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1.
Amino acid sequence of the b subunit of human factor XIII, a protein composed of ten repetitive segments 总被引:10,自引:0,他引:10
Factor XIII is a plasma protein that participates in the final stages of blood coagulation. The complete amino acid sequence of the b subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gt11 cDNA library prepared from human liver mRNA was screened with an affinity-purified antibody against the b subunit of human factor XIII. Nine positive clones were isolated from 2 X 10(6) phage and plaque-purified. The largest cDNA insert was sequenced and shown to contain 2180 base pairs coding for a portion of the leader sequence (19 amino acids), the mature protein (641 amino acids), a stop codon (TGA), a 3' noncoding region (187 nucleotides), and a poly(A) tail. When the b subunit of human factor XIII was digested with cyanogen bromide, nine peptides were isolated by gel filtration and reverse-phase high-performance liquid chromatography. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 299 amino acid residues were identified. These amino acid sequences were in complete agreement with the amino acid sequence predicted from the cDNA. The b subunit of factor XIII contained 10 repetitive homologous segments, each composed of about 60 amino acids and 4 half-cystine residues. Each of these repeated segments is a member of a family of repeats present in human beta 2-glycoprotein I, complement factor B, and haptoglobin alpha 1 chain. Three potential Asn-linked carbohydrate attachment sites were also identified in the b subunit of factor XIII. 相似文献
2.
Taylor RM Baniulis D Burritt JB Gripentrog JM Lord CI Riesselman MH Maaty WS Bothner BP Angel TE Dratz EA Linton GF Malech HL Jesaitis AJ 《The Journal of biological chemistry》2006,281(48):37045-37056
The catalytic core of the phagocyte NADPH oxidase is a heterodimeric integral membrane protein (flavocytochrome b (Cyt b)) that generates superoxide and initiates a cascade of reactive oxygen species critical for the host inflammatory response. In order to facilitate structural characterization, the present study reports the first direct analysis of human phagocyte Cyt b by matrix-assisted laser desorption/ionization and nanoelectrospray mass spectrometry. Mass analysis of in-gel tryptic digest samples provided 73% total sequence coverage of the gp91(phox) subunit, including three of the six proposed transmembrane domains. Similar analysis of the p22(phox) subunit provided 72% total sequence coverage, including assignment of the hydrophobic N-terminal region and residues that are polymorphic in the human population. To initiate mass analysis of Cyt b post-translational modifications, the isolated gp91(phox) subunit was subject to sequential in-gel digestion with Flavobacterium meningosepticum peptide N-glycosidase F and trypsin, with matrix-assisted laser desorption/ionization and liquid chromatography-mass spectrometry/mass spectrometry used to demonstrate that Asn-132, -149, and -240 are genuinely modified by N-linked glycans in human neutrophils. Since the PLB-985 cell line represents an important model system for analysis of the NADPH oxidase, methods were developed for the purification of Cyt b from PLB-985 membrane fractions in order to confirm the appropriate modification of N-linked glycosylation sites on the recombinant gp91(phox) subunit. This study reports extensive sequence coverage of the integral membrane protein Cyt b by mass spectrometry and provides analytical methods that will be useful for evaluating posttranslational modifications involved in the regulation of superoxide production. 相似文献
3.
From a human-leukocyte cDNA library, we cloned cDNA encoding a novel protein, which has a significant homology with the b subunit of ATP synthase (proton-transporting ATPase, F1F0-ATPase; EC3.6.1.34) derived from Anabaena sp. strain PCC 7120. The cDNA has an open reading frame of 1314 nucleotides corresponding to 438 amino acids. The coding sequence was 37.9% identical over 57 amino acid with b subunit of ATP synthase. The 34-amino-acid region of the predicted peptide sequence displays a coiled-coil motif that could form a complex with some other protein(s). We designated this novel gene as ATP-BL because of its homology to the b subunit of ATP synthase. The ATP-BL locus was mapped by fluorescence in situ hybridization (FISH) and radiation hybrid mapping to the q24 region of chromosome 16. 相似文献
4.
