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1.
Synaptic membranes from human and swine brains were solubilized with 8 M urea and the proteins were reduced and alkylated. A protein was isolated from both sources and had identical amino acid compositions and molecular weights as determined by electrophoresis on polyacrylamide-sodium dodecylsulfate gels and by ion-exchange chromatography and gel filtration on Bioglas 1000. The apparent molecular weight of the protein was 53 000 on the acrylamide-sodium dodecylsulfate gels. Neither neutral sugars nor sialic acid was a significant component of the protein. When the proteins were digested with trypsin and the resultant peptides subjected to chromatography (n-butanol/acetic acid/water) and electrophoresis (pH 3.7) the peptide maps were identical. The protein comprises 1-2 percent of the total synaptosomal protein. With regard to amino acid composition, molecular weight, peptide map characteristics, behavior on DEAE-cellulose columns, electrophoretic mobility and sugar content, the synaptic protein is quite similar to the monomer of swine tubulin. 相似文献
2.
The H1 histones of the unicellular green alga Chlamydomonas reinhardtii were extracted from isolated nuclei, fractionated by high performance liquid chromatography, and analyzed by two-dimensional electrophoresis, peptide mapping, and N-terminal sequencing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 5% perchloric acid extracts of isolated C. reinhardtii nuclei revealed two H1 proteins (H1A and H1B). Two-dimensional gel analysis did not reveal heterogeneity of either algal H1 protein, but did detect differences in the hydrophobic amino acid content of the C. reinhardtii H1A and H1B. Digestion of H1A and H1B with V8 protease revealed two distinctly different peptide maps. C. reinhardtii H1 peptide maps were not at all similar to those of Pisum H1, but algal and pea H2B peptide maps did show some peptides in common. Seventeen amino acid residues were obtained from C. reinhardtii H1A amino terminal sequencing, while the H1B N-terminus was blocked. A search of protein data bases revealed no sequence homology of the H1A N-terminus with any known protein. Chlamydomonas histones fractionated by high performance liquid chromatography revealed minor components (histone variants) for H2A and H2B. The amino acid composition of Chlamydomonas lysine-rich histones was compared to those of various other unicellular algae. 相似文献
3.
Peptide mapping of heterogeneous protein samples. 总被引:5,自引:0,他引:5
A simple two-dimensional electrophoretic method for peptide mapping of heterogeneous protein samples is presented. The reduced and denatured proteins of the mixture are separated in a first dimension by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. After completion of the electrophoresis, the whole gel lane is equilibrated in stacking gel buffer and is transferred at right angles onto a second slab gel. A protease solution is overlayed on the gel lane and a partial proteolysis of the proteins to be analyzed is performed during the stacking phase of the second electrophoresis. The second electrophoresis resolves the characteristic pattern of peptides of each individual protein as a series of spots located below the original position of the undigested protein. The peptide maps of the following samples are presented as examples: protein P23 and P23* of bacteriophage T4, membranes of Dictyostelium discoideum, membranes of human erythrocytes, and 35S-labeled proteins of D. discoideum synthesized in vivo or in a cell-free wheat germ extract. In complex samples, up to 20 individual proteins can be analyzed at once and a protein comprising only 1% of the total sample generates a clearly identifiable peptide pattern. Good reproducibility of the patterns obtained allows the comparison of samples of different origins. 相似文献
4.
E. Kaltschmidt M. Dzionara D. Donner H. G. Wittmann 《Molecular & general genetics : MGG》1967,100(4):364-373
Summary The proteins of E. coli ribosomes were separated by a specially developed type of preparative polyacrylamide gel electrophoresis and proteins corresponding to 16 of the separated bands have so far been isolated. The amino acid compositions and the tryptic peptide maps of these proteins show certain degree of similarity as well as distinct differences. Determination of molecular weights revealed a wide range: The lowest molecular weight was 9,000 and the highest 41,000 for the proteins so far studied.The similarities between various ribosomal proteins in their amino acid compositions and their peptide maps on one hand and the wide range in their molecular weights on the other hand can be explained by a hypothesis involving gene duplications. 相似文献
5.
