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1.
Poppy Datta Sudipa Chakrabarty Amit Chakrabarty Abhijit Chakrabarti 《Journal of biosciences》2007,32(2):1147-1151
We have shown the differential interactions of the erythroid skeletal protein spectrin with the globin subunits of adult haemoglobin (HbA); these indicate a preference for α-globin over that for β-globin and intact HbA in an adenosine 5′-triphosphate (ATP)-dependent manner. The presence of Mg/ATP led to an appreciable decrease in the binding affinity of the α-globin chain to spectrin and the overall yield of globin-spectrin cross-linked complexes formed in the presence of hydrogen peroxide. Similar effects were also seen in the presence of 2-,3-diphosphoglycerate (2,3 DPG), the other important phosphate metabolite of erythrocytes. The binding affinity and yield of cross-linked high molecular weight complexes (HMWCs) formed under oxidative conditions were significantly higher in α-globin compared with intact haemoglobin, HbA and the β-globin chain. The results of this study indicate a possible correlation of the preferential spectrin binding of the α-globin chain over that of the β-globin in the haemoglobin disorder β-thalassaemia. 相似文献
2.
Chakrabarti A Bhattacharya S Ray S Bhattacharyya M 《Biochemical and biophysical research communications》2001,282(5):1189-1193
Spectrin is a large, worm-like cytoskeletal protein that is abundant in all cell types. The denatured heme enzyme, horseradish peroxidase showed significant decrease in the reactivation yield, after 30 min of refolding, in presence of increasing concentrations of spectrin from that in the absence. This indicated that spectrin could bind denatured HRP and inhibit their refolding. In presence of 1 mM ATP and 10 mM MgCl(2) the spectrin binding of denatured HRP is abolished. This activity of decreasing the reactivation yield was found to be ATP-dependent and the denatured enzyme after 30 min refolding in the presence of spectrin, pretreated with Mg/ATP, showed about 40% increase in the reactivation yield compared to the same in absence of spectrin. Fluorescence spectroscopic studies indicated binding of ATP to native spectrin showing concentration-dependent quenching of tryptophan fluorescence by ATP. The apparent dissociation constant of binding of ATP to spectrin was estimated to be 1.1 mM. A high affinity binding of spectrin with denatured HRP has been characterized (K(d) = 16 nM). Since these properties are similar to those of established molecular chaperone proteins, these data indicate that spectrin might have a chaperone-like function in erythrocytes. 相似文献
3.
Y Al-Abed S VanPatten H Li J A Lawson G A FitzGerald K R Manogue R Bucala 《Molecular medicine (Cambridge, Mass.)》2001,7(9):619-623
BACKGROUND: Typically, a diagnosis of diabetes mellitus is based on elevated circulating blood glucose levels. In an attempt to discover additional markers for the disease and predictors of prognosis, we undertook the characterization of HbA1d3 in diabetic and normal patients. MATERIAL AND METHODS: PolyCAT A cation exchange chromatography and liquid chromatography-mass spectroscopy was utilized to separate the alpha- and beta-globin chains of HbA1d3 and characterize their presence in normal and diabetic patients. RESULTS: We report the characterization of HbA1d3 as a glutathionylated, minor hemoglobin subfraction that occurs in higher levels in diabetic patients (2.26 +/- 0.29%) than in normal individuals (1.21 +/- 0.14%, p < 0.001). The alpha-chain spectrum displayed a molecular ion of m/z 15126 Da, which is consistent with the predicted native mass of the HbA0 alpha-globin chain. By contrast, the mass spectrum of the beta-chain showed a mass excess of 307 Da (m/z = 16173 Da) versus that of the native HbA0 beta-globin chain (m/z = 15866 Da). The native molecular weight of the modified beta-globin chain HbA0 was regenerated by treatment of HbA1d3 with dithiothreitol, consistent with a glutathionylated adduct. CONCLUSIONS: We propose that HbA1d3 (HbSSG) forms normally in vivo, and may provide a useful marker of oxidative stress in diabetes mellitus and potentially other pathologic situations. 相似文献
4.
5.
