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1.
Monkey pituitary somatotropin has been studied by zero-order, second-order, and circular dichroism spectroscopy. Difference absorption spectra have also been generated during proteolytic digestion of the hormone. The molar extinction coefficient of the native protein was found to be 23,800 +/- 550 (M-1 cm-1) at 276.6 nm. A comparison of the conformations of monkey and human pituitary somatotropins indicates a close relationship between the two molecules, including alpha-helix contents of 55 +/- 5%. 相似文献
2.
Reaction of human chorionic somatomammotropin and human pituitary growth hormone with tetranitromethane at 0 degrees C 总被引:2,自引:0,他引:2
The comparative reactivity of the eight tyrosine residues which occur at homologous positions in human chorionic somatomammotropin and human pituitary growth hormone has been investigated by their reaction with tetranitromethane at 0 °C. The derivatives were characterized by circular dichroism spectra, spectrophotometric titrations, rate of tryptic digestion, and immunodiffusion. Pigeon crop-sac stimulating activities were fully retained in these derivatives. The extent of modification for human chorionic somatomammotropin and human pituitary growth hormone was 2.5 and 4.2 out of 8 residues, respectively. The location of each modified tyrosine residue in the derivatives was determined by amino acid analysis of isolated nitrated peptides after cyanogen bromide cleavage and enzymatic digestion. It was found that tyrosine-143 was highly reactive in the pituitary hormone but unreactive in the placental hormone. 相似文献
3.
Equine pituitary somatotropin (growth hormone) has been studied by zero-order and second-order absorption spectroscopy, and by circular dichroism. Difference absorption spectra have also been generated during proteolytic digestion of the hormone. The molar extinction coefficient of the native protein was found to be 16,050 +/- 330 M-1 cm-1 at 278.1 nm. Comparison of the conformations of equine somatotropin and somatotropins isolated from several other mammalian species indicates a close structural relationship between these molecules. With the increasing number of species which have been studied, it is becoming evident that with regard to conformation, the somatotropins can be subdivided into at least three major groups. 相似文献
4.
Carlos Pendn J. Pedro Martínez-Barber Manuela Ortíz Manuel M. Valdivia 《Protein expression and purification》1996,7(4):389-394
Somatolactin, a pituitary hormone belonging to the growth hormone/prolactin family, is produced in the intermediate lobe of teleost pituitary. To date, the functions of this new hormone and the target tissues are unknown. ASolea senegalensissomatolactin (ssSL) cDNA has previously been cloned and isolated. Here we have inserted this cDNA into a pET-3a plasmid in order to produce recombinant ssSL inE. coliBL21 (DE3) cells. The protein induced was isolated from inclusion bodies by a solubilization–renaturation procedure originally developed to generate native disulfide bonds, to get putative active proteins. The recombinant somatolactin was further purified to homogeneity by gel filtration on FPLC. The estimated molecular weight of 26 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis agrees well with the molecular mass calculated from the translated cDNA sequence and with native somatolactin (SL). The recombinant protein showed electrophoretic mobility identical to that of one of the native forms of SL secretedin vitroby cultured pituitaries from sole. Another native SL expressed inS. senegalensisrepresented a glycosylated modified hormone as shown byN-glycosidase treatment. Further, recombinant SL was recognized by an anti-native SL antibody and used to generate polyclonal sera reactive with the native pituitary hormone. To date, this represents the first recombinant SL protein isolated in sufficient quantities for biophysical and biochemical investigation and for studies on its physiological actions. 相似文献
5.
Two thrombin fragments of reduced-carbamidomethylated human somatotropin representing the full primary structure of the native hormone (residues 1-134 and 135-191) have been found to form a recombinant molecule with properties similar to those of reduced-carbamidomethylated human somatotropin as shown by circular dichroism spectroscopy, two receptor-binding assays, and radioimmunoassay. In contrast, the homologous thrombin fragments of reduced-carbamidomethylated sheep hormone (residues 1-133 and 134-191) do not undergo recombination. Furthermore, neither the reduced-alkylated nor the reduced and nonalkylated C-terminal thrombin fragment of sheep hormone is able to interact with the reduced-carbamidomethylated N-terminal thrombin fragment of human hormone, under conditions which favor the recombination of the two human somatotropin fragments. 相似文献
6.
J Clarkson F Korber T Christensen F Junker J Pedersen F B Hansen 《Journal of molecular biology》1989,208(4):719-721
Single crystals of natural sequence human growth hormone have been grown from media containing ethanol, acetone or paraldehyde. Recombinant growth hormone in its native and desamidated form and pituitary hormone have been crystallized. A full native set of diffraction data extending to 3.5 A resolution has been obtained with synchrotron radiation for crystals of recombinant human growth hormone grown from ethanol. The identity of the material in these crystals has been established by anion-exchange chromatography. 相似文献
7.
Isolation of prolactin from equine pituitary glands has been described. It has a potency of 42 IU/mg in the pigeon crop-sac test and consists of 199 amino acids. The hormone has only four half-cystine residues in contrast to other mammalian prolactins which have six residues. From NH2-terminal sequence analysis and amino acid composition of cyanogen bromide fragments, the NH2-terminal disulfide loop is missing in the equine prolactin molecule. Circular dichroism spectra indicate that the α-helical content of equine prolactin appears to be lower (50%) than that found in the ovine hormone (65%). 相似文献
8.
