共查询到20条相似文献,搜索用时 15 毫秒
1.
H S Kim X Y Wei P Ruth E Perez-Reyes V Flockerzi F Hofmann L Birnbaumer 《The Journal of biological chemistry》1990,265(20):11858-11863
A rabbit skeletal muscle dihydropyridine (DHP) receptor can be purified as an alpha 1-alpha 2-delta-beta-gamma complex, of which alpha 2 and delta are disulfide bonded. This complex has Ca2+ channel activity when incorporated into lipid bilayers. We reported recently that expression of alpha 1 in murine L cells (LCa cells) leads to appearance of both DHP binding and Ca2+ currents, and that we failed to detect alpha 2 by immunoblotting. LCa cell Ca2+ channel currents resembled those in rabbit skeletal muscle in their sensitivity to both voltage and the DHP agonist Bay K 8644, but differed in that they responded to depolarization much more slowly. We now report details of the molecular cloning of the cDNA encoding the 1857-amino acid long alpha 1 transfected into the L cells and results from studies on expression of beta, as well as, on allosteric regulation of DHP binding to these cells. The alpha 1 cDNA was cloned by a combination of cDNA library screening (5355 base pairs) and chemical synthesis (508 base pairs). Using rabbit labeled beta cDNA, which cross-reacts with murine beta mRNA, we failed to observe cross-hybridizing beta mRNA in LCa cells. Using a labeled single stranded 200-base long rabbit alpha 2 cDNA that cross-reacts with mouse alpha 2 mRNA, we likewise failed to observe cross-hybridizing alpha 2 mRNA in LCa cells and hence confirmed the absence of an endogenous murine alpha 2 in these cells. Using LCa cell membranes as DHP receptor source we found the binding of the DHP antagonist (+)-[3H]PN200-110 to be regulated by both verapamil and diltiazem as it is in rabbit skeletal muscle membranes. However, we noted a difference; at concentrations above 10(-6) M, verapamil inhibited residual DHP binding in LCa but not in skeletal muscle membranes. We conclude that neither alpha 2 nor beta are essential for expression of alpha 1 on the cell surface, or for its functioning as a voltage-gated Ca2+ channel, or for its allosteric regulation of DHP binding by Ca2+ channel antagonists. The studies neither exclude roles for gamma and delta, nor for alpha 2 or beta in determining more subtle properties of this channel. 相似文献
2.
Yamaguchi S Okamura Y Nagao T Adachi-Akahane S 《The Journal of biological chemistry》2000,275(52):41504-41511
The dihydropyridine (DHP)-binding site has been identified within L-type Ca(2+) channel alpha(1C) subunit. However, the molecular mechanism underlying modulation of Ca(2+) channel gating by DHPs has not been clarified. To search for novel determinants of high affinity DHP binding, we introduced point mutations in the rat brain Ca(2+) channel alpha(1C) subunit (rbCII or Ca(v)1.2c) based on the comparison of amino acid sequences between rbCII and the ascidian L-type Ca(2+) channel alpha(1) subunit, which is insensitive to DHPs. The alpha(1C) mutants (S1115A, S1146A, and A1420S) and rbCII were transiently expressed in BHK6 cells with beta(1a) and alpha(2)/delta subunits. The mutation did not affect the electrophysiological properties of the Ca(2+) channel, or the voltage- and concentration-dependent block of Ca(2+) channel currents produced by diltiazem and verapamil. However, the S1115A channel was significantly less sensitive to DHP antagonists. Interestingly, in the S1115A channel, DHP agonists failed to enhance whole-cell Ca(2+) channel currents and the prolongation of mean open time, as well as the increment of NP(o). Responsiveness to the non-DHP agonist FPL-64176 was also markedly reduced in the S1115A channel. When S1115 was replaced by other amino acids (S1115D, S1115T, or S1115V), only S1115T was slightly sensitive to S-(-)-Bay K 8644. These results indicate that the hydroxyl group of Ser(1115) in IIIS5-S6 linker of the L-type Ca(2+) channel alpha(1C) subunit plays a critical role in DHP binding and in the action of DHP Ca(2+) channel agonists. 相似文献
3.
