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PubMedDate not supplied

Specificities of rabbit antisera to multiple antigen (MAP) peptides.

Two multiple antigen peptides consisting of 6 and 7 aminoa cid residues, respectively, plus a 12-residue fragment, used as a control, all linked to a polylysine core, were used as immunogens in rabbits in order to obtain an immune response. Rabbit antisera against such polymers were then tested in ELISA against a panel of antigens in order to analyze the specificities of the resulting antibodies. The responses were different for all three immunogens, being partially or totally directed, for two of the three compounds, including the 12-residue control MAP peptide, against the polylysyl core, which is considered as non immunogenic. The third MAP polymer was practically unable to elicit an immune response.

Amino Acid Sequence↗
PubMed2001

Improvement of an in-gel tryptic digestion method for matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mapping by use of volatile solubilizing agents.

The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.

Amino Acid Sequence↗
PubMed1976

The molybdenum--iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide 'mapping'.

The molybdenum- and iron-containing protein components of nitrogenase purified from Klebsiella pneumoniae, Azotobacter vinelandii, Azotobacter chroococcum and Rhizobium japonicum bacteroids all gave either one or two protein-staining bands after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, depending on the commercial brand of sodium dodecyl sulphate used. The single band obtained with K. pneumoniae Mo-Fe protein when some commercial brands of sodium dodecyl sulphate were used in the preparation of the electrode buffer was resolved into two bands by the addition of 0.01% (v/v) dodecanol to the buffer. Protein extracted from the two bands obtained after electrophoresis of K. pneumoniae Mo-Fe protein gave unique and distinct peptide 'maps' after tryptic digestion. Undissociated Mo-Fe protein contained both sets of tryptic peptides. These data are consistent with Mo-Fe protein from K. pneumoniae being composed of non-identical subunits. Amino acid analyses of the subunit proteins revealed some clear differences in amino acid content, but the two subunits showed close compositional relatedness, with a different index [Metzer, H., Shapiro, M.B., Mosiman, J.E. & Vinton, J.G. (1968) Nature (London) 219, 1166-1168] of 4.7.

Amino Acids↗
PubMed1981

Biochemical characterization of Ia alloantigens in guinea pigs. II. Comparative peptide mapping of Ia antigens from B cells, T cells, and macrophages.

Radioactive Ia.4 molecules were prepared from 3H- or 14C-labeled splenocytes, selected PEL, or bronchoalveolar macrophages (M phi). Studies in the accompanying paper indicated that incorporation into Ia.4 in these 3 populations is due to B cells, T cells, and macrophages, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to isolate the 58,000 m.w. form of Ia.4. The eluted molecule was then reduced, and the resultant alpha and beta subunits were isolated by a second separation on SDS-PAGE. Alpha (or beta) chains from 1 population labeled with 3H-amino acids was mixed with alpha (or beta) chains obtained from a population representing a different cell type that was labeled with 14C-amino acids and the mixture was digested with trypsin. Double-label (3H/14C) comparative peptide mapping was performed using high-pressure liquid chromatography to separate the peptides. Eighteen to 20 peaks of radioactivity were resolved from alpha chains, and 12 to 15 from beta chains. No reproducible differences were observed when comparing alpha or beta chains of T cells and macrophages, or those of B cells and macrophages. These results indicate that the primary structure of Ia.4 molecules is identical on the 3 cell types in question. The implications of having a T cell bearing the same Ia that it recognized on a macrophage in conjunction with antigen is discussed.

Amino Acids↗
PubMed1977

[Quantitative dansyl micromethod of amino acid analysis. Study of peptide maps of ribonuclease tryptic hydrolysate].

Some variants of the quantitative dansyl method in combination with thin-layer chromatography on polyamide plates have been compared, and principles of its optimization have been demonstrated. Sensitivity of the method is about 10(-11) mole, the error does not exceed 10%. Data are given on the analysis by the dansyl method of peptides of the tryptic hydrolysate of bovine pancreatic ribonuclease after their separation by peptide mapping procedure.

Amino Acids↗
PubMed1986

Applications of chemical cleavage procedures to the peptide mapping of neurofilament triplet protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A procedure for examining possible sequence homology in the triplet neurofilament proteins using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system is described. Five different chemical reagents (cyanogen bromide, BNPS-skatole, hydroxylamine, formic acid, and nitrothiocyanobenzoic acid) have been used for peptide mapping studies. Potential applications of this technique are discussed.

Amino Acid Sequence↗
PubMed1985

Avian adductor profundus muscle: characterization of a pure slow tonic region by histochemical, monoclonal antibody and peptide mapping studies.

The fibre type composition of the avian adductor profundus (AP) muscle which is composed of a thick white posterior part (Post. AP) and a thin red anterior part (Ant. AP) was investigated. Using the histochemical ATPase technique, monoclonal antibody analysis of myosin and C-protein isoforms, and electrophoretic and peptide mapping analyses of myosin, we have established that the Post. AP is composed of essentially pure slow tonic fibres similar to those of the anterior latissimus dorsi muscle (ALD). The Ant. AP, on the other hand, is shown to contain a mixture of slow and fast fibres, the latter giving immunocytochemical reactions atypical of the fast fibres. The larger size of the Post. AP in comparison with the ALD muscle should provide significantly more tissue for biochemical studies of tonic fibres than was previously available.

Adenosine Triphosphatases↗
PubMed1984

Monoclonal antibodies and peptide mapping reveal structural similarities between the subunits of the glycine receptor of rat spinal cord.

The glycine receptor of rat spinal cord is an oligomeric membrane glycoprotein of molecular mass 250,000 daltons that contains three polypeptides of 48,000, 58,000, and 93,000 daltons. Monoclonal antibodies (mAbs) were prepared against the affinity-purified glycine receptor protein by using 125I-labeled receptor preparations for the detection of positive hybrids. From nine monoclonal antibodies obtained, six recognized denatured receptor polypeptides blotted to nitrocellulose paper. Two of these antibodies bound to more than one glycine receptor polypeptide: mAb GlyR 4a stained the 48,000- and 58,000-dalton polypeptides, and mAb GlyR 7a stained the 48,000- and 93,000-dalton polypeptides. Common antigenic determinants thus are shared by the different subunits of the glycine receptor. Complementary results were obtained by peptide mapping of 125I-labeled glycine receptor polypeptides with various proteases. A set of peptide fragments of the same apparent molecular mass was produced from the different glycine receptor polypeptides by using V8 protease, chymotrypsin, and elastase. These data suggest that the subunits of the glycine receptor have significant homology within their primary structure and may have evolved from a common ancestor receptor polypeptide.

Amino Acid Sequence↗
PubMed1982

Peptide map comparisons of similar serologically defined HLA-DR antigens isolated from different lymphoblastoid cell lines.

In these studies, we have examined the possibility that DR subtypes, closely related to each other in structure, compose the major DR allotypic groups. The structures of DR molecules, isolated from pairs of cell lines that had the same serologically defined HLA-DR type, were compared by peptide mapping. HLA-DR molecules isolated from pairs of cell lines in which both members were either DR1, DR2, or DR7 were identical. However, DR molecules isolated from cell lines LG-29 (DR5) and LG-38 (DR5) displayed two distinct differences in their small (beta) subunits. This number of differences, two of 15 peptides or 15%, is much fewer than is observed between DR allotypes (approximately 50%) and suggests that at least two subtypes exist within the DR5 allotype family.

Animals↗
PubMed1979

Peptide map comparison of the proteins of infectious bursal disease virus.

The genome of infectious bursal disease virus consists of two segments of double-stranded RNA of 2.5 X 10(6) and 2.2 X 10(6) molecular weight. Polyacrylamide gel electrophoresis of purified virus resolved four structural polypeptides: VP-1 (90,000), VP-2 (41,000), VP-3 (35,000), and VP-4 (28,000). Peptide map comparisons of radioiodinated virion proteins indicated no precursor-product relationship between them. The possible relationship between the size of the virus genome and the number and sizes of different viral proteins is discussed.

Genes, Viral↗
PubMed1981

[35S]-methionine labelled polypeptides from secondary mouse kidney fibroblasts: coordinates and one dimensional peptide maps of some major polypeptides.

A total of 1147 [35S]-methionine labelled polypeptides (826 acidic (IEF) and 321 basic (NEPHGE)) from asynchronous secondary mouse kidney fibroblasts have been separated using high resolution two dimensional gel electrophoresis. Beside numbering the spots, we give for the major polypeptides their coordinates (M.W. and relative mobility respect to beta-actin (IEF) or NEPHGE polypeptide 9 (NEPHGE)). By using one dimensional peptide mapping it has been possible to find homologies between several major mouse and HeLa cell polypeptides.

Animals↗
PubMed1984

Structural analysis of human adult and fetal alkaline phosphatases by cyanogen bromide peptide mapping.

The adult and fetal forms of human intestinal alkaline phosphatase (ALPase; orthophosphoric-monoester phosphohydrolase, EC 3.1.3.1) are indistinguishable by a variety of analytical procedures. However, they differ electrophoretically and can be differentiated by binding studies with monoclonal antibodies. In this report, these two enzymes along with placental and liver ALPases are compared by the technique of CNBr peptide mapping, and the role of carbohydrate in generating these patterns is investigated. NaDodSO4/PAGE of CNBr digests of radiolabeled ALPases from fetal and adult intestine shows that these two isozymes share five of seven common-sized CNBr fragments. Placental ALPase shares only one common-sized fragment with either intestinal enzyme. Liver ALPase has no CNBr fragments in common with any of the others. These data indicate that fetal intestinal ALPase is not a heterodimer of one subunit each of intestinal ALPase and placental ALPase as has been postulated. CNBr digests of neuraminidase-treated enzymes reveal a change of mobility of only one CNBr band in each of fetal intestinal, placental, and liver ALPases, indicating the presence of sialic acid residues in these fragments. Periodic acid/Schiff reagent staining (specific for carbohydrate) of CNBr digests of fetal and adult intestinal ALPases reacts with only one band in each enzyme, which is the same band from the fetal enzyme shown to contain sialic acid. However, fetal and adult intestinal ALPases each contain at least one CNBr fragment of unique size that is apparently nonglycosylated.

Alkaline Phosphatase↗
PubMedDate not supplied

[Peptide mapping of the major proteins in 2 strains of influenza virus].

The protein composition of two influenza A virus strains from different serological groups was studied. For preliminary separation of envelope and core proteins the virus was treated with nonionic detergent Triton X-100 solution followed by chromatography in ultragel AcA 34. After their separation by disk electrophoresis and treatment with trypsin they were labeled with tritium and peptide analysis was carried out. Differences in peptide maps between the appropriate light and heavy chains of hemagglutinins of the strains under study were demonstrated. Differences in the composition of nucleoprotein and matrix protein were insignificant.

Animals↗
PubMed1985

Peptide mapping of the insulin-binding site of the 130-kDa subunit of the insulin receptor by means of a novel cleavable radioactive photoprobe.

A radioactive photoaffinity probe for the insulin receptor was prepared by derivatizing insulin at its B29 lysine with a novel crosslinking reagent having a cleavable azo linkage. Insulin receptors purified from human placental membranes were photoaffinity labeled with this probe. The photolabeled receptor was treated with dithionite to cleave the azo linkage, thereby removing the insulin ligand and transferring the radioactivity to the receptor protein. The radioactive labeled subunit was isolated and digested with elastase for peptide mapping and separation by high performance liquid chromatography. Results obtained indicated that it will be feasible to use this new photoaffinity probe to obtain radioactive peptides representing the insulin-binding site(s) on the receptor subunit.

