interaction with the signaling scaffold proteins, betaarrestin and RACK1. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, encompasses the entire N-terminal RACK1 Interaction Domain (RAID1) together with a portion of the beta-arrestin binding site. (1)H NMR and CD analyses indicate that this region has propensity to form a helical structure. The leucine-rich hydrophobic grouping essential for RACK1 interaction forms a discrete hydrophobic ridge located along a single face of an amphipathic alpha-helix with Arg34 and Asn36, which also play important roles in RACK1 binding. The Asn22/Pro23/Trp24/Asn26 grouping, essential for RACK1 interaction, was located at the N-terminal head of the amphipathic helix that contained the hydrophobic ridge. RAID1 is thus provided by a distinct amphipathic helical structure. We suggest that the binding of PDE4D5 to the WD-repeat protein, RACK1, may occur in a manner akin to the helix-helix interaction shown for G(gamma) binding to the WD-repeat protein, G(beta). A more extensive section of the PDE4D5 N-terminal sequence (Thr11-Ala85) is involved in beta-arrestin binding. Several residues within the RAID1 helix contribute to this interaction however. We show here that these residues form a focused band around the centre of the RAID1 helix, generating a hydrophobic patch (from Leu29, Val30 and Leu33) flanked by polar/charged residues (Asn26, Glu27, Asp28, Arg34). The interaction with beta-arrestin exploits a greater circumference on the RAID1 helix, and involves two residues (Glu27, Asp28) that do not contribute to RACK1 binding. In contrast, the interaction of RACK1 with RAID1 is extended over a greater length of the helix and includes Leu37/Leu38, which do not contribute to beta-arrestin binding. A membrane-permeable, stearoylated Val12-Ser49 38-mer peptide disrupted the interaction of both beta-arrestin and RACK1 with endogenous PDE4D5 in HEKB2 cells, whilst a cognate peptide with a Glu27Ala substitution selectively failed to disrupt PDE4D5/RACK1 interaction. The stearoylated Val12-Ser49 38-mer peptide enhanced the isoprenaline-stimulated PKA phosphorylation of the beta(2)-adrenergic receptors (beta(2)AR) and its activation of ERK, whilst the Glu27Ala peptide was ineffective in both these regards. 相似文献
15.
Enhancer-origin interaction in plasmid R6K involves a DNA loop mediated by initiator protein 总被引:34,自引:0,他引:34
Initiation of DNA replication from ori beta of plasmid R6K requires the presence of the ori gamma sequence in cis. We demonstrate that binding of initiator protein to the seven strong, tandem binding sites in gamma increases binding of the protein at the very weak binding site present in ori beta by cooperativity at a distance. The gamma-beta interaction via the initiator results in a DNA loop, as revealed by the novel technique of cyclization enhancement and as confirmed by exonuclease III protection, electron microscopy, and chemical footprinting. The protein-mediated gamma-beta interaction in vitro suggests that the cooperative interaction of gamma-bound protein with the beta sequence by DNA looping is an early step in the initiation of DNA replication at the beta origin of R6K. 相似文献
16.
Heterotrimeric guanine nucleotide-binding proteins are important mediators in signal transduction and function by transmitting information from membrane-bound receptors to effectors. Because these proteins are membrane bound and contain covalent lipid modifications, detergents are required for solubilization and purification. It was discovered that the interaction between the beta5 subunit and the gamma2 subunit was disrupted in two detergents, cholate and Chaps (3-[(3-cholamidopropyl) dimethylammonio]-1-propansulfonate). A beta5gamma2 column was constructed in which recombinant betagamma dimers were immobilized on a FLAG antibody column via a hexahistidine-FLAG-tagged gamma2 subunit, gamma2HF. Greater than 95% of the beta5 subunit was specifically eluted from the immobilized gamma2HF subunit using a cholate gradient from 0.05 to 1.0% and greater than 40% of the beta5 subunit was eluted using a Chaps gradient from 0.05 to 1.0%. In contrast, the beta1, beta2, and beta3 subunits remained bound to the gamma2HF subunit in all concentrations of Chaps and cholate. Genapol C-100, Triton X-100, and polyoxyethylene-10-lauryl ether did not interfere with any of the four beta subunits' ability to interact with the gamma2 subunit. These data suggest that the beta5 subunit is not stable in bile acid or Chaps-type detergents (i.e., Chapso, glycocholate, deoxycholate). Therefore, alternative detergents should be used to extract dimers containing the beta5 subunit. 相似文献
17.
