共查询到20条相似文献,搜索用时 46 毫秒
1.
J P Burbach H H Van Tol V M Wiegant R A Van Ooijen R A Maes 《The Journal of biological chemistry》1985,260(11):6663-6669
The peptides that represent the major components with alpha-endorphin- and gamma-endorphin-like immunoreactivity in the rat neurointermediate lobe were purified to homogeneity and chemically characterized. Rat neurointermediate lobes were extracted by boiling and homogenization in acetic acid. Peptide purification was based on gel filtration, followed by two high-pressure liquid chromatography steps. Pools containing peptides with the size and immunochemical properties of alpha- and gamma-endorphins were resolved by reverse-phase high-pressure liquid chromatography into multiple immunoreactive components. The major forms were finally purified by paired-ion high-pressure liquid chromatography. The amino acid compositions of these peptides fitted the beta-endorphin sequences 1-16 and 1-17. Tryptic mapping, aminopeptidase M digestion, chromatographic characterization, and immunoreactivity to an antiserum recognizing the N alpha-acetylated terminus of endorphins showed that these peptides were indistinguishable from N alpha-acetyl-alpha-endorphin (N alpha-acetyl-beta-endorphin 1-16), and N alpha-acetyl-gamma-endorphin (N alpha-acetyl-beta-endorphin 1-17). The NH2-terminal residue of the peptides was identified by mass spectrometry as N alpha-acetyltyrosine, substantiating the identity of the peptides. The results demonstrate the existence of N alpha-acetylated alpha- and gamma-endorphin as endogenous peptides in the neurointermediate lobe of the rat pituitary gland. In view of their occurrence and biological properties they should be considered significant members of the pro-opiomelanocortin family. 相似文献
2.
Peptides recognized by rabbit antibodies to vasoactive intestinal peptide (VIP) were extracted from diisopropyl fluorophosphate-treated rat basophilic leukemia (RBL) cells and resolved by filtration on Sephadex G-25 in 50 mM acetic acid. The immunoreactive VIPs of RBL cells eluted from Sephadex G-25 at 35-41%, 53-60%, and 69-73% bed volume, but not at 63-68% as for the neuropeptide VIP1-28. The two forms of immunoreactive VIP larger than VIP1-28 reacted with antibodies to both VIP1-9 and VIP10-28, but the smallest was bound only by antibodies to VIP10-28. The smallest immunoreactive VIP was purified by ion-exchange and reverse-phase high-performance liquid chromatography, and the amino acid sequence was determined to be that of VIP10-28 with asparagine-free acid at the carboxyl terminus rather than the amide of VIP neuropeptide. Challenge of RBL cells with 1 microM ionophore A23187 at 37 degrees C released VIP10-28 rapidly to a mean of 75% at 5 min and 77% at 30 min. The VIP generated and released by mast cells thus consists of a mixture of peptides that all differ structurally from the neuropeptide VIP. 相似文献
3.
4.
F J Joubert 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1975,356(12):1901-1908
Toxin CM-13b was purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose. The toxin comprises 65 amino acid residues and is cross-linked by five disulphide bridges. The complete amino acid sequence of toxin CM-13b was elucidated. The reduced and S-carboxymethylated toxin was digested with trypsin and chymotrypsin and the peptides purified by DEAE-cellulose chromatography and chromatography or electrophoresis on paper. The amino acid sequences of the intact toxin and its constituent peptides were determined by the Edman-Begg procedure, either through the use of the automatic sequenator or by manual manipulation. The chymotryptic digest provided the necessary overlapping peptides for aligning the tryptic peptides. The primary structure of toxin CM-13b shows a high degree of homology with that of protein S4C11 from Naja melanoleuca venom[1], but their toxicities are very different. 相似文献
5.