The human class II major histocompatibility complex protein HLA-DR1 has been expressed in Escherichia coli as denatured alpha and beta subunits and folded in vitro to form the native structure. DR1 folding yields are 30-50% in the presence or absence of tight-binding antigenic peptides. The protein produced in this manner is soluble and monomeric with the expected apparent molecular weight. It reacts with conformation-sensitive anti-DR antibodies and exhibits peptide-dependent resistance to SDS-induced chain dissociation and to proteolysis as does the native protein. The observed peptide specificity and dissociation kinetics are similar to those of native DR produced in B-cells and finally the protein exhibits circular dichroism spectra and cooperative thermal denaturation as expected for a folded protein. We conclude that the recombinant DR1 has adopted the native fold. We have folded DR1 in the absence of peptide and isolated a soluble, peptide-free alphabeta-heterodimer. The empty DR1 can bind antigenic peptide but exhibits altered far UV-circular dichroism and thermal denaturation relative to the peptide-bound form. 相似文献
9.
10.
Y N Sinha T A Gilligan 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1984,177(3):465-474
Extracts of mouse and rat adenohypophyses have been analyzed for a low-molecular-weight variant of growth hormone (GH) known to occur in humans, the so-called "20K"-GH (mol wt = 20,000 vs 22,000 for traditional human GH). Pituitary proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostained with an antiserum raised against murine GH. Reactive and unreactive bands in close vicinity of the major GH band were fingerprinted by a peptide-mapping technique that reveals only the tyrosine-containing peptides, but can be applied to fingerprint single protein bands within gels. We found a protein band 2000 mol wt smaller than the major murine GH in both mouse and rat pituitary glands whose fingerprint resembled that of major GH. The peptide-mapping results are consistent with the interpretation that the internal deletion of amino acid residues most likely is in the same region of the molecule as in the human 20K-GH. Unlike the human 20K-GH, however, the murine counterpart showed no cross-reactivity with an antiserum raised against 22K-GH in the test system used here. 相似文献
11.
Pituitary somatotropin (growth hormone) from the sturgeon (Acipenser gulden-stadti) has been studied by zero-order and second-order absorption spectroscopy, as well as by circular dichroism. Difference absorption spectra have also been generated during proteolytic digestion of the hormone. The molar extinction coefficient of the native protein was found to be 15,000 +/- 110 M-1 cm-1 at 278.5 nm. Comparison of the conformations of sturgeon somatotropin and somatotropins isolated from several mammalian species, including bovine and human, indicates a close relationship between these molecules. Such similarities may be related to the relatively high biopotency of this fish hormone in mammalian assay systems. 相似文献
12.
A human plasma retinol-binding protein (RBP) mutant, named RBP-S, has been designed and produced in which the six native cysteine residues, involved in the formation of three disulfide bonds, have been replaced with serine. A hexa-histidine tag was also added to the C-terminus of RBP for ease of purification. The removal of the disulfide bonds led to a decrease in the affinity of RBP for all trans-retinol. Data indicates all-trans-retinol binds RBP and RBP-S with Kd = 4 x 10(-8) M and 1 x 10(-7) M, respectively, at approximately 20 degrees C. RBP-S has reduced stability as compared to natural RBP below pH 8.0 and at room temperature. Circular dichroism in the far-UV shows that there is a relaxation of the RBP structure upon the removal of its disulfide bonds. Circular dichroism in the near-UV shows that in the absence of the disulfide bonds, the optical activity of RBP is higher in the 310-330 nm than in the 280-290 nm range. This work suggests that the three native disulfide bonds aid in the folding of RBP but are not essential to produce a soluble, active protein. 相似文献
13.
T A Bewley 《Biochemistry》1977,16(2):209-215
The reduction and alkylation of the two disulfide bonds in a preparation of human pituitary growth hormone which had been previously modified by limited proteolysis with the enzyme plasmin have been studied. Quantitative and selective reduction of the carboxyl-terminal disulfide, as well as total reduction of both disulfides, has been achieved in the absence of denaturants. Circular dichroism spectra of the various reduced and reduced-alkylated derivatives have provided sufficient information to allow an estimation of the individual contributions of each disulfide bond to the total optical activity of the protein. These contributions were found to represent a significant portion of the total optical activity between 290 and 250 nm. The carboxyl-termimal bond exhibits negative dichroism with an apparent center near 258 nm ([theta]M,258nm = 2100 deg cm2 dmol-1). By comparison, the contribution of the remaining disulfide is red-shifted to 273 nm, is also negative in sign, and somewhat more intense ([theta]M,273nm = 3200 deg cm2 dmol-1). Circular dichroism measurements have also been used to approximate the rate of reduction of the protein. 相似文献
14.
