共查询到20条相似文献,搜索用时 31 毫秒
1.
The kinetics of folding from random coils to two-chain coiled coils of beta beta-tropomyosin was studied by stopped-flow CD (SFCD) in the backbone region (222 nm). Two species were studied: the reduced form and the doubly disulfide cross-linked form. The proteins were totally unfolded in 6M urea-saline buffer, then refolded by tenfold dilution into benign buffer. In the refolding medium, they spontaneously recover the two-chain coiled-coil structure. Reduced beta beta refolds in at least two stages: one or more fast phases (< 0.04 s), in which an intermediate with 71% of the equilibrium ellipticity forms, followed by a slower time-resolvable phase that completes the folding. The slow phase is first order, signifying that dimerization occurs in the fast phase. The time constant of the slow phase is 2 s at 20 degrees C and requires activation parameters of delta S not equal to = -7 +/- 0.3 cal/mol.K, delta H not equal to = 15 +/- 1 kcal/mol. These results are very similar to those previously found for the reduced genetic variant alpha alpha-tropomyosin. In contrast, refolding of doubly disulfide cross-linked beta beta is complete within the dead time (< 0.04 s), whereas the singly cross-linked alpha alpha species also displays a slow phase. The opposite process, unfolding reduced beta beta from the coiled-coil state, is complete within the dead time, as in the alpha alpha variant. 相似文献
2.
Equilibrium thermal denaturation curves (by circular dichroism) are reported for doubly cross-linked beta beta tropomyosin two-chain coiled coils. Cross-linking was performed by reaction of sulfhydryls with either ferricyanide or 5,5'-dithiobis(2-nitrobenzoate) (NbS2). The extent of reaction was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and either by titration of residual sulfhydryls with NbS2 (ferricyanide cross-linking) or by determination of mixed disulfide (protein-S-SbN) through reaction with dithiothreitol (NbS2 cross-linking). The results indicate approximately 90% conversion to molecules with interchain cross-links at both C-36 and C-190. Thermal unfolding curves are compared with those obtained previously for non-cross-linked species. The curves are indistinguishable up to approximately 40 degrees C. Above approximately 40 degrees C, the doubly cross-linked species is more stable, but the transition is less steep. This relationship is also compared with that found between alpha alpha tropomyosin (a similar coiled coil made of a genetic variant chain having a sulfhydryl only at C-190) and its singly cross-linked derivative. Thermal curves for alpha alpha and beta beta non-cross-linked species are very similar, alpha alpha being somewhat more stable. For cross-linked alpha alpha, however, the curve sags at temperatures somewhat below the region of principal cooperative loss of helix, the latter occurring at higher temperature but with the same steepness as in the non-cross-linked case. The sag has been ascribed to a "pretransition" in the region of C-190. Thus, doubly and singly cross-linked species differ in that the former show no pretransition and decreased steepness in the principal transition.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
The kinetics of chain exchange in two-chain coiled coils: alpha alpha- and beta beta-tropomyosin. 总被引:3,自引:0,他引:3
Measurements are presented on the time course of chain exchange among two-chain alpha-helical coiled coils of rabbit tropomyosin. All experiments are in a regime (temperature, protein concentration) in which coiled-coil dimers are the predominant species. Self-exchange in alpha alpha-tropomyosin was investigated by mixing alpha alpha species with alpha* alpha*, the asterisk designating an alpha-chain whose lone sulfhydryl (C190) has been blocked by carboxyamidomethylation. The overall process alpha alpha + alpha* alpha* in equilibrium with 2 alpha alpha* is followed by measurement of the fraction (h) of alpha alpha* species as a function of time. Similarly, self-exchange in beta beta-tropomyosin is examined by measurements of the overall process: beta beta + beta* beta* in equilibrium with 2 beta beta*, in which beta* signifies a beta-chain blocked at both sulfhydryls (C36 and C190). The observed time course for both chains is well fit by the first-order equation: h (t) = h (infinity) (1-e-k1t), with h (infinity) congruent to 0.5. This long-time limit is as expected for self-exchange, and agrees with experiments that attain equilibrium after slow cooling of thermally dissociated and unfolded chains. The simplest consonant mechanism is chain exchange by rate-limiting dissociation of dimers followed by random reassociation. Kinetic analysis shows k1 to be the rate constant for the chain dissociation step, a quantity not previously measured for any coiled coil. This rate constant for beta beta species is about an order of magnitude greater than for alpha alpha. In both, the activation enthalpy and entropy are very large, suggesting that activation to an extensively (greater than 50%) unfolded species necessarily precedes dissociation. Experiments are also reported for overall processes: alpha alpha + beta* beta* in equilibrium with 2 alpha beta* and alpha* alpha* + beta beta in equilibrium with 2 alpha* beta. Results are independent of which chain is blocked. Again h (infinity) congruent to 0.5, in agreement with equilibrium experiments, and the time course is first order. The rate constants and activation parameters are intermediate between those for self-exchange. 相似文献
4.