Reverse-phase high-pressure liquid chromatography has been used for the purification of some large cyanogen bromide peptides from flavocytochrome b2 fragment alpha. Acetonitrile gradients at acid and/or neutral pH using mu Bondapak C18 columns were useful for the smaller peptides (43 and 67 residues). The two larger ones, alpha CB1 and alpha CB2, could only be separated from each other by trifluoroacetic acid/1-propanol gradients on mu Bondapak-CN columns. The various systems tested are presented and compared. The elucidation of the amino acid sequence of alpha CB2 (95 residues), alpha CB3 (67 residues) and alpha CB4 (43 residues) is described. The fragments were digested with trypsin, chymotrypsin and Staphylococcus aureus V8 protease as necessary. Fragment alpha CB2 was also cleaved at the unique tryptophanyl bond with cyanogen bromide. Peptides were fractionated by Sephadex chromatography, thin-layer finger-printing and/or high-pressure liquid chromatography. Peptides were sequenced mostly in the liquid phase sequenator. The cyanogen bromide peptides could be ordered using information obtained previously, as well as additional data obtained in this work. Together with the previous elucidation of cytochrome b2 core sequence and of the hinge region [Guiard, B. and Lederer, F. (1976) Biochimie (Paris) 58, 305--316; Ghrir, R. and Lederer, F. (1981) Eur. J. Biochem. 120, 279--287], the present results enable us to present the complete sequence of fragment alpha (314 residues) with only three overlaps missing between cyanogen bromide peptides. Sequence comparisons with other known flavoproteins do not indicate any noticeable similarity. Structural predictions indicate an alteration of alpha helices and beta structure. The possibility that the non-heme-binding portion of fragment alpha could constitute a flavin-binding domain is discussed. 相似文献
6.
The complete amino acid sequence of the kappa-chain of the mouse myeloma protein MOPC 21 was established. The protein was reduced and alkylated with iodo[2-(14)C]acetic acid, and 21 tryptic peptides were isolated, mainly by paper electrophoresis and paper chromatography. Three large tryptic peptides (of 35, 36 and 42 residues), which were difficult to isolate in this manner, were obtained pure and in excellent yields by a combination of Sephadex G-50 gel filtration in 1% (w/v) NH(4)HCO(3) and chromatography on a DEAE-cellulose column in ammonium acetate buffer, pH8.1. Peptides overlapping the tryptic peptides were isolated from a chymotryptic digest. The chain is 214 residues long. Microheterogeneity of two peptides was observed and is believed to be due to deamidation. It was not excluded that such deamidation could occur in serum from which the protein was isolated. The sequence is compared with the sequences of two other mouse kappa-chains, and with the human kappa-chain basic sequences. 相似文献
7.
Purification of Escherichia coli 50 S ribosomal proteins by high performance liquid chromatography 总被引:1,自引:0,他引:1
The 50 S subunit proteins from the Escherichia coli ribosome were purified by size-exclusion, ion-exchange or reversed phase high performance liquid chromatography (HPLC) avoiding any precipitation or desalting procedures during isolation. Best resolution of this complex protein mixture was achieved by reversed phase chromatography on supports with short alkyl chains and C18 hydrocarbon-bonded phases; 23 out of the 32 proteins from the 50 S subunit were purified as shown by two-dimensional gel electrophoresis, amino acid analysis and direct micro-sequencing. Protein recoveries varied between 25 and 84% as determined by amino acid analysis. Ribosomal proteins of other organisms can be separated under similar conditions. 相似文献
8.
Peptide inhibitors of angiotensin I-converting enzyme in digests of gelatin by bacterial collagenase. 总被引:2,自引:0,他引:2
Peptide inhibitors of angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) were produced by digesting gelatin with bacterial collagenase. The inhibitors were isolated from the digests with a combination of alcohol fractionation, treatment with Amberlite CG-50 column, gel filtration through Sephadex G-25, and Dowex 50 column and paper chromatography. Nine peptide fractions were purified to apparent homogeneity judging by thin-layer and ion-exchange column chromatography, and amino acid composition. Amino acid sequences of the peptides were determined: 2 were found to be mixtures of peptides and the sequence of another was only partially determined. Six of the peptides were potent inhibitors of the converting enzyme, while the other three were less active. 6 peptides were substrates for the enzyme. The enzyme released a dipeptide, Ala-Hyp from one peptide and was strongly inhibited by this dipeptide. The remainder of the parent peptides was a less effective inhibitor. 相似文献
9.
