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1.
Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea
Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise
combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression
of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the
highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant
reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression.
However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that
specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions
between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into
the structural change of ATP synthase during catalysis. 相似文献
2.
Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise
combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression
of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the
highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant
reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression.
However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that
specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions
between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into
the structural change of ATP synthase during catalysis. 相似文献
3.
The α1 subunit coding for the human brain type E calcium channel (Schneider et al., 1994) was expressed in Xenopus oocytes in the absence, and in combination with auxiliary α2δ and β subunits. α1E channels directed with the expression of Ba2+ whole-cell currents that completely inactivated after a 2-sec membrane pulse. Coexpression of α1E with α2bδ shifted the peak current by +10 mV but had no significant effect on whole-cell current inactivation. Coexpression of α1E with β2a shifted the peak current relationship by −10 mV, and strongly reduced Ba2+ current inactivation. This slower rate of inactivation explains that a sizable fraction (40 ± 10%, n= 8) of the Ba2+ current failed to inactivate completely after a 5-sec prepulse. Coinjection with both the cardiac/brain β2a and the neuronal α2bδ subunits increased by ≈10-fold whole-cell Ba2+ currents although coinjection with either β2a or α2bδ alone failed to significantly increase α1E peak currents. Coexpression with β2a and α2bδ yielded Ba2+ currents with inactivation kinetics similar to the β2a induced currents, indicating that the neuronal α2bδ subunit has little effect on α1E inactivation kinetics. The subunit specificity of the changes in current properties were analyzed for all four β subunit
genes. The slower inactivation was unique to α1E/β2a currents. Coexpression with β1a, β1b, β3, and β4, yielded faster-inactivating Ba2+ currents than currents recorded from the α1E subunit alone. Furthermore, α1E/α2bδ/β1a; α1E/α2bδ/β1b; α1E/α2bδ/β3; α1E/α2bδ/β4 channels elicited whole-cell currents with steady-state inactivation curves shifted in the hyperpolarized direction. The
β subunit-induced changes in the properties of α1E channel were comparable to modulation effects reported for α1C and α1A channels with β3≈β1b > β1a≈β4≫β2a inducing fastest to slowest rate of whole-cell inactivation.
Received: 27 March 1997/Revised: 10 July 1997 相似文献
4.
A pH-variation study of jack bean (Canavalia ensiformis) urease steady-state kinetic parameters and of the inhibition constant of boric acid, a urease competitive inhibitor, was performed using both noninhibitory organic (MES, HEPES and CHES) and inhibitory inorganic (phosphate) buffers, in an effort to elucidate the functions exercised in the catalysis by the ionizable groups of the enzyme active site. The results obtained are consistent with the requirement for three groups utilized by urease with pK(a)s equal to 5.3+/-0.2, 6.6+/-0.2 and 9.1+/-0.4. Based on the appearance of the ionization step with pK(a)=5.3 in v(max)-pH, K(M)-pH and K(i)-pH profiles, we assigned this group as participating both in the substrate binding and catalytic reaction. As shown by its presence in v(max)-pH and K(M)-pH curves, the obvious role of the group with pK(a)=9.1 is the participation in the catalytic reaction. One function of the group featuring pK(a)=6.6, which was derived from a two-maxima v(max)-pH profile obtained upon increasing phosphate buffer concentration, an effect the first time observed for urease-phosphate systems, is the substrate binding, another possible function being modulation of the active site structure controlled by the ionic strength. It is also possible that the pK(a)=6.6 is a merger of two pK(a)s close in value. The study establishes that regular bell-shaped activity-pH profiles, commonly reported for urease, entail more complex pH-dependent behavior of the urease active site ionizable groups, which could be experimentally derived using species interacting with the enzyme, in addition to changing solution pH and ionic strength. 相似文献
5.
6.