Elongation factor 1 beta gamma from Artemia. Purification and properties of its subunits 总被引:7,自引:0,他引:7
The guanine nucleotide exchange factor, elongation factor 1 beta gamma (EF-1 beta gamma) has been purified from Artemia cysts using an improved method. The protein consists of two distinct polypeptides with relative molecular masses of 26,000 (EF-1 beta) and 46,000 (EF-1 gamma). A nucleoside diphosphate phosphotransferase activity often found in EF-1 beta gamma preparations has been completely separated from the actual guanine nucleotide exchange stimulatory activity of EF-1 beta gamma, thus indicating that nucleotide diphosphate phosphotransferase is not an intrinsic property of EF-1 beta. Both EF-1 beta gamma and EF-1 beta have been shown to stimulate the following three reactions to a comparable degree: (a) exchange of GDP bound to EF-1 alpha with exogenous GDP; (b) EF-1 alpha-dependent binding of Phe-tRNA to ribosomes; (c) poly(U)-dependent poly(phenylalanine) synthesis. However, a significantly higher nucleotide exchange rate was observed in the presence of EF-1 beta gamma compared to EF-1 beta alone. Concerning elongation factor 1 gamma (EF-1 gamma) the following observations were made. In contrast to EF-1 beta, pure EF-1 gamma is rather insoluble in aqueous buffers, but the tendency to precipitate can be partially suppressed by the addition of detergents. In particular, EF-1 gamma partitions solely into the detergent phase of Triton X-114 solutions. EF-1 gamma is also more susceptible to spontaneous, specific fragmentation. It is remarkably that about 5% of the cellular pool of EF-1 beta gamma was found to be present in membrane fractions, under conditions where no EF-1 alpha was detectable in these fractions. Furthermore it was noted that EF-1 beta gamma copurified strongly with tubulin on DEAE-cellulose. Moreover, it was observed that from a mixture of EF-1 beta gamma and tubulin, EF-1 gamma coprecipitates with tubulin using a non-denaturating immunoprecipitation technique. These findings suggest that EF-1 gamma has a hydrophobic domain and interacts with membrane and cytoskeleton structures in the cell. 相似文献
18.
Simó B Perelló L Ortiz R Castiñeiras A Latorre J Cantón E 《Journal of inorganic biochemistry》2000,81(4):275-283
The interaction of copper (II), zinc(II) and cadmium(II) with Trimethoprim (2,4-diamino-5-(3',4',5'-trimethoxybenzyl) pyrimidine) has been studied. The crystal structures of [Zn(Trim)2Cl2] (2) and [Cd(Trim)Cl2(CH3OH)]n (4) are reported. Compound (2) exhibits a distorted tetrahedral environment around the metal center and crystallizes in the triclinic space group P1 with a=10.2397(6), b=10.4500(6), c=16.3336(16) A, alpha=96.141(8), beta=106.085(5), gamma=96.551(5) degrees and Z=2. In complex (4), the Cd(II) centers are bridged sequentially by two chlorine ions to form infinite chains and present a six-coordinated environment; the compound crystallizes in the monoclinic P2(1)/C space group with a=13.958(5), b=7.532(2), c=18.390(2) A, alpha=90, beta=97.32(5), gamma=90 degrees and Z=4. In both structures the Trimethoprim acts as a monodentate ligand through the pyrimidinic nitrogen N(1) atom. The characterization of the Cu(Trim)2(CH3O)(ClO4) complex through EPR and magnetic measurements suggests a binuclear or polinuclear nature, with bridging methoxo groups. The complexes were screened for their activity against several bacteria, showing activity similar to that of trimethoprim. 相似文献
19.
Receptor stimulation of nucleotide exchange in a heterotrimeric G protein (alphabetagamma) is the primary event-modulating signaling by G proteins. The molecular mechanisms at the basis of this event and the role of the G protein subunits, especially the betagamma complex, in receptor activation are unclear. In a reconstituted system, a purified muscarinic receptor, M2, activates G protein heterotrimers alphai2beta1gamma5 and alphai2beta1gamma7 with equal efficacy. However, when the alpha subunit type is substituted with alphao, alphaobeta1gamma7 shows a 100% increase in M2-stimulated GTP hydrolysis compared with alphaobeta1gamma5. Using a sensitive assay based on betagamma complex stimulation of phospholipase C activity, we show that both beta1gamma5 and beta1gamma7 form heterotrimers equally well with alphao and alphai. These results indicate that the gamma subunit interaction with a receptor is critical for modulating nucleotide exchange and is influenced by the subunit-type composition of the heterotrimer. 相似文献
20.
Stewart J Hingorani MM Kelman Z O'Donnell M 《The Journal of biological chemistry》2001,276(22):19182-19189
The beta sliding clamp encircles the primer-template and tethers DNA polymerase III holoenzyme to DNA for processive replication of the Escherichia coli genome. The clamp is formed via hydrophobic and ionic interactions between two semicircular beta monomers. This report demonstrates that the beta dimer is a stable closed ring and is not monomerized when the gamma complex clamp loader (gamma(3)delta(1)delta(1)chi(1)psi(1)) assembles the beta ring around DNA. delta is the subunit of the gamma complex that binds beta and opens the ring; it also does not appear to monomerize beta. Point mutations were introduced at the beta dimer interface to test its structural integrity and gain insight into its interaction with delta. Mutation of two residues at the dimer interface of beta, I272A/L273A, yields a stable beta monomer. We find that delta binds the beta monomer mutant at least 50-fold tighter than the beta dimer. These findings suggest that when delta interacts with the beta clamp, it binds one beta subunit with high affinity and utilizes some of that binding energy to perform work on the dimeric clamp, probably cracking one dimer interface open. 相似文献