Unique peptide maps of the three largest proteins specified by the flavivirus Kunjin.

Tryptic digests of four polypeptides found in Kunjin virus-infected Vero cells, NV5, NV4, V3, and NV3, were compared by peptide mapping. The polypeptides to be analyzed were labeled with radioactive methionine and separated by electrophoresis through polyacrylamide gels containing sodium dodecyl sulfate. Because infection of Vero cells by Kunjin virus does not inhibit host cell protein synthesis, radioactively labeled viral polypeptides prepared from infected cells migrate coincidentally during sodium dodecyl sulfate-gel electrophoresis with some of the labeled host proteins. Thus, the genuine viral methionine-containing peptides in tryptic digests of viral proteins have been identified by co-analyzing polypeptides from [3H]methionine-labeled uninfected cells and [35S]methionine-labeled infected cells and determining the 35S/3H ratio in the peptides resolved in two dimensions on thin-layer chromatography plates. The peptide map of NV3 demonstrated that it is host coded, whereas NV5, NV4, and V3 have unique peptide maps and, therefore, account for approximately one-half of the coding potential of Kunjin virus RNA.     

Chemical studies on the cysteine and terminal peptides in tryptic digests of actin

1. On exhaustive digestion of carboxymethylated actin in 6m-urea solutions with carboxypeptidase A, 1 mole of phenylalanine was liberated/43000g. of protein. At a lower urea concentration and in the absence of urea, carboxymethyl-cysteine (CMCys) was also liberated. 2. Three cysteine-containing peptides were identified by the study of peptide ;maps' of tryptic digests of actin treated with thiol reagents. 3. The three peptides, each containing one residue of CMCys, were isolated from tryptic digests of carboxymethylated actin by ion-exchange chromatography. 4. One of these peptides was possibly the N-terminal peptide and contained about 17-18 residues; another was CMCys-Asp-Ile-Asp-Ile-Arg; the other, CMCys-Phe, was the C-terminal tryptic peptide. 5. The chemical evidence suggests that the actin molecule consists of a single polypeptide chain of molecular weight about 44000.     

Ferrate oxidation of Escherichia coli DNA polymerase-I. Identification of a methionine residue that is essential for DNA binding

Treatment of Escherichia coli DNA polymerase-I with potassium ferrate (K2FeO4), a site-specific oxidizing agent for the phosphate group-binding sites of proteins, results in the irreversible inactivation of enzyme activity as judged by the loss of polymerization as well as 3'-5' exonuclease activity. A significant protection from ferrate-mediated inactivation is observed in the presence of DNA but not by substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme also exhibits loss of template-primer binding activity, whereas its ability to bind substrate triphosphates is unaffected. In addition, comparative high pressure liquid chromatography tryptic peptide maps obtained before and after ferrate oxidation demonstrated that only five peptides of the more than 60 peptide peaks present in the tryptic digest underwent a major change in either peak position or intensity as a result of ferrate treatment. Amino acid analyses and/or sequencing identified four of these affected peaks as corresponding to peptides that span residues 324-340, 437-455, 456-464, and 512-518, respectively. However, only the last peptide, which has the sequence: Met-Trp-Pro-Asp-Leu-Gln-Lys, was significantly protected in the presence of DNA. This latter peptide was also the only peptide whose degree of oxidation correlated directly with the extent of inactivation of the enzyme. Amino acid analysis indicated that methionine 512 is the target site in this peptide for ferrate oxidation. Methionine 512, therefore, appears to be essential for the DNA-binding function of DNA polymerase-I from E. coli.     

Interspecies homology of RNA tumor virus proteins.

We report the application of a highly sensitive column chromatographic technique to the comparison of tryptic peptide maps of some RNA tumor virus proteins. By combining microbore ion-exchange chromatography with a sensitive fluorescent assay using o-phthalaldehyde, we obtained high-resolution peptide maps starting with only microgram amounts of protein. Our discovery of coincident peptides from the 15,000 and 30,000 molecular weight proteins from murine and feline leukemia viruses supports serological evidence for interspecies antigenic determinants; coincident peptides were also found for the 10,000 molecular weight proteins from these viruses, although immunochemical data did not reveal interspecies determinants. The relatively large number of coeluting peptides found in the 15,000 and 10,000 molecular weight proteins is strong evidence for the existence of homology.     

