共查询到20条相似文献,搜索用时 15 毫秒
1.
The luteinizing hormone isolated from sperm-whale pituitary was separated into two subunits, alpha- and beta-, by ion-exchange chromatography on sulfoethyl-Sephadex. The hormone subunits were reconstituted, carboxymethylated and cleaved by BrCN and proteolytic enzymes. In order to block tryptic hydrolysis at lysine residues the alpha-subunit was subjected to maleylation. Large-sized fragments of BrCN were cleaved by chymotrypsin and trypsin, while large-sized fragments of trypsin were split by chymotrypsin. The resulting peptides were separated by gel filtration on Sephadex, ion-exchange chromatography on Aminex A-5 and thin-layer partition chromatography on cellulose. The amino acid sequence of the peptides was determined by the Edman method, using identification of the N-terminal amino acids in a reaction with dansyl chloride or dimethylaminoazobenzene-4-isothiocyanate. It was shown that the alpha-subunit of the luteinizing hormone is a peptide chain consisting of 96 amino acid residues with covalently linked carbon chains at asparagine residues at positions 56 and 82. The N-terminal amino acid of the alpha-subunit is phenylalanine, the C-terminal amino acid is serine. The alpha-subunit is heterogeneous at the N-end, i. e. beside phenylalanine it contains threonine and trace amounts of proline, aspartate, glutamate and glycine. 相似文献
2.
Bernard MP Lin W Cao D Myers RV Xing Y Moyle WR 《The Journal of biological chemistry》2004,279(43):44438-44441
Twenty residues of the human choriogonadotropin (hCG) beta-subunit that are wrapped around alpha-subunit loop 2 like a "seatbelt" stabilize the heterodimer and enable the hormone to distinguish lutropin (LHR), follitropin, and thyrotropin receptors. The N-terminal portion of the seatbelt contains a small disulfide-stabilized loop needed for heterodimer assembly and is thought to mediate hCG-LHR interactions. To test the latter notion, we compared the LHR binding and signal transduction activities of hCG analogs in which the alpha-subunit C terminus (alphaCT) was cross-linked to residues in the small seatbelt loop. Analogs having an intersubunit disulfide between a cysteine in place of alphaCT residue alphaSer-92 and cysteines substituted for loop residues betaArg-94, betaArg-95, or betaSer-96 had high activities in LHR binding and signaling assays despite the fact that both portions of the hormone are thought to be essential for hCG activity. Use of a larger probe blocked hormone activity when the alphaCT was cross-linked to cysteines in place of residues betaArg-95 and betaAsp-99, but not to cysteines in place of residues betaArg-94, betaSer-96, or betaThr-97. This suggested that the side chains of residues betaArg-95 and betaAsp-99, which face in the same outward direction from the heterodimer, are nearer than the others to the LHR interface. The finding that residue 95 can be cross-linked to small alphaCT probes without eliminating hormone activity indicates its side chain does not participate in essential LHR contacts. We suggest that contacts between the small seatbelt loop and the LHR, if any, involve its backbone atoms and possibly the side chain of residue betaAsp-99. 相似文献
3.
Inhibition of human choriotropin binding to receptor by human choriotropin alpha peptides. A comprehensive synthetic approach 总被引:2,自引:0,他引:2
M C Charlesworth D J McCormick B Madden R J Ryan 《The Journal of biological chemistry》1987,262(28):13409-13416
Synthetic overlapping peptides of the alpha-subunit of human chorionic gonadotropin (hCG) were made by solid-phase peptide synthesis employing a comprehensive synthetic approach. The entire primary structure of the alpha-subunit was synthesized as a series of nine consecutive peptides, each 15 residues in length, and overlapping with its two adjacent neighbors by 5 residues on each side. Receptor binding activity of each synthetic peptide was measured by the inhibition of binding of 125I-labeled hCG to rat ovarian receptor. Peptides alpha 21-35, alpha 31-45, alpha 71-85, and alpha 81-92 were shown to compete for binding with native hCG, thus demonstrating that at least two regions on the alpha-subunit may be part of the binding site(s) of the hormone. The low affinity of the peptides (10(-5)-10(-6) M) compared to native hormone (10(-10) M) for receptor is not unexpected due to the probability of discontinuous and multiple sites involved in receptor binding. An ultrapure preparation of hCG alpha-subunit also had low affinity (10(-5), suggesting that conformational changes upon combination with beta-subunit to form dimer or changes in conformation after binding are necessary for high affinity interaction. These results correlate with previous predictions of binding sites based on studies employing chemical and enzymatic modifications of intact hormone and show that synthetic peptide strategies are helpful in the elucidation of protein structure and function. 相似文献
4.