Identification of alpha 2-macroglobulin as a cytokine binding plasma protein. Binding of interleukin-1 beta to "F" alpha 2-macroglobulin 总被引:6,自引:0,他引:6
Treatment of normal human plasma with methylamine resulted in the discovery of an interleukin-1 beta(IL-1 beta) binding protein. The protein was labeled with 125I-IL-1 beta and the relative molecular mass (Mr) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein-IL-1 beta complex had a Mr of approximately 400,000 in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis but became dissociated when exposed to beta-mercaptoethanol. The 125I-IL-1 beta labeled protein complex could be immunoprecipitated from plasma by using an anti-alpha 2-macroglobulin (alpha 2M) antiserum. Similarly, a monoclonal antibody (mAb) specific for electrophoretically fast ("F")alpha 2M was able to adsorb the 125I-IL-1 beta labeled complex from plasma. The mAb was also capable of adsorbing "F" alpha 2M-125I-IL-1 beta complexes from binary reaction mixtures, but failed to adsorb free 125I-IL-1 beta. Experiments carried out with purified plasma alpha 2M established that IL-1 beta became bound to alpha 2M only upon reaction with trypsin or methylamine, which results in the appearance of free thiol groups in alpha 2M ("F" alpha 2M). There was no binding of IL-1 beta to the native form of alpha 2M (electrophoretically slow or "S" alpha 2M), which lacks free thiol groups. Pretreatment of "F" alpha 2M with N-ethylmaleimide or [ethylenebis(oxyethylenenitrilo)] tetraacetic acid prevented complex formation between "F" alpha 2M and IL-1 beta. In contrast, the yield of "F" alpha 2M IL-1 beta complex formation was increased severalfold in the presence of 2.5 mM Zn2+. These findings indicate that "F" alpha 2M interacts with IL-1 beta through a thiol-disulfide exchange reaction. Zn2+ may play a major role in bringing together the reactive domains of the adjoining peptide backbones into proper orientation. The ready complex formation between "F" alpha 2M and the pleiotropic cytokine IL-1 beta suggests a novel biological role for "F" alpha 2M, since "F" alpha 2M-IL-1 beta complexes, but not "F" alpha 2M alone, retained IL-1-like activity in the thymocyte costimulator bioassay. 相似文献
4.
Subunit composition of the purified dihydropyridine binding protein from skeletal muscle 总被引:1,自引:0,他引:1
The dihydropyridine (DHP) receptor from rabbit skeletal muscle has been characterized by affinity labeling and purification. Two procedures were used for purification: one that was a procedure modified from that of Curtis and Catterall (1984) and one that employed an anti alpha 1 monoclonal antibody (Mab) affinity column. In addition, both digitonin and CHAPS solubilizations were utilized with each purification technique. The major findings are as follows: (1) In contrast to the behavior in digitonin, neither the 52K (beta) nor the 140K (alpha 2) polypeptide quantitatively copurifies with the 170K (alpha 1) polypeptide when the purification is carried out in CHAPS. This has been shown by use of both wheat germ and monoclonal antibody columns. The digitonin-extracted receptor complex bound to the Mab affinity column loses alpha 2 and beta when the digitonin is replaced by CHAPS, and when the complex is bound to a WGA column, a CHAPS wash causes dissociation of alpha 1, beta, and gamma from alpha 2. Loss of binding of dihydropyridines occurs with the CHAPS wash but can be partially restored by the addition of the CHAPS wash to the material eluted from the column with N-acetylglucosamine. (2) Although both detergents solubilized greater than 80% of the polypeptides associated with the DHP binding site, the ability of these proteins to bind dihydropyridines is reduced more by CHAPS treatment than by digitonin treatment, raising the possibility that subunit interactions contribute to high-affinity binding. Alternatively, CHAPS may remove tightly bound lipids necessary for binding or cause irreversible denaturation of the binding site.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
5.