Role of terminal nonhomologous domains in initiation of human red cell spectrin dimerization 总被引:4,自引:0,他引:4
Human erythrocyte spectrin is an antiparallel heterodimer comprised of a 280 kDa alpha subunit and a 246 kDa beta subunit which further associates into tetramers in the red cell membrane cytoskeleton. Lateral association of the flexible rodlike monomers involves a multiple-step process that is initiated by a high affinity association near the actin-binding end of the molecule (dimer nucleation site). In this study, recombinant alpha and beta proteins comprising two or four "spectrin type" motifs with and without adjacent, terminal nonhomologous domains were evaluated for their relative contributions to dimer initiation, and the thermodynamic properties of these heterodimer complexes were measured. Sedimentation equilibrium studies showed that in the absence of the heterologous subunit, individual recombinant proteins formed weak homodimers (K(d) > 0.3 mM). When 2-motif (alpha20-21 and beta1-2) and 4-motif (alpha18-21 and beta1-4) recombinants lacking the terminal nonhomologous domains were paired with the complementary protein, high affinity heterodimers were formed in sedimentation equilibrium analysis. Both the alpha20-21/beta1-2 complex and the alpha20-21EF/betaABD1-2 complex showed stoichiometric binding with similar binding affinities (K(d) approximately 10 nM) using isothermal titration calorimetry. The alpha20-21/beta1-2 complex showed an enthalpy of -10 kcal/mol, while the alpha20-21EF/betaABD1-2 complex showed an enthalpy of -13 kcal/mol. Pull-down assays using alpha spectrin GST fusion proteins showed strong associations between all heterodimer complexes in physiological buffer, but all heterodimer complexes were destabilized by the presence of Triton X-100 and other detergents. Complexes lacking the nonhomologous domains were destabilized to a greater extent than complexes that included the nonhomologous domains. The detergent effect appears to be responsible for the apparent essential role of the nonhomologous domains in prior reports. Taken together, our results indicate that the terminal nonhomologous domains do not contribute to dimer initiation nor are they required for formation of high affinity spectrin heterodimers in physiological buffers. 相似文献
6.
Biophysical characterization of the alpha-globin binding protein alpha-hemoglobin stabilizing protein 总被引:6,自引:0,他引:6
Gell D Kong Y Eaton SA Weiss MJ Mackay JP 《The Journal of biological chemistry》2002,277(43):40602-40609
Alpha-hemoglobin stabilizing protein (AHSP) is a small (12 kDa) and abundant erythroid-specific protein that binds specifically to free alpha-(hemo)globin and prevents its precipitation. When present in excess over beta-globin, its normal binding partner, alpha-globin can have severe cytotoxic effects that contribute to important human diseases such as beta-thalassemia. Because AHSP might act as a chaperone to prevent the harmful aggregation of alpha-globin during normal erythroid cell development and in diseases of globin chain imbalance, it is important to characterize the biochemical properties of the AHSP.alpha-globin complex. Here we provide the first structural information about AHSP and its interaction with alpha-globin. We find that AHSP is a predominantly alpha-helical globular protein with a somewhat asymmetric shape. AHSP and alpha-globin are both monomeric in solution as determined by analytical ultracentrifugation and bind each other to form a complex with 1:1 subunit stoichiometry, as judged by gel filtration and amino acid analysis. We have used isothermal titration calorimetry to show that the interaction is of moderate affinity with an association constant of 1 x 10(7) m(-1) and is thus likely to be biologically significant given the concentration of AHSP (approximately 0.1 mm) and hemoglobin (approximately 4 mm) in the late pro-erythroblast. 相似文献
7.
Functional role of the distal valine (E11) residue of alpha subunits in human haemoglobin 总被引:2,自引:0,他引:2
We have expressed human alpha-globin to a high level in Escherichia coli as a fusion protein, purified it and removed the N-terminal leader sequence by site-specific proteolysis with blood coagulation factor Xa. The apo globin has been refolded and reconstituted with haem and native beta-globin to form fully functional haemoglobin (Hb) with properties identical to those of native human Hb. By site-directed mutagenesis we have altered the distal residues of the alpha subunits and compared the functional properties of these mutant proteins. The rates of various ligands binding to these proteins in the R-state have been reported by Mathews et al. Here, we present the oxygen equilibrium curves of three E11 alpha mutants and the crystal structures of two of these mutants in the deoxy form. Replacing the distal valine residue of alpha-globin with alanine, leucine or isoleucine has no effect on the oxygen affinity of the protein in either quaternary state, in contrast to the equivalent mutations of beta subunits. The crystal structure of the valine E11 alpha----isoleucine mutant shows that the larger E11 residue excludes water from the haem pocket, but causes no significant movement of other amino acid residues. We conclude that the distal valine residue of alpha-globin does not control the oxygen affinity of the protein by sterically hindering ligand binding. 相似文献
8.