Genetic diversity in natural populations of mammalian reoviruses: tryptic peptide analysis of outer capsid polypeptides of murine, bovine, and human type 1 and 3 reovirus strains.

We have studied the structural relationships between the outer capsid polypeptides of eight murine, bovine, and human isolates of type 1 and 3 mammalian reoviruses. Our results show that the outer capsid polypeptides of reoviruses isolated from different mammalian species, in different years and different geographical areas, have both conserved and unique methionine-containing tryptic peptides. We found that tryptic peptides from mu 1C polypeptides of two human, one murine, and two bovine type 3 isolates and one human and two bovine type 1 reoviruses are highly conserved. Our data show that only one tryptic peptide pattern of the mu 1C polypeptide (encoded by the M2 gene) was present in reoviruses isolated from the three different mammalian species. The mu 1C polypeptide of the type 3 Dearing strain contained one tryptic peptide not found in any other reovirus isolate examined. In marked contrast to the mu 1C polypeptides, the sigma 3 polypeptides (encoded by the S4 gene) of three type 1 and three type 3 isolates were divided into two patterns based on significant differences in their tryptic peptides. In addition, at least seven tryptic peptides were conserved among the sigma 3 polypeptides of all virus strains examined. The sigma 3 polypeptide of the type 3 Dearing strain was distinguishable from the sigma 3 polypeptides of all other strains examined. The one mu 1C and two sigma 3 tryptic peptide patterns were found to occur interchangeably in isolates of type 1 or type 3. About 1/3 of the tyrosine-containing tryptic peptides of sigma 1 polypeptides of four type 3 isolates examined were conserved. Comparison of peptide differences in sigma 1 polypeptides of these isolates showed that each had one or more unique tryptic peptides, suggesting that the S1 genes coding for these polypeptides had undergone genetic drift or, alternatively, that there are at least two tryptic peptide patterns present among the sigma 1 polypeptides of these isolates. Our results suggest that genetic drift and reassortment are the most likely explanation for the extensive genetic diversity found in natural populations of mammalian reoviruses.     

The primary structure of the cytotoxin alpha-sarcin

The primary structure of the cytotoxin alpha-sarcin was determined. Eighteen of the 19 tryptic peptides were purified; the other peptide has arginine only. The complete sequence of 17 of the peptides was determined; the sequence of the remaining peptide was determined in part. The sequence of the 39 NH2-terminal residues was obtained by automated Edman degradation. The carboxyl-terminal amino acids were identified after carboxypeptidase treatment. The assignment of the amino acids in the tryptic peptides was confirmed and their alignment established from the sequence of the secondary tryptic peptides obtained after cleavage of citraconylated alpha-sarcin, from the sequence of a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine peptide, from the sequence of a chymotryptic peptide, and from the sequence of a peptide obtained with Staphylococcus aureus V8 protease. alpha-Sarcin contains 150 amino acid residues; the molecular weight is 16,987. There are disulfide bridges between cysteine residues at positions 6 and 148 and between residues 76 and 132.     

Amphiphilic peptides with arginines and valines for the delivery of plasmid DNA

A non-toxic and efficient gene carrier is one requirement for clinical gene therapy. In this study, amphiphilic peptides composed of arginines and valines were synthesized and characterized as plasmid DNA (pDNA) carriers. The peptides have a cationic region containing 1-4 arginines and a hydrophobic region containing 6 valines. The arginine-valine peptides (RV peptides) formed micelles in aqueous solution with a critical micelle concentration (CMC) of 1.35 mg/ml. In gel retardation assay, the RV peptides retarded all pDNA at weight ratios (pDNA:RV peptide) of 1:3 for R1V6, 1:2 for R2V6 and R3V6, and 1:1 for R4V6. A heparin competition assay showed that the R3V6 peptide formed tighter complexes with pDNA than poly-L-lysine (PLL). In vitro transfection assay into HEK293 cells showed that the R1V6 and R2V6 peptides had the highest transfection efficiencies at 1:30 weight ratios (pDNA:RV peptide), while the R3V6 and R4V6 peptides had the highest efficiencies at 1:20 weight ratios. Under optimal conditions, the R3V6 peptide had the highest transfection efficiency of all the RV peptides and PLL. MTT assay showed that the RV peptides did not have any detectable toxicity to cells. Therefore, the RV peptide may be useful for the development of non-toxic gene carriers.     