K. L. Larsen H. J. S. Christensen F. Mathiesen L. H. Pedersen W. Zimmermann 《Applied microbiology and biotechnology》1998,50(3):314-317
The conversion of soluble starch to cyclomaltohexaose (α-CD), cyclomaltoheptaose (β-CD), cyclomaltooctaose (γ-CD) and cyclomaltononaose
(δ-CD) by cyclodextrin glycosyltransferases (E.C. 2.4.1.19) from Bacillus spp. and bacterial isolates was studied. The results show that δ-CD was formed by all the enzymes investigated in the range
of 5%–11.5% of the total amount of α-, β-, γ-, and δ-CD produced.
Received: 17 February 1998 / Received revision: 18 May 1998 / Accepted: 21 May 1998 相似文献
7.
Mutation in splicing consensus sequences causes lack of TCR membrane expression due to exon excision
T-cell antigen receptor (TCR) membrane-negative T-cell mutants can be divided into two groups: 1) those which lack one of
the six TCR polypeptides and 2) those which contain a mutated TCR chain. The present experiments reveal a new mechanism for
the development of TCR membrane-negative T-cell variants: mutations in splicing consensus motifs causing excision or misreading
of an entire exon (exon 3 of the TCRAC or TCRBC genes). C27.15 cells transcribe a TCR α chain consisting of TCRAVJCexon1Cexon2-encoded amino acids plus six new amino acids. The assembly defect seems to be that the truncated α chain does not interact
with CD3 δ molecules; consequently, no TCR αβ/CD3 δεγε complexes are formed. E6.E12 cells transcribe a TCR β chain composed
of TCRBVDJCexon1Cexon2-encoded amino acids plus twenty-seven new amino acids, which seem not to form a transmembrane region. The truncated β chain
does associate with CD3 γε heterodimers, yet no TCR αβ/CD3 δεγε complexes are made. This may be due either to low assembly
of TCR β/CD3 γε trimers or to lack of access of the mutated TCR β/CD3 γε trimers to the TCR α/CD3 δε compartment in the endoplasmic
reticulum.
Received: 25 September 1996 / Revised: 7 November 1996 相似文献
8.
Beauséjour M Noël D Thibodeau S Bouchard V Harnois C Beaulieu JF Demers MJ Vachon PH 《Apoptosis : an international journal on programmed cell death》2012,17(6):566-578
In human intestinal epithelial crypt (HIEC) cells, the PI3-K/Akt-1 pathway is crucial for the promotion of cell survival and
suppression of anoikis. Class I PI3-K consists of a complex formed by a catalytic (C) and regulatory (R) subunit. Three R
(p85α, β, and p55γ) and four C (p110α, β, γ and δ) isoforms are known. Herein, we analyzed the expression of PI3-K isoforms
in HIEC cells and determined their roles in cell survival, as well as in the β1 integrin/Fak/Src-mediated suppression of anoikis.
We report that: (1) the predominant PI3-K complexes expressed by HIEC cells are p110α/p85β and p110α/p55γ; (2) the inhibition
and/or siRNA-mediated expression silencing of p110α, but not that of p110β, γ or δ, results in Akt-1 down-activation and consequent
apoptosis; (3) the expression silencing of p85β or p55γ, but not that of p85α, likewise induces Akt-1 down-activation and
apoptosis; however, the impact of a loss of p55γ on both Akt-1 activation and cell survival is significantly greater than
that from the loss of p85β; and (4) both the p110α/p85β and p110α/p55γ complexes are engaged by β1 integrin/Fak/Src signaling;
however, the engagement of p110α/p85β is primarily Src-dependent, whereas that of p110α/p55γ is primarily Fak-dependent (but
Src-independent). Hence, HIEC cells selectively express PI3-K isoform complexes, translating into distinct roles in Akt-1
activation and cell survival, as well as in a selective engagement by Fak and/or Src within the context of β1 integrin/Fak/Src-mediated
suppression of anoikis. 相似文献
9.
