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1.
We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins. 相似文献
2.
3.
Characterization of B and H blood-group active glycosphingolipids from human B erythrocyte membranes
Peter Hanfland 《Chemistry and physics of lipids》1975,15(2):105-124
Two blood group B active glycosphingolipids (B-I and B-II) previously isolated and highly purified from human B erythrocytes [21] were analysed first by degradation with α-D-galactosidase from coffee beans, α-L-fucosidase from bovine kidney and with 0,1 N trichloracetic acid; the native B-glycolipids as well as their degradation products were then investigated by methylation analysis with combined gas chromatography-mass spectrometry, by thin layer chromatography, twodimensional immunodiffusion and by the hemagglutination inhibition technique. Together with the results obtained by mass spectrometry of permethylated glycolipids [26] the following structures were elucidated: α-D-galactopyranosyl-(1 → 3)-[α-L-fucopyranosyl-(1 → 2)]-D-galactopyranosyl-(1 → 4)-N-acetyl-D-glucosaminosyl-(1 → 3)-D-galactopyranosyl-(1 → 4)-D-glucopyranosyl-(1 → 1)-ceramide for the B-I glycosphingolipid and α-D-galactopyranosyl-(1 → 3)-[α-L-fucopyranosyl-(1 → 2)]-D-galactopyranosyl-(1 → 4)-N-acetyl-D-glucosaminosyl-(1 → 3)-D-galactopyranosyl-(1 → 4)-N-acetyl-D-glucosaminosyl-(1 → 3)-D-galactopyranosyl-(1 → 4)-D-glucopyranosyl-(1 → 1)-ceramide for the B-II glycosphingolipid. A H active glycolipid fraction from B erythrocytes further purified by thin layer chromatography was also investigated by methylation analysis. The pattern of its partially methylated alditol acetates was essentially the same as that of the α-galactosidase treated and permethylated B-I glycolipid. It also exhibited strongly precipitating and hemagglutination inhibiting H properties as well as the two α-galactosidase treated B-I and B-II glycosphingolipids. Based upon these data the following tentative structure was proposed: α-L-fucopyranosyl-(1 → 2)-D-galactopyranosyl-(1 → 4)-N-acetyl-D-glucosaminosyl-(1 → 3)-D-galactopyranosyl-(1 → 4)-D-glucopyranosyl-(1 → 1)-ceramide. Gas chromatographic analysis revealed sphingosine and lignoceric, nervonic and behenic acids to be the main components of the ceramide residues of the three glycosphingolipids. From the data presented the H active substance very probably can be regarded as the immediate precursor of the B-I glycosphingolipid from human B erythrocyte membranes. 相似文献
4.
Neurotransmitter-caused increase in [3H]inositol incorporation into phosphatidylinositol de novo synthesis vs exchange 总被引:1,自引:0,他引:1
[3H]inositol and 32Pi were simultaneously incorporated into rat parotid phosphatidylinositol. The ratio of [3H]/32Pi incorporation dropped dramatically following stimulation with muscarinic or alpha-adrenergic agonists and returned to control values following the addition of appropriate antagonists. The drop in [3H]/32Pi ratio can be explained by a rapid increase in de- novo synthesis of phosphatidylinositol following its receptor-mediated breakdown. The change in this ratio also provided evidence for the existence of CDP-DG + inositol in equilibrium phosphatidylinositol exchange reaction in the intact tissue. 相似文献
5.
- 1.
- 1. The net uptake of α-aminoisobutyric acid (AIB) in Ehrlich ascites tumor cells has been studied under a variety of transmembrane concentration gradients of Na+, K+ and AIB itself. 相似文献
6.
Ben M. De Rooij Jan N.M. Commandeur Jane R. Ramcharan Hilda C.P. Schuilenburg Ben L.M. Van Baar Nico P.E. Vermeulen 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,685(2):1402
Epichlorohydrin (ECH) is used in many industrial processes. Different toxic effects of ECH were found in rodents. The metabolism of ECH was investigated before in rats using [14C]ECH. The aim of this investigation was the development of non-radioactive quantitative analytical methods for measuring two urinary metabolites of ECH, namely 3-chloro-2-hydroxypropylmercapturic acid (CHPMA) and α-chlorohydrin (α-CH). The identity of CHPMA and α-CH excreted in urine of rats treated with 5 to 35 mg/kg ECH was confirmed by GC-MS. The quantitative analysis of CHPMA, involving ethyl acetate extraction from acidified urine and subsequent methylation and analysis by gas chromatography-flame photometric detection (GC-FPD), showed a method limit of detection of 2 μg/ml. The analysis of α-CH, based on ethyl acetate extraction and subsequent analysis by GC-ECD, showed a method limit of detection of 2 μg/ml. CHPMA and α-CH derivatives could be determined quantitatively down to concentrations of 0.5 and 0.4 μg/ml urine, respectively, by selected-ion monitoring GC-MS under EI conditions. Cumulative urinary excretion of CHPMA and α-CH by rats treated with ECH were found to be 31 ± 10 and 1.4 ± 0.6% (n = 13) of the ECH dose, respectively. For CHPMA, the dose-excretion relationship suggested partially saturated ECH metabolism. For α-CH, the dose-excretion relationship was linear. With fractionated urine collection it was found that approximately 74 and 84% of the total cumulative excretion of CHPMA and α-CH, respectively, took place within the first 6 h after administration of ECH. From these investigations it is concluded that the GC-FPD and GC-ECD based methods developed are sufficiently sensitive to measure urinary excretion of CHPMA and α-CH in urine from rats administered 5 to 35 mg/kg ECH. It is anticipated that the analysis of CHPMA and α-CH based on GC-MS may be sufficiently sensitive to investigate urinary excretion from humans occupationally exposed to ECH. 相似文献
7.
采用基因工程方法制取人胸腺素α原获得成功。用20ug/ml植物血球凝集素(PHA)和500U/ml重组人白细胞介素2(IL-2)联合刺激人胎儿胸腺细胞,从中提取总RNA,经反转录PCR获得了人胸腺素α原cDNA;将之克隆入pUC19中,序列测定表明与已报道序列一致,进一步将之亚克隆入原核表达载体pBV220,转化大肠杆菌DH5a.观察到在不改变氨基酸编码的前提下,增加胸腺素a原上游引物中A、T含量,可以明显提高胸腺素α原的表达量,同时,不同培养基对它的表达也有影响。胸腺素α原在大肠杆菌中以可溶形式表达,不需复性。初步活性测定显示,它可明显刺激人外周血淋巴细胞E-玫瑰花结形成率。重组人胸腺素α原在大肠杆菌中表达,为其临床应用及基础研究奠定了基础。 相似文献
8.
Mark A. Brown Alan Carne Geoffrey K. Chambers 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1996,113(4):731-736
α2-Macroglobulin (α2-M), a large molecular mass proteinase-binding protein, was identified in plasma from tuatara (Sphenodon), a rare reptile endemic to New Zealand. In this genus, α2-M constitutes 11–13% of total plasma protein (∼2.2–3.9 mg/ml). Analysis of blood samples collected at approximately monthly intervals from individual tuatara indicated that the plasma level of α2-M remains fairly constant. The subunits of tuatara α2-M have an apparent molecular mass of ∼160 kDa as determined by SDS-polyacrylamide gel electrophoresis and the intact protein is an oligomer that contains inter-chain disulfide bonds. N-terminal sequence analyses of tuatara α2-M revealed a distinct similarity to α-macroglobulins of other vertebrates and that at least two types of α2-M subunits are present in plasma of tuatara. 相似文献
9.
A 250-kilodalton cellular protein is induced by progestins in two human breast cancer cell lines MCF7 and T47D 总被引:1,自引:0,他引:1
We have studied the effect of R5020, a synthetic progestin, on the biosynthesis of cellular proteins extracted from the MCF7 and T47D human breast cancer cells, using gel electrophoresis. R5020 stimulates the synthesis, as measured after [35S]-methionine labelling, and the accumulation, as shown by silver staining, of a protein of molecular weight approximately equal to 250,000. The increase of the labelled 250-kilodalton protein was rapid (3 hours) and after 3 days this protein represented approximately equal to 6% of the total cellular proteins (approximately equal to 1 microgram/150,000 cells). The induction of the 250-kilodalton protein was obtained by physiologically active concentrations of several progestins and high concentrations of 5 alpha-dihydrotestosterone but not by estradiol or dexamethasone. It was inhibited by R486 , a progestin antagonist, but not by flutamide, an androgen antagonist. These results indicate a mediation by the progesterone receptor. The 250-kilodalton protein appears to be an excellent probe to study in cell culture the mechanism of action of progestin on human cells. 相似文献
10.
A successful approach has been developed for the sequencing of apolipoprotein B based upon the procedure of Cleveland et al. [(1977) J. Biol. Chem. 252, 1102-1106] involving limited proteolysis in the presence of sodium dodecyl sulfate. Staphylococcus aureus protease was employed to produce large peptides which were isolated in relatively pure form by preparative gel electrophoresis. Two peptides were partially sequenced using spinning-cup microsequencing techniques. The sequences are: Peptide R2-5, -Ala-Leu-Val-Gly-Ile-Asn- Gly-Glu-Ala-Asn-Leu-Asp-Phe-Leu-Asn-Ile-Pro-Leu-Arg-Ile-Pro-Pro- Met-Arg-(Arg)-; Peptide R3-1, -Leu-Val-Ala-Lys-Pro-Ser-Val-Ser-Val-Glu- Phe-Val-Thr-Asn-Met-Gly-Ile-Ile-Pro-Lys-Phe-Ala-Arg-. Several stretches of residues suitable for the construction of oligonucleotide probes have been identified. 相似文献