High-performance liquid chromatography on an Asahipak GS-320 column using isocratic elution with 0.1 M acetic acid has proven effective for fractionation of peptides of molecular weights lower than 3000. This technique enabled the separation of the peptides derived from digestion of native ribonuclease F1 by trypsin and chymotrypsin in combination with conventional gel filtration through Sephadex G-25 and reversed-phase HPLC. Amino acid analysis of the cystine-containing peptides thus obtained revealed the disulfide linkages Cys-6-Cys-102 and Cys-24-Cys-84 in this protein. The behavior of a number of peptides in the HPLC on an Asahipak GS-320 column is described and the separation mechanism is discussed. 相似文献
10.
Yuhei Morita Katsuzo Shimizu Nobuko Kada 《Bioscience, biotechnology, and biochemistry》2013,77(5):671-677
In a continuation of the structural studies on Japanese-radish peroxidase a. the products resulting from the action of pepsin on performic acid-oxidized apo-peroxidase a have been examined by ion-exchange chromatography on a Dowex 50W-X2 column, followed by gelfiltration chromatography on a Sephadex G-25 column and by high voltage paper electrophoresis. Seven peptides have been isolated in purified forms in yields of 6 to 39 per cent, and their amino acid compositions have been determined. 相似文献
11.
F J Joubert 《European journal of biochemistry》1976,64(1):219-232
Three toxins (CM-8, CM-11, and CM-13a) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose. Whereas toxin CM-8 and CM-11 comprise 60 amino acid residues, toxin CM-13a contains 61 residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have been elucidated. The reduced and S-carboxymethylated toxins were digested with trypsin and chymotrypsin and the peptides purified by ion-exchange chromatography, gel filtration and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequencer or by manual manipulation, was employed to obtain the sequence of the intact toxins and the pure peptides. The chymotryptic digests provided the necessary overlapping peptides which allowed the alignment of the tryptic peptides. The properties of the three toxins were compared with those of the cytotoxin group. The toxicities the serological properties, the sequences and the invariant amino acid residues of toxin CM-8 and CM-11 resemble the corresponding properties of the cytotoxin group. The sequence and serological properties of toxin CM-13a show that it is related to the cytotoxin group, but its toxicity is much lower than those encountered in the cytotoxin group. 相似文献
12.
Acid extracts of Xenopus laevis skin were fractionated by gel filtration on Sephadex G50 ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC). Peptides related to thyrotropin releasing hormone (TRH) were identified in the eluted fractions by trypsin digestion and radioimmunoassay (RIA) using antibodies to the TRH tripeptide pGlu-His-Pro amide or to a TRH-related pentapeptide pGlu-His-Pro-Gly-Lys. In addition to the tripeptide hormone, evidence was obtained for the presence of peptides containing 10-20 amino acid residues which were extended on the NH2-terminal or COOH-terminal side of TRH. The peptides extending on the NH2-terminal side predominated and were shown to comprise 5 components present in differing concentrations, indicating that the processing sites in the TRH prohormone vary in their susceptibility to proteolysis. Evidence was also obtained for the presence of small amounts of the TRH-related pentapeptide pGlu-His-Pro-Gly-Lys. Using similar procedures it was demonstrated that TRH extended peptides were present in bovine hypothalamus. In this species the peptides extended at the NH2-terminus of TRH occurred in similar concentrations to the peptides extended at the COOH-terminus. The results show that processing of the TRH prohormone in Xenopus and ox leads to the formation of peptides intermediate in size between the prohormone and the tripeptide amide; the TRH extended peptides occur in significant quantity and in Xenopus are formed with a high degree of specificity. 相似文献
13.
L R Ferguson G W Rewcastle J M Lello P G Alley R N Seelye 《Analytical biochemistry》1984,143(2):325-332
A method has been developed for extraction and purification of the major bile acids in human feces, and for their quantitative estimation using high-pressure liquid chromatography. Freeze-dried fecal material was extracted with alkaline ethanol and, after removal of neutral steroids, was subjected to thin-layer chromatography, followed by reversed-phase C18 silica cartridge (Sep-Pak) purification. The mixture was further separated into free, and glyco and tauro conjugates by ion-exchange chromatography. Subsequent resolution of individual bile acids was accomplished by HPLC using a counterion pairing method. 相似文献
14.