Liver microsomes contain two distinct NADPH-Monooxygenases with NH2-terminal segments homologous to the flavin containing NADPH-monooxygenase of Pseudomonas fluorescens

Two NADPH-reductase preparations (FAD-containing monooxygenases) were isolated from rabbit liver microsomes, referred to as from 1 and from 2. Purification was achieved by means of anion-exchange, cation-exchange and hydroxylapatite chromatography in the presence of cholate and Nonidet P-40. Affinity chromatography on 2', 5'-ADP Sepharose was used to increase the purity and to concentrate the enzyme. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, form 1 exhibited a single band at Mr 58,500 and form 2 at Mr 58,000. The NH2- terminus of form 1 is blocked, whereas the NH2-terminus of form 2 is homologous to the NADPH-phydroxybenzoate hydrolase from Pseudomonas fluorescens. The latter and the form 2 enzyme share 11 identical residues in the NH2-terminal segment of 15 residues. Both forms were subjected to tryptic cleavages and peptide mapping. Sequence analysis of the peptides obtained indicated that forms 1 and 2 are similar but not identical proteins. A tryptic peptide, homologous to residues 3 to 32 of form 2 enzyme was isolated from the form 1 protein. This segment has 24 residues that are identical to the form 2 and contains the consensus sequence Gly-X-Gly-X-X-Gly, found in most FAD binding proteins. These results indicate that the NADPH-monooxygenase system consists of at least two distinct proteins representing different gene products.     

The amino acid sequence of the alpha chain of the major haemoglobin of the rat (Rattus norvegicus).

1. A partial amino acid sequence of the alpha chain from the rat (Wistar, Rattus norvegicus) major haemoglobin is reported. The soluble tryptic peptides prepared from aminoethylated alpha-globin were separated by peptide 'mapping'. Sequencing of the tryptic peptides was carried out by the dansyl-Edman method and by the overlapping of smaller peptide fragments derived from secondary enzymic digestion. The insoluble 'core' peptides were further digested with chymotrypsin, thermolysin and pepsin to give smaller soluble peptides for sequencing. The tryptic peptides were ordered on the basis of their homology with the corresponding peptides of human alpha chain. 2. The proposed sequence is compared with that obtained by using an automated sequencer [Garrick et al. (1975) Biochem. J. 149, 245-258]. The differences in sequence resulting from the two methods are discussed. 3. It is suggested that the externally situated cysteine (residue 13) is responsible for the observed inhibition of crystallization of rat haemoglobin at alkaline pH. 4. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50047 (9 pages) at the British Library (Linding Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from which copies can be obtained on the terms given in Biochem. J. (1975) 145, 5.     

Evidence that the Yb subunits of hepatic glutathione transferases represent two different but related families of polypeptides

Three soluble rat liver glutathione (GSH) transferases A, C and one referred to as 'D', all of which are dimers of Yb subunits [Bass et al. (1977) Biochim. Biophys. Acta, 492, 163-175], have been compared with respect to C-terminal amino acids and tryptic peptide maps. GSH transferases A and 'D' gave different tryptic peptide maps and different C-terminal amino acids, lysine and proline respectively. In each case the number of tryptic peptides is about half of that expected from their lysine and arginine content, and there are 2 mol C-terminal amino acid/mol enzyme. This indicates that GSH transferases A and 'D' represent two different Yb homodimers, which we refer to here as Y1bY1b and Y2bY2b respectively. GSH transferase C is the corresponding heterodimer Y1bY2b since it gives all the tryptic peptides which arise from GSH transferase A and GSH transferase 'D' and also contains both C-terminal lysine and proline. These results provide a structural basis to similar conclusions drawn by Mannervik and Jensson [(1980) J. Biol. Chem. 257, 9909-9912] based on enzymic and immunological comparisons. Tryptic peptide maps show that GSH transferases A and 'D' have considerable homology since there are 23 peptides common to both, 12 peptides unique to A and 8 peptides unique to 'D'. Even so GSH transferase A is selectively induced by a phenobarbitone regime. It is, therefore, concluded that Y1b and Y2b are derived from separate but related genes. A similar conclusion has been drawn concerning the Ya and Yc subunits [Beale et al. (1982) Eur. J. Biochem. 126, 459-463], and a comparison of amino acid compositions, presented here, further suggests a genetic relationship between both pairs of subunits.     