High-mobility group chromosomal proteins of wheat 总被引:4,自引:0,他引:4
S Spiker 《The Journal of biological chemistry》1984,259(19):12007-12013
Four proteins have been extracted from purified chromatin of wheat embryos with 0.35 M NaCl. These proteins are soluble in 2% (w/v) trichloroacetic acid and thus meet the original operational requirements to be classified as "high-mobility group" (HMG) chromosomal proteins. The proteins have been characterized by one- and two-dimensional electrophoresis, amino acid analysis, and peptide mapping. Three of the proteins (HMGb, c, and d) share the mammalian HMG characteristic of being rich in both acidic and basic amino acid residues. Unlike their putative mammalian counterparts, these plant HMG proteins contain less than 7 mol % proline. The fourth wheat protein (HMGa) is rich in both proline and in basic amino acid residues. This wheat protein, however, contains only about half the proportion of acidic residues found in mammalian HMG proteins--a characteristic also found in the trout testis HMG protein, H6. Comparative peptide maps show that none of the wheat HMG proteins are degradation products of other HMG proteins or the H1 histones. The peptide maps have not, however, been useful in establishing homologies with mammalian HMG proteins. Wheat HMG proteins are released from DNase I-treated nuclei and co-isolate with micrococcal nuclease-sensitive chromatin fractions. Similar observations concerning the HMG proteins of vertebrate animals have been considered consistent with a role for these proteins as structural components of actively transcribed chromatin. 相似文献
6.
报道了一个通过有限酶切蛋白质产生多肽片段的方法.蛋白质经单向SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离和用考马斯亮蓝短暂染色后,切下所需的蛋白质带,将其放入另一个SDS-PAGE凝胶的样品槽内,在电泳过程中该蛋白质被蛋白酶如蛋白酶V8降解,所产生的多肽片段随之被分离.电泳结束后,将多肽片段电印迹至聚偏二氟乙烯(polyvinylidene difluoride,PVDF)膜上.这些多肽片段从PVDF膜上切下后可以直接被用于分析氨基酸序列.该方法能广泛适用于分析一般蛋白质和N端被修饰蛋白质的氨基酸序列. 相似文献
7.
J R Davie 《Analytical biochemistry》1985,144(2):522-526
A method to obtain peptide maps of basic proteins on acetic acid/urea (AU) -polyacrylamide minislab gels is presented. Basic proteins such as the histones are digested with Staphylococcus aureus V8 protease in the stacking gel (pH 4) of an AU-polyacrylamide minislab gel. As the peptides are resolved in the AU minislab gel on the basis of charge and size, it is possible to separate peptides containing modified amino acids from the unmodified, parent peptide. The peptide(s) containing the modified residue may be identified following electrophoresis on a second-dimension sodium dodecyl sulfate-polyacrylamide minislab gel. This procedure will be useful for comparing histone variants and for the study of histone modifications. 相似文献
8.
Ralph L. Nachman Eric A. Jaffe Barbara Ferris 《Biochemical and biophysical research communications》1980,92(4):1208-1214
Factor VIII antigen from platelet intracellular granules was immunoprecipitated using a monospecific rabbit antibody to normal plasma factor VIII antigen. The factor VIII antigen in the immunoprecipitate was isolated on sodium dodecyl sulfate polyacrylamide gels, radiolabeled with 125I, trypsinized, and subjected to peptide mapping using two dimensional high voltage electrophoresis and thin layer chromatography. The platelet protein was compared to purified plasma factor VIII antigen. The two dimensional tryptic 125I peptide map of platelet granule factor VIII antigen was similar but not identical to the plasma factor VIII antigen peptide map. The platelet and plasma proteins shared approximately 34 radioactive peptide spots. Seven plasma factor VIII antigen peptides were not detected in platelet factor VIII antigen. The reason for the structural differences of plasma and platelet granule factor VIII antigen are unknown. The possibility is raised that proteolysis has altered the platelet protein in vitro. It is also possible that factor VIII antigen synthesized by megakaryocytes differs from the plasma protein. 相似文献
9.
