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1.
The rates of dissociation and recombination of the subunits of human luteinizing hormone have been measured under a variety of conditions by exploiting the fact that the fluorescence probe 1,8-anilinonaphthalene sulfonate binds to the native hormone but not to its subunits. The first-order dissociation rate is strongly dependent on temperature, pH, and urea concentration. Similarly, the rate of recombination depends on pH and temperature and is independent of subunit concentration in the range from 10 to 40 μm. At 37 °C, the subunits dissociate below pH 4 and recombine above pH 5. The results presented herein should be useful for developing improved methods of purifying human luteinizing hormone and its subunits.  相似文献   

2.
A large change in quantum yield of the fluorescent probe 1,8-anilinonaphthalene sulfonate is produced when it combines with the glycoprotein hormone, human chorionic gonadotropin. A method of analyzing for the hormone in the presence of its subunits has been developed based on the finding that the subunits have no effect on 1,8-anilinonaphthalene sulfonate fluorescence. Quantitative rates of dissociation and recombination can be obtained with very small concentrations of hormone since fluorescence measurements are fast and sensitive. The effects of temperature, pH, and urea concentration on the rate of human chorionic gonadotropin dissociation have been measured. The rates of recombination of subunits have been studied as a function of temperature, pH, and KCl concentration. Human chorionic gonadotropin is stable in water to pH 12 and pH 4.5 at 37 °C.  相似文献   

3.
The fluorescence of 1,8-anilinonaphthalene sulfonate (ANS) was enhanced in the presence of ovine lutropin (oLH). Fluorescence titration curves were sigmoidal with 50% saturation between 200 and 500 μm. Exclusion chromatography experiments indicated that the hormone self-associates to form dimers in the presence of excess ANS. By contrast, the isolated a and β subunits of oLH caused a much smaller enhancement of the fluorescence of ANS and did not self-associate in its presence. Dissociation of the intact hormone into its subunits was accompanied by 1) a loss in the ability to enhance ANS fluorescence, 2) the appearance of a negative differential absorption spectrum whose magnitude indicated the increased solvent-exposure of at least two tyrosines, and 3) a loss in conformational rigidity as evidenced by a decrease in polarization (P) of tyrosyl fluorescence from ~0.17 to ~0.13. Similar rates of dissociation were obtained by all three measurements and the first order rate constant at pH 3.6 and 37 °C under conditions of low ionic strength was k = 0.18 min?1; at high ionic strength, e.g., 0.5 m KC1, dissociation was incomplete even after prolonged incubation. Acid-dissociated subunits recombined readily in 0.5 m acetate buffer, pH 5.3, and the recovery of the intrinsic absorption and fluorescence properties as well as the ability to enhance ANS fluorescence ranged between 70 and 90%. Titration of the isolated α and β subunits with acid or GdmCl had little or no effect on P, suggesting that residual secondary or tertiary structure is either absent, very stable, or its disruption does not alter the rigidity of the tyrosyl environment. The relatively high P for oLH-β (0.17) suggests a conformation which is rigid compared with oLH-α (0.13). P for both subunits decreased smoothly with increasing temperature between 20 and 70 °C. By contrast, oLH exhibited a thermal transition near 50 °C characterized by a drop in P from a value near that of β to a value near that of a as the subunits dissociated. Because α has more tyrosines with a higher average quantum yield, its fluorescence would be expected to dominate that of the hormone or of an equimolar mixture of subunits. Thus, most of the conformation changes which accompany dissociation and recombination appear to occur in the α subunit.  相似文献   

4.
J C Lee  L C Yeh  P M Horowitz 《Biochimie》1991,73(9):1245-1247
Binding studies of yeast 40S ribosome with bis (1,8-anilinonaphthalenesulfonate) (bis-ANS) revealed the binding of 3-4 molecules of bis-ANS per ribosome with a dissociation constant (Kd) of 1.45 microM. Binding of AUG to the 40S subunits resulted in a concentration-dependent decrease in the bis-ANS fluorescence without displacing all of the bound bis-ANS from the ribosomes. The residual bis-ANS fluorescence at saturation with AUG corresponds to about 3 molecules of bis-ANS per ribosome. Thus AUG displaces one of the bound bis-ANS molecules. The data suggest that AUG binds at a hydrophobic site on the yeast 40S subunit.  相似文献   