M. Loui Thomas Rajan A. Badwe Ramakant K. Deshpande Urmila C. Samant Shubhada V. Chiplunkar 《Cancer immunology, immunotherapy : CII》2001,50(4):218-225
The mechanism responsible for tissue specific localization of γδ T cell subsets is not well understood. In order to explain
the sequestration of specific γδ T cell subsets in the peripheral blood and tumor tissue of patients with esophageal cancer,
we examined the function and expression of adhesion molecules on these cells. A hierarchy in the expression of adhesion molecules
was observed. In vitro activated γδ T cells showed dominant expression of LFA-1 (CD11a), VLA-α4 (CD49d), intermediate expression
of VLA-α5 (CD49e) and L-selectin (CD62L), but low expression of CD44v6 and αEβ7 (CD103). It was observed that the γδ T cells use LFA-1, L-selectin and CD44v6 to bind to squamous cell carcinoma (SCC) cells,
whereas they adhere to fibroblast cells using LFA-1, VLA-α4 and VLA-α5. Vδ1 T cell subsets from the peripheral blood γδ T
cells utilize a larger array of adhesion molecules, namely LFA-1, VLA-α4, VLA-α5, L-selectin and αEβ7, to bind to SCC cells compared to the restricted usage of LFA-1, L-selectin and CD44v6 by the Vδ2 T cells. Flow cytometric
analysis of tumor infiltrating lymphocytes from the esophageal tumors confirmed the selective accumulation of Vδ1+γδ T cells in the tumor compartment. It thus appears that adhesion molecules expressed on these lymphocytes play an important
role in the recruitment and retention of Vδ1 T cells in the tumor milieu.
Received: 27 November 2000 / Accepted: 1 March 2001 相似文献
10.
Nishimura K Yamauchi N Chowdhury VS Torii M Hattori MA Kaneto M 《Cell and tissue research》2011,345(2):275-284
Peroxisome proliferator-activated receptors (PPARs) play an important role in different compartments of the female reproductive
system in rodents and humans. However, expressional profiles and physiological functions of PPARs in the endometrium prior
to the placentation are not well understood. In this study, we determined expressional profiles of the PPARs during early
pregnancy. Immunocytochemistry revealed that both PPARα and PPARβ/δ were strongly detected in the endometrial stroma on days
4.5–6.5 of pregnancy, which is just a starting time of implantation. Delayed implantation animal model showed that the expressions
of PPARα and PPARβ/δ occurred after the initiation of implantation in the endometrial stroma. Moreover, an in vitro decidualization
model further revealed that the expression of PPARα increased in the cultured rat endometrial stromal cells at 24 h after the decidualization treatment, but the expression of
PPARβ/δ was delayed and increased at 48 h after the treatment. PPARγ was expressed in the endometrial stroma and its expression decreased
significantly at 2.5 days post-coitum and maintained a low level of expression during the period of implantation. These results
indicate that PPARα is expressed and induced by the initiation of implantation, prior to the expression of PPARβ/δ in decidualized
endometrium. Increasing expression of PPARγ during fertilization and its decline during the period of implantation further
suggest that PPARs may play important roles during early pregnancy. 相似文献
11.
Tomohiro Yamaguchi Youichi Suzuki Ryuichi Katakura Takusaburo Ebina Junkichi Yokoyama Yoshiaki Fujimiya 《Cancer immunology, immunotherapy : CII》1998,47(2):97-103
γδT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the
activation and proliferation of circulating γδT cells should be fully understood prior to their adoptive transfer to cancer
patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified γδT cells isolated from
glioblastoma patients. γδT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) αβγ, but the levels of IL-2Rβ
or γ expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal γδT
cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations
of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response,
this activity was completely or partially abrogated by anti-IL-2Rβ, or anti-IL-2Rγ antibodies, but not by anti-IL-2Rα antibodies.
Incubation of γδT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific
autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific
activity could be significantly blocked by anti-IL-2Rγ and anti-IL-2R-β mAb, but not by anti-IL-2Rα mAb. Thus, in contrast
to IL-2, IL-15 activates tumor-specific γδT cells through the components of IL-2Rβ and IL-2Rγ, but not IL-2Rα. These enhanced
in vitro tumor-specific and proliferative responses of γδT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic
use of γδT cells in cancer patients.
Received: 23 January 1998 / Accepted: 20 May 1998 相似文献
12.
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14.