Xing Y Lin W Jiang M Cao D Myers RV Bernard MP Moyle WR 《The Journal of biological chemistry》2004,279(43):44427-44437
Efforts to identify the manner in which human choriogonadotropin (hCG) contacts lutropin receptors (LHR) have been stymied by the complex structure of the hormone and the likelihood that it contacts the receptor at multiple sites. During studies of hCG assembly in mammalian cells, we found that addition of a cysteine to the long disordered beta-subunit COOH terminus (betaCT) enabled it to become cross-linked by a disulfide to cysteines that are substituted for residues in loop alpha2 or in the alpha-subunit COOH terminus (alphaCT). This created a "knob" on the alpha-subunit at the location of the cysteine. Knobs of various sizes and charges were useful for probing surfaces of the alpha-subunit thought previously to contact the LHR. Attachment of the betaCT to residues in loop alpha2 facing loops beta1 and beta3 reduced hormone activity only a few fold revealing that this surface does not participate in essential high affinity receptor contacts, a finding inconsistent with our earlier view of the hCG-LHR complex. In contrast, this approach showed that the opposite surface of loop alpha2 appeared to be nearer the receptor interface. Although attachment of knobs to portions of the alphaCT reduced hormone activity substantially, this finding was difficult to interpret. As discussed, this procedure should be adapted readily to other proteins and may facilitate the introduction of fluorophores, enzymes, or other reagents at specific sites on protein surfaces. It may also permit one to cross-link proteins or to obscure specific protein surfaces during the development of "Trojan Horse" therapeutics. 相似文献
5.
Xing Y Lin W Jiang M Myers RV Cao D Bernard MP Moyle WR 《The Journal of biological chemistry》2001,276(50):46953-46960
Most heterodimeric proteins are stabilized by intersubunit contacts or disulfide bonds. In contrast, human chorionic gonadotropin (hCG) and other glycoprotein hormones are secured by a strand of their beta-subunits that is wrapped around alpha-subunit loop 2 "like a seatbelt." During studies of hCG synthesis in COS-7 cells, we found that, when the seatbelt was prevented from forming the disulfide that normally "latches" it to the beta-subunit, its carboxyl-terminal end can "scan" the surface of the heterodimer and become latched by a disulfide to cysteines substituted for residues in the alpha-subunit. Analogs in which the seatbelt was latched to residues 35, 37, 41-43, and 56 of alpha-subunit loop 2 had similar lutropin activities to those of hCG; that in which it was latched to residue 92 at the carboxyl terminus had 10-20% the activity of hCG. Attachment of the seatbelt to alpha-subunit residues 45-51, 86, 88, 90, and 91 reduced lutropin activity substantially. These findings show that the heterodimer can form before the beta-subunit has folded completely and support the notions that the carboxyl-terminal end of the seatbelt, portions of alpha-subunit loop 2, and the end of the alpha-subunit carboxyl terminus do not participate in lutropin receptor interactions. They suggest also that several different architectures could have been sampled without disrupting hormone activity as the glycoprotein hormones diverged from other cysteine knot proteins. 相似文献
6.