G L Rosenquist K Maruthainar D G Smyth 《Biochemical and biophysical research communications》1986,134(1):14-20
Lipotropin, beta-endorphin and a series of peptides related to beta-endorphin were extracted from rat antrum and resolved by gel filtration, ion exchange chromatography and high pressure liquid chromatography; the concentrations of the peptides were determined by radioimmunoassay. The major peptide with beta-endorphin immunoreactivity present in the antrum was lipotropin but it was accompanied by substantial quantities of beta-endorphin in its biologically active form; in addition there were minor quantities of a number of inactive beta-endorphin related peptides. The experiments demonstrate that in rat antrum gastrin can be accompanied by both active and inactive forms of beta-endorphin. The implications of post-translational processing mechanisms common to gastrin and beta-endorphin are discussed. 相似文献
15.
Amino acid sequence around the active-site serine residue in the acyltransferase domain of goat mammary fatty acid synthetase. 总被引:2,自引:1,他引:1
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
J Mikkelsen P H?jrup M M Rasmussen P Roepstorff J Knudsen 《The Biochemical journal》1985,227(1):21-27
Goat mammary fatty acid synthetase was labelled in the acyltransferase domain by formation of O-ester intermediates by incubation with [1-14C]acetyl-CoA and [2-14C]malonyl-CoA. Tryptic-digest and CNBr-cleavage peptides were isolated and purified by high-performance reverse-phase and ion-exchange liquid chromatography. The sequences of the malonyl- and acetyl-labelled peptides were shown to be identical. The results confirm the hypothesis that both acetyl and malonyl groups are transferred to the mammalian fatty acid synthetase complex by the same transferase. The sequence is compared with those of other fatty acid synthetase transferases. 相似文献
16.
In vitro poly(ADP-ribosyl)ation of seminal ribonuclease 总被引:1,自引:0,他引:1
The site of in vitro ADP-ribosylation of seminal ribonuclease was determined. Seminal enzyme was found to be a good receptor of [14C]ADP-ribose residues under the reaction conditions used. The recovery of [14C]ADP-ribosylated RNase was about 65% after purification. After tryptic digestion of modified enzyme, a fraction containing [14C]ADP-ribosylated peptides was separated from the others by ion-exchange chromatography on M82 resin. Radioactive peptides were then purified by affinity chromatography on anti-poly(ADP-ribose)IgG-Sepharose. High performance liquid chromatography of a mixture obtained after pronase digestion of purified ADP-ribosylated peptides revealed only one radioactive peptide whose amino acid composition corresponded to a peptide that has equimolar quantities of aspartic acid, serine, and glycine. Carboxypeptidase Y digestion of this peptide showed that its amino acid sequence was Asp-Ser-Gly. Only position 14-16 of seminal RNase corresponded to this sequence. The chemical stability of the ADP-ribose/enzyme linkage indicated that aspartic acid 14 is the modification site in seminal RNase. 相似文献
17.
Major conjugated bile acids of human bile have been resolved by high-pressure liquid chromatography. The elutions are carried out in two stages on Corasil II or μPorasil columns; first, an alkaline solvent system (2-propanol/ethyl acetate/water/7n ammonium hydroxide, 260:600:50:3) was used for separation into groups: tauro-dihydroxy derivatives, taurocholate, glyco-dihydroxy derivatives, and glycocholate. The fraction containing glyco-dihydroxy conjugates was separated by rechromatography in acetonitrile/acetic acid, 400:10, and the fraction containing tauro-dihydroxy conjugates could be partially resolved by rechromatography in acetonitrile/acetic acid/formic acid (97%)/water, 500:10:5:10. Three samples of prepared human bile have been similarly treated. 相似文献
18.