Hiroaki Sawai Takeshi Itoh Kazumi Kokaji Kazuo Shinozuka 《Journal of molecular evolution》1997,45(3):209-215
Oligomerization of α-adenosine 5′-phosphorimidazolide (α-ImpA) has been done in an aqueous solution using a uranyl-ion catalyst
or a poly(U) template as a model process of prebiotic synthesis of RNA with α-glycosidic linkage. α-Oligoriboadenylates up
to hexamer were formed from α-ImpA by the uranyl-ion catalyst. 3′-5′ Linkage was mainly formed in the oligomerization. The
poly(U) template mediated the oligomerization of α-ImpA, but to a very low extent. The yield and chain length of the resulting
α-oligomers were far lower than those of the corresponding β-oligomer formation under the same conditions. Physico-chemical
properties of α-oligoriboadenylates are presented along with those of the corresponding β-oligoriboadenylates. The results
indicate that β-RNA is more advantageous than α-RNA from the points of their synthesis and properties.
Received: 10 February 1997 / Accepted: 31 March 1997 相似文献
15.
Lee S Jia B Pham BP Shao Y Kwak JM Cheong GW 《Extremophiles : life under extreme conditions》2012,16(1):87-93
Sarcosine oxidase (SOX) catalyzes the oxidation of the methyl group in sarcosine and transfer of the oxidized methyl group
into the one-carbon metabolic pool. Here, we separately cloned and expressed α and β subunit of SOX from Thermococcus kodakarensis KOD1 (TkSOX) in Escherichia coli and the recombinant proteins were purified to homogeneity. Gel filtration chromatography and transmission electron microscopy
analysis showed that the α subunit formed a dimeric structure and behaved as an NADH dehydrogenase; β subunit was a tetramer
that had sarcosine oxidase and l-proline dehydrogenase activity. The TkSOX complex assembled into the hetero-octameric (αβ)4 form and had NADH dehydrogenase activity. Gold-label analysis indicated that α and β subunits were oriented in the alternative
form. Based on these results, we suggested that TkSOX was a multifunctional enzyme and that each subunit and (αβ)4 complex may separately exist as a function enzyme in different conditions. 相似文献
16.
Commercial formulations of hexachlorocyclohexane (HCH) consist of a mixture of four isomers: α, β, γ, and δ. All four isomers
are toxic and recalcitrant pollutants. β-HCH is more problematic due to its longer persistence in the environment. Sphingomonas sp. BHC-A was able to degrade not only α-, γ-, and δ-HCH but also β-HCH. To clone a gene responsible for the degradation
of β-HCH, a Tn5 mutation was introduced into BHC-A, and one mutant BHC-A45 defective in β-HCH degradation was selected. Sequencing analysis
showed this mutant had a Tn5 insertion at the site of one haloalkane dehalogenase gene, designated linB2. linB2 was overexpressed in Escherichia coli and the 32-kDa product LinB2 showed the conversion activity of not only β-HCH to β-2,3,4,5,6-pentachlorocyclohexanol (β-PCHL)
but also β-PCHL to β-2,3,5,6-tetrachloro-1,4-cyclohexanediol. 相似文献
17.
Pedro J. Silva Baltazar de Castro W. R. Hagen 《Journal of biological inorganic chemistry》1999,4(3):284-291
The sulfhydrogenase complex of Pyrococcus furiosus is an αβγδ heterotetramer with both hydrogenase activity (borne by the αδ subunits) and sulfur reductase activity (carried
by the βγ subunits). The β-subunit contains at least two [4Fe-4S] cubanes and the γ-subunit contains one [2Fe-2S] cluster
and one FAD molecule. The δ-subunit contains three [4Fe-4S] cubanes and the α-subunit carries the NiFe dinuclear center. Only
three Fe/S signals are observed in EPR-monitored reduction by dithionite, NADPH, or internal substrate upon heating. All other
clusters presumably have reduction potentials well below that of the H+/H2 couple. Heat-induced reduction by internal substrate allows, for the first time, EPR monitoring of the NiFe center in a hyperthermophilic
hydrogenase, which passes through a number of states, some of which are similar to states previously defined for mesophilic
hydrogenases. The complexity of the observed transitions reflects a combination of temperature-dependent activation and temperature-dependent
reduction potentials.