Unique glycosylation of three keratan sulfate proteoglycan isoforms

Recent work demonstrates isoforms of bovine corneal keratan sulfate proteoglycan containing structurally unique core proteins of 25 and 37 kDa (Funderburgh, J., and Conrad, G. (1990) J. Biol. Chem. 265, 8297-8303). In the current study, two forms (37A and 37B) of the 37-kDa protein were separated by ion-exchange chromatography after removal of keratan sulfate with endo-beta-galactosidase. Keratan sulfate linkage sites in core proteins were labeled with UDP-[3H]galactose using galactosyltransferase. Labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by tryptic digestion and reversed-phase chromatography. The 37A protein has three keratan sulfate-linkage sites, and the 37B and 25-kDa proteins each contain one linkage site. Reversed-phase tryptic maps of the three proteins differed in total peptide profile and in glycosylated peptides labeled with periodate-[3H]-NaBH4. Tryptic mapping of the two 37-kDa isoforms after deglycosylation showed differences in total tryptic peptides, in peptides labeled with [14C]iodoacetic acid, and in peptides recognized by antibodies to a mixture of the 37-kDa cores. Antibody to a synthetic peptide with N-terminal sequence obtained from mixed 37-kDa cores reacted exclusively with the 37B isoform. These results show that bovine corneal keratan sulfate proteoglycan has three different core proteins each with distinct glycosylation and unique primary structure.     

Peptide mapping of multiple forms of cyclic nucleotide phosphodiesterase

Purified multiple forms of 3':5'-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17) were analyzed using two-dimensional tryptic pep]tide mapping of radioiodinated peptides. Comparisons of peptide maps of rat liver insulin-sensitive phosphodiesterase (PDE) with rat brain calmodulin-sensitive PDE suggest that some peptides co-migrate (31-43% co-migration). However, except for a single peptide, bovine retinal rod outer segment PDE, peptide maps appear unrelated to the other two forms (7-12% co-migration). In contrast, peptide maps of a 36,000-dalton proteolysis product of calmodulin-sensitive PDE are highly related to the peptide maps of a rat brain calmodulin-sensitive holoenzyme (73% co-migration). These results suggest that the multiple PDE forms are distinct molecular entities.     

Acetoxy drug: protein transacetylase of buffalo liver-characterization and mass spectrometry of the acetylated protein product

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3-3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K(m) (1667 microM) and V(max) (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3-3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3-3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3-3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3-3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys(51), Lys(82), Lys(123), Lsy(181), Lys(191) and Lys(210)) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions.     

Conservation of an ATP-binding domain among RecA proteins from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r.

The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N3ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the [alpha-32P]8N3ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in each of the digests and these peptides eluted identically with the tryptic peptide T31 of the E. coli K-12 RecA protein, which was the unique site of 8N3ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10(7) years.     

Albumin-like proteins from the pituitary glands of Dahl salt-resistant rats.

Dahl selectively bred rats for susceptibility (S strain) or resistance (R strain) to the hypertensive effect of high salt (NaCl) diet. Pituitary glands of R rats accumulate large amounts of four unique proteins not seen in S rats. These proteins were called R1, R2, R3, and R4 in order of decreasing electrophoretic mobility. Albumin, R4, R2, and R1 all bound to an affinity column for albumin (Cibacron blue 3G-A dye coupled to agarose) and were eluted in that order by a KSCN gradient. It was shown by crossed immunoelectrophoresis that R1 and R2 cross-react with plasma albumin. Peptide maps or tryptic digest of R1 and albumin showed that the majority of peptides generated were identical. It was not possible to incorporate labeled amino acid into albumin or the albumin-like R proteins with pituitary incubates, indicating that albumin-like proteins were not synthesized de novo by pituitary glands. R rat pituitary glands showed much greater protease (arginine esterase) activity than did S. This suggests that R proteins are formed locally in the pituitary gland of R proteins are formed locally in the pituitary gland of R rats by cleavabe of specific peptide bonds in albumin. The function of these endogenous albumin fragments is unknown, but albumin fragments produced in vitro by other investigators are known to potentiate bradykinin, a hypotensive peptide.     