Comparative assessment of the ligand and metal ion binding properties of integrins alpha9beta1 and alpha4beta1 总被引:2,自引:0,他引:2
Pepinsky RB Mumford RA Chen LL Leone D Amo SE Riper GV Whitty A Dolinski B Lobb RR Dean DC Chang LL Raab CE Si Q Hagmann WK Lingham RB 《Biochemistry》2002,41(22):7125-7141
Integrins alpha9beta1 and alpha4beta1 form a distinct structural class, but while alpha4beta1 has been subjected to extensive study, alpha9beta1 remains poorly characterized. We have used the small molecule N-(benzenesulfonyl)-(L)-prolyl-(L)-O-(1-pyrrolidinylcarbonyl)tyrosine (3) to investigate the biochemical properties of alpha9beta1 and directly compare these properties with those of alpha4beta1. Compound 3 has a high affinity for both integrins with K(D) values of < or =3 and 180 pM for alpha9beta1 in 1 mM Mn2+ (activating) and 1 mM Ca2+ and 1 mM Mg2+ (nonactivating) conditions and < or =5 and 730 pM for alpha4beta1 under the corresponding conditions. Ca2+ treatment promoted the binding of 3 to both integrins (EC50 = 30 microM Ca2+ in both cases). Compound 3 binding to both integrins was also stimulated by the addition of the activating monoclonal antibody TS2/16. These findings indicate that the mechanisms by which metal ions and TS2/16 regulate ligand binding to alpha9beta1 and alpha4beta1 are similar. The binding of 3 to both integrins induced the mAb 9EG7 LIBS epitope, a property consistent with occupancy of the receptor's ligand binding site by 3. But whereas EGTA treatment inhibited the binding of 9EG7 to alpha4beta1, it stimulated the binding of 9EG7 to alpha9beta1. The 9EG7 and TS2/16 effects point to contributions of the beta1-chains on binding. Cross-linking data revealed that the integrin alpha-chains are also involved in binding the small molecule, as stable linkages were observed on both the alpha9 chain of alpha9beta1 and the alpha4 chain of alpha4beta1. Extensive structure-activity analyses with natural and synthetic ligands indicate distinct features of the ligand binding pockets. Most notable was the estimated >1000-fold difference in the affinity of the integrins for VCAM-1, which binds alpha4beta1with an apparent K(D) of 10 nM and alpha9beta1 with an apparent K(D) of >10 microM. Differences were also seen in the binding of alpha9beta1 and alpha4beta1 to osteopontin. Compound 3 competed effectively for the binding of VCAM-1 and osteopontin to both integrins. While these studies show many similarities in the biochemical properties of alpha9beta1 and alpha4beta1, they identify important differences in their structure and function that can be exploited in the design of selective alpha9beta1 and alpha4beta1 inhibitors. 相似文献
6.
To gain further insight into the quaternary structures of methemoglobin derivatives in the low-spin state, the interaction of fully liganded valency hybrid human hemoglobins with IHP was studied by proton NMR spectroscopy. Upon addition of IHP to (alpha CO beta + N3-)2, the same resonances as the previously reported IHP-induced NMR peaks for azidomethemoglobin (alpha + N3-beta +N3-)2 appeared, whereas the binding of IHP did not significantly affect the NMR spectra for (alpha + N3-beta CO)2. The binding of IHP also brought about more pronounced spectral changes for (alpha CO beta + Im)2 and (alpha CO beta + H2O)2 than for (alpha + Im beta CO)2 and (alpha + H2O beta CO)2. Therefore, the IHP-induced NMR peaks for azidomethemoglobin are attributed to the beta heme methyl group. Such IHP-induced beta heme methyl resonances were also observed for (alpha NO beta + N3-)2, which undergoes quaternary structural change, analogously to the R-T transition by the binding of IHP. From the above results, it was suggested that the IHP-induced heme methyl resonances for azidomethemoglobin and (alpha CO beta +N3-)2 may also be associated with the quaternary structure of these Hbs, implying the presence of the IHP-induced "T-like" state in low-spin metHb A. 相似文献
7.