Recombination products composed of the native beta-subunit and an alpha-subunit with an enzymatically shortened C-terminal region showed a diminished (less than 5 amino acids removed) or - in the case of des-(88-92)-alpha/native beta - a completely abolished ability to bind to testicular LH/hCG receptors of the rat. An antigenic determinant which is present in native hCG but not in the isolated subunits was not or incompletely expressed in the modified hormone species. Antigenic determinants which are characteristic for the isolated alpha-subunit, however, were not affected by removal of the C-terminal residues 88-92. The immunologic experiments indicate that hCG containing an alpha-subunit with a shortened C-terminal region differs from native hCG in its conformation. These conformational changes are probably responsible for the loss in receptor-binding ability. 相似文献
7.
The residues 90-92 can be split off from the C-terminal region of the isolated alpha-subunit of choriogonadotropin (residues 88--92: -Tyr-Tyr-His-Lys-Ser-OH) by means of serine carboxypeptidase (des-Lys91,Ser92-alpha-subunit; des-(90-92)-alpha-subunit). However, when choriogonadotropin is digested by serine carboxypeptidase, only the residues 143-145 (-Leu-Pro-Gln-OH) form the C-terminus of the beta-subunit are released (des-(143-145)-choriogonadotropin). Depending on the pH conditions, glutamine 145 and the residues 143-145, respectively, are liberated by digestion of the isolated beta-subunit (des-Gln145-beta-subunit and des-(143-145)-beta-subunit, respectively). The present study provides evidence that the C-termini of both the isolated subunits and those in choriogonadotropin are probably arranged on the surface of the molecules. The biological activity of des-(143-145)-choriogonadotropin is not significantly decreased. The immunological activity, however, is reduced when measured by complement fixation. In comparison to the native hormone, a four-fold amount of des-(143-145)-choriogonadotropin has to be applied to obtain highest complement fixation. The conformation of des-(143-145)-choriogonadotropin does not seem to differ from that of the native hormone, when estimated both by CD measurements and by Ans-choriogonadotropin fluorescence. The respective determinant therefore seems to depend, at least to some extent, on the sequence of the C-terminal region of the beta-subunit of the hormone; complement fixation, however, does not seem to be affected significantly, when the des-(143-145)-beta-subunit is compared with the native beta-subunit using an antiserum against the native beta-subunit. This provides evidence that this C-terminal determinant is possibly more immunogenic at the hormone than at the isolated beta-subunit. The biological activity of recombined choriogonadotropin in vivo as well as in vitro is markedly reduced when serine 92 is removed from the C-terminus of the alpha-subunit (des-Ser92,Lys91-alpha-native beta-subunit: 36% residual activity in vivo). Biological activity is lost when the residues 88-90 are removed by digestion of the des-Ser92,Lys91-alpha-subunit with carboxypeptidase A. Recombination products between a modified alpha-and the native beta-subunit show a reduced Anschoriogonadotropin fluorescence (des-Lys91,-Ser92-alpha + native beta-subunit: 52%; des-(88-92)-alpha- + native beta-subunit: 23%). The Ans-induced aggregation of choriogonadotropin, however, also takes place in those recombination products which display a low Ans-choriogonadotropin fluorescence, indicating that the reduction is probably not caused by a portion of the molecules losing their binding sites for Ans. Therefore the diminished Ans-choriogonadotropin fluorescence seems to signal small conformational changes. The CD spectra of the native and the des-(90-92)-alpha-subunit, however, seem not to differ significantly. It is shown that the release of amino acids from the C-terminus of the alpha-subunit causes a disturbance of the interaction between the subunits. This seems to prevent an effective conformational change of the beta-subunit which probably is a prerequisite for the binding of the hormone to the receptors of Leydig cells. 相似文献
8.