Y. Terry Lee Ki Soon Kim Colleen Byrnes Jaira F. de Vasconcellos Seung-Jae Noh Antoinette Rabel Emily R. Meier Jeffery L. Miller 《PloS one》2013,8(7)
Based upon the lack of clinical samples available for research in many laboratories worldwide, a significant gap exists between basic and clinical studies of beta-thalassemia major. To bridge this gap, we developed an artificially engineered model for human beta thalassemia by knocking down beta-globin gene and protein expression in cultured CD34+ cells obtained from healthy adults. Lentiviral-mediated transduction of beta-globin shRNA (beta-KD) caused imbalanced globin chain production. Beta-globin mRNA was reduced by 90% compared to controls, while alpha-globin mRNA levels were maintained. HPLC analyses revealed a 96% reduction in HbA with only a minor increase in HbF. During the terminal phases of differentiation (culture days 14–21), beta-KD cells demonstrated increased levels of insoluble alpha-globin, as well as activated caspase-3. The majority of the beta-KD cells underwent apoptosis around the polychromatophilic stage of maturation. GDF15, a marker of ineffective erythropoiesis in humans with thalassemia, was significantly increased in the culture supernatants from the beta-KD cells. Knockdown of beta-globin expression in cultured primary human erythroblasts provides a robust ex vivo model for beta-thalassemia. 相似文献
9.
Characterization of the binding of the anti-sickling compound, BW12C, to haemoglobin. 总被引:1,自引:0,他引:1
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The anti-sickling agent BW12C [Beddell, Goodford, Kneen, White, Wilkinson & Wootton (1984) Br. J. Pharmacol. 82, 397-407] was designed to left-shift the oxygen saturation curve of haemoglobin (HbA) by preferential binding to the oxy conformation at a single site between the terminal amino groups of the alpha-chains through Schiff's base formation, ionic and hydrophobic interactions. In the present work, Schiff's base linkages formed with [14C]BW12C were reduced with NaBH4 and the alpha- and beta-globin chains separated. Under oxy conditions at a molar ratio of 2:1, the covalently bound BW12C is localized almost exclusively on a single alpha-chain; tryptic digestion confirms the terminal amino group (alpha 1-valine) as the reaction site, in accord with the design hypothesis. However, about half the labelled BW12C is released on tetramer disruption, suggesting the presence of additional non-covalent binding. Under deoxy conditions, alpha- and beta-chains are labelled approximately equally, and at higher molar ratios additional binding in both oxy and deoxy conditions is seen. Isoelectric-focusing studies under oxy conditions show a complex pattern of modified bands for both HbA and HbA1c (blocked beta-terminal amino groups) but no modification for HbA carbamylated at both alpha- and beta-terminal amino groups or at the alpha-chains only, again confirming the alpha-terminal amino region as the main interaction site. Equilibrium dialysis measurements under oxy conditions indicate two strong binding sites with a binding constant of less than 10(-6) M and a number of weaker binding sites. The present data thus confirm that BW12C binds at the intended locus but reveal additional non-covalent binding at an undefined site, and weaker binding through Schiff's base formation with other amino groups. 相似文献
10.
