Molecular Mass Peptides Research Articles

Chromosomal Localization and cDNA Cloning of the Genes (DDB1 and DDB2) for the p127 and p48 Subunits of a Human Damage-Specific DNA Binding Protein

DDB is a damage-specific DNA binding protein whose binding activity is absent from a minority of cell strains from individuals with xeroderma pigmentosum Group E, a human hereditary disease characterized by defective nucleotide excision DNA repair and an increased incidence of skin cancer. The binding activity from Hela cells is associated with polypeptides of M r 124,000 and 41,000 as determined by SDS-polyacrylamide gels. This report describes the isolation of full-length human cDNAs encoding each polypeptide of DDB. The predicted peptide molecular masses based on open reading frames are 127,000 and 48,000. When expressed in an in vitro rabbit reticulocyte system, the p48 subunit migrates with an M r of 41 kDa on SDS-polyacrylamide gels, similarly to the peptide purified from Hela cells. There is no significant homology between the derived p48 peptide sequence and any proteins in current databases, and the derived peptide sequence of pl27 has homology only with the monkey DDB p127 (98% nucleotide identity and only one conserved amino acid substitution. Using a fluorescence in situ hybridization technique, the DDB p127 locus (DDB1) was assigned to the chromosomal location 11q12-q13, and the DDB p48 locus (DDB2) to 11p11-p12.

Amino acid sequence of the minor isomorph of the crustacean hyperglycemic hormone (CHH-II) of the Mexican crayfish Procambarus bouvieri (Ortmann): presence of a D-amino acid.

The primary structure of the neurohormone crustacean hyperglycemic hormone (CHH-II) was determined by means of enzymatic digestions, manual Edman degradation, and mass spectrometry. CHH-II is a 72 residue peptide (molecular mass 8388 Da), with six cysteines forming three disulfide bridges that connect residues 7-43, 23-39, and 26-52. The peptide has blocked N- and C-termini, and lacks tryptophan, histidine, and methionine. The CHH-I and CHH-II of Procambarus bouvieri have identical sequences and elicit levels of hyperglycemia that are not distinguishable. The difference between the two isomorphs consists in a posttranslational modification of a L-Phe in CHH-I to a D-Phe in CHH-II at the third position from the N-terminus.

Electrofiltration of solutions of amino acids or peptides

Summary - Enhanced transfer according to the isoelectric point (pl) of amino acids and peptides (molecular mass 130-6 000 9 mol-1) was achieved using laboratory filtration in the presence of an electric field. The permeate was enriched with arginine and lysine of negative electrophoretic mobility, while the retentate was enriched with the amino acid characterized by the lowest pl value, aspartic acid. A model which assumes no retention of amino acids by ultrafiltration (UF) membranes describes reasonably weil their transmission to the permeate. The peptide mixture permeate was enriched with peptides which were either positively or negatively charged according to the electric field direction. The effect was significant even under low electric field. The model did not quantily satisfactorily the experimental transmission probably because of significant retention of sorne peptides by the ultrafiltration membranes. The present study shows that electrofiltration (EF) may be a useful and efficient process for achieving selective separation of charged biological molecules provided that further work is aimed at a better understanding of which mechanisms rule the retention in EF and of the effect of process variables (flux, electric field, conductivity, charge of the molecules). electrofiltration 1amino acid 1 peptide Nomenclature: Cp, Cp amino acid concentration of permeate, retentate, 9 1-1; E, electric field, Y m-'; J, permeation flux, m3 m-2 s-'; Mp molecular mass; MMCO, molecular mass eut-off; N, Avogadro number (6.023 x 1023); pl, isoelectric pH; Rm, membrane hydraulic resistance, m-'; Rf, hydraulic resistance of fouling layer, m-1; Tr, transmission rate; TP, transmembrane pressure, Pa; r, molecule radius, m; U, electrophoretic mobility, m2 S-1 y-1; Z, number of charges; E, charge of electron, Cb; <p, sieving coefficient; Il, dynamic viscosity of feed, Pa s; IlP, dynamic viscosity of permeate, Pa s; p, density, kg m~.

Analysis of Protein Hydrolyzates. 1. Use of Poly(2-hydroxyethylaspartamide)-Silica Column in Size Exclusion Chromatography for the Fractionation of Casein Hydrolyzates

A poly(2-hydroxyethylaspartamide)-silica column (PHEA), with a 0.05 M formic acid mobile phase, was tested for the size exclusion chromatography (SEC) of amino acids and small peptides (molecular mass lower than 1000 Da). The PