共查询到20条相似文献,搜索用时 31 毫秒
1.
R Knorr W Danho E E Büllesbach H G Gattner H Zahn G L King C R Kahn 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1982,363(12):1449-1460
An analogue of porcine insulin which differs from the native molecule in that the amino-acid residue B22-L-arginine is replaced by its D-enantiomer has been synthesized. The [D ArgB22]B-chain was synthesized by the segment condensation method and purified as the di-S-sulfonate by ion exchange chromatoggraphy on SP-Sephadex at pH 3.5. Combination with native porcine sulfhydryl A-chain gave [DArgB22]insulin which was purified by ion exchange chromatography on SP-Sephadex at pH 4.5 with a linear NaCl gradient. The biological activity of this analogue as measured by glucose oxidation in rat epididymal adipocytes was 2%. Thymidine incorporation into DNA of human fibroblast was 16%. The immunoreactivity using antipork insulin antibody in a double antibody immunoassay was 4%. The receptor-binding affinity as measured by radioreceptor assays was 2% with cultured human fibroblasts and 1% with rat adipocytes. These results suggest that the L-configuration at B22-arginine is essential for retaining the biological, immunological and receptor-binding properties of the hormone. 相似文献
2.
目的 :获得具有生物学活性的重组人生长激素 (rhGH)。方法 :PBV -GH/DH5α菌体经超声破菌、反复洗涤后获得包涵体。将包涵体变性、复性 ,用硫酸铵盐析 ,离子交换层析和凝胶层析进行纯化。产物经SDS -PAGE、HPLC、N末端 15个氨基酸序列检测验证。结果 :终产物rhGH纯度达 98.2 % ,比活性大于 3.0IU/mg。分子量为 2 2kDa ,N末端氨基酸序列与DNA序列推导的氨基酸序列完全一致。结论 :从自构建的PBV -GH/DH5α工程菌中获得高纯度、高活性重组人生长激素。其纯化工艺为中试生产提供可靠依据。 相似文献
3.
A crown-gall tumor growth factor (TGF-II) isolated from bean leaves inoculated with Agrobacterium tumefaciens strain 13333 is shown to be γ-aminobutyric acid (GABA). This identification is based on the comparative behavior of purified TGF-II and authentic GABA with respect to elution from preparative ion exchange and molecular sieve columns, ninhydrin reaction, TLC, co-chromatography on an automated amino acid analyzer, MS analysis and biological activity. GABA is detected by bioassay only in bean leaves infected with the bacterium and is in growth limiting supply when only a few tumors are present per leaf. GABA promotes tumor growth when as little as 1 ng is applied per leaf. 相似文献
4.
The effects of various chemical and enzymatic treatments on the biological activity of porcine luteinizing hormone-releasing hormone (LH-RH) are described. This experiment was performed before the elucidation of the structure of LH-RH. LH-RH activity was abolished by the following endopeptidases: chymotrypsin, subtilisin, papain, and thermolysin, but not by pepsin or trypsin. Exopeptidases did not affect LH-RH activity, but a purified preparation of pyrolidone carbosylpeptidase did. The amino acid sequence of LH-RH/FSH-RH was established to be (pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Amine. This decapeptide lacks both the Amine terminus and the COOH terminus. Its Amine-terminal dipeptide sequence,(pyro)Glu-His, is similar to that of tyrotropin-releasing hormone. The lack of inactivation by the exopeptidases is in good agreement with these findings. Treatment with various chemical reagents showed that tyrosine, histidine, tryptophan, and arginine in LH-RH are important for its biological activity. Nitrous acid and Edman degradation did not inactivate LH-RH. These results are also in agreement with the determined structure of LH-RH. This hormone showed a high follicle-stimulating hormone-releasing hormone (FSH-RH) activity. The inactivation of LH-RH was always accompanied by a loss of FSH-RH activity. These experiments also shed some light on the structure-activity relationship of this hormone. 相似文献
5.
A H?gset O R Blingsmo O Saether V T Gautvik E Holmgren M Hartmanis S Josephson O S Gabrielsen J O Gordeladze P Alestr?m 《The Journal of biological chemistry》1990,265(13):7338-7344
Human parathyroid hormone (hPTH) is a peptide hormone consisting of 84 amino acids (hPTH(1-84)). Employing the promoter and signal sequence of Staphylococcus aureus-protein A we have expressed hPTH in Escherichia coli. The expressed proteins are excreted to the growth medium, allowing for rapid and easy purification of the desired products. By amino acid sequence analysis and mass spectrometry, we have shown that the major excreted product is correctly processed human identical hPTH(1-84). The purified recombinant hPTH(1-84) stimulates adenylate cyclase activity in rat osteosarcoma cell membranes to exactly the same extent as synthetic parathyroid hormone standards, indicating that the recombinant product has full biological activity. 相似文献
6.