Levasseur DN Ryan TM Reilly MP McCune SL Asakura T Townes TM 《The Journal of biological chemistry》2004,279(26):27518-27524
A new recombinant, human anti-sickling beta-globin polypeptide designated beta(AS3) (betaGly(16) --> Asp/betaGlu(22) --> Ala/betaThr(87) --> Gln) was designed to increase affinity for alpha-globin. The amino acid substitutions at beta22 and beta87 are located at axial and lateral contacts of the sickle hemoglobin (HbS) polymers and strongly inhibit deoxy-HbS polymerization. The beta16 substitution confers the recombinant beta-globin subunit (beta(AS3)) with a competitive advantage over beta(S) for interaction with the alpha-globin polypeptide. Transgenic mouse lines that synthesize high levels of HbAS3 (alpha(2)beta(AS3)(2)) were established, and recombinant HbAS3 was purified from hemolysates and then characterized. HbAS3 binds oxygen cooperatively and has an oxygen affinity that is comparable with fetal hemoglobin. Delay time experiments demonstrate that HbAS3 is a potent inhibitor of HbS polymerization. Subunit competition studies confirm that beta(AS3) has a distinct advantage over beta(S) for dimerization with alpha-globin. When equal amounts of beta(S)- and beta(AS3)-globin monomers compete for limiting alpha-globin chains up to 82% of the tetramers formed is HbAS3. Knock-out transgenic mice that express exclusively human HbAS3 were produced. When these mice were bred with knock-out transgenic sickle mice the beta(AS3) polypeptides corrected all hematological parameters and organ pathology associated with the disease. Expression of beta(AS3)-globin should effectively lower the concentration of HbS in erythrocytes of patients with sickle cell disease, especially in the 30% percent of these individuals who coinherit alpha-thalassemia. Therefore, constructs expressing the beta(AS3)-globin gene may be suitable for future clinical trials for sickle cell disease. 相似文献
11.
The normal expression of human beta globin is critically dependent upon the constitutively high stability of its encoding mRNA. Unlike with alpha-globin mRNA, the specific cis-acting determinants and trans-acting factors that participate in stabilizing beta-globin mRNA are poorly described. The current work uses a linker-scanning strategy to identify a previously unknown determinant of mRNA stability within the beta-globin 3' untranslated region (3'UTR). The new determinant is positioned on an mRNA half-stem opposite a pyrimidine-rich sequence targeted by alphaCP/hnRNP-E, a factor that plays a critical role in stabilizing human alpha-globin mRNA. Mutations within the new determinant destabilize beta-globin mRNA in intact cells while also ablating its 3'UTR-specific interaction with the polyfunctional RNA-binding factor nucleolin. We speculate that 3'UTR-bound nucleolin enhances mRNA stability by optimizing alphaCP access to its functional binding site. This model is favored by in vitro evidence that alphaCP binding is enhanced both by cis-acting stem-destabilizing mutations and by the trans-acting effects of supplemental nucleolin. These studies suggest a mechanism for beta-globin mRNA stability that is related to, but distinct from, the mechanism that stabilizes human alpha-globin mRNA. 相似文献
12.
Effect of nucleotide on the binding of N,N'-p-phenylenedimaleimide-modified S-1 to unregulated and regulated actin 总被引:3,自引:0,他引:3
In our previous study [Chalovich, J. M., Greene, L. E., & Eisenberg, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4909-4913], myosin subfragment 1 that was modified by having its two reactive thiol groups cross-linked by N,N'-p-phenylenedimaleimide (pPDM) was found to resemble the myosin subfragment 1-adenosine 5'-triphosphate (S-1.ATP) complex in its interaction with actin. In the present study, we examined the effect of actin on adenosine 5'-diphosphate (ADP) trapped at the active site of pPDM.S-1. Our results indicate first that, in the presence of actin, ADP is no longer trapped at the active site but exchanges rapidly with free nucleotide. Different pPDM.S-1.nucleotide complexes were then formed by exchanging nucleotide into the active site of pPDM.S-1 in the presence of actin. The binding of pPDM.S-1.ATP or pPDM.S-1.PPi to actin is virtually identical with that of unmodified S-1 in the presence of ATP. Specifically, at mu = 18 mM, 25 degrees C, pPDM.S-1.ATP or pPDM.S-1.PPi binds to unregulated actin with the same affinity as does S-1.ATP, and this binding does not appear to be affected by troponin-tropomyosin. On the other hand, pPDM.S-1.ADP and pPDM.S-1 with no bound nucleotide both show a small, but significant, difference between their binding to actin and the binding of S-1.ATP; pPDM.S-1 and pPDM.S-1.ADP both bind about 2- to 3-fold more strongly to unregulated actin than does S-1.ATP.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
Multiple binding modes of substrate to the catalytic RNA subunit of RNase P from Escherichia coli. 总被引:2,自引:2,他引:0
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M1 RNA that contained 4'-thiouridine was photochemically cross-linked to different substrates and to a product of the reaction it governs. The locations of the cross-links in these photochemically induced complexes were identified. The cross-links indicated that different substrates share some contacts but have distinct binding modes to M1 RNA. The binding of some substrates also results in a substrate-dependent conformational change in the enzymatic RNA, as evidenced by the appearance of an M1 RNA intramolecular cross-link. The identification of the cross-links between M1 RNA and product indicate that they are shared with only one of the three cross-linked E-S complexes that were identified, an indication of noncompetitive inhibition by the product. We also examined whether the cross-linked complexes between M1 RNA and substrate(s) or product are altered in the presence of the enzyme's protein cofactor (C5 protein) and in the presence of different concentrations of divalent metal ions. C5 protein enhanced the yield of certain M1 RNA-substrate cross-linked complexes for both wild-type M1 RNA and a deletion mutant of M1 RNA (delta[273-281]), but not for the M1 RNA-product complex. High concentrations of Mg2+ increased the yield of all M1 RNA-substrate complexes but not the M1 RNA-product complex. 相似文献
14.