A comparison of molecular properties of hepatic triglyceride lipase and lipoprotein lipase from human post-heparin plasma 总被引:7,自引:0,他引:7
Hepatic triglyceride lipase was isolated from human post-heparin plasma by the method of Ehnholm et al. using modifications which increased the specific activity 12-fold to approximately 3,000 mumol of free fatty acid/h/mg of protein. Lipoprotein lipase with similar specific activity was prepared from the same plasma samples using heparin and concanavalin A affinity chromatography. The molecular weight of hepatic triglyceride lipase (69,000) was slightly greater than that of lipoprotein lipase (67,000) as determined by polyacrylamide electrophoresis in sodium dodecyl sulfate-containing buffers. These proteins had identical amino acid compositions, terminal amino acid residues, and tryptic peptide maps. However, the differences previously described regarding optima of pH and ionic strength and the requirement for apolipoprotein CII (only for lipoprotein lipase) were maintained in the highly purified state. It was found that both proteins contain approximately 8% carbohydrate. Antisera prepared in goats selectively precipitated each activity. Other antisera prepared in chickens reacted with both enzymes, suggesting a common antigenic determinant. 相似文献
10.
A number of hydrophobic proteins have been separated and purified to varying degrees from synaptic membranes derived from bovine brain. The proteins, which have been obtained using preparative acrylamide gel electrophoresis, have been analyzed for molecular weight, amino acid composition, peptide mapping, N-terminal amino acids, and for their ability to bind calcium and ATP. A number of the proteins bound calcium, the greatest binding being associated with a component having a molecular weight of 1.5 · 104, a binding capacity of 4 calcium/molecule, and a Km of 1.5 · 10?5 M. An acidic tryptic peptide derived from this protein was evidently responsible for the calcium-binding. ATP binding appeared to be confined largely to the higher molecular weight proteins. From the peptide mapping there appears to be a similar acidic component in a number of the proteins exhibiting calcium-binding. ATP-binding was associated mainly with the high molecular weight proteins, particularly those which consisted of numerous basic tryptic peptides. 相似文献
11.
A combination of new mass spectrometric methods that can be used to determine the primary structures of proteins, including post-translational modifications, with unprecedented speed and accuracy is described. Structural characterization of alpha-crystallins from bovine lenses has been used to illustrate the methods. The molecular weights of alpha-crystallins fractionated, but not to homogeneity, by reversed-phase HPLC were determined with an uncertainty of 0.01% which is at least 100 times more accurate than is possible using conventional methods. This information was used to identify the primary gene product as well as its phosphorylated and truncated forms. Molecular weight maps of proteolytic digests of these proteins were determined by directly coupled capillary HPLC fast atom bombardment-mass spectrometry. From these maps, the entire amino acid sequence was confirmed, and the phosphorylated peptide was identified. The MS/MS daughter ion mass spectrum of the phosphorylated peptide provided sufficient information to determine which residue was phosphorylated. Because protein structure, including post-translational modifications, is determined on the basis of molecular weight, this method has broad application and will be useful for a variety of diverse and challenging problems in protein structure elucidation. 相似文献
12.
Similarities between stratum corneum basic protein and histidine-rich protein II from newborn rat epidermis 总被引:2,自引:0,他引:2
The stratum corneum basic protein and histidine-rich protein II were each isolated from newborn rat epidermis and compared by biochemical and immunologic methods. The proteins were indistinguishable by immunodiffusion using antiserum elicited to either protein. The migration of the proteins on SDS-polyacrylamide gel electrophoresis was identical giving a molecular weight of 49 000. These proteins, which have similar but unusual amino acid compositions, give very similar tryptic peptide maps. Both proteins aggregate with keratin filaments to form macrofibrils. These results suggest that histidine-rich protein II and stratum corneum basic protein are the same protein. We suggest that this protein be called histidine-rich basic protein. 相似文献
13.