5.
The rate of formation of human chorionic gonadotropin from its alpha and beta subunits has been measured at neutral pH and 37 degrees C as a function of subunit concentration, using the fluorescence probe, 1,8-anilinoaphthalene-sulfonate (ANS), to monitor the reaction. The subunits were prepared by acid dissociation of the intact hormone (pH less than or equal to 2, 37 degrees C). Following neutralization, the rate of appearance of ANS fluorescence was identical with the rate of recovery of receptor binding activity and both of these properties were completely recovered. Kinetic data obtained over a 100-fold range of subunit concentrations (1.5 to 146 muM) were not compatible with a simple second-order reaction scheme, but required at least one additional step. The data were best fit by a model in which the subunits reversibly form an intermediate complex (alpha + beta in equilibrium alphabeta) which then undergoes a conformational rearrangement to form the native structure (alphabeta leads to H). Ultraviolet difference absorption measurements suggest that most of the change in the environment of the tyrosyl residues occurs during this second step.  相似文献   

6.
The α and β subunits of highly potent ovine follitropin have been isolated by dissociation in 8 m urea, pH 7.5, and chromatography on DEAE-Sephadex A25. The isolated subunits display microheterogeneity on polyacrylamide gel electrophoresis and have very low activity in follitropin-specific radioreceptor and radioimmunoassays. The tryptophan fluorescence spectra of native follitropin and the isolated β subunit are different. The recombinant of follitropin α + β subunit had the same activity as the native hormone in the radioimmunoassay, but its activity in the radioreceptor and in vivo bioassay was about 65% of the intact hormone. Substitution of the follitropin α by ovine lutropin α subunit (prepared by a method not involving urea) to form the recombinant restored full activity in all the three assays investigated. The formation of recombined hormone proceeds at a rapid rate and is almost complete by 6 h. The α and β subunits of ovine follitropin differ from each other in amino acid composition. No significant differences were apparent in their carbohydrate composition. The amino acid composition of the ovine follitropin α and lutropin α subunits are very similar. The oxidized α subunit has phenylalanine at its NH2-terminus while aspartic acid is present at this position in the oxidized β subunit.  相似文献   

7.
The molecular behavior of hog thyroglobulin in acid solutions has been examined by fluorescence, absorption, proton binding, and velocity centrifugation. The precipitation of thyroglobulin which normally occurs in the pH region near its isoelectric point was prevented by reducing the salt concentration to 0.01 m and the protein concentrations to the lowest levels needed for measurements. The rate of denaturation is very slow near pH 5.0 but increases rapidly with decreasing pH. The molecular properties of acid-denatured thyroglobulin, though still retaining some aspects of its native tertiary structure, are quite different from those of the native protein. In the molecular transition: (a) buried tryptophanyl residues become exposed, (b) the anomalous dissociation of certain groups becomes normalized, (c) subunits are formed, and (d) the molecular form of thyroglobulin and its subunits becomes partially unfolded.  相似文献   

8.
Exposure of aqueous solutions of native human choriogonadotropin (hCG), asialo-hCG (A-hCG), and chemically deglycosylated hCG (DG-hCG) to heat treatment revealed significant differences in their stability. Solutions of hCG and A-hCG were rapidly inactivated above 50 degrees C. On the other hand, solutions of DG-hCG were comparatively more stable under similar conditions as shown by the retention of significant receptor binding, immunological, and hormonal antagonistic activities. Heated solutions (100 degrees C) of hCG and A-hCG quickly lost their ability to enhance the fluorescence of the probe 1-anilino-8-naphthalenesulfonate (1,8-ANS) indicating dissociation into subunits. DG-hCG solutions were more stable in this respect suggesting significant preservation of conformational features required for the interaction with 1,8-ANS. Solutions of hCG and A-hCG which had been thermally denatured (100 degrees C, 10 min) required almost 48 h at 37 degrees C to regain complete ANS binding ability as well as receptor binding activity. Under the same conditions, heated solutions of DG-hCG completely regained these abilities in less than 2 h. A similar pattern was observed with acid (pH 2.0)-dissociated hCG, A-hCG, and DG-hCG. While heated solutions of hCG had no effect on the action of native hCG in vitro, heated DG-hCG solutions still retained their ability to antagonize the cyclic AMP accumulation or steroidogenesis induced by native hCG in rat interstitial cells. Thus, removal of carbohydrate residues (approximately 75% loss) from hCG renders the hormone more resistant to thermal denaturation.  相似文献   

9.
The molecular properties of the salt-induced partly folded acidic state of apomyoglobin as well as myoglobin were investigated by fluorescence and circular dichroism of the extrinsic fluorophore 1,8-anilinonaphthalenesulfonate. The occurrence of a fluctuating tertiary structure ("molten globule") at acidic pH in the presence of salt was suggested by the disappearance of the dichroic activity of the fluorophore bound to the partly folded protein. Moreover, the structure of the intermediate is not influenced by the presence of heme, thus suggesting that heme is not crucial in the early stage of myoglobin folding.  相似文献   

10.
The stimulation of cyclic AMP accumulation and testosterone synthesis in isolated rat Leydig cells by ovine and bovine ICSH, their subunits prepared by a new, mild procedure (dissociation of subunits at pH 3 and salt fractionation) and the recombined hormones have been studied. Whereas the isolated subunits exhibit less than 0.2% of the potency of the native hormones, recombination of the subunits results in full restoration of the biological activity. In contrast to this, recombination of the subunits prepared by a countercurrent distribution method resulted in only partial regeneration of the biological activity. The ovine hormone was found to be twice as active as the bovine ICSH. Both heterologous hybrids of the subunits of the ovine and bovine hormones were significantly more active than the bovine hormone. The utility of the isolated rat Leydig cell system as a rapid, sensitive bioassay for ICSH is also discussed.  相似文献   

11.
An improved procedure is described for the isolation of the flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida as well as methods for the separation of its subunits in native form and their recombination to reconstitute the original flavocytochrome. Under appropriate conditions, the reconstitution is stoichiometric and results in complete recovery of the catalytic activity of the flavocytochrome. The separated flavoprotein subunit shows only 2% of the catalytic activity of the original enzyme on p-cresol and is characterized by converging lines in bisubstrate kinetic analysis, while the intact and reconstituted enzymes show parallel line kinetics in steady-state experiments. van't Hoff plots of the dependence of the dissociation constant of the subunits of PCMH on temperature show a break near 15 degrees C. Above this temperature, KD is characterized by a positive delta H value of 12.6 kcal mol-1; below 15 degrees C, the dissociation is essentially temperature independent. The subunit dissociation is strongly dependent on ionic strength in the oxidized form of PCMH but not in the reduced form of the enzyme. Reduction also lowers the KD significantly, while substrates and nonoxidizable competitive inhibitors lower the dissociation constant even further, suggesting a conformation change. Combination of the subunits to form PCMH entails a small but measurable change in the absorption spectra of the component proteins.  相似文献   

12.
Wu Y  Ji SR  Wang HW  Sui SF 《Biochemistry. Biokhimii?a》2002,67(12):1377-1382
C-Reactive protein (CRP) is composed of five identical noncovalently linked monomers and characterized as an important acute-phase protein. The CRP subunit obtained by denaturing treatments, which is termed modified CRP, has also been widely studied. In the current work, we found that there exists some degree of natural dissociation of CRP in stock solution. This dissociation is critically dependent on the absence of Ca2+. Low pH could enhance the dissociation of CRP, while ionic strength has little effect. Anilinonaphthalenesulfonate (ANS) fluorescence detections indicate that the exposure of hydrophobic surface increases during the dissociation. Acidic pH conditions also induce an increase in ANS fluorescence. This suggests that hydrophobic interactions between CRP subunits may contribute to the study of its pentameric structure. Surface plasmon resonance experiments indicate that monomeric CRP does not specifically bind to phosphatidylcholine-containing membrane as native CRP does. Electron microscopy shows that monomeric CRP binds to negatively charged lipid through electrostatic forces, and such lipid may induce the dissociation of CRP due to the acidic pH in the diffuse double layer near the membrane.  相似文献   