F(1)F(o) ATP synthases function by a rotary mechanism. The enzyme's peripheral stalk serves as the stator that holds the F(1) sector and its catalytic sites against the movement of the rotor. In Escherichia coli, the peripheral stalk is a homodimer of identical b subunits, but photosynthetic bacteria have open reading frames for two different b-like subunits thought to form heterodimeric b/b' peripheral stalks. Chimeric b subunit genes have been constructed by substituting sequence from the Thermosynechococcus elongatus b and b' genes in the E. coli uncF gene, encoding the b subunit. The recombinant genes were expressed alone and in combination in the E. coli deletion strain KM2 (Deltab). Although not all of the chimeric subunits were incorporated into F(1)F(o) ATP synthase complexes, plasmids expressing either chimeric b(E39-I86) or b'(E39-I86) were capable of functionally complementing strain KM2 (Deltab). Strains expressing these subunits grew better than cells with smaller chimeric segments, such as those expressing the b'(E39-D53) or b(L54-I86) subunit, indicating intragenic suppression. In general, the chimeric subunits modeled on the T. elongatus b subunit proved to be more stable than the b' subunit in vitro. Coexpression of the b(E39-I86) and b'(E39-I86) subunits in strain KM2 (Deltab) yielded F(1)F(o) complexes containing heterodimeric peripheral stalks composed of both subunits. 相似文献
5.
M Revington D T McLachlin G S Shaw S D Dunn 《The Journal of biological chemistry》1999,274(43):31094-31101
In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface. 相似文献
6.
A dimer of 156-residue b subunits forms the peripheral stator stalk of eubacterial ATP synthase. Dimerization is mediated by a sequence with an unusual 11-residue (hendecad) repeat pattern, implying a right-handed coiled coil structure. We investigated the potential for producing functional chimeras in the b subunit of Escherichia coli ATP synthase by replacing parts of its sequence with corresponding regions of the b subunits from other eubacteria, sequences from other polypeptides having similar hendecad patterns, and sequences forming left-handed coiled coils. Replacement of positions 55-110 with corresponding sequences from Bacillus subtilis and Thermotoga maritima b subunits resulted in fully functional chimeras, judged by support of growth on nonfermentable carbon sources. Extension of the T. maritima sequence N-terminally to position 37 or C-terminally to position 124 resulted in slower but significant growth, indicating retention of some capacity for oxidative phosphorylation. Portions of the dimerization domain between 55 and 95 could be functionally replaced by segments from two other proteins having a hendecad pattern, the distantly related E subunit of the Chlamydia pneumoniae V-type ATPase and the unrelated Ag84 protein of Mycobacterium tuberculosis. Extension of such sequences to position 110 resulted in loss of function. None of the chimeras that incorporated the leucine zipper of yeast GCN4, or other left-handed coiled coils, supported oxidative phosphorylation, but substantial ATP-dependent proton pumping was observed in membrane vesicles prepared from cells expressing such chimeras. Characterization of chimeric soluble b polypeptides in vitro showed their retention of a predominantly helical structure. The T. maritima b subunit chimera melted cooperatively with a midpoint more than 20 degrees C higher than the normal E. coli sequence. The GCN4 construct melted at a similarly high temperature, but with much reduced cooperativity, suggesting a degree of structural disruption. These studies provide insight into the structural and sequential requirements for stator stalk function. 相似文献
7.
8.