The study of enolase in man-mouse somatic hybrids confirms synteny between ENO1 and the markers on human chromosome 1 (AK2, PGM1, Pep-C) and synteny between ENO2 and the markers on human chromosome 12 (LDHB, Pep-B). The study also shows that the different enolase bands observed in mouse cell strains (Cl1D, R4, A9, 3T3), in hamster cell strains (CH, V79/4, A3), and in 3 of the different bands observed in human fibroblasts have a dimeric structure. The formation of these enolase bands depends on genes at two different loci alpha and beta. The hamster cell line CH (HGPRT) showed a rare enolase phenotype with a two-banded pattern in the intermediate region, a triple-banded pattern in the slow region, and one single isozyme in the fast region. This hamster strain is heterozygous for the first locus and homozygous for the second one. The relationship between these different enolase bands is as follow: in the slow "a" zone, alpha1alpha1,alpha1 alpha2,alpha2alpha2; in the intermediate "i" zone, alpha1beta1, alpha2beta1; and, in the fast "b" zone, beta1beta1. It appears that the frequency of heterozygotes for the alpha or beta loci in man is very low. Of 32 unrelated fibroblast strains investigated, none was found to be heterozygous for the alpha or beta locus. 相似文献
8.
D Rampe H S Kim A E Lacerda L Birnbaumer A M Brown 《Biochemical and biophysical research communications》1990,169(3):825-831
We examined the binding of the 1,4-dihydropyridine (DHP) [3H]PN200-110 to membranes from a fibroblast cell line transfected with the alpha 1 subunit (DHP receptor) of the L-type Ca2+ channel from rabbit skeletal muscle. Binding site affinity (KD) and density (Bmax) were 1.16 +/- 0.31 nM and 142 +/- 17 fmoles/mg protein, respectively. This affinity corresponded closely with that observed in native skeletal muscle. The Ca2+ channel antagonists diltiazem and MDL 12,330A stimulated [3H]PN200-110 binding in a dose-dependent manner while flunarizine, quinacrine and trifluoperazine inhibited binding. Surprisingly, D600 also stimulated [3H]PN200-110 binding in a dose-dependent and stereoselective manner. It is concluded that the fibroblast cells used in this study provide a unique system for interactions of the Ca2+ channel ligands with the alpha 1 subunit of the skeletal muscle L-type Ca2+ channel. 相似文献
9.
J C Bonner A L Goodell J A Lasky M R Hoffman 《The Journal of biological chemistry》1992,267(18):12837-12844
The mechanism by which the platelet-derived growth factor (PDGF)-binding protein, alpha 2-macroglobulin (alpha 2M), modulates PDGF bioactivity is unknown, but could involve reversible PDGF-alpha 2M binding. Herein we report that greater than 70% of 125I-PDGF-BB or -AB complexed to alpha 2M was dissociated by SDS-denaturation followed by SDS-polyacrylamide gel electrophoresis, i.e. most of the binding was noncovalent. Reduction of the PDGF.alpha 2M complex following denaturation dissociated the cytokine from alpha 2M by greater than 90%, suggesting covalent disulfide bond formation. Approximately 50% of the growth factor was dissociated by lowering the pH from 7.5 to 4.0. 125I-PDGF-BB bound alpha 2M in a time-dependent manner (t1/2 = approximately 1 h), reaching equilibrium after 4 h. The 125I-PDGF.BB/alpha 2M complex dissociated more slowly (t1/2 = approximately 2.5 h). "Slow" and "fast" alpha 2M bound nearly equal amounts of PDGF-AB or -BB. Trypsin treatment converted PDGF-BB/alpha 2M complex to the fast conformation but did not release bound 125I-PDGF-BB. All PDGF-isoforms (AA, -AB, and -BB) competed for binding with 125I-PDGF-BB binding to slow alpha 2M and fast alpha 2M-methylamine by 65-80%. Other cytokines that bind alpha 2M (transforming growth factor-beta 1 and -beta 2, tumor necrosis factor-alpha, basic fibroblast growth factor, interleukin -1 beta, and -6) did not compete for 125I-PDGF-BB binding slow alpha 2M, but transforming growth factor-beta 1 and basic fibroblast growth factor inhibited 125I-PDGF-BB binding alpha 2M-methylamine by 30-50%. The reversible nature of the PDGF.alpha 2M complex could allow for targeted PDGF release near mesenchymal cells which possess PDGF receptors. 相似文献
10.
Kinetics of carbon monoxide binding to fully and partially reduced human hemoglobin valency hybrids.
