共查询到20条相似文献,搜索用时 15 毫秒
1.
Integrin conformational changes mediate integrin activation and signaling triggered by intracellular molecules or extracellular ligands. Even though it is known that αβ transmembrane domain separation is required for integrin signaling, it is still not clear how this signal is transmitted from the transmembrane domain through two long extracellular legs to the ligand-binding headpiece. This study addresses whether the separation of the membrane-proximal extracellular αβ legs is critical for integrin activation and outside-in signaling. Using a disulfide bond to restrict dissociation of the α-subunit Calf-2 domain and β-subunit I-EGF4 domain, we were able to abolish integrin inside-out activation and outside-in signaling. In contrast, disrupting the interface by introducing a glycosylation site into either subunit activated integrins for ligand binding through a global conformational change. Our results suggest that the interface of the Calf-2 domain and the I-EGF4 domain is critical for integrin bidirectional signaling. 相似文献
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Donaldson IJ Shefta J Lawson CA Bushnell JR Morgan AW Isaacs JD Carpenter D Shaw MA Rooth I Quinnell RJ Zumla AM Ollier WR Chintu CZ Muyinda GP Hill AS Boylston AW 《Immunogenetics》2002,53(10-11):884-893
This study investigated polymorphisms of genes in two regions of the T-cell antigen receptor beta-subunit (TCRB) locus, including BV9S2P, and BV6S7 in a 5' linkage group, and BV8S3, BV24S1, BV25S1, BV18S1, BV2S1, BV15S1 and BV3S1 in a 3' linkage group. These loci have been genotyped in individuals from five regions in Africa, including The Gambia, Nigeria, Cameroon, Tanzania, and Zambia, and in individuals from northern Britain, northern India, and Papua New Guinea (PNG). In the 3' linkage group, 11 unique haplotypes were identified in the combined African populations; two equally frequent haplotypes represent the majority of African chromosomes. One haplotype was found in all four regions studied. This is the most frequent haplotype in the northern British, northern Indian and PNG populations. Although present, it is infrequent in the African populations. A North-South gradient in the frequency of a common African haplotype was observed. The distribution did not represent that of a known disease. Evidence suggests that malaria is not responsible for selection of these haplotypes. Overall, this study highlights large differences in the genetic constitution of the TCRB locus between Africans and other populations. 相似文献
4.
Gabriella B. Vannelli Tullio Barni Gianni Forti Andres Negro-Vilar Wylie Vale Mario Serio Giuseppe C. Balboni 《Cell and tissue research》1992,269(2):221-227
Summary The localization of inhibin -subunit in the human testis was studied at the light- and electron-microscope level with immunostaining techniques. Antibodies against specific fragments of porcine and human inhibin -subunits were utilized. At light microscopy, inhibin -subunit immunoreactivity was detected in Sertoli cells, spermatocytes and in some Leydig cells. At electron microscopy, gold labeling was found in the cisternae of the Golgi apparatus and in the endoplasmic reticulum of Sertoli and Leydig cells. Gold labeling for inhibin was also found in coated vesicles in the cytoplasm of Sertoli cells as well as in coated pits and coated vesicles in the cytoplasm of some spermatocytes. The results of the present study suggest that, in the human testis, inhibin is produced by Sertoli and Leydig cells and is taken up by spermatocytes, on which it might act in a paracrine manner. 相似文献
5.
S. V. Ponomarenko 《Journal of Evolutionary Biochemistry and Physiology》2007,43(5):467-475
The ATP-synthase γ-subunit (FoF1) belongs to the rotor part of this oligomeric complex. Catalytic hydrolysis of adenosine triphosphate (ATP) is accompanied by rotation of γ-polypeptide inside the sphere formed by six subunits (αβ)3 of the enzyme. The γ-subunit regulates ATPase and ATP-synthase activities of the FoF1. In the present work, evolutionary and reverse changes of this regulatory polypeptide and their effect on properties of the enzyme are studied. It is suggested that elongation of the γ-subunit globular part had resulted from the atpC intragene duplication in the process of adaptive evolution. The evolved fragment participates in light regulation of the chloroplast ATP-synthase. 相似文献
6.