Current ideas on unfolding equilibria in two-chain, coiled-coil proteins are examined by studies of a species of beta beta tropomyosin that is sulfhydryl blocked at C190 and disulfide cross-linked at C36 (.beta-beta.). The desired species is produced by a seven-step process: (1) Rabbit skeletal muscle, comprising predominantly alpha alpha and alpha beta species, is oxidized with ferricyanide, cross-linking both species at C190. (2) The product is carbamylated at C36 of beta chains, using cyanate in denaturing medium at pH 6. (3) All C190 cross-links are reduced with dithiothreitol (DTT). (4) All C190 sulfhydryls are permanently blocked by carboxyamidomethylation. (5) Chromatography on carboxymethylcellulose in denaturing medium is used to separate C190-blocked alpha chains from C190-blocked, C36-carbamylated beta chains. (6) The latter are decarbamylated in denaturing medium by raising the pH to 8.0. (7) The C190-blocked beta chains are renatured and cross-linked at C36 by ferricyanide. The procedure and the quality of the final product are judged by NaDodSO4/polyacrylamide gel electrophoresis, titration of free sulfhydryls, and electrophoretic analysis of trypsin digestion products. Thermal unfolding curves are reported for the resulting pure .beta-beta. species and for its DTT-reduction product. The latter (.beta beta.) show equilibrium thermal unfolding curves that are very similar to those of the parent beta beta noncross-linked species. The .beta-beta. cross-linked species unfolds in a single-phase, cooperative transition with a melting temperature intermediate between the pretransition and posttransition shown by its cross-linked counterpart, the C190 cross-linked, C36-blocked species (.beta-beta.), which was studied earlier. These transitions are compared with one another and with that of the doubly cross-linked species, beta-(-)beta, in the light of two extant physical models for such transitions. The all-or-none segments model successfully rationalizes the data qualitatively for the .beta-beta. and .beta-beta. species if the usual postulates of greater inherent stability of the amino vs the carboxyl end of the molecule and of strain at each cross-link are accepted. However, the same model then requires that the beta-(-)beta species be the least stable of the three, whereas experiment shows the opposite, thus falsifying the all-or-none segments model. The continuum-of-states model is also qualitatively in accord with data on the .beta-beta. and .beta-beta. species.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
5.
A method has been developed for exploring the quaternary fine structure of oligomeric proteins by crosslinking studies and applied to bovine heart mitochondrial F1-ATPase. The F1 was first labeled with 1-fluoro-2,4-dinitro-[14C]benzene, subsequently reduced with sodium hydrosulfite, and finally cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Gel electrophoresis in the chemically modified protein in the presence of sodium dodecyl sulfate and mercaptoethanol showed the existence of a 105-115-kilodalton molecular species in addition to the five monomeric subunits of F1. This cross-linked species could be alpha 2, alpha beta, or beta 2. Isolation of the cross-linked species and titration with 5,5'-dithiobis-(2-nitrobenzoic acid) showed the absence of sulfhydryl group. Therefore, the cross-linked species must be the dimer beta 2. After digestion of the purified beta 2 with pepsin, a single radioactive peptide was isolated. Determination of the amino acid sequence of this peptide and comparison of its radioactivity with the total radioactivity on beta-subunits show that it was formed exclusively by cross-linking Lys162 of one beta-subunit with Glu199 of another beta-subunit. The observation that two beta-subunits can be cross-linked by a rigid phenylenediamine bridge of 5.7- or 4.3-A length is difficult to reconcile with the widely assumed structure of F1 with the alpha- and beta-subunits occupying alternate corners of a planar hexagon, but is consistent with the structure in which a triangular set of three beta-subunits sits above a triangular set of three alpha-subunits in a staggered conformation. 相似文献
6.