The kinetics of carbon monoxide binding following fast reduction of the valency hybrids alpha2+betaCO2 and alphaCO2beta+2 by hydrated electrons have been studied at different degrees of reduction. The results show that at pH 6.0 and 7.0 reduction of one heme group yields a species which reacts fast with carbon monoxide (rate constant of the order of 10(6) M-1S-1). At pH 6.0 the intermediates alphaCO2beta2 and alpha2betaCO2 bind carbon monoxide with a rate characteristic of the T state. At pH 7.0 alphaCO2beta2 is for the greater part in the T state, while in the case of alpha2betaCO2 the R and the T state are about equally populated. 相似文献
11.
Berjukow S Marksteiner R Gapp F Sinnegger MJ Hering S 《The Journal of biological chemistry》2000,275(29):22114-22120
The role of the inactivated channel conformation in the molecular mechanism of Ca(2+) channel block by the 1,4-dihydropyridine (DHP) (+)-isradipine was analyzed in L-type channel constructs (alpha(1Lc); Berjukow, S., Gapp, F., Aczel, S., Sinnegger, M. J., Mitterdorfer, J., Glossmann, H., and Hering, S. (1999) J. Biol. Chem. 274, 6154-6160) and a DHP-sensitive class A Ca(2+) channel mutant (alpha(1A-DHP); Sinnegger, M. J., Wang, Z., Grabner, M., Hering, S., Striessnig, J., Glossmann, H., and Mitterdorfer, J. (1997) J. Biol. Chem. 272, 27686-27693) carrying the high affinity determinants of the DHP receptor site but inactivating at different rates. Ca(2+) channel inactivation was modulated by coexpressing the alpha(1A-DHP)- or alpha(1Lc)-subunits in Xenopus oocytes with either the beta(2a)- or the beta(1a)-subunit and amino acid substitutions in L-type segment IVS6 (I1497A, I1498A, and V1504A). Contrary to a modulated receptor mechanism assuming high affinity DHP binding to the inactivated state we observed no clear correlation between steady state inactivation and Ca(2+) channel block by (+)-isradipine: (i) a 3-fold larger fraction of alpha(1A-DHP)/beta(1a) channels in steady state inactivation at -80 mV (compared with alpha(1A-DHP)/beta(2a)) did not enhance the block by (+)-isradipine; (ii) different steady state inactivation of alpha(1Lc) mutants at -30 mV did not correlate with voltage-dependent channel block; and (iii) the midpoint-voltages of the inactivation curves of slowly inactivating L-type constructs and more rapidly inactivating alpha(1Lc)/beta(1a) channels were shifted to a comparable extent to more hyperpolarized voltages. A kinetic analysis of (+)-isradipine interaction with different L-type channel constructs revealed a drug-induced inactivated state. Entry and recovery from drug-induced inactivation are modulated by intrinsic inactivation determinants, suggesting a synergism between intrinsic inactivation and DHP block. 相似文献
12.
T Ohkusa A D Carlos J J Kang H Smilowitz N Ikemoto 《Biochemical and biophysical research communications》1991,175(1):271-276
We have investigated a) the effects of the dihydropyridines (DHPs) nifedipine and nimodipine on depolarization-induced (T-tubule-mediated) Ca2+ release in the vesicles consisting of the complex of the T-tubule and SR, and b) the binding of [3H]nimodipine to these vesicles. These DHPs inhibited the slow but not the fast phase of depolarization-induced release, both of which are mediated via the T-tubule. The DHPs have no effect on caffeine-induced release in which T-tubules are not involved. There are two classes of DHP binding sites: one, with high affinity and small capacity, and another, exhibiting low affinity and a much larger capacity. The inhibition paralleled the low affinity binding of DHP with no correlation with the high affinity binding. These results suggest that the low affinity DHP binding sites located probably in the DHP receptor, rather than the high affinity DHP binding site, are responsible for the inhibition of e-c coupling. 相似文献
13.