一种快速构建cRNA标准曲线检测基因表达方法的建立 总被引:6,自引:1,他引:6
为了建立一种适于实验室乃至常规定量检测mRNA表达的、可快速构建cRNA标准曲线的方法,设计带有T7启动子序列和PolyT序列的引物对目的基因和内参照进行PCR,克隆入载体作为体外合成cRNA的模板,快速构建cRNA标准.结果表明:该曲线的线性范围至少达6个数量级,相关系数为0.99.该法快速、简便,适用于所有靶基因. 相似文献
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Johanna Fehr Dorke Meyer Patricia Widmayer Heike Claudia Borth Frauke Ackermann Beate Wilhelm Thomas Gudermann Ingrid Boekhoff 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》2007,193(1):21-34
Although chemotaxis has been proposed to guide sperm to egg throughout the animal kingdom, sperm attractants released from
mammalian eggs have not been identified. Since the G protein subunit α-gustducin is accepted as a marker of chemosensitive
cells, attempts were made to explore whether α-gustducin is also expressed in spermatozoa of mammals. Immunohistochemical
approaches using an anti-α-gustducin-specific antibody revealed the most intense immunoreactivity in differentiating spermatids.
Further evidence for the α-gustducin expression was obtained analyzing testicular and sperm-derived tissue preparations in
western blot analyses. To elucidate whether α-gustducin is retained in mature spermatozoa, epididymal mouse and rat sperm
were subjected to immunocytochemistry as well as immunogold electron microscopy. A specific staining was obtained within the
circumference of the midpiece-localized mitochondria, on the axoneme and the outer dense fibers surrounding the microtubules
of this region, whereas no labeling was detectable in the end piece regions. The analysis of ejaculated bovine and human sperm
revealed a comparable segmental distribution pattern for α-gustducin. Although a possible function for α-gustducin has yet
to be determined, the axonemal-associated localization within the midpiece and principal piece of different mammalian spermatozoa
raises the possibility that this G protein α-subunit may process intracellular signals controlling sperm motility.
Johanna Fehr and Dorke Meyer contributed equally to this work. 相似文献
9.
The β-subunit of high-voltage-activated (HVA) calcium channels is essential for the regulation of expression and gating. On
the other hand, various reports have suggested that β subunits play no role in the regulation of low-voltage-activated T-type
channels. In addition there has been no clear demonstration of a physical interaction between the α-subunit of T-type channel
with β-subunit. In this study, we systematically investigated the interaction between CaVα and CaVβ. The four CaVβ isoforms were expressed in a bacterial system and purified into homogeneity, whereas the ten types of CaVα alpha interaction domain (AID) peptides were chemically synthesized. All possible combinations of CaVα and CaVβ were then tested for by in vitro immunoassays. We describe here the identification of a new interaction between CaV3.3 and CaVβ proteins. This interaction is of low affinity compared to that between the AID of the HVA α-subunit and the alpha-binding
pocket (ABP) site of the β-subunit. The AID peptide of HVA channel exerted no effect on the CaV3.3-CaVβ interaction, thus demonstrating that another site not in the ABP of CaVβ protein played a role in binding with CaV3.3. This is the first demonstration of an α-β subunit interaction in a T-type calcium channel. 相似文献
10.
Hongbo Shen ;Yanping Yang ;Feifei Wang ;Ying Zhang ;Naihao Ye ;Shengfeng Xu ;Honghai Wang 《Acta biochimica et biophysica Sinica》2009,(5):379-388
The increasing emergence of drug-resistant tuberculosis (TB) poses a serious threat to the control of this disease. It is in urgent need to develop new TB drugs. Tryptophan biosynthetic pathway plays an important role in the growth and replication of Mycobacterium tuberculosis (Mtb). The β-subunit of tryptophan synthase (TrpB) catalyzes the last step of the tryptophan biosynthetic pathway, and it might be a potential target for TB drug design. In this study, we overexpressed, purified, and characterized the putative TrpB-encoding gene Rv1612 in Mtb H37Rv. Results showed that Mtb His-TrpB optimal enzymatic activity is at pH 7.8 with 0.15 M Na^+ or 0.18 M Mg^2+ at 37℃. Structure analysis indicated that Mtb TrpB exhibited a typical β/α barrel structure. The amino acid residues believed to interact with the enzyme cofactor pyridoxal-5'-phosphate were predicted by homology modeling and structure alignment. The role of these residues in catalytic activity of the Mtb His-TrpB was confirmed by site-directed mutagenesis. These results provided reassuring structural information for drug design based on TrpB. 相似文献
11.
Morera FJ Alioua A Kundu P Salazar M Gonzalez C Martinez AD Stefani E Toro L Latorre R 《FEBS letters》2012,586(16):2287-2293
The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca(2+)-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein-protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. 相似文献
12.