1. Myeline proteins in bovine peripheral nerve migrated as two main band-(BF and BR protein) and one faint middle band (BM protein) on sodium dodecyls sulfate-polyacrylamide gel electrophoresis. The relative mobility of these two main bands differed from those of myelin proteins in the central nervous system. 2. The acid extract of the myelin fraction from bovine peripheral nerve was separated into one main peak and two minor peaks on a Sephadex G-75 column. The major component of the second minor peak was the BM protein; the major component of the main peak was the BF protein. The BR protein was not extractable by acid solution. 3. Molecular weights of the BF, the BM and the BR protein were determined as around 13 000, 20 000 and 28 000, respectively, by sodium dodecylsulfate-polyacrylamide gel electrophoresis. 4. The amino acid composition of the BF protein was quite different from the encephalitogenic protein and the Folch-Lees type proteolipid protein in the central nervous system. However the BM protein showed similar amino acid composition to the encephalitogenic protein. 5. The tryptic peptide maps of the BF protein and of the encephalitogenic protein were quite different. The results suggested that the amino acid sequences of these two proteins are different and that they contain no common tryptophan-containing peptide. 相似文献
14.
Location and purification of enzymes on polyacrylamide gels using dialyzable fluorescent markers 总被引:1,自引:0,他引:1
A method for the location of proteins/enzymes by polyacrylamide gel electrophoresis using dialyzable low-molecular-weight fluorescent peptide markers is described. The markers prepared by treating the peptic digest of casein with fluorescamine showed several bands on gel electrophoresis which helped in locating the proteins. The located desired protein could subsequently be purified by extraction. 相似文献
15.
Isoforms of corneal keratan sulfate proteoglycan 总被引:6,自引:0,他引:6
Bovine corneal keratan sulfate proteoglycan was found to contain three major protein components. Two proteins (37 and 25 kDa) were released from the proteoglycan by endo-beta-galactosidase, N-glycanase, or chemical deglycosylation. A smaller protein (20 kDa), not covalently linked to keratan sulfate, co-purified with the proteoglycan by conventional and high performance ion exchange chromatography, by ethanol precipitation, and by affinity purification on columns of monoclonal antibody to keratan sulfate, but could be separated from the proteoglycan by gel filtration chromatography in dissociative agents. The three proteins produced different fragmentation patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after digestion with V8 protease, and each had unique two-dimensional tryptic peptide maps. The N-terminal amino acid sequence of the core proteins differed. In addition, the proteoglycans containing these proteins differed in molecular size, suggesting different levels of glycosylation of the two core proteins. Similarity of the core proteins was suggested by similar amino acid composition, similarities in tryptic maps, and antigenic cross-reactivity. Corneal keratan sulfate proteoglycan, therefore, seems to occur in two different, but related, forms whose core proteins may represent members of a homologous family. 相似文献
16.
Signal peptides used in biosynthesis of proteins are cleaved at a very specific site by signal peptidase during posttranslational translocation of cytoplasmic proteins across the membrane. In some cases, however, there can be cleavage at nonspecific sites, giving rise to heterogeneity in the mature protein, which manifests itself as either elongation or truncation of the N terminus of the mature protein. When used as biopharmaceutical therapeutics, such heterogeneities may be a cause for concern, depending on the nature of the heterogeneity. This article describes the determination of such heterogeneity by peptide mapping in both the heavy chain and the light chain (LC) of a Chinese hamster ovary (CHO) cell-expressed monoclonal antibody (mAb). The peptide map method described here was capable of detecting the extended N-terminal peptides at levels as low as 1% relative to the peak area of the intact N-terminal peptide. The LC of a mAb product was truncated at its N termini by two amino acid residues at approximately 3-4% levels, resulting from alternate signal peptide cleavage. This article describes the quantitation of this truncation by liquid chromatography-mass spectrometry (LC-MS) peptide mapping. Also described is analysis and characterization of LC truncation by reduced and denatured capillary electrophoresis in sodium dodecyl sulfate (CE-SDS). The truncated mAb, which was devoid of the two N-terminal amino acids, was engineered and shown to migrate as the “pre-LC” peak in reduced CE-SDS assay. The amount of the pre-LC peak recovered from the CE-SDS assay was shown to correlate with the amount of truncated peptide observed from the reduced and alkylated peptide map of the engineered mAb. 相似文献
17.