A microtiter plate assay measuring the binding of cells expressing integrins alpha4beta1 or alpha4beta7 to VCAM-1 and MAdCAM-1, expressed as Ig fusion proteins, was used to explore the interplay between the variables of integrin beta-chain, identity and density of ligand, and identity and concentration of activating cations. Both Mn2+ and Mg2+ supported binding of either integrin to either ligand. Ca2+ supported only the binding of alpha4beta1 to VCAM-Ig. Cation concentrations required for half-maximal binding (EC50) ranged from 0.8-280 microM for Mn2+ and 0.8-30 mM for Mg2+, being thus 2-3 logs lower for Mn2+ compared to Mg2+ independent of ligand. EC50 values for binding of alpha4beta1 to VCAM-Ig were 30-45-fold lower compared to MAdCAM-Ig, while alpha4beta7 showed an opposite 3-15-fold selectivity for MAdCAM-Ig over VCAM-Ig. The density of ligand required for adhesion via alpha4beta1 was markedly lower with Mn2+ versus Mg2+, and with VCAM-Ig versus MAdCAM-Ig. These results were interpreted in terms of a coupled equilibrium model, in which binding of activating metal ions and of integrin ligands each stabilizes activated integrin. We conclude that Mn2+ and Mg2+ bind to common regulatory sites with different affinities, producing similar activated states of the integrin. The resulting activated alpha4beta1 binds more strongly to VCAM-Ig versus MAdCAM-Ig by 30-45-fold, while similarly activated alpha4beta7 binds more strongly to MAdCAM-Ig versus VCAM-Ig by 3-15-fold. Inhibition studies showed that Ca2+ also binds to regulatory sites on both integrins. However, the Ca2+-activated state of alpha4beta1 is distinct from that achieved by Mn2+ and Mg2+, possessing increased selectivity for binding to VCAM-1 versus MAdCAM-1. 相似文献
14.
W Borth B Scheer A Urbansky T A Luger L Sottrup-Jensen 《Journal of immunology (Baltimore, Md. : 1950)》1990,145(11):3747-3754
Human alpha 2-macroglobulin (H alpha 2M) is a major IL-1 beta binding plasma protein. The characteristics of the H alpha 2M IL-1 beta complex formation suggested, that cleavage of the internal thiol ester in other members of the alpha-macroglobulin family (alpha M) could enable these proteins to bind IL-1 beta. Characterization of optimal conditions for binding 125I IL-1 beta to H alpha 2M showed that H alpha 2M-IL-1 beta complex formation could be obtained over a pH range of 6.3 to 9 in the presence of some metal cations (i.e., Zn2+, Cd2+, Cu2+, Ni2+). Other divalent metal cations (i.e., Mn2+, Mg2+, Ca2+) were without effect. Time kinetic studies showed that binding of IL-1 beta to H alpha 2M was complete within 200 min and that H alpha 2M-IL-1 beta complexes became increasingly resistant to dissociation by boiling in SDS as a function of incubation time. Human pregnancy zone protein, rat alpha 1-, alpha 2-macroglobulin (R alpha 1M, R alpha 2M), all homologous with H alpha 2M, were tested for their ability to bind IL-1 beta. In each instance, alpha M-IL-1 beta complex formation was observed only after treatment of alpha M with methylamine, a primary amine that causes cleavage of the internal thiol ester in alpha M and the appearance of free thiol groups. Similarly, for each of these proteins, complex formation was increased several fold in the presence of Zn2+. Competition experiments using cytokines or proteins of similar molecular mass as IL-1 beta established that only unlabeled IL-1 beta was effective in inhibiting binding of 125I IL-1 beta to H"F" alpha 2M. Acylation of H"F" alpha 2M by diethylpyrocarbonate blocked the binding of IL-1 beta when analyzed by native PAGE. Deacylation of H"F" alpha 2M with hydroxylamine partially restored the binding capacity of H"F" alpha 2M further supporting the involvement of histidyl residues in the Zn2(+)-dependent binding of IL-1 beta. Reduced thioredoxin, but not its alkylated form, from Escherichia coli readily releases H"F" alpha 2M bound IL-1 beta under conditions that did not lead to reduction of disulfide bonds in H"F" alpha 2M. The action of thioredoxin also augmented IL-1-like activity in two independent bioassays suggesting that H"F" alpha 2M bound IL-1 beta is partially biologically inactive or latent. These results suggest that "activated" alpha M exert a modulating role for IL-1 beta by exposing specific binding sites, which are inaccessible in the native proteins.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
15.