Purification and partial characterization of inhibin from porcine follicular fluid 总被引:15,自引:0,他引:15
J Rivier J Spiess R McClintock J Vaughan W Vale 《Biochemical and biophysical research communications》1985,133(1):120-127
Inhibin, a protein of gonadal origin that suppresses the basal secretion of follicle stimulating hormone by anterior pituitary cells has been purified from porcine follicular fluid. Using several RP-HPLC steps and gel filtration under denaturing conditions, we obtained a fraction approximately ten thousand fold purified which showed one band on SDS PAGE and in the same experiment two bands after reduction (MW ca 14K and ca 18K) suggesting a molecular weight of 32K for inhibin. Edman degradation of isolated inhibin and carboxymethylated chain A indicated that the first 6 residues were H-Ser-Thr-Ala-Pro-Leu-Pro-; by subtraction, the first 3 residues of chain B could be deduced to be H-Gly-Leu-Glu-. EC50 was ca 0.3 ng/ml or 10 pM in our in vitro pituitary cell culture assay. Antibodies to residues 1-6 were raised which could immunoneutralize purified inhibin activity in an in vitro assay. 相似文献
15.
Human pituitary growth hormone: immunochemical investigations of biologically active fragments 总被引:6,自引:0,他引:6
Guinea pig antisera to human growth hormone were tested for their ability to recognize the two biologically active fragments of the hormone produced by human plasmin digestion and a synthetic active fragment. A precipitin line was obtained with native human growth hormone, plasmin-treated human growth hormone, and its NH2-terminal fragment (residues 1–134). In the microcomplement-fixation and radioimmunoassay experiments, the NH2-terminal plasmin fragment (residues 1–134) showed a greater immunoreactivity than the COOH-terminal plasmin fragment (residues 141–191). This, in turn, was more active than the synthetic fragment (residues 95–136). 相似文献
16.
Bertucci C Barsotti MC Raffaelli A Salvadori P 《Biochimica et biophysica acta》2001,1544(1-2):386-392
Derivatisation of lysine residues in human albumin was performed in vitro by reaction with penicillin G. This modification reaction has been reported to occur in patients treated with high dosages of the antibiotic. The structure of the modified protein was characterised by mass spectrometry and circular dichroism. The number of the lysine residues involved depends on the time of incubation and on the drug/protein molar ratio. The secondary structure of the modified protein does not change significantly with respect to the native protein. Furthermore, the binding properties of the modified albumin were characterised by CD spectroscopy. Phenylbutazone, diazepam and bilirubin, known to bind to specific binding areas, were used as markers. A decrease of the affinity to the high-affinity binding sites was observed after the modification. 相似文献
17.
In polyacrylamide gel electrophoresis and isoelectric focusing, somatotropin produced by recombinant DNA technology is as heterogeneous as highly purified native pituitary somatotropin. This heterogeneity is not attributable to different degrees of deamination of a single molecular species. In addition to the main protein of 22 kDa, the cloned products contain traces of interchain disulphide dimers of somatotropin. The quantitative amino acid analyses of the three cloned somatotropins investigated are neither identical nor do they correspond to the analysis of the native pituitary hormone. Moreover, there are discrepancies between the amino acid compositions of the hormones studied and the generally recognized amino acid analysis for human somatotropin. 相似文献
18.
The unfolding of human plasma alpha 1-acid glycoprotein (AGP) induced by heat or guanidine hydrochloride was studied under equilibrium conditions. In thermal unfolding, an intermediate state was detected by the appearance of unusual positive difference absorption bands in the 287-295-nm region, which occurred at lower temperatures than the common denaturation bands at 284 and 291 nm. The formation of this intermediate species apparently involves a local conformational change that perturbs the environment of tryptophyl residues, without affecting the secondary structure of the protein as judged from circular dichroism spectra. On the other hand, denaturation of the glycoprotein induced by guanidine hydrochloride seemed to follow a two-state model with no evidence of any intermediate species; however, the analysis of the transition curve indicated that the change in the accessibility to solvent of amino acid residues of AGP upon unfolding is significantly lower than those observed for other proteins. According to these results, it is proposed that part of the polypeptide chain in native AGP, namely, that from residue 122 to the C-terminus, may be "loosely" folded. 相似文献
19.
Porcine pituitary lactogenic hormone has been examined by exclusion chromatography, analytical ultracentrifugation, free-boundary electrophoresis, solubility behavior, circular dichroism, and fluorescence spectra. The results have been compared with previous studies of ovine pituitary lactogenic hormone. These two hormones were found to resemble one another very closely by all criteria studied. While the homology between these lactogenic hormones and growth hormones is strongly reflected in the sharing of certain physicochemical properties, a number of distinct structural differences between these two closely related evolutionary lines are now becoming apparent. 相似文献
20.
A 11-residue segment, sequence positions 135-145, has been cleaved selectively from the human somatotropin molecule by limited digestion of the hormone with trypsin at pH 6.0 in 90% (v/v) glycerol. Differences between the tertiary structures of the native and trypsin-modified hormones have been demonstrated by circular dichroism spectroscopy. Despite these conformational differences, the biological properties of intact hormone and its modified form appeared to be very similar, as tested by two receptor-binding assays, radioimmunoassay, and the rat tibia test. 相似文献