Ashim C. Roy Wu-Xiang Liao Ying Chen Sabaratnam Arulkumaran Shan S. Ratnam 《Molecular and cellular biochemistry》1996,165(2):151-153
Seven new point mutations have been identified from LH -subunit gene by PCR-mediated SSCP, and sequencing. One mutation was found changing amino acid from Gln102 to Ser102. The remaining six mutations, which did not change the codings, were in complete linkage disequilibrium. SSCP can be used in the diagnosis of LH-related disorders. 相似文献
13.
Abdulaev NG Zhang C Dinh A Ngo T Bryan PN Brabazon DM Marino JP Ridge KD 《Journal of biomolecular NMR》2005,32(1):31-40
Heterologous expression systems are often employed to generate sufficient quantities of isotope-labeled proteins for high-resolution NMR studies. Recently, the interaction between the prodomain region of subtilisin and an active, mutant form of the mature enzyme has been exploited to develop a cleavable affinity tag fusion system for one-step generation and purification of full-length soluble proteins obtained by inducible prokaryotic expression. As a first step towards applying high-resolution NMR methods to study heterotrimeric G-protein α-subunit (Gα) conformation and dynamics, the utility of this subtilisin prodomain fusion system for expressing and purifying an isotope-labeled Gα chimera (∼40 kDa polypeptide) has been tested. The results show that a prodomain fused Gα chimera can be expressed to levels approaching 6–8 mg/l in minimal media and that the processed, mature protein exhibits properties similar to those of Gα isolated from natural sources. To assay for the functional integrity of the purified Gα chimera at NMR concentrations and probe for changes in the structure and dynamics of Gα that result from activation, 15N-HSQC spectra of the GDP/Mg2+ bound form of Gα obtained in the absence and presence of aluminum fluoride, a well known activator of the GDP bound state, have been acquired. Comparisons of the 15N-HSQC spectra reveals a number of changes in chemical shifts of the 1HN, 15N crosspeaks that are discussed with respect to expected changes in the protein conformation associated with Gα activation. 相似文献
14.
Basudev Shome Albert F. Parlow Wan-Kyng Liu Hyun S. Nahm Ted Wen Darrell N. Ward 《The protein journal》1988,7(4):325-339
A collaborative study from two laboratories has been undertaken to re-evaluate the human follitropin β-subunit sequence (hFSHβ), since areas of uncertainty remain in the wake of two earlier reports. The first report was by Shome and Parlow (1974). The second, by Saxena and Rathnam (1976), proposed revisions for sequence not definitively placed in the first study, as well as some differences in other placements. We have re-examined the sequence of the hFSHβ with more recent methodology. This has led to revision of certain areas of the sequence and resolution of differences between the two earlier proposals. Specifically, an-Ile-Ser- is established at 21–22, Asp at 41, Arg at 44, Lys at 46, and Glu at 111. These were areas of disagreement in the earlier proposals. A definitive placement of the residues around tryptophan-27 has now been obtained by three laboratories. C-terminal heterogeneity was observed with subunits ending at residue 107, 109, or 111. N-terminal heterogeneity has been observed in all preparations examined to date. A significant population of molecules with a proteolytic nick between residues 38–39 is noted. This is very likely an artifact of the collection and processing. The preparations examined in the present studies showed no evidence of residues 112–118 proposed by Saxena and Rathnam. 相似文献
15.
Jianhe Hu Minglu Xu Bolin Hang Lan Wang Qing Wang Junjie Chen Tao Song Dengfeng Fu Ziliang Wang Sanhu Wang Xingyou Liu 《World journal of microbiology & biotechnology》2011,27(4):767-771
In this study, a novel 18-residue linear antimicrobial peptide derived from the central part of the bovine hemoglobin ??-subunit was identified. The peptide was purified by a combination of cationic exchange and reversed-phase high-performance liquid chromatography. The sequence was determined to be VNFKLLSHSLLVTLASHL. The theoretical molecular weight of this peptide was calculated to be 1992.38 Da, which is the same as that determined (1992.401 Da) by matrix-assisted laser desorption ionization mass spectrometry. Sequence analysis showed that there is a high degree of homology in this peptide among hemoglobin ??-subunits of bovine, sheep, deer, porcine, and human. In a radial-diffusion plate assay, this purified peptide exhibited antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans. 相似文献
16.