Beta beta homodimers exist in native rabbit skeletal muscle tropomyosin and increase after denaturation-renaturation. 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《Protein science : a publication of the Protein Society》网站下载免费的PDF全文](/ch/ext_images/free.gif)
M. E. Holtzer S. G. Kidd D. L. Crimmins A. Holtzer 《Protein science : a publication of the Protein Society》1992,1(3):335-341
Native tropomyosin from rabbit skeletal muscle (RSTm) consists mainly of alpha alpha and alpha beta coiled coils (alpha/beta approximately 3-4/1). In some extant studies, no beta beta molecules have been found. In this study, RSTm from several different preparations was disulfide cross-linked, both preparation and cross-linking being done under nondenaturing conditions. The cross-linked product was assayed for the presence of beta beta molecules cross-linked at both C36 and C190 (beta = beta). In such cross-linked RSTm, 3-8% beta = beta is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, C4 reversed-phase high-performance liquid chromatography, and a free-solution capillary electrophoresis experiment. This percentage becomes approximately 4-10% beta beta when corrected for incomplete double cross-linking and is independent of protein concentration (0.1-10.0 mg/mL), indicating that the observed beta beta species are not artifacts due to intermolecular cross-linking. Upon denaturation and subsequent renaturation either by heating to 55 degrees C or by incubating at 45 degrees C followed by quenching to room temperature, or by guanidine hydrochloride exposure followed by phased renaturation by dialysis, the fraction of beta beta increases, indicating that the reassociation favors homodimer formation somewhat over random association. This result differs from the random association observed when the sulfhydryl on one of the chains is carboxyamidomethylated (Holtzer, M.E., Breiner, T., & Holtzer, A., 1984, Biopolymers 23, 1811-1833), and from the overwhelming heterodimer preferences reported for tropomyosins from other organisms (Lehrer, S.S., Qian, Y., & Hvidt, S., 1989, Science 246, 926-928; Lehrer, S.S. & Qian, Y., 1990, J. Biol. Chem. 265, 1134-1138). 相似文献
7.
A scanning calorimetric study of unfolding equilibria in homodimeric chicken gizzard tropomyosins. 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Using both circular dichroism (CD) and differential scanning calorimetry (DSC), several laboratories find that the thermal unfolding transitions of alpha alpha and beta beta homodimeric coiled coils of rabbit tropomyosin are multistate and display an overall unfolding enthalpy of near 300 kcal (mol dimer)(-1). In contrast, an extant CD study of beta beta and gamma gamma species of chicken gizzard tropomyosin concludes that their unfolding transitions are simple two-state transitions, with much smaller overall enthalpies (98 kcal mol(-1) for beta beta and 162 kcal mol(-1) for gamma gamma). However, these smaller enthalpies have been questioned, because they imply a concentration dependence of the melting temperatures that is far larger than observed by CD. We report here DSC studies of the unfolding of both beta beta and gamma gamma chicken gizzard homodimers. The results show that these transitions are very similar to those in rabbit tropomyosins in that 1) the overall unfolding enthalpy is near 300 kcal mol(-1); 2) the overall delta C(rho) values are significantly positive; 3) the various transitions are multistate, requiring at least two and as many as four domains to fit the DSC data. DSC studies are also reported on these homodimeric species of chicken gizzard tropomyosin with a single interchain disulfide cross-link. These results are also generally similar to those for the correspondingly cross-linked rabbit tropomyosins. 相似文献
8.