13.
The alpha and beta subunits of human follitropin were isolated in a high state of purity. The tryptophan fluorescence of the native hormone and the isolated beta subunit are different. The N-terminus of the alpha and beta subunits was identified as valine and aspartic acid respectively. While recombination of the isolated alpha and beta subunits restores the electrophoretic mobility of the intact hormone, its receptor binding activity cannot be fully regenerated. Substitution of the human follitropin alpha by an ovine lutropin alpha subunit, to form a recombinant with the follitropin beta subunit, generates a complex with 2-3 receptor binding activity of the native human follitropin and the same activity as ovine follitropin. Acylation of the intact hormone does not disrupt the quaternary structure but leads to complete inactivation. Acylation studies with the subunits suggests the crucial role of the epsilon-amino groups of the alpha subunit in determining biological activity.  相似文献   

14.
Two related compounds, 1,8-anilinonaphthalenesulfonate (1,8-ANS) and bis(1,8-anilinonaphthalenesulfonate) (Bis-ANS), are useful fluorescent probes for hydrophobic areas on protein molecules. Using fluorescence, we examined the binding of these compounds to bovine brain tubulin and found that Bis-ANS and 1,8-ANS bound to tubulin with Ki values of 2 and 25 microM, respectively. Bis-ANS potently inhibited the polymerization of tubulin into microtubules in vitro. In the presence of microtubule-associated protein 2, half-maximal inhibition of assembly was obtained at 3 microM Bis-ANS. In the presence of tau protein, half-maximal inhibition was obtained at 15 microM Bis-ANS. Surprisingly, 1,8-ANS, even at 200 microM, did not inhibit assembly. Scatchard analysis indicated one binding site for Bis-ANS on tubulin. Previous reports of 1,8-ANS binding to tubulin may have been influenced by the presence of Bis-ANS which until recently was a common contaminant of commercial supplies. Because of its intense fluorescence in addition to its potent inhibitory effects, Bis-ANS appears to be a useful probe to study microtubule assembly and other interactions involving tubulin.  相似文献   

15.
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   

16.
The association and dissociation rates of pituitary porcine luteinizing hormone (pLH) and equine LH (eLH) at oxidizing potential were slow and those of equine choriogonadotropin (eCG) were even much slower. At reducing potential mimicking endoplasmic reticulum condition, association of pLH subunits was observed in less than 5 min instead of 24 h at oxidizing potential. At neutral pH and 37 degrees C, DTNB and 2-nitro-5-thiocyanobenzoic acid (NTCB) were found to react with two cysteine residues (i.e., one S-S bridge) in pLH. The temperature dependence of the NTCB reaction on pLH was found to be similar to that of the dissociation of the hormone (Tm approximately 75 degrees C). The tight correlation between the reaction of two cysteines and dissociation of the subunits of pLH and eLH strongly suggests that transient opening of one fragile disulfide bridge is required for heterodimer assembly. Moreover, the absence of cysteine reaction with eCG indicates that its bulky carbohydrate chains exert a negative influence on the opening of this bridge leading to considerably diminished association-dissociation rates of its subunits.  相似文献   

17.
The dissociation of pyruvate oxidase (PO) caused by pressure up to 220 MPa at various conditions was explored by measuring the intrinsic fluorescence spectra and polarization. At 5 degrees C and pH 7.6 the standard volume change (deltaV0) and free energy upon dissociation of the enzyme is -220 ml/mol and 29.83 kCal/mol, respectively. It was found that FAD was irreversibly removed during the pressure-dissociation of the enzyme. A much smaller standard volume change (-153 ml/mol) and lower free energy (24.92 kCal/mol) of apo-pyruvate oxidase (apo-PO) compared with the native enzyme indicated that FAD played very important role in stabilizing the enzyme and significantly influenced the standard volume change. The substrate pyruvic acid can significantly stabilize the enzyme against pressure in spite the standard volume for the enzyme in this case has a big increase relative to the native enzyme. The comparison of the intrinsic fluorescence of the native and the activated enzyme obtained by limited proteolysis indicated that the physical separation of alpha-peptide from the enzyme only occurred when the subunits were dissociated from each other under pressure.  相似文献   