Localization of the insulin-binding site to the cysteine-rich region of the insulin receptor alpha-subunit 总被引:9,自引:0,他引:9
C C Yip H Hsu R G Patel D M Hawley B A Maddux I D Goldfine 《Biochemical and biophysical research communications》1988,157(1):321-329
Affinity-purified insulin receptor was photoaffinity labeled with a cleavable radioactive insulin photoprobe. Exhaustive digestion of the labeled alpha-subunit with endoproteinase Glu-C produced a major radioactive fragment of 23 kDa as a part of the putative insulin-binding domain. This fragment could contain either residues 205-316 or 518-633 of the alpha-subunit. Rat hepatoma cells and Chinese hamster ovary cells were transfected with cDNA encoding a human insulin receptor mutant with a deletion of the cysteine-rich region spanning amino acid residues 124-319. Insulin binding by these cells was not increased in spite of high numbers of the mutant insulin receptors being expressed. A panel of monoclonal antibodies which was specific for the receptor alpha-subunit and inhibited insulin binding immunoprecipitated the photolabeled 23-kDa receptor fragment but not the receptor mutant. A synthetic peptide containing residues 243-251 was specifically bound by agarose-insulin beads. We therefore suggest that the 23-kDa fragment contains residues 205-316, and that insulin binding occurs, in part, in the cysteine-rich region of the alpha-subunit. 相似文献
9.
M Fabry E Schaefer L Ellis E Kojro F Fahrenholz D Brandenburg 《The Journal of biological chemistry》1992,267(13):8950-8956
We have used a preparation of soluble human insulin receptor ectodomain and a novel photoreactive, biotinylated derivative of insulin (4-azidosalicyloyl(B1-biocytinyl-B2-lysine)-insulin) to identify a new hormone contact site within the extracellular domain of the insulin receptor. The ectodomain was photoaffinity-labeled and digested to completion with trypsin, and the resulting tryptic fragment was purified by either HPLC or by streptavidin-affinity chromatography. The amino terminus of the fragment was identified as Gly390 within the alpha-subunit. These results suggest that residues that are carboxyl-terminal to the cysteine-rich domain, in addition to previously identified regions within the amino terminus of the alpha-subunit, contribute to the insulin binding site. The implications of these results for the de novo folding of the insulin receptor to constitute the hormone binding site are discussed. 相似文献
10.
Kimura T Tabuchi Y Takeguchi N Asano S 《The Journal of biological chemistry》2002,277(23):20671-20677
The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the non-catalytic beta-subunit. Correct assembly between the alpha- and beta-subunits is essential for the functional expression of H(+),K(+)-ATPase. The beta-subunit contains nine conserved cysteine residues; two are in the cytoplasmic domain, one in the transmembrane domain, and six in the ectodomain. The six cysteine residues in the ectodomain form three disulfide bonds. In this study, we replaced each of the cysteine residues of the beta-subunit with serine individually and in several combinations. The mutant beta-subunits were co-expressed with the alpha-subunit in human embryonic kidney 293 cells, and the role of each cysteine residue or disulfide bond in the alpha/beta assembly, stability, and cell surface delivery of the alpha- and beta-subunits and H(+),K(+)-ATPase activity was studied. Mutant beta-subunits with a replacement of the cytoplasmic and transmembrane cysteines preserved H(+),K(+)-ATPase activity. All the mutant beta-subunits with replacement(s) of the extracellular cysteines did not assemble with the alpha-subunit, resulting in loss of H(+),K(+)-ATPase activity. These mutants did not permit delivery of the alpha-subunit to the cell surface. Therefore, each of these disulfide bonds of the beta-subunit is essential for assembly with the alpha-subunit and expression of H(+),K(+)-ATPase activity as well as for cell surface delivery of the alpha-subunit. 相似文献
11.
Bovine liver cDNA clones encoding a precursor of the alpha-subunit of the mitochondrial ATP synthase complex 总被引:2,自引:0,他引:2
G A Breen 《Biochemical and biophysical research communications》1988,152(1):264-269
cDNA clones encoding a precursor of the alpha-subunit of the mitochondrial ATP synthase complex have been isolated from a bovine liver cDNA library using the alpha-subunit gene from Saccharomyces cerevisiae as a probe. Analyses of the nucleotide sequence of these cDNA clones reveal that the bovine liver ATP synthase alpha-subunit is initially synthesized as a precursor with an aminoterminal extension 43 amino acids in length. This aminoterminal presequence contains seven basic residues, no acidic residues, and seven polar uncharged serine and threonine residues. A single mRNA species, approximately 1.85 kb in length, was detected for the ATP synthase alpha-subunit precursor in both bovine liver and heart. 相似文献
12.