T R Boyle J Campana L J Sweet J E Pessin 《The Journal of biological chemistry》1985,260(14):8593-8600
Insulin receptors purified from human placental membranes by gel-filtration and insulin-agarose affinity chromatography were found to be composed of eight different high molecular weight complexes as identified by nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The subunit stoichiometry of these different high molecular weight forms of the insulin receptor were determined by comparisons of silver-stained gel profiles with the autoradiograms of 125I-insulin specifically cross-linked to the alpha subunit and [gamma-32P]ATP specifically autophosphorylated beta subunit gel profiles. Two-dimensional SDS-polyacrylamide gel electrophoresis in the absence and presence of reductant confirmed the subunit stoichiometries as alpha 2 beta 2, alpha 2 beta beta 1, alpha 2 (beta 1)2, alpha 2 beta, alpha 2 beta 1, alpha 2, alpha beta, and beta, where alpha is the Mr = 130,000 subunit, beta is the Mr = 95,000 subunit, and beta 1 is the Mr = 45,000 subunit. Treatment of the insulin receptor preparations with oxidized glutathione or N-ethylmaleimide prior to SDS-polyacrylamide gel electrophoresis increased the relative amount of the alpha 2 beta 2 complex concomitant with a total disappearance of the alpha 2 beta, alpha 2 beta 1, alpha 2, and free beta forms. The effects of oxidized glutathione were found to be completely reversible upon extensive washing of the treated insulin receptors. In contrast, the effects of N-ethylmaleimide were totally irreversible by washing, consistent with known sulfhydryl alkylating properties of this reagent. The formation of these lower molecular weight insulin receptor subunit complexes was further demonstrated to be due to SDS/heat-dependent intramolecular sulfhydryl-disulfide exchange occurring within the alpha 2 beta 2 complex. These studies demonstrate that the largest disulfide-linked complex (alpha 2 beta 2) is the predominant insulin receptor form purified from the human placenta with the other complexes being generated by proteolysis and by internal subunit dissociation. 相似文献
9.
The guanine nucleotide binding proteins (G proteins) that couple hormone and other receptors to a variety of intracellular effector enzymes and ion channels are heterotrimers of alpha, beta, and gamma subunits. One way to study the interfaces between subunits is to analyze the consequences of chemically cross-linking them. We have used 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linking reagent that reacts with sulfhydryl groups, to cross-link alpha to beta subunits of Go and Gi-1. Two cross-linked products are formed from each G protein with apparent molecular masses of 140 and 122 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both bands formed from Go reacted with anti-alpha o and anti-beta antibody. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is anomalous since the undenatured, cross-linked proteins have the same Stokes radius as the native, uncross-linked alpha beta gamma heterotrimer. Therefore, each cross-linked product contains one alpha and one beta subunit. Activation of Go by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not prevent cross-linking of alpha to beta gamma, consistent with an equilibrium between associated and dissociated subunits even in the presence of GTP gamma S. The same cross-linked products of Go are formed in brain membranes reacted with BMH as are formed in solution, indicating that the residues cross-linked by BMH in the pure protein are accessible when Go is membrane bound. Analysis of tryptic peptides formed from the cross-linked products indicates that the alpha subunit is cross-linked to the 26-kDa carboxyl-terminal portion of the beta subunit. The cross-linked G protein is functional, and its alpha subunit can change conformation upon binding GTP gamma S. GTP gamma S stabilizes alpha o to digestion by trypsin (Winslow, J.W., Van Amsterdam, J.R., and Neer, E.J. (1986) J. Biol. Chem. 261, 7571-7579) and also stabilizes the alpha subunit in the cross-linked product. Cross-linked G o can be ADP-ribosylated by pertussis toxin. This ADP-ribosylation is inhibited by GTP gamma S with a concentration dependence that is indistinguishable from that of the control, uncross-linked G o. These two kinds of experiments indicate that alpha o is able to change its conformation even though it cannot separate completely from beta gamma. Thus, although dissociation of the subunits accompanies activation of G o in solution, it is not obligatory for a conformational change to occur in the alpha subunit. 相似文献
10.