Characterization of Cholecystokinin from the Human Brain 总被引:1,自引:0,他引:1
Laurence J. Miller Ian Jardine† Edward Weissman Vay Liang W. Go David Speicher‡ 《Journal of neurochemistry》1984,43(3):835-840
Human forms of cholecystokinin have not previously been characterized chemically. In this study, we have extracted and purified the predominant molecular form of cholecystokinin present in human cerebral cortex. The peptide was characterized by amino acid analysis, automated peptide sequencing, and fast atom bombardment mass spectrometry. It appears to be identical to porcine cholecystokinin-octapeptide, with the sequence of Asp-Tyr(SO3)-Met-Gly-Trp-Met-Asp-Phe(NH2). This structural identity is consistent with the observations that the peptide in human brain and porcine cholecystokinin-octapeptide are recognized similarly by a battery of antisera to porcine cholecystokinin; that they coelute from several chromatographic systems, including gel filtration, ion exchange, and reversed-phase; and that they possess similar biological activities. 相似文献
7.
M Schleyer T Trah I Schleyer E Kley 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1981,362(7):875-881
Human and porcine somatotropin were compared by different methods. Both hormones show biological activity in the Greenspan assay. Molecular masses, N- and C-terminal amino acids as well as the chromatographic patterns on ion exchange cellulose at room temperature are identical. Chromatography in the cold results in different behaviour of the two hormones. Polyacrylamide gel electrophoresis and electrofocusing also show characteristic differences. The quantitative amino acid analyses of the two hormones are different. The N-terminal amino acid sequences estimated up to position 20 show identity only in 13 positions. 相似文献
8.
The preparation of two highly purified lipolytically active hog pituitary peptides, called P-LF II C and P-LF II D is described. The two peptides are free of other pituitary hormone activities. In isolated rat and porcine adipose tissue, both fractions are lipolytically much more active than every other lipolytic active pituitary peptide described to date. By fraction P-LF II D, the first pituitary peptide was isolated which has lipolytic activity in isolated rat adipose tissue than corticotropin, the lipolytically most active pituitary hormone known so far. On isolated porcine adipose tissue, fraction P-LF II D as well as P-LF II C showed without doubt higher activity than corticotropin. 相似文献
9.
C H Li D Chung J Izdebski T Majewski T F Mowles 《International journal of peptide and protein research》1988,31(2):201-204
Growth hormone has been purified to homogeneity from blue fox pituitary glands. It has 191 amino acids with two disulfide bridges and a single tryptophan residue. The somatotropin activity is only 8% when compared with the bovine hormone in the receptor-binding assay. From radioimmunoassay data using baboon antisera to porcine or bovine growth hormone, the fox hormone has 14-17% immunoreactivity of bovine or porcine hormone. 相似文献
10.
Peter Böhlen Paul Brazeau Fred Esch Nicholas Ling William B. Wehrenberg Roger Guillemin 《Regulatory peptides》1983,6(4):343-353
Peptides with high intrinsic activity to release growth hormone from pituitary cells in tissue cultures were isolated from two different human pancreatic tumors that had caused acromegaly. Homogeneous peptides were obtained after gel filtration and two steps of reverse-phase high-performance liquid chromatography. From one tumor a 44-residue peptide (human pancreas growth hormone releasing factor, hpGRF-44) was isolated, together with two shorter fragments of reduced bioactivity having 40 and 37 amino acid residues (hpGRF-40, hpGRF-37). In contrast, the other tumor contained only one form of GRF which proved to be identical to hpGRF-40. These hpGRFs are indistinguishable from partially purified preparations of hypothalamic growth hormone releasing factor of human, porcine and murine origins with respect to biological activity and are very similar in their physicochemical properties (molecular weight, retention behavior on reverse-phase HPLC, absence of sulfhydryl groups). One of the pancreatic tumors also contained two forms of immunoreactive somatostatin. One form, after isolation and partial microsequencing, was identified as somatostatin-14 with a structure identical to that of the peptide found in other species. The second form has tentatively been identified as somatostatin-28 on the basis of chromatographic behavior. 相似文献
11.