McLachlin DT Coveny AM Clark SM Dunn SD 《The Journal of biological chemistry》2000,275(23):17571-17577
The b subunit dimer of the Escherichia coli ATP synthase, along with the delta subunit, is thought to act as a stator to hold the alpha(3)beta(3) hexamer stationary relative to the a subunit as the gammaepsilonc(9-12) complex rotates. Despite their essential nature, the contacts between b and the alpha, beta, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b(24-156), a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F(1) sector or to complete F(1)F(0) was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to alpha and beta), and 109 and 110 (to alpha only). Mass spectrometric analysis of peptide fragments derived from the b(24-156)A92C cross-link revealed that cross-linking took place within the region of alpha between Ile-464 and Met-483. This result indicates that the b dimer interacts with the alpha subunit near a non-catalytic alpha/beta interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F(0) in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b(24-156)A92C and beta as well as b(24-156)I109C and alpha are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex. 相似文献
9.
Complete primary structure of the transacylase (E2b) subunit of the human branched chain alpha-keto acid dehydrogenase complex 总被引:4,自引:0,他引:4
Y Nobukuni H Mitsubuchi F Endo I Matsuda 《Biochemical and biophysical research communications》1989,161(3):1035-1041
We isolated from a placental cDNA library by immunoscreening a cDNA clone encoding the transacylase (E2b) precursor of the human branched chain alpha-keto acid dehydrogenase (BCKDH) complex. The cDNA insert consists of 2,649 base pairs with an open reading frame of 1,431 base pairs which can be translated into 477 amino acids and a 3'-untranslated region of 1,205 base pairs. The deduced amino acid sequence includes a leader peptide of 56 amino acid residues, a lipoyl-bearing domain, a E3-binding domain and an inner core domain. A mature human E2b subunit is likely to contain 421 amino acid residues with a calculated Mr 46,322. The nucleotide sequence of the open reading frame and the deduced amino acid sequence of the human E2b shows 91.6% and 92.0% homology with those of the bovine E2b subunit, respectively. 相似文献
10.
The effects of mutation of residue Ala-128 of the b subunit of Escherichia coli ATP synthase to aspartate on the structure of the subunit and its interaction with the F(1) sector were analyzed. Determination of solution molecular weights by sedimentation equilibrium ultracentrifugation revealed that the A128D mutation had little effect on dimerization in the soluble b construct, b(34-156). However, the mutation caused a structural perturbation detected through both a 12% reduction in the sedimentation coefficient and also a reduced tendency to form intersubunit disulfide bonds between cysteine residues inserted at position 132. Unlike the wild-type sequence, the A128D mutant was unable to interact with F(1)-ATPase. These results indicate that the A128D mutation caused a structural change in the C-terminal region of the protein, preventing the binding to F(1) but having little or no effect on the dimeric nature of b. 相似文献
11.
Mature seeds of lentil (Lens culinaris Medik.) were previously reported to contain an insecticidal cysteine-rich peptide, likely of the albumin-1 subunit b type. The purpose of this work was to determine the amino acid sequence of this insecticidal lentil peptide in an Eston lentil extract by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), after reduction of the disulfide bridges, alkylation of the cysteine residues and hydrolysis by pronase, trypsin, chymotrypsin and endoproteinase Asp-N. Sequences of key fragments were supported by monoisotopic mass measurements and by sequence ions from collision-induced dissociation (CID) experiments with a MALDI-TOF/TOF analyzer (MS/MS analysis). The new 37 amino acid sequence revealed strong similarities to a histidine-containing pea PA1b peptide and to soybean leginsulins but with a unique segment of RSSA in the middle. The lentil PA1b peptide sequence agreed completely with that derived from a L. culinaris genomic DNA sequence. 相似文献
12.