The modification of the carboxyl groups of the subunits of bovine luteinizing hormone to neutral derivatives by carbodiimide-mediated coupling with glycine methyl ester has been studied. The modified alpha subunit, which has 8 residues of glycine methyl ester incorporated, will no longer recombine with native beta (hormone-specific) subunit, but the modified beta subunit, with 6 to 7 glycine methyl esters incorporated, will recombine with native alpha to yield a partially active hormone. Derivatization of the intact hormone results in dissociation to subunits together with formation of a major side product which is covalently cross-linked. Significant cross-linked product was not obtained during modification of individual subunits, thus indicating an orientation between an activated carboxyl group(s) and a nucleophile(s) in the intact hormone which favors coupling. Separation of subunits from the derivatized, noncross-linked fraction by countercurrent distribution reveals a heterogeneous preparation of the modified alpha subunit which also will not recombine with either a native or modified beta subunit. The beta subunit from the modified intact hormone was indistinguishable from the modified isolated beta subunit in amino acid composition and in ability to recombine with native alpha subunit. The results are consonant with data from this and other laboratories in which various modifications of the alpha chain, the subunit common to the glycoproteins, more seriously affect recombination than similar modifications of the beta subunits. The number of carboxyl groups modified in each subunit is compatible with but not in total agreement with assignments of amides reported from sequence studies. 相似文献
11.
Cross-linking reagents have been used to link covalently adjacent subunits of solubilized spinach chloroplast coupling factor 1, which is a latent ATPase. 1,5-Difluoro-2,4-dinitrobenzene, dimethyl-3,3'-dithiobispropionimidate, and dimethylsuberimidate are able to form bridges of 3 to 11 A between amino groups, and hydrogen peroxide and the o-phenanthroline-cupric ion complex catalyze the oxidation of intrinsic sulfhydryl groups. The five individual subunit bands (alpha, beta, gamma, delta, and epsilon) and several new aggregate bands can be separated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The same four fastest moving aggregate bands, as characterized by their mobilities, migrate more slowly than the heaviest subunit band and appear with all of the cross-linkers employed. The subunit composition of the aggregate bands has been determined through the use of the reversible cross-linkers, dimethyldithiobispropionimidate, (o-phenanthroline)2Cu(II), and H2O2, and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which aggregates are separated in the first dimension, the disulfide cross-links are cleaved, and the individual subunits present in the aggregates are separated in the second dimension. The subunits are detected by Coomassie brilliant blue staining and by labeling some of the sulfhydryl groups of the gamma and epsilon subunits with radioactive N-ethylmaleimide. The results obtained indicate that the alpha and beta subunits can cross-link directly with each of the other subunits, that two beta subunits are adjacent, and that gamma epsilon, gamma epsilon 2, alpha delta, and beta delta aggregates are present. A minimal subunit stoichiometry consistent with these results is alpha 2 beta 2 gamma delta epsilon 2. A possible structural model of the coupling factor is derived from the data. Similar, but less extensive, experiments have been carried out with the heat-activated coupling factor (which is an ATPase); no differences in the spatial arrangement of subunits are detected from the two-dimensional gel electrophoresis analysis of the cross-linked aggregates. 相似文献
12.
Chemical and immunochemical characteristics of tropomyosins from striated and smooth muscle 总被引:21,自引:2,他引:19
1. On electrophoresis in dissociating conditions the tropomyosins isolated from skeletal muscles of mammalian, avian and amphibian species migrated as two components. These were comparable with the alpha and beta subunits of tropomyosin present in rabbit skeletal muscle. 2. The alpha and beta components of all skeletal-muscle tropomyosins contained 1 and 2 residues of cysteine per 34000g respectively. 3. The ratio of the amounts of alpha and beta subunit present in skeletal muscle tropomyosins was characteristic for the muscle type. Muscle consisting of slow red fibres contained a greater proportion of beta-tropomyosin than muscles consisting predominantly of white fast fibres. 4. Mammalian and avian cardiac muscle tropomyosins consisted of alpha-tropomyosin only. 5. Mammalian and avian smooth-muscle tropomyosins differed both chemically and immunologically from striated-muscle tropomyosins. 6. Antibody raised against rabbit skeletal alpha-tropomyosin was species non-specific, reacting with all other striated muscle alpha-tropomyosin subunits tested. 7. Antibody raised against rabbit skeletal beta-tropomyosin subunit was species-specific. 相似文献
13.