Pettersson J Mossberg AK Svanborg C 《Biochemical and biophysical research communications》2006,345(1):260-270
HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of apo alpha-lactalbumin and oleic acid, formed in casein after low pH treatment of human milk. This study examined if HAMLET-like complexes are present in casein from different species and if isolated alpha-lactalbumin from those species can form such complexes with oleic acid. Casein from human, bovine, equine, and porcine milk was separated by ion exchange chromatography and active complexes were only found in human casein. This was not explained by alpha-lactalbumin sequence variation, as purified bovine, equine, porcine, and caprine alpha-lactalbumins formed complexes with oleic acid with biological activity similar to HAMLET. We conclude that structural variation of alpha-lactalbumins does not preclude the formation of HAMLET-like complexes and that natural HAMLET formation in casein was unique to human milk, which also showed the highest oleic acid content. 相似文献
12.
Donna R. Curtis J. Hugh McDowell Paul A. Hargrave 《Preparative biochemistry & biotechnology》2013,43(2):83-102
A general method is described which allows the identification and preparation of peptides containing any amino acid of interest. The method has been applied to isolation of the methionyl peptides from a peptic digest of oxidized bovine rhodopsin. The peptide digestion mixture is first partially separated by ion exchange column chromatography. Location of peptides containing the desired amino acid is performed by amino acid analysis of acid hydrolyzed column fractions by high voltage paper electrophoresis. Peptides are further purified and prepared by peptide mapping, elution, and amino acid analysis using inexpensive high capacity techniques. Peptide sequencing is performed by a manual dansyl-Edman method well adapted for rapidly processing large numbers of samples. The methods are particularly well suited for detection and preparation of peptides containing amino acids for which there is no specific detection method. 相似文献
13.
PHI--a new brain-gut peptide 总被引:3,自引:0,他引:3
K Tatemoto 《Peptides》1984,5(2):151-154
The detection of the C-terminal amide structure in porcine intestinal extracts has led to the discovery of a 27 amino acid residue peptide designated PHI (PHI-27, peptide HI). The peptide was found to have structural homologies to vasoactive intestinal peptide (VIP) and growth hormone-releasing factor (GRF). Subsequent studies have revealed that PHI exhibits a variety of biological activities which resemble those of VIP. Moreover, it was found that the peptide is able to inhibit the binding of VIP to its receptors, and to stimulate cyclic AMP production. PHI is present in both brain and gut in high concentrations and probably acts as a neurotransmitter or neuromodulator rather than a hormone. A comparison of the amino acid sequences of porcine, human and bovine PHI indicated that human PHI differs from the porcine peptide in two positions (12 and 27), and bovine PHI differs in one position (10). The amino acid sequence (deduced from the cDNA sequence) of the VIP precursor recently obtained from human neuroblastoma cells also contains an identical sequence to the newly-isolated human PHI from human colonic extracts. PHI has thus been shown to be co-synthesized with VIP in the same precursor molecule. 相似文献
14.
Using the pUBJ10 plasmid containing the modified bovine growth hormone (bGH) cDNA, large production has been attempted in E. coli BL21 strain. The bGH was highly expressed upto the level of 35% of total cell proteins by IPTG induction and temperature shift to 40°C. The recombinant bGH (rbGH) was isolated from inclusion bodies by solubilization in 10 M urea and followed by DEAE-TOYOPEARL 650C ion exchange and Sephadex G-100 column chromatography. The pUBJ10-derived bGH was eluted at 25.28 min similar to the standard bGH (at 25.18 min) by reverse-phase HPLC. The analysis of N-terminal amino acid showed that the mature bGH has glutamic acid as a first amino acid in agreement with DNA sequencing data. The biological activity was indirectly measured by radioreceptor assay and compared with a pituitary-derived bGH. 相似文献
15.
K Nokihara 《Peptides》1990,11(2):185-191
To evaluate the chemically synthesized materials, two cardiodilatins, CDD-126 and CDD-88/CDD(39-126), a precursor of atrial hormone and a related fragment, were isolated from porcine atria by immunoaffinity chromatography or alginic acid adsorption followed by an ion exchange high performance liquid chromatography. The chemical synthesis was carried out using an automated peptide synthesizer. After cleavage and refolding, the crude CDDs were directly characterized by the novel method of primary structure determination using electroblotting and microsequencing. The purified synthetic CDDs were identical with the natural ones in physicochemical and immunochemical properties, as well as their biological actions both in vivo and in vitro. 相似文献
16.