Zerria K Bebbiche N Barbouche MR Dellagi K Fathallah DM 《Archives de l'Institut Pasteur de Tunis》2002,79(1-4):11-17
The leukocyte beta2 integrin CR3 (CD11/CD18), is a surface heterodimeric glycoprotein that functions as a divalent cation-dependent adhesive complex. It mediates several important cell-substrate and cell-cell adhesive interactions among which the interaction with vascular endothelial cells that lead to leukocyte transmigration. We have isolated cDNA clones-coding for the rat complement receptor type 3 (CR3) alphaM subunit (CD11b) from a cDNA library. The cDNA sequence showed respectively 89.4% and 74.6% homology with its mouse and human counterpart. We have expressed the sequence coding for the VA module or Von Willebrand type domain (A-domain) and produced it in E. coli as a soluble recombinant fusion protein with GST. Simultaneously, we have cloned DNA fragments specific to the rat ICAM-1 domain 1 and domain 3 and expressed each clone in E. coli as recombinant soluble (rs) fusion proteins with GST. Recombinant CD11b A-domain was released from the fusion protein by thrombin cut. Purified ICAM-1 fusion peptides and CD11b A-domain were used to develop a direct binding assay that showed a specific binding between the rat ICAM-1 Ig like domain 3 and CD11b A-domain. These data demonstrate that the IgSF modules can be produced as a soluble recombinant fusion protein and used to study direct binding to the VA module displayed by members of the integrin superfamily. 相似文献
13.
There are currently 25 recognized species of the chipmunk genus Tamias. In this study we sequenced the complete mitochondrial cytochrome b (cyt b) gene of 23 Tamias species. We analyzed the cyt b sequence and then analyzed a combined data set of cyt b along with a previous data set of cytochrome oxidase subunit II (COII) sequence. Maximum-likelihood was used to further test the fit of models of evolution to the cyt b data. Other sciurid cyt b sequence was added to examine the evolution of Tamias in the context of other sciurids. Relationships among Tamias species are discussed, particularly the possibility of a current sorting event among taxa of the southwestern United States and the extreme divergences among the three subgenera (Neotamias, Eutamias, and Tamias). 相似文献
14.
S Kindler B Schulz M Goedert C C Garner 《The Journal of biological chemistry》1990,265(32):19679-19684
Full length cDNA clones encoding microtubule-associated proteins (MAP) 2b and 2c from rat brain have been isolated and sequenced. The cDNA fragments spanning the coding regions for both MAP2b and MAP2c were assembled and expressed in Escherichia coli. The mobility of these bacterial expressed proteins in sodium dodecyl sulfate gels is identical to that of MAP2b and MAP2c from rat brain. The protein sequence of rat MAP2b has been compared to the full length sequence from mouse and the partial sequence from human high molecular weight MAP2. This comparison has revealed that MAP2b is composed of several highly conserved domains flanked by domains with extensive sequence divergence. Two of the conserved domains, found either at the NH2 or COOH terminus, overlap with the binding domain for the regulatory subunit of the cAMP-dependent protein kinase II and the microtubule-binding domain, respectively. A third homologous domain of unknown function lies in a central region of MAP2b. Secondary structure prediction suggests that the portion of MAP2b which extends from the microtubule surface is composed of an extensive number of alpha-helices separated by small turns which may account for the extended yet flexible structure of MAP2. Interestingly, the 4000-base pair deletion from the middle of MAP2b which generates MAP2c not only removes these helices, but also this third highly conserved MAP2b domain. 相似文献
15.
16.
Mutants of Escherichia coli H+-ATPase defective in the delta subunit of F1 and the b subunit of F0 总被引:5,自引:0,他引:5
Complete nucleotide sequence of the genes for subunits of the H+ ATPase of E.coli has been determined and several hybrid plasmids carrying various portions of these genes have been constructed. Genetic complementation and recombination tests of about forty mutants of E.coli defective in the ATPase were performed using these plasmids for identifying the locations of the mutations. Two mutants defective in the delta subunit and a novel type of mutant defective in the b subunit of F0 were identified. The delta subunit mutants showed no proton conduction, suggesting that this subunit has an important role for the proton conduction. The ATPase of the b subunit mutant has a normal activity of proton channel portion, which phenotype is clearly different from that of mutants of the b subunit reported previously. 相似文献
17.