Received: 10 December 1998 / Accepted: 16 February 1999 相似文献
18.
Zhigang Wang Laura Martins Walther R. Ellis Jr. L. Que Jr. 《Journal of biological inorganic chemistry》1997,2(1):56-64
One- and two-dimensional NMR experiments have been carried out on different forms of myohemerythrin (MHr), a monomeric 13.9-kDa
oxygen carrier, focusing on paramagnetically shifted proton resonances. Compared to the corresponding forms of octameric hemerythrin
(Hr), all of the MHr forms exhibit spectra with better resolution and signal-to-noise ratios. The metMHr spectra allow the
differentiation of the signals from the Nδ-H protons of the five Nε-coordinated His ligands and those from the bridging Asp and Glu ligands. The 1D spectra of deoxyMHr exhibit a number of relatively
sharp features including three solvent-exchangeable peaks that account for five protons. One of these His N-H protons exchanges
more slowly with solvent than the other four and is assigned to His 54, which, by analogy to the crystal structure of deoxyHr,
is the only His ligand that is hydrogen-bonded to an amino acid residue, Glu24 in this case. One-dimensional NOE results on
the non-exchangeable signals clearly show the connectivities among the α and β protons of the bridging Asp111, and the α,
β, and γ protons of the bridging Glu58 ligands. One-dimensional NOE experiments performed on the N-H proton signals of the
coordinated His ligands, together with the COSY results, help to identify the geminal β protons of the His ligands. Upon the
binding of N3
– to one of the Fe(II) sites in deoxyMHr, the overlapping His Nδ-H proton signals observed in the deoxyMHr spectrum are resolved into individual signals; these have been correlated to the
corresponding signals in deoxyMHr by saturation transfer experiments. Similarly, all five His N-H protons are resolved in
the 1H NMR spectrum of the deoxy form of the single point mutant L103N MHr. However, all five N-H protons readily exchange with
solvent, indicating that the mutation affects the hydrogen-bonding interaction between His54 and Glu24.
Received: 20 May 1996 / Accepted: 24 October 1996 相似文献
19.
I. N. Konareva 《Neurophysiology》2011,42(4):286-293
In a group including 72 adults of both sexes, we studied correlations between the estimates of the so-called coronary-prone
personality type (type A) diagnosed using the Jenkins questionnaire and the spectral powers (SPs) of the frequency components
(rhythms) of background EEGs recorded in the resting state (leads C3 and C4 according to the 10-20 system). Despite natural
high interindividual variability, estimates that characterized the subject as belonging to the behavioral type A corresponded,
on average, to relatively low SPs of the δ, θ, and α EEG components, intermediate values of the β1 rhythm SP and coefficient of reactivity of the α rhythm, and higher SPs of the high-frequency (β2 and γ) rhythms. Estimates characterizing type B personality corresponded to significantly higher δ-rhythm SPs, intermediate
SPs of the θ and α rhythms, and smaller SPs of the β and γ rhythms. The interhemisphere asymmetry coefficient for the α rhythm
was usually negative in type-A individuals and positive in the cases of types B and AB. The peculiarities observed are probably
determined, to a certain extent, by the fact that both the characteristics of the behavioral types of the personality and
the amplitude parameters of EEG rhythms depend significantly on inherited (in particular neurochemical) factors. Such peculiarities
of the neurodynamic constitution of the individual are determined, to a considerable extent, by the specificity of organization
and functioning of a few neurotransmitter (in particular aminergic) and neurohumoral systems. 相似文献
20.
Marylène Bertrand André Brack 《International journal of peptide research and therapeutics》1997,4(4-6):387-389
Summary Alternating poly(Glu-Leu) exhibits a random coil structure in pure water at neutral pH. The addition of 0.5 equiv of Ca2+ induces a coil-to-β-sheet transition and the addition of 0.15 equiv of Fe3+ induces a coil-to-α-helix transition. Conformational competition between these two structures was studied by mixing preformed
β-sheets and α-helices in different proportions. Circular dichroism spectra clearly show that β-sheets are favored at the
expense of α-helices in β-sheet-rich mixtures. 相似文献