Makanji Y Walton KL Wilce MC Chan KL Robertson DM Harrison CA 《The Journal of biological chemistry》2008,283(24):16743-16751
Inhibins A and B negatively regulate the production and secretion of follicle-stimulating hormone from the anterior pituitary, control ovarian follicle development and steroidogenesis, and act as tumor suppressors in the gonads. Inhibins regulate these reproductive events by forming high affinity complexes with betaglycan and activin or bone morphogenetic protein type II receptors. In this study, the binding site of inhibin A for betaglycan was characterized using inhibin A mutant proteins. An epitope for high affinity betaglycan binding was detected spanning the outer convex surface of the inhibin alpha-subunit. Homology modeling indicates that key alpha-subunit residues (Tyr(50), Val(108), Thr(111), Ser(112), Phe(118), Lys(119), and Tyr(120)) form a contiguous epitope in this region of the molecule. Disruption of betaglycan binding by the simultaneous substitution of Thr(111), Ser(112), and Tyr(120) to alanine yielded an inhibin A variant that was unable to suppress activin-induced follicle-stimulating hormone release by rat pituitary cells in culture. Together these results indicate that a high affinity interaction between betaglycan and residues Val(108)-Tyr(120) of the inhibin alpha-subunit mediate inhibin A biological activity. 相似文献
13.
The amino acid sequence of the alpha-subunit of the gonadotropins, lutropin and follitropin from bullfrog, Rana catesbeiana, has been determined. The alpha-subunit was identified in both hormones by the amino acid composition and ovulation activity of lutropin in the Xenopus ovary, by means of reconstituted hormones in various combinations. The amino acid sequences of two identical alpha-subunits from lutropin and follitropin were determined or deduced by different strategies. The alpha-subunit of those gonadotropins have 97 amino acid residues, the longest among the known alpha-subunits of gonadotropins, and one arginine insertion at position 29. Ten cysteine residues and two sugar-chain binding sites at Asn57 and Asn83 are completely conserved among the species. The molecular mass of this subunit is 11,026 Da not including the sugar chains. The bullfrog alpha-subunit has approximately 70% sequence identity with mammalian alpha-subunits. 相似文献
14.
G S Monastyrskaia N E Broude A M Melkov Iu V Smirnov I V Malyshev 《Bioorganicheskaia khimiia》1987,13(1):20-26
The nucleotide sequence of the cDNA, containing coding region of the alpha-subunit of the pig kidney Na+, K+-ATPase, was determined. The region contains 3063 b.p. coding for 1021 amino acid residues. In the course of processing, five amino acid residues are cleaved to yield the mature Na+, K+-ATPase alpha-subunit containing 1016 amino acid residues. 相似文献
15.
Kenji Watanabe Makoto Shimoyamada Takuo Onizuka Hideo Akiyama Motohiro Niwa Tomoe Ido Yoji Tsuge 《DNA sequence》2004,15(4):251-261
The primary amino acid sequence of alpha-subunit in ovomucin (OVM) from hen thick egg white was determined. The 2087 amino acid residues with a relative molecular mass of 230.9 kDa along the full length of the alpha-subunit were represented. The alpha-subunit contains domains, arranged from the N- to C-terminals in the following order: D1-D2-D'-D3-R (central region)-D4-C1-CK (Cystine-knot), in a manner similar to the arrangement of D, C and CK domains in human pre-pro-von Willebrand factor (hpp-vWF) and hMUC2. The alpha-subunit showed identities on amino acid sequences with hpp-vWF and hMUC2 at 33 and 41% in the N-terminal region and 30 and 38% in the C-terminal region, respectively. The numbers and positions of cysteine residues were highly conserved among alpha-subunit, hpp-vWF and hMUC2. However, R showed no virtual sequence homology with the corresponding regions in two proteins. It was estimated that alpha-subunit was not part of a large peptide of OVM, but was independently synthesized from beta-subunit. 相似文献
16.
17.