The electrophoretic mobility of radioiodinated follitropin (FSH) alpha and beta subunits as well as the alpha beta dimer changed markedly depending on the concentration of reducing agents such as dithiothreitol. The changes were more dramatic in the beta subunit than in the alpha subunit. 125I-FSH, complexed to the receptor on porcine granulosa cells or in Triton X-100 extracts, was cross-linked with a cleavable (nondisulfide) homobifunctional reagent, solubilized in sodium dodecyl sulfate without reducing agents, and electrophoresed. The cross-linked sample revealed three bands of high molecular mass, in addition to the hormone subunit and dimer bands. The band of lightest mass, 110 kDa, was the major band and the other two of 76 and 62 kDa were barely noticeable. Upon reduction with dithiothreitol, the 110-kDa band decreased while the 76- and 62-kDa bands increased, indicating the existence of disulfides between components of the 110-kDa complex. Formation of the disulfide-linked complexes requires 125I-FSH, specifically bound to the hormone receptor and cross-linking, and can be prevented with an excess of native FSH but not human choriogonadotropin. Complex formation was independent of blocking free sulfhydryl groups with N-ethylmaleimide. When the cross-linked complexes were reduced in the gel matrix and analyzed on fresh gels, the 76- and 62-kDa complexes were generated from the 110-kDa band, indicating the loss of two components. The lost components were estimated to be at 14 and 34 kDa. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22-, 14-, and 34-kDa components to the FSH alpha beta dimer. The results of reduction of the cross-linked complexes demonstrate the existence of disulfide linkage between the three components. 相似文献
14.
Characterization of transducin from bovine retinal rod outer segments. The role of sulfhydryl groups 总被引:5,自引:0,他引:5
The properties and functions of the sulfhydryl groups of transducin were examined by 5,5' -dithiobis-(2-nitrobenzoic acid) titration and N-ethylmaleimide modification. The T beta gamma subunit of transducin contained a total of six free sulfhydryl groups and two were reactive under native conditions. Both reactive sulfhydryl groups were located in the beta polypeptide. The functions of transducin were not affected by the modification of these two sulfhydryl groups. The T alpha subunit of transducin contained three accessible sulfhydryl groups under both native and denaturing conditions. When 1.3 sulfhydryl groups were covalently modified by N-ethylmaleimide, the GTPase activity, the guanosine 5' -(beta, gamma-imido)triphosphate (Gpp(NH)p) uptake, and the rhodopsin-binding property of transducin were inhibited. The binding of Gpp(NH)p to T alpha blocked two of the three sulfhydryl groups from chemical modification and increased the reactivity of the remaining one. Modification of this specific sulfhydryl group of T alpha -Gpp(NH)p inhibited the exchange of the bound Gpp(NH)p for GTP. However, the modified T alpha-Gpp(NH)p was able to activate cGMP phosphodiesterase in solution and on positively charged liposomes. These findings demonstrated that a conformational change of T alpha occurs upon the binding of Gpp(NH)p and a specific sulfhydryl group of T alpha plays an important role in the activation of transducin in retinal rod outer segments. 相似文献
15.