García MC 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,825(2):111-123
The study of the effect of mobile phases on sensitivity in the analysis of peptides and proteins by high-performance liquid chromatography (HPLC)-electrospray mass spectrometry (ESI-MS) has been the aim of this review. Reversed-phase chromatography (RPLC) is the chromatographic mode most suitable for coupling with ESI-MS since mobile phases containing organic modifiers are used. The analysis of proteins and peptides by RPLC mostly involves the use of trifluoroacetic acid (TFA) as an ion-pairing agent despite its being a strong suppressor of the MS signal. Different studies reporting the effects of using other ion-pairing agents (other perfluorinated acids, acetic acid, formic acid, etc.) and buffers (ammonium acetate, ammonium formate, ammonium bicarbonate, morpholine, etc.) in RPLC-ESI-MS of proteins and peptides did not yield a single strong candidate that could generally replace TFA. The enhancement in sensitivity with other reagents observed in some cases strongly depended on the analyte, the experimental conditions used, and the mass spectrometer and, usually, it did not compensate for the loss in separation resolution related to TFA. The examples of direct coupling of affinity, size-exclusion, or ion-exchange chromatography (IEC) to ESI-MS are very limited because of incompatibilities related to the use of mobile phases containing high salt concentrations. To overcome this problem, an intermediate desalting step is needed. Multidimensional chromatography, microdialysis, and ion-capture modules can be used to couple these chromatographic modes with ESI-MS. Multidimensional chromatography with RPLC as a second dimension has most often been used. Although most examples involve the trap and analysis in the second dimension of a certain part of the first separation, some comprehensive analyses of the entire sample in the second dimension have also appeared. 相似文献
19.
绵羊生殖道抗菌肽 总被引:2,自引:0,他引:2
以屠宰场收集的新鲜、健康、雌性绵羊生殖器官为原材料.采用乙酸浸提、透析、Sephadex G-50凝胶过滤层析和反相高效液相色谱(RP-HPLC)等方法分离纯化绵羊生殖道抗菌肽.以G+、G-和真菌为抗菌活性检测指示菌株,利用薄层琼脂糖孔穴扩散法、微量肉汤稀释法进行抗菌活性检测.对分离纯化所得纯品进行分子质量质谱测定、纯度鉴定、N端测序,并对其性质进行研究.结果表明:分离纯化所得两个绵羊生殖道抗菌肽分子质量分别为4820.47 u和4012.5 u,N端部分氨基 酸序列分别为AYVLDEPKP和YDSGA.对G+细菌(S. aureus ATCC2592、Streptococcu ATCC55121)、G-细菌(E. coli ATCC25922)、真菌(C. albicans ATCC2002)均具有良好的抑菌活性.对家兔红细胞无溶血活性,对人血液凝固无影响.目前未见有从绵羊生殖道分离纯化得到抗菌肽的报道,并且这一研究结果进一步证实抗菌肽在多种动物生殖道天然免疫防御方面起着重要作用. 相似文献
20.
A highly effective procedure for fragmenting the Neurospora crassa plasma membrane H+-ATPase and purifying the resulting peptides is described. The enzyme is cleaved with trypsin to form a limit digest containing both hydrophobic and hydrophilic peptides, and the hydrophobic and hydrophilic peptides are then separated by extraction with an aqueous ammonium bicarbonate solution. The hydrophilic peptides are fractionated by Sephadex G-25 column chromatography into three pools, and the individual peptides in each pool are purified by high-performance liquid chromatography. The hydrophobic peptides are dissolved in neat trifluoroacetic acid (TFA), diluted with chloroform-methanol (1:1), and the hydrophobic peptide solution thus obtained is then fractionated by Sephadex LH-60 column chromatography in chloroform-methanol (1:1) containing 0.1% TFA. The recoveries in all of the above procedures are greater than 90%. The N-terminal amino acid sequences of three of the hydrophobic H+-ATPase peptides purified by this methodology have been determined, which establishes the position of these peptides in the 100,000 Da polypeptide chain by reference to the published gene sequence, and documents the sequencability of the hydrophobic peptides purified in this way. This methodology should facilitate the identification of a variety of amino acid residues important for the structure and function of the H+-ATPase molecule. Moreover, the overall strategy for working with the protein chemistry of the H+-ATPase should be applicable to other amphiphilic integral membrane proteins as well. 相似文献