High sequence coverage by in-capillary proteolysis of native proteins and simultaneous analysis of the resulting peptides by nanoelectrospray ionization-mass spectrometry and tandem mass spectrometry

Losses of proteolytic peptides during extraction and/or purification procedures succeeding in-gel or in-solution digests of proteins frequently occur in the course of protein identification investigations. In order to overcome this disadvantage, the method of in-capillary digest was developed: native proteins were incubated in the presence of endoproteases in the electrospray capillary and the resulting peptides were analyzed by nanoelectrospray-mass spectrometry during the ongoing proteolysis. In-capillary digest of apomyglobin by use of trypsin in a molar ratio of 25:1 yielded complete degradation already after 15 min. The sequence coverage based on formation of molecular ions was 100% and peptide ions could be fragmented by collision-induced dissociation and sequenced. When myoglobin was incubated in the electrospray capillary with trypsin in a molar ratio of 500:1, a clear shift from molecular ions and miscleaved peptide ions to the expected final tryptic peptide ions was observed over a 2 h period. The peptide spectra obtained from tryptic in-capillary proteolysis of bovine serum albumin and apotransferrin, respectively, gave rise to sequence coverages of more than 40% for both proteins. The data obtained from the peptide maps as well as from collision-induced dissociation (CID) of selected peptides were more than sufficient for protein identification by database searches. An elephant milk protein preparation was used to demonstrate the application of in-capillary proteolysis on protein mixtures. Tryptic digest, simultaneous analysis of the proteolytic peptides by use of CID, and subsequent sequencing allowed the identification of lactoferrin, alphas1-casein, beta-casein, delta-casein, and kappa-casein by homology search.     

Subunit structure of higher plant glyceraldehyde-3-phosphate dehydrogenases (EC 1.2.1.12 and EC 1.2.1.13).

In a previous publication (Cerff, R. (1979) Eur. J. Biochem., 94, 243--247) we demonstrated that chloroplast NADP-linked glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) from higher plants consists of two separate isoenzymes with apparent subunit compositions A2B2 (isoenzyme 1) and A4 (isoenzyme 2), where Subunits A and B are distinguished by slightly different molecular weights (A smaller than or approximately to B). In the present study we compare isoenzymes 1 and 2 from Sinapis alba and Hordeum vulgare on the basis of antigenic cross-reactivity, tryptic peptides, and amino acid composition. Isoenzymes 1 and 2 show immunochemical identity. They also have very similar tryptic peptide maps and amino acid compositions. This strongly suggests that Subunits A and B of the NADP-linked enzyme are very similar in primary sequence. As opposed to this, cytoplasmic NAD-specific glyceraldehyde-3-P dehydrogenase (EC 1.2.1.12) does not cross-react with antisera raised against the NADP-linked enzyme. Furthermore, tryptic peptide maps of the NAD-specific enzyme show little or no similarity with those of the NADP-linked enzyme. This indicates that the subunits of the NADP-linked enzyme and the subunit of the NAD-specific enzyme are different proteins coded by separate genes. The differences in the amino acid compositions between the two species corresponds to a SdeltaQ value of 21, suggesting some sequence resemblance and a common phylogenetic origin.     

Die Proteinstruktur des RNS-Bakteriophagen fr

Summary The coat protein of the RNA containing bacteriophage f_r has been hydrolyzed and its amino acid composition determined (Table 1). Furthermore, the protein was split with trypsin and the tryptic peptides were separated by column chromatography on Dowex 1 (Figure 1) and purified by paper chromatography and electrophoresis.The amino acid composition of all but one tryptic peptide are given in Table 2. The large peptide T₁₃ which is much more difficult to purify than all other peptides, was isolated by several methods. Its amino acid composition is shown in Table 3. All tryptic peptides are compiled in Table 4.Amino acid sequences have been fully or partially determined for 9 tryptic peptides (Table 5) and the others are presently being investigated.These findings are compared with the results from other RNA phages, especially f₂. It is concluded from the available data that the relationship between the coat proteins of the RNA phages is similar to that between the various naturally occurring strains of tobacco mosaic virus whose amino acid sequences are known.

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Herrn Prof.G. Melchers zum 60. Geburtstag gewidmet.     

Comparative studies on partial microsequence-analysis of Bence-Jones proteins

1. Sequence analyses of Bence-Jones proteins up to 15 amino acids from the N-terminus provide decision of subgroups. 2. Investigation of primary structure of 3 Bence-Jones proteins, monomer and dimer of a kappa type, is limited at position 9-10 using DABITC reagent, whereas DABITC/PITC double coupling method allows sequencing up to 20-21 amino acids. 3. Sequencing of tryptic peptides allows only determination of 6-9 amino acids. 4. Microsequencing of tryptic peptide T13a shows the allotypic variant, inv b+, of Bence-Jones proteins TRA and GAN.