Purification and characterization of an erythroid cell-specific factor that binds the murine alpha- and beta-globin genes. 总被引:7,自引:4,他引:3
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An erythroid cell-specific nuclear factor that binds tightly to a sequence motif (5'-GATAAGGA-3') shared by many erythroid cell-specific promoters was purified to homogeneity by DNA sequence affinity chromatography. Visualization of the purified factor, which we term EF-1, showed a simple pattern comprising a polypeptide doublet with Mrs of 18,000 and 19,000. We confirmed that these species account for EF-1-binding activity by eluting the polypeptides from sodium dodecyl sulfate-polyacrylamide gels and renaturing the appropriate binding activity. Using the purified polypeptides, we mapped seven factor-binding sites that are dispersed across the murine alpha- and beta-globin genes. The murine alpha-globin gene is flanked by at least two EF-1-binding sites. One site is centered at nucleotide (nt) -180 (with respect to the alpha-globin cap site). A fivefold-weaker site is located downstream of the alpha-globin poly(A) addition site, at nt +1049. We mapped five EF-1-binding sites near the murine beta-globin gene. The strongest site was centered at nt -210. Four additional sites were centered at nt -266 (adjacent to the binding site of a factor present in both murine erythroleukemia and Raji cells), -75 (overlapping the beta-globin CCAAT box), +543 (within the second intervening sequence), and -111. 相似文献
15.
16.
Interaction between myosin and F-actin. Correlation with actin-binding sites on subfragment-1 总被引:3,自引:0,他引:3
F-Actin bindings to subfragment-1 (S-1) and S-1 after limited proteolysis by trypsin (S-1t) were studied in the absence and presence of ATP by means of ultracentrifugation. No significant difference in the affinities for F-actin was observed between S-1 and S-1t in the absence of ATP. In contrast, the affinity for F-actin in the presence of ATP was decreased about 50 times by the limited proteolysis of the S-1 heavy chain. The S-1 whose SH1 and SH2 groups were cross-linked by N,N'-p-phenylenedimaleimide bound F-actin weakly. The affinity for F-actin was similar to that of unmodified S-1 in the presence of ATP and was also decreased markedly by limited proteolysis of the cross-linked S-1. Reciprocals of the dissociation constant of acto-S-1 complex decreased markedly with increase of ionic strength in the presence of ATP, but decreased only slightly at the rigor state. All these results are consistent with our proposal that S-1 has two different actin binding sites, as reported previously (Katoh, T., Imae, S., & Morita, F. (1984) J. Biochem. 95, 447-454). The mechanism of activation of S-1 ATPase by F-actin is discussed. 相似文献
17.
CACCC and GATA-1 sequences make the constitutively expressed alpha-globin gene erythroid-responsive in mouse erythroleukemia cells.