The mechanism of ATP synthase: a reassessment of the functions of the b and a subunits 总被引:12,自引:0,他引:12
A model for the mechanism of ATP synthase was proposed previously (Cox, G.B., Jans, D.A., Fimmel, A.L., Gibson, F. and Hatch, L. (1984) Biochim. Biophys. Acta 768, 201-208) in which the b subunit of the Fo of Escherichia coli rotated. The driving force was proposed to be an interaction between two charged residues in the membrane, namely, Lys-23 of the b subunit and Asp-61 of the c subunit. To test this proposal the Lys-23 of the b subunit was replaced by threonine using site-directed mutagenesis. The resulting mutant, although it had an impairment in the assembly of the F1F0-ATPase, was normal with respect to oxidative phosphorylation. The role of the a subunit, which had been previously proposed to be a structural one, was reassessed by examination of the possible secondary and tertiary structure of the analogous proteins from several sources. Not only did these subunits appear to have very similar structures, but in each there was a highly conserved helical arm on one of the transmembrane helices which could form a proton channel if it interacted with the Asp-61 of the c subunit. A revised model is therefore presented in which five transmembrane helices from the a subunit and two from the b subunit are surrounded by a ring of c subunits. The highly conserved nature of the structures of the a, b and c subunits from various organisms suggests that the model may have relevance for ATP synthases from bacterial plasma membranes, mitochondria and chloroplasts. 相似文献
18.
T Tsuji F Yamamoto Y Miura K Takio K Titani S Pawar T Osawa S Hakomori 《The Journal of biological chemistry》1990,265(12):7016-7021
19.
The evolutionary origin of photosynthetic reaction centers has long remained elusive. Here, we use sequence and structural analysis to demonstrate an evolutionary link between the cytochrome b subunit of the cytochrome bc(1) complex and the core polypeptides of the photosynthetic bacterial reaction center. In particular, we have identified an area of significant sequence similarity between a three contiguous membrane-spanning domain of cytochrome b, which contains binding sites for two hemes, and a three contiguous membrane-spanning domain in the photosynthetic reaction center core subunits, which contains binding sites for cofactors such as (bacterio)chlorophylls, (bacterio)pheophytin and a non-heme iron. Three of the four heme ligands in cytochrome b are found to be conserved with the cofactor ligands in the reaction center polypeptides. Since cytochrome b and reaction center polypeptides both bind tetrapyrroles and quinones for electron transfer, the observed sequence, functional and structural similarities can best be explained with the assumption of a common evolutionary origin. Statistical analysis further supports a distant but significant homologous relationship. On the basis of previous evolutionary analyses that established a scenario that respiration evolved prior to photosynthesis, we consider it likely that cytochrome b is the evolutionary precursor for type II reaction center apoproteins. With a structural analysis confirming a common evolutionary origin of both type I and type II reaction centers, we further propose a novel "reaction center apoprotein early" hypothesis to account for the development of photosynthetic reaction center holoproteins. 相似文献
20.
F0 part of the ATP synthase from Escherichia coli. Influence of subunits a, and b, on the structure of subunit c 总被引:1,自引:0,他引:1
Four different sets of proteoliposomes were prepared from F0, subunit c, a complex of subunits a and c (ac complex) and an ac complex supplemented with subunit b. Only liposomes containing intact F0 or all subunits of F0 were active in proton translocation and F1 binding [Schneider, E. and Altendorf, K. (1985) EMBO J. 4, 515-518]. The conformation of subunit c in the different preparations was analyzed by labelling the proteoliposomes with the hydrophobic photoactivatable reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). Subsequent isolation and Edman degradation of this polypeptide revealed distinct radioactive labelling patterns over the entire amino acid sequence. In the F0 complex and in the ac complex subunit c retains a labelling pattern which is related to that found in TID-labelled membrane vesicles of Escherichia coli [Hoppe et al. (1984) Biochemistry 23, 5610-5616]. In the absence of subunit a, considerably more and different amino acid residues of subunit c are modified. The labelling data are discussed in relation to structural aspects of F0 and functional properties of proteoliposomes reconstituted with F0 or individual subunits. 相似文献