Generation of oligomeric insulin receptor forms by intramolecular sulfhydryl-disulfide exchange. Involvement of masked sulfhydryl groups 总被引:2,自引:0,他引:2
H Kr?mer A Deger R Koch R Rapp M Hinz U Weber 《Biological chemistry Hoppe-Seyler》1987,368(5):471-479
Insulin receptors from rat liver membranes were labelled with a 125I-labelled photoreactive insulin analogue or by iodination using lactoperoxidase and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Under nonreducing conditions different receptor forms with Mr 400,000 (alpha 2 beta 2), 360,000 (alpha 2 beta beta'), 330,000 (alpha 2 beta' beta'), 320,000 (alpha 2 beta), 280,000 (alpha 2 beta'), 240,000 (alpha 2), 210,000 (alpha beta), 165,000 (alpha beta') and 115,000 (alpha) were detected. The subunit composition of these receptor forms was determined by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence and presence of dithioerythritol. During denaturation in sodium dodecyl sulfate in the absence of reductants, the Mr 400,000 receptor form (alpha 2 beta 2) was converted into the Mr 320,000 (alpha 2 beta) and Mr 240,000 (alpha 2) receptor form. This conversion was prevented either by N-ethylmaleimide, oxidants, or low pH. In contrast, alkylation of the receptor with N-ethylmaleimide under non-denaturing conditions did not prevent the appearance of intermediate-sized receptor forms. Furthermore, the inhibition of receptor cleavage by N-ethylmaleimide during denaturation was also observed when the amount of free sulfhydryl groups was reconstituted by the addition of an unlabelled and non-alkylated receptor sample to the alkylated and photoaffinity-labelled receptor. These results suggest, that the generation of different oligomeric receptor forms detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis is due at least in part to the cleavage of one or both beta-subunits from the insulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
16.
Photoaffinity labeling of the follitropin receptor 总被引:1,自引:0,他引:1
A photoactivatable derivative of human follitropin was used to identify the follitropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycine, and radioiodinated. The 125I-labeled hormone (125I-hormone) derivative associated with the same number of receptors as 125I-hormone itself, but with a slightly lower Ka, 1.12 X 10(10) M-1 compared with 1.4 X 10(10) M-1 for the 125I-hormone. The binding could be blocked with untreated hormone. Its alpha and beta subunits could be cross-linked to produce alpha beta dimer by photolysis. When the 125I-hormone derivative bound to the cells was photolyzed for crosslinking and the products resolved by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, two new bands (106 and 61 kDa) of lower electrophoretic mobility appeared in addition to the alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis, and the 125I-hormone derivative specifically bound to cells bearing the receptor. Binding could be blocked by excess untreated follitropin but not with human choriogonadotropin and thyrotropin. Under nonreducing conditions, one major band (104 kDa) of cross-linked complexes appeared. Upon reduction with dithiothreitol and second-dimensional electrophoresis, the 104-kDa band produced two smaller complexes of 75 and 61 kDa, indicating the loss of two components and the existence of intercomponent disulfides. Successful production of the 104-kDa complex requires blocking of free sulfhydryl groups with N-ethylmaleimide. It is, however, independent of various protease inhibitors or the temperature and the time period of hormone incubation with cells or the plasma membrane fraction. The mass estimates and the interaction with the hormone of the photoaffinity-labeled components are discussed. 相似文献
17.
Three cysteine-containing tryptic peptides were isolated and sequenced from mitochondrial 4-aminobutyrate aminotransferase using DABIA (4-dimethylaminoazobenzene-4-iodoacetamide) as specific labeling reagent for sulfhydryl groups. The enzyme is a dimer made up of two identical subunits, but four out of the six cysteinyl residues/dimer form disulfide bonds when treated with iodosobenzoate to yield inactive enzyme species. To identify the cysteinyl residues undergoing reversible oxidation/reduction, the S-DABIA-labeling patterns of the fully reduced (active) and fully oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced enzyme contained three labeled peptides. If the enzyme was treated with iodosobenzoate prior to reaction with DABIA and tryptic digestion, only one labeled peptide was detected and identified (peptide I), indicating that the two missing cysteinyl-containing peptides (peptides II, III) have been oxidized. The sulfhydryl groups undergoing oxidation/reduction were found to be intersubunit, based on SDS/polyacrylamide gel electrophoresis results. The loss of catalytic activity of 4-aminobutyrate aminotransferase by oxidation of sulfhydryl residues is related to constraints imposed at the subunit interface by the insertion of disulfide bonds. 相似文献
18.