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Although the human alpha-globin and beta-globin genes are co-regulated in adult life, they achieve the same end by very different mechanisms. For example, a transfected beta-globin gene is expressed in an inducible manner in mouse erythroleukemia (MEL) cells while a transfected alpha-globin gene is constitutively expressed at a high level in induced and uninduced MEL cells. Interestingly, when the alpha-globin gene is transferred into MEL cells as part of human chromosome 16, it is appropriately expressed in an inducible manner. We explored the basis for the lack of erythroid-responsiveness of the proximal regulatory elements of the human alpha-globin gene. Since the alpha-globin gene is the only functional human globin gene that lacks CACCC and GATA-1 motifs, we asked whether their addition to the alpha-globin promoter would make the gene erythroid-responsive in MEL cells. The addition of each of these binding sites to the alpha-globin promoter separately did not result in inducibility in MEL cells. However, when both sites were added together, the alpha-globin gene became inducible in MEL cells. This suggests that erythroid non-responsiveness of the alpha-globin gene results from the lack of erythroid binding sites and is not necessarily a function of the constitutively active, GC rich promoter. 相似文献
18.
Binding of the N-terminal 70-kDa (70K) fragment of fibronectin to fibroblasts blocks assembly of intact fibronectin and is an accurate indicator of the ability of various agents to enhance or inhibit fibronectin assembly. Such binding is widely thought to be to already assembled fibronectin. We evaluated this hypothesis with fibronectin-null mouse fibroblasts plated on laminin-1 in the absence of intact fibronectin. As a proteolytic fragment or recombinant protein, 70K bound fibronectin-null cells specifically in linear arrays that extended outwards from the periphery of spread cells. At early time points, these arrays were similar to those formed by intact fibronectin. 70K arrays formed within 5 min following ligand addition at concentrations as low as 5 nM, indicating rapid and high affinity binding. Bound 70K was extractable with Triton X-100 or deoxycholate but became insoluble when cross-linked with a membrane-impermeable agent into large SDS-stable complexes. Intact fibronectin, in contrast, became progressively non-extractable in the absence of cross-linking. The detergent-resistant arrays of cross-linked 70K localized to tips of cellular extensions and partially overlapped with alpha6 and beta1 integrin subunits at the base of the extensions. alpha5 did not localize with 70K arrays, but became progressively co-localized with assemblies of intact fibronectin over time. These results support a model in which the 70-kDa region of fibronectin binds to linearly arrayed cell surface molecules of adherent cells to initiate assembly, display of the arrays is controlled by the integrin that mediates adhesion, and fibronectin-binding integrins promote fibronectin-fibronectin interactions during progression of assembly. 相似文献
19.
N E O'Leary I R Cheema K Moore W B Mehard M G Buse 《Biochemical and biophysical research communications》1987,144(1):329-336
Histone-H1 purified from rat skeletal muscle is a relatively potent inhibitor of peptide chain initiation in a cell free system, the rabbit reticulocyte lysate (50% inhibition at approximately 0.4 microM). H1 does not inhibit formation of the ternary complex nor its attachment to 40S ribosomes; the data are compatible with H1 binding to mRNA. The inhibition shows mRNA selectivity: translation of beta-globin mRNA is more affected than that of alpha-globin mRNA and hepatic albumin mRNA more than total hepatic mRNA. Whether or not histone-H1 plays a role in translational regulation in intact cells is conjectural, it may serve as a useful model for protein-mRNA interactions. 相似文献
20.
Complexes formed from A13+ or Be2+ and fluoride inhibit the single-stranded DNA-dependent ATPase activity of RecA protein. In contrast, poly(dT)-RecA-ADP complexes, which are inactive for cleavage of LexA protein, become fully active in the presence of AlF4- or BeF3- ions. These data suggest that fluoride complexes of aluminum and beryllium (called herein X) convert RecA-ADP complexes, which bind weakly to single-stranded DNA, into RecA-ADP-X complexes, which bind tightly to single-stranded DNA, the ADP-X moiety behaving as a nonhydrolyzable analogue of ATP. We propose that AlF4- and BeF3- ions act as analogues of inorganic phosphate by binding to the site of the gamma-phosphate of ATP on RecA-ADP complexes, hence mimicking the single-stranded DNA-RecA-ADP-Pi transition state. We conclude that the elementary reaction that switches RecA protein from a high affinity single-stranded DNA binding state to a low affinity single-stranded DNA binding state is not ATP hydrolysis per se but Pi release. 相似文献