18.
A strongly fluorescent 5-dimethylamino-1-naphthalene sulfonate (dansyl) derivative of bovine thyrotropin has been prepared. The dye-conjugated hormone is bioactive and shares, essentially unchanged, the membrane binding and adenylate cyclase stimulatory activities of the native hormone. Binding of 125I-labeled dansyl-thyrotropin to thyroid plasma membranes is sensitive to inhibition by gangliosides and, as is the case for the binding of 125I-thyrotropin, galactosyl-N-acetylgalactosaminyl[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GDIb) is the most potent binding inhibitor. Gangliosides interact with dansyl-thyrotropin, causing a large increase of the quantum yield and a 5- to 10-nm blue shift of the emission maximum of the hormone-bound naphthalene chromophore; gangliosides cause no change in the fluorescent properties of the free dye. The fluorescence enhancement caused by gangliosides can be specifically reversed by unlabeled thyrotropin. The effect of gangliosides on dansyl-thyrotropin fluorescence is strongly salt-dependent; salts cannot, however, reverse the formation of the dansyl-thyrotropin.ganglioside complex once it has formed. The salt data suggest that the association of the ganglioside with dansyl-thyrotropin is dominated by electrostatic interactions, but that salt-independent, short range interactions, most likely hydrophobic, dominate the dissociation of the dansyl-thyrotropin-ganglioside adduct. Sucrose gradient centrifugation, ultracentrifugation, and fluorescence polarization data indicate that the gangliosides are micellar in nature under the conditions of these experiments. Acid titration of dansyl-thyrotropin causes a marked quenching of dansyl fluorescence which in part reflects dissociation of the hormone into its constituent alpha and beta subunits. In the presence of GDIb, but not N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GDIa), pH-dependent quenching and subunit dissociation are essentially eliminated. Circular dichroism results and fluorescence polarization studies support the interpretation that the ganglioside interaction causes a conformational change in the thyrotropin molecule. The acid titration data together with differences in the ability of gangliosides to influence the tyrosine fluorescence of the thyrotropin molecule indicate that different gangliosides induce different conformational perturbations in the thyrotropin molecule.  相似文献   

19.
The subunits of ovine lutropin prepared by acid dissociation and salt precipitation were characterized by end group analysis, tryptic peptide mapping, SDS gel electrophoresis and biological activity. No evidence of internal peptide cleavage was found in the alpha subunit. The subunits possessed low activity. The alpha and beta subunits recombined effectively to generate a complex that had full receptor binding activity and in vitro biological activity. The recombinants of subunits prepared by countercurrent distribution showed only 50% activity in both assays. The salt precipitation method alpha subunit could be completely reduced and reoxidized in the absence of denaturants. The reoxidized alpha subunit combines with the native beta subunit generating full activity. However, this recombined hormone tends to lose activity with time, suggesting that the reoxidation may not fully restore the native structur of the reduced alpha subunit. The native lutropin alpha subunit effectively combined with follitropin beta subunit generating complete follitropin activity.  相似文献   

20.
In this paper, the influence of Ca2+ on the adsorption properties of 1,8-anilinonaphthalenesulfonate (ANS) and analogous probes to sonicated vesicles of phosphatidylcholine was studied by means of spectrofluorometry. The fluorescence of ANS added to the vesicle dispersion increases with the Ca2+ concentration in the inner media but remains constant when Ca2+ concentration is changed in the outside solution. However, the fluorescence decreases when large anions such as ClO4- are present in the external solution. Ca2+ inside large liposomes promotes a similar behaviour to that found in sonicated vesicles when they are osmotically contracted in hypertonic media. The results can be interpreted in terms of Ca2+ adsorption on the inner interface and a cooperative interaction between the monolayers.  相似文献   

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