The kinetics for electron transfer have been measured for samples of hemoglobin valency hybrids with initially one type of subunit, alpha or beta, in the oxidized state. Incubation of these samples under anaerobic conditions tends to randomize the type of subunit that is oxidized. With a time coefficient of a few hours at pH 7, 25 degrees C, the Hb solution (0.1 mm heme) approaches a form with about 60% of beta chains reduced, indicating a faster transfer rate in the direction alpha to beta. There was no observable electron transfer for samples saturated with oxygen. The electron transfer occurs predominantly between deoxy and aquo-met subunits, both high spin species. Furthermore, electron transfer does not depend on the quaternary state of hemoglobin. Incubation of oxidized cross-linked tetramer Hb A with deoxy Hb S also displayed electron transfer, implying a mechanism via inter-tetramer collisions. A dependence on the overall Hb concentration confirms this mechanism, although a small contribution of transfer between subunits of the same tetramer cannot be ruled out. These results suggest that in vivo collisions between the Hb tetramers will be involved in the relative distribution of the methemoglobin between subunits in association with the reductase system present in the erythrocyte. 相似文献
19.
DNA binding activity of chicken progesterone receptor B form (PRB) and A form (PRA) has been examined. This activity is strongly dependent upon the presence of thiols in the buffer. Stability studies showed that PRB was more sensitive to oxidation than was PRA. Receptor preparations were fractionated by DNA-cellulose chromatography to DNA-positive and DNA-negative subpopulations, and sulfhydryl groups were quantified on immunopurified receptor by labeling with [3H]-N-ethylmaleimide. Labeling of DNA-negative receptors with [3H]-N-ethylmaleimide showed 21-23 sulfhydryl groups on either PRA or PRB form when the proteins were reduced and denatured. A similar number was seen without reduction if denatured DNA-positive receptor species were tested. In contrast, the DNA-negative PRB had only 10-12 sulfhydryl groups detectable without reduction. A similar number (12-13 sulfhydryl groups) was found for PRA species that lost DNA binding activity after exposure to a nonreducing environment in vitro. We concluded that the naturally occurring receptor forms unable to bind to DNA, as well as receptor forms that have lost DNA binding activity due to exposure to a nonreducing environment in vitro, contain 10-12 oxidized cysteine residues, likely present as disulfide bonds. Since we were unable to reduce the disulfide bonds when the native DNA-negative receptor proteins were treated with dithiothreitol (DTT), we speculate that irreversible loss of DNA binding activity of receptor in vitro is due to oxidation of cysteine residues that are not accessible to DTT in the native state. 相似文献
20.
Hemoglobin A, cross-linked between Lys 99 alpha 1 and Lys 99 alpha 2, was used to obtain a partially oxidized tetramer in which only one of the four hemes remains reduced. Because of the absence of dimerization, asymmetric, partially oxidized derivatives are stable. This is evidenced by the fact that eight of the ten possible oxidation states could be resolved by analytical isoelectric focusing. A triply oxidized hemoglobin population HbXL+3 was isolated whose predominant component was (alpha + alpha +, beta + beta 0). This triferric preparation was examined as a possible model for the triliganded state of ferrous HbA. The aquomet and cyanomet derivatives were characterized by their CD spectra and their kinetic reactions with carbon monoxide. CD spectra in the region of 287 nm showed no apparent change in quaternary structure upon binding ligand to the fourth, ferrous heme. The spectra of the oxy and deoxy forms of the cyanomet and aquomet derivatives of HbXL+3 differed insignificantly and were characteristic of the normal liganded state. Upon addition of inositol hexaphosphate (IHP), both the oxy and deoxy derivatives of the high-spin triaquomet species converted to the native deoxy conformation. In contrast, IHP had no such effect on the conformation of the low-spin cyanomet derivatives of HbXL+3. The kinetics of CO combination as measured by stopped-flow and flash photolysis techniques present a more complex picture. In the presence of IHP the triaquomet derivative does bind CO with rate constants indicative of the T state whether these are measured by the stopped-flow technique or by flash photolysis.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献