Bieri M Mobbs JI Koay A Louey G Mok YF Hatters DM Park JT Park KH Neumann D Stapleton D Gooley PR 《Biophysical journal》2012,102(2):305-314
AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the β-subunit, for which there are two isoforms (β(1) and β(2)). Muscle-specific β(2)-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous β(1)-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of β(2)-CBM is concurrent with an increase in flexibility in β(2)-CBM, which may account for the affinity differences between the two isoforms. In contrast to β(1)-CBM, unbound β(2)-CBM showed microsecond-to-millisecond motion at the base of a β-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this β-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from β(2)-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the β(2)-CBM mutant. Insertion of a threonine into the background of β(1)-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms. 相似文献
17.
J. Lowe 《生物化学与生物物理学报:生物膜》2004,1661(1):40-46
We have previously demonstrated that Na+, K+-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the α subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of α isoforms (α1 and α2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na+, K+-ATPase activity 2.6-fold more sensitive to ouabain (I50=1.0±0.1 μM) than the activity of innervated membranes (I50=2.6±0.2 μM). As depicted in [3H]ouabain binding experiments, when the [3H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na+, K+-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of α1 and α2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na+, K+-ATPase α-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell. 相似文献
18.
Giraldez T Rojas P Jou J Flores C Alvarez de la Rosa D 《American journal of physiology. Renal physiology》2012,303(3):F328-F338
Amiloride-sensitive epithelial Na(+) channels (ENaCs) can be formed by different combinations of four homologous subunits, named α, β, γ, and δ. In addition to providing an apical entry pathway for transepithelial Na(+) reabsorption in tight epithelia such as the kidney distal tubule and collecting duct, ENaCs are also expressed in nonepithelial cells, where they may play different functional roles. The δ-subunit of ENaC was originally identified in humans and is able to form amiloride-sensitive Na(+) channels alone or in combination with β and γ, generally resembling the canonical kidney ENaC formed by α, β, and γ. However, δ differs from α in its tissue distribution and channel properties. Despite the low sequence conservation between α and δ (37% identity), their similar functional characteristics provide an excellent model for exploring structural correlates of specific ENaC biophysical and pharmacological properties. Moreover, the study of cellular mechanisms modulating the activity of different ENaC subunit combinations provides an opportunity to gain insight into the regulation of the channel. In this review, we examine the evolution of ENaC genes, channel subunit composition, the distinct functional and pharmacological features that δ confers to ENaC, and how this can be exploited to better understand this ion channel. Finally, we briefly consider possible functional roles of the ENaC δ-subunit. 相似文献
19.
Masayuki Okuyama Masashi Miyamoto Ichiro Matsuo Shogo Iwamoto Ryo Serizawa Masanari Tanuma 《Bioscience, biotechnology, and biochemistry》2017,81(8):1503-1511
The recombinant catalytic α-subunit of N-glycan processing glucosidase II from Schizosaccharomyces pombe (SpGIIα) was produced in Escherichia coli. The recombinant SpGIIα exhibited quite low stability, with a reduction in activity to <40% after 2-days preservation at 4 °C, but the presence of 10% (v/v) glycerol prevented this loss of activity. SpGIIα, a member of the glycoside hydrolase family 31 (GH31), displayed the typical substrate specificity of GH31 α-glucosidases. The enzyme hydrolyzed not only α-(1→3)- but also α-(1→2)-, α-(1→4)-, and α-(1→6)-glucosidic linkages, and p-nitrophenyl α-glucoside. SpGIIα displayed most catalytic properties of glucosidase II. Hydrolytic activity of the terminal α-glucosidic residue of Glc2Man3-Dansyl was faster than that of Glc1Man3-Dansyl. This catalytic α-subunit also removed terminal glucose residues from native N-glycans (Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2) although the activity was low. 相似文献
20.
Sylvie Gerber Jean-Michel Rozet Dominique Bonneau Eric Souied Jean Weissenbach Jean Frezal Arnold Munnich Josseline Kaplan 《Human genetics》1995,95(4):382-384
Stargardt's disease is an autosomal recessive infantile macular degeneration of unknown origin whose gene has been recently mapped to chromosome 1p21-p13 by linkage analysis in eight multiplex families. Since the cone-specific -subunit of the transducin gene (GNAT2) has been mapped to chromosome 1p13, we tested GNAT2 as the disease-causing gene in our series. Using a novel intragenic polymorphism, we show here that GNAT2 is most probably located centromeric to the genetic interval encompassing the disease gene (D1S424-D1S236, location score = 3.54). In addition, single-strand conformation polymorphism and sequence analyses of the eight exons of the GNAT2 gene was performed in our probands. No evidence of a deleterious base substitution was observed in any affected individual. Taken together, these results support the exclusion of GNAT2 as the causal disease gene of Stargardt's disease. 相似文献