Bovine cDNA probes for the beta-subunit of follicle-stimulating hormone beta (FSH beta) and the alpha-subunit of the glycoprotein hormones identify genetic variation (polymorphic restriction fragments) near these genes in sheep. The inheritance of the polymorphic restriction fragments was studied in half-sibling pedigrees generated by mating heterozygous (B+) rams to non-carrier (++) ewes so that the co-inheritance or genetic linkage to the Booroola (FecB) locus and the alpha- and beta-subunits of FSH could be analysed. Genetic recombination was observed between the FSH beta locus and the FecB locus in all five families studied and between the alpha-subunit and the FecB locus in the two families studied. We conclude that the FecB mutation does not lie within the FSH beta- or alpha-subunit genes encoding the heterodimeric hormone FSH, and that the high concentrations of FSH observed in carrier ewes must result from indirect actions of the FecB mutation on the synthesis, processing, storage, release or metabolism of FSH. 相似文献
18.
In order to elucidate the effect of single amino acid substitutions on the conformation of the tryptophan synthase alpha-subunit from Escherichia coli in solution, 1H NMR spectra of the wild-type and mutant proteins were measured at various pHs. Two of the four His C2-proton resonances of the alpha-subunit were assigned to two His residues at positions 92 and 146 by using a mutant protein with Thr substituted for the His at position 92. The replacement did not affect the conformation of the protein significantly. The proton resonances of all the Tyr residues in the aromatic region could be picked up from other resonance peaks, employing the wild-type alpha-subunit deuterated at all of the Phe residues. On comparison of the spectra of the wild-type protein with those of the mutant protein with Met substituted for the Glu at position 49, it was concluded that the substitution affects only the residues close to the substituted residue at acidic pH but that a larger part of the protein is affected at alkaline pH. NOE experiments showed that the five Tyr residues, four of which are located in the proximity of position 49, are close to one another. The present results are discussed in the light of the conformational stability of the protein. 相似文献
19.
Structural difference of the insulin receptors from circulating monocytes and erythrocytes 总被引:1,自引:0,他引:1
A McElduff R J Comi G Grunberger 《Biochemical and biophysical research communications》1985,133(3):1175-1180
We compared insulin receptors obtained from cells widely used in human studies, the circulating monocytes and erythrocytes. Biochemically, these receptors possess both binding (alpha-subunit) and tyrosine kinase (beta-subunit) activities similar to insulin receptors from other sources. Subtle differences in molecular weight, however, were detected between the alpha-subunits of these two cell types when analyzed by NaDodSO4-PAGE. Crosslinked [125I]insulin-labeled alpha-subunit of the monocyte insulin receptor was of higher apparent molecular weight than the alpha-subunit derived from red cells. Neuraminidase treatment of the alpha-subunits from each cell type indicated more sialic acid residues were present on the monocyte than the red cell alpha-subunit. The structural properties of the insulin receptors of human circulating cells are similar but not identical to insulin receptors of other characterized systems. 相似文献
20.
The glycoprotein hormone common alpha-subunit can be induced in HeLa and other nontrophoblastic tumor cell lines by sodium butyrate. This report demonstrates that production of alpha-subunit can be further modulated by theophylline, especially in conjunction with butyrate. This synergism was not observed with other phosphodiesterase inhibitors such as xanthine, caffeine, theobromine, or methylisobutylxanthine. Induction by a combination of the short chain fatty acid plus the methylxanthine results from a decrease in the lag time after effector addition as well as a change in the rate of subunit accumulation. The increase in alpha-subunit is correlated with an increase in the levels of alpha-subunit mRNA, suggesting that induction is manifest at a pretranslational stage. The production of alpha-subunit was only marginally affected in cultures treated with 8-Br-cAMP or forskolin. Intracellular levels of cAMP were increased approximately threefold by methylisobutylxanthine, twofold by theophylline, fourfold by forskolin, and about 50% by butyrate, yet significant induction was achieved only by butyrate and theophylline. Taken together, these data suggest that the synergism between butyrate and theophylline is not mediated by cAMP. 相似文献