Affinity Labels↗
PubMed2004

Differential antibody responses to Plasmodium falciparum-derived B-cell epitopes induced by diepitope multiple antigen peptides (MAP) containing different T-cell epitopes.

Epitopes of universal character are needed when designing subunit vaccines against infectious diseases such as malaria. We have compared the immunogenicity of B-cell epitopes from the Plasmodium falciparum antigen repeats DPNANPNV (PfCS protein) and VTEEI (Pf332) when assembled with four different universal T-cell epitopes in diepitope multiple antigen peptides (MAP). T-epitopes employed were from P. falciparum antigens (CS.T3, [T(*)]4 and EBP3) or from the Clostridium tetani toxin (P2). In association with either of the T-epitopes, the genetic unresponsiveness to the B-epitopes was successfully bypassed. Our results show that the immunogenicity of a T-epitope alone does not necessarily predict the ability of the T-epitope to provide T-cell help when combined with other epitopes in an immunogen. Further, the nature of the immune responses in terms of total IgG antibodies and their subclass distribution, T-cell proliferation and IFN-gamma production, varied with the T-epitope and mouse strain, which may indicate the need for inclusion of a combination of different universal T-epitopes in a future malaria subunit vaccine.

Animals↗
PubMed1986

Dihydroorotase from Escherichia coli. Cloning the pyrC gene and production of tryptic peptide maps.

We have inserted a 1.7-kilobase pair Escherichia coli DNA fragment containing the 1-kilobase pair pyrC gene into the high copy number plasmid pKC16. Dihydroorotase expressed by the pyrC plasmid in E. coli constituted 6.3% of the soluble protein in frozen cell paste. Pure dihydroorotase derived from this frozen cell paste was compared with pure enzyme derived from an E. coli strain lacking the pyrC plasmid: tryptic peptide maps from the two dihydroorotase preparations, produced using reverse-phase high performance liquid chromatography, were indistinguishable. We conclude that the entire pyrC gene is present on the hybrid plasmid and that the dihydroorotase produced from this plasmid is identical to the wild type.

Amidohydrolases↗
PubMed1981

Structure of murine Ia antigens. Two dimensional electrophoretic analyses and high pressure liquid chromatography tryptic peptide maps of products of the I-A and I-E subregions and of an associated invariant polypeptide.

We demonstrate that an invariant polypeptide, first described by Jones et al. (21), co-immunoprecipitates with our Ia molecules, that its interaction with Ia polypeptides varies with haplotype, and that it is not a precursor of the Aalpha, Abeta, Ealpha, or Ebeta. polypeptides. We also show that the polypeptides that we have previously characterized are contaminated with very little, if any, invariant protein. Further, we have used our high-pressure liquid chromatography tryptic peptide map technique to formally map the genes encoding Aalpha, Abeta, and Ebeta to the I-A subregion using recombinant and F1 hybrid mice.

Animals↗
PubMed1979

Human adenine phosphoribosyltransferase. Affinity purification, subunit structure, amino acid composition, and peptide mapping.

Adenine phosphoribosyltransferase (EC 2.4.2.7) has been purified 55,000-fold from normal human erythrocytes. The native molecular weight of the enzyme is 38,200 as determined by sedimentation equilibrium centrifugation. The subunit molecular weight is 18,000 as determined by sodium dodecyl sulfate gel electrophoresis and 17,000 as determined by gel filtration in guanidine hydrochloride, suggesting that the enzyme is a dimer in its native state. Cross-linking the enzyme with dimethylsuberimidate confirms the dimeric structure and peptide mapping data suggested that the subunits are quite similar if not identical. The amino acid composition reveals that 33% of the residues are hydrophobic.

Adenine Phosphoribosyltransferase↗