H J M?gert O Hill H D Zucht S Martin M Meyer W G Forssmann K Adermann 《Biochemical and biophysical research communications》1999,259(1):141-148
Guanylin and uroguanylin are structurally related intestinal peptide hormones which were purified from a limited number of mammals and are capable of activating the particulate guanylate cyclase-C. Although the biological functions of guanylin and uroguanylin are not yet clarified in detail, they are involved in the regulation of the intestinal water and electrolyte balance. In order to verify the general importance of this hormone system in mammals, we cloned the corresponding cDNAs from pig. Here, we present the nucleotide sequences and the deduced amino acid sequences representing porcine guanylin and uroguanylin. The expression patterns of the corresponding genes, as shown by Northern hybridization and RT-PCR analysis, resemble those of the human homologues. Further, we demonstrate the bioactivity of both porcine peptide hormones by inducing the intracellular cGMP production in human T84 cells and by ion transport experiments using porcine intestinal mucosa in the Ussing chamber. 相似文献
17.
N alpha-Acyl amino acid releasing enzyme (NAARE), an enzyme cleaving acetylMet-Ala at the Met-Ala bond was purified from rat brain cytosol to apparent homogeneity by salt precipitation, gel filtration, and several steps of ion exchange. Levels of NAARE exceeded acylase measured with acetylmethionine in all brain regions and subcellular fractions examined: 60% was associated with cytosol and the remainder with debris or the crude nuclear and mitochondrial-synaptosomal subfractions. Activity was highest in pituitary and was approximately 0.5-0.6 that of liver or kidney. The purified enzyme preferentially hydrolyzed acetylmethionyl peptides: Km for acetylMet-Ala was 0.93; Vmax, 3.5 nmol-1 (kcat, 1185) with pH optimum of 8.9 as compared with 8.2 for acylases measured in cytosol. The purified enzyme was devoid of acylase and common exo- and endopeptidase contamination. Structure-activity relationships examined with synthetic formylated or acetylated peptides indicated no significant effects for di- or tripeptides if the second substituent was Ala, Ser, Asn, or Thr, but the activity was reduced 0.5-fold for Leu, a branched-chain amino acid. No hydrolysis was observed for polypeptides with five or more residues having N-terminal acetylated Tyr (enkephalin) or Ser (alpha-melanocyte-stimulating hormone, thymosin alpha 1), supporting the notion that the enzyme plays a role only in turnover of smaller peptides formed perhaps as a result of endopeptidase cleavage of proteins or polypeptides containing acetylated Met at the N terminus. 相似文献
18.
基因工程人胰岛素原和胰岛素的分离纯化及性质研究 总被引:2,自引:0,他引:2
E. coli DH 5 alpha cells harboring a plasmid pWR 590-BCA 4 for fused human proinsulin production were cultured. The fused human proinsulin was isolated from the fermented cells and then subjected it to cleavage with BrCN. The cleaved product was then converted to crude proinsulin-S-sulfonate using oxidative sulfitolysis. The isolation of human proinsulin-S-sulfonate was accomplished by ion exchange chromatography on QAE-sephadex A-25, followed by gel filtration on sephadex G-50. The purified human proinsulin-S-sulfonate was folded using a disulfide interchange method. The folding mixture was then chromatographed on sephadex G-50 and purified proinsulin was obtained. The proinsulin was then converted to human insulin and C-peptide by a combination cleavage with trypsin and carboxypeptidase B. The total yield of human insulin was about 5 mg/L The Zinc insulin crystals were obtained with amorphous human insulin using citrate method. The amino acid composition N-terminal sequences as well as C-terminal amino acid residues are in agreement with expected results. The hypoglycemic activity of purified human insulin is 26-27 U/mg, as judged by mouse convulsion assay, and the RIA activity is about 99% of that of porcine insulin. 相似文献
19.
20.
M Otani H Yamauchi T Meguro S Kitazawa S Watanabe H Orimo 《Journal of biochemistry》1976,79(2):345-352
Calcitonin was extracted from the pericardium and esophagus of eel in quantities sufficient to permit purification and chemical characterization. Homogeneous calcitonin could be isolated by a six-step fractionation starting from acetone powder of the organs. The fractionation procedure consisted of acid extraction, gel filtration on Sephadex G-75, chromatography on SP-Sephadex C-25, gel filtration on the Sephadex G-50, chromatography on carboxymethylcellulose, and gel filtration on Sephadex G-50. Fractionation of the hormone was monitored by assay of its biological activity and from its behaviour on thin layer chromatography and polyacrylamide gel disc electrophoresis. The hormone contained 32 amino acid residues, like calcitonins from other species of animals, but its amino acid composition was different from those of previously characterized hormones. Eel calcitonin possessed almost the same, or higher, biological activity as the salmon or chicken hormone, which show the highest specific activity among calcitonins so far isolated. 相似文献