Correlations between the energetics of cooperativity and quaternary structural probes have recently been made for the intermediate ligation states of Hb [Daugherty et al. (1991) Proc. Natl. Acad. Sci. US 88, 1110-1114]. This has led to a "molecular code" which translates configurations of the 10 ligation states into switch points of quaternary transition according to a "symmetry rule"; T-->R quaternary structure change is governed by the presence of at least one heme-site ligand on each of the alpha beta dimeric half-molecules within the tetramer [see Ackers et al. (1992) Science 255, 54-63, for summary]. In order to further explore this and other features of the cooperative mechanism, we have used oxygen binding to probe the energetics and cooperativities for the vacant sites of the cyanomet ligation species. We have also probed structural aspects of all eight cyanomet ligation intermediates by means of sulfhydryl reaction kinetics. Our oxygen binding results, obtained from a combination of direct and indirect methods, demonstrate the same combinatorial aspect to cooperativity that is predicted by the symmetry rule. Overall oxygen affinities of the two singly-ligated species (alpha +CN beta)(alpha beta) and (alpha beta +CN)(alpha beta) were found to be identical (pmedian = 2.4 Torr). In contrast, the doubly-ligated species exhibited two distinct patterns of oxygen equilibria: the asymmetric species (alpha +CN beta +CN)(alpha beta) showed very high cooperativity (nmax = 1.94) and low affinity (pmedian = 6.0 Torr), while the other three doubly-ligated species showed diminished cooperativity (nmax = 1.23) and considerably higher oxygen affinity (pmedian = 0.4 Torr). Extremely high oxygen affinities were found for the triply-ligated species (alpha +CN beta +CN)(alpha beta +CN) and (alpha +CN beta +CN)(alpha +CN beta) (pmedian = 0.2 Torr). Their oxygen binding free energies are considerably more favorable than those of the alpha and beta subunits within the dissociated alpha beta dimer, demonstrating directly the quaternary enhancement effect, i.e., enhanced oxygen affinity at the last binding step of tetramer relative to the dissociated protomers. Oxygen binding free energies measured for the alpha subunit within the isolated (alpha beta +CN) dimer and for the beta subunit within the isolated (alpha +CN beta) dimer sum to the free energy for binding two oxygens to normal hemoglobin dimers (-16.3 +/- 0.2 versus -16.7 +/- 0.2, respectively), arguing against cooperativity in the isolated dimer. Correlations were established between cooperative free energies of the 10 cyanomet ligation microstates and the kinetics for reacting their free sulfhydryl groups.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
16.
17.
D Kirchhofer J Grzesiak M D Pierschbacher 《The Journal of biological chemistry》1991,266(7):4471-4477
alpha v beta 1 and alpha v beta 3 are two related members of the integrin family of cell surface receptors both of which interact with their ligands through the Arg-Gly-Asp recognition sequence, alpha v beta 1 and alpha v beta 3 share the same cation-binding subunit, alpha v, suggesting a similar cation requirement for both integrins. Instead, we observed that Ca2+ exerts different effects on their binding function. The attachment of alpha v beta 3-loaded liposomes to vitronectin and the alpha v beta 3-mediated adhesion of U 251 cells to an Arg-Gly-Asp-containing peptide was supported equally well by Ca2+ and Mg2+. However, IMR 32 cells which bind to Arg-Gly-Asp-containing peptides through alpha v beta 1 adhered in Mg2+ but not in Ca2+. In agreement, Ca2+ did not support the attachment of alpha v beta 1-loaded liposomes to the macromolecular ligand fibronectin or the binding of alpha v beta 1 to Gly-Arg-Gly-Asp-Ser-Pro-Lys-Sepharose in affinity chromatography experiments. Furthermore, in the presence of a constant Mg2+ concentration, Ca2+ had opposite effects on the two receptors in that it inhibited the alpha v beta 1-mediated adhesion of IMR 32 cells to the peptide substrate while enhancing alpha v beta 3-mediated adhesion of U251 cells. The Ca2+ effects occurred at physiological cation concentrations and therefore, our data suggest a physiological role for Ca2+ as a regulator of integrin function and indicate a possible involvement of the beta subunits in cation binding. 相似文献
18.
C Benassayag G Vallette N Cittanova E Nunez M F Jayle 《Biochimica et biophysica acta》1975,412(2):295-305
In polyacrylamide gels, highly purified rat alpha1-fetoprotein shows a molecular heterogeneity, i.e. a "slow" and a "fast" moving fraction. We have isolated by electrophoretic fractionation and subsequent elution these two forms of alpha1-fetoprotein, and we have studied comparatively the binding parameters for estradiol-17beta of whole alpha1-fetoprotein preparations and of the isolated forms. We have shown that the number of binding sites per molecule of whole alpha1-fetoprotein is always, in our experimental conditions, a fractional number, inferior to unity (0.3). Furthermore, the analysis of the binding parameters of the "two forms" of alpha1-fetoprotein allows discrimination between different classes of binding sites. For the "slow" fraction, the number of predominant binding sites per molecule of protein is close to unity (0.7-0.9), whereas for the "fast" fraction, a very low fractional value is found (0.1). The corresponding association constants are reproducibly different for the two fractions: Ka = 0.1.10(8) M-1 for the "slow" alpha1-fetoprotein, and Ka = 0.7.10(8) M-1 for the "fast" alpha1-fetoprotein. Traces of a very high affinity (10(9) M-1) minor class of binding sites are demonstrated in the "slow" fraction. These results point to the existence of a molecular population of alpha1-fetoprotein, some forms of which have a strong or very strong affinity, and some a negligible affinity, for estrogens. 相似文献
19.
《The Journal of general physiology》1994,103(4):605-623
To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K- pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+. 相似文献
20.
The properties of the nucleotide binding sites in the isolated beta and alpha subunits of H(+)-ATPase from Bacillus PS3 (TF1) have been examined by studying the EPR properties of bound VO(2+), which is a paramagnetic probe for the native Mg2+ cation cofactor. The amino acid ligands of the VO2+ complexes with the isolated beta subunit, with the isolated alpha subunit, with different mixtures of both alpha and beta subunits, and with the catalytic alpha 3 beta 3 gamma subcomplex have been characterized by a combination of EPR, ESEEM, and HYSCORE spectroscopies. The EPR spectrum of the isolated beta subunit with bound VO2+ (1 VO2+/beta) is characterized by (51)V hyperfine coupling parameters (A( parallel) = 168 x 10(-)(4) cm(-)(1) and A( perpendicular) = 60 x 10(-)(4) cm(-)(1)) that suggest that VO2+ binds to the isolated beta subunit with at least one nitrogen ligand. Results obtained for the analogous VO2+ complex with the isolated alpha subunit are virtually identical. ESEEM and HYSCORE spectra are also reported and are similar for both complexes, indicating a very similar coordination scheme for VO2+ bound to isolated alpha and beta subunits. In the isolated beta (or alpha) subunit, the bound VO2+ cation is coordinated by one nitrogen ligand with hyperfine coupling parameters A( parallel)((14)N) = 4.44 MHz, and A( perpendicular)((14)N) = 4.3 MHz and quadrupole coupling parameters e(2)()qQ approximately 3.18 MHz and eta approximately 1. These are typical for amine-type nitrogen ligands equatorial to the VO2+ cation; amino acid residues in the TF1 beta and alpha subunits with nitrogen donors that may bind VO2+ are reviewed. VO2+ bound to a mixture of alpha and beta subunits in the presence of 200 mM Na2SO4 to promote the formation of the alpha 3 beta 3 hexamer has a second nitrogen ligand with magnetic properties similar to those of a histidine imidazole. This situation is analogous to that in the alpha 3 beta 3 gamma subcomplex and in the whole TF1 enzyme [Buy, C., Matsui, T., Andrianambinintsoa, S., Sigalat, C., Girault, G., and Zimmermann, J.-L. (1996) Biochemistry 35, 14281-14293]. These data are interpreted in terms of only partially structured nucleotide binding sites in the isolated beta and alpha subunits as compared to fully structured nucleotide binding sites in the alpha 3 beta 3 heterohexamer, the alpha 3 beta 3 gamma subcomplex, and the whole TF1 ATPase. 相似文献