A polypeptide cardiotoxin containing 60 amino acids with 4 disulfide bonds has been synthesized by the "fragment solid-phase" method. The identity of the synthetic product with native cardiotoxin was established by chromatography on Sephadex G-50, carboxymethyl-cellulose column chromatography, thin layer chromatography, disc gel electrophoresis, amino acid analysis, end group analysis, peptide mapping, circular dichroism spectra, and four biological tests. 相似文献
18.
Computer prediction of peptide maps: assignment of polypeptides to human and mouse mitochondrial DNA genes by analysis of two-dimensional-proteolytic digest gels. 总被引:10,自引:2,他引:8
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D C Wallace J H Yang J H Ye M T Lott N A Oliver J McCarthy 《American journal of human genetics》1986,38(4):461-481
We have prepared a computer program that predicts complete and partial peptide maps from amino acid sequences. The program fragments amino acid sequences at designated cleavage sites and calculates the molecular weight and relative labeling of each peptide. These data are graphed as log molecular weight of the original protein (X-axis) vs. log molecular weight of the component peptides (Y-axis). The program is interactive, permitting adjustment of a number of graphic parameters and alteration of the position of proteins in the first dimension to accommodate aberrations in protein mobility. The program has been used to predict the V8 protease peptide maps of the 13 open reading frames (ORFs) identified in the human and the mouse mitochondrial DNA (mtDNA) sequences. The results were compared to the V8 protease peptide maps obtained for mouse and human mitochondrially synthesized proteins by two-dimensional proteolytic digest gels. A high correlation was observed between the predicted and observed peptide maps. These results suggest the assignment of several proteins to mtDNA genes. 相似文献
19.
Protein samples prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis are preferentially cleaved at aspartyl-prolyl peptide bonds upon heating at 110 degrees C. The presence of aspartyl-prolyl peptide bonds in a protein can therefore be detected by gel electrophoresis of heated samples and the resulting peptides mapped. The method of heat cleavage also works well with proteins in bands cut from electrophoresed gels using modified stacking conditions in the second electrophoresis. An immunoblotting procedure for peptide mapping of nanogram quantities of specific proteins in complex mixtures is demonstrated. Peptide maps produced by aspartyl-prolyl peptide bond cleavage of fructose-1,6-bisphosphatases from different sources show the effectiveness of the above techniques and suggest a conservation of aspartyl-prolyl peptide bonds in pig kidney and mouse and rat liver fructose-1,6-bisphosphatases. 相似文献
20.
J. A. Reig J. M. Ramos M. Cozar J. S. Aguilar M. Criado J. Monreal 《Journal of neurochemistry》1982,39(2):507-511
Abstract: Starting from a pellet of beef brain myelin insoluble in chloroform/ methanol (2:1, vol/vol) (Wolfgram protein fraction), a pure W2 protein with apparent molecular weight of 52,000 was isolated by a simple preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. A comparative chemical analysis was carried out between purified W2 and a standard tubulin. Glutamic acid and arginine were the N-terminals detected. Similar peptide maps and amino acid composition were also found in both proteins. Immunological cross-reactivity was detected when W2 protein was tested against antitubulin serum. These results suggest that W2 protein could have a tubulin-like protein nature that is associated with the myelin membrane and could play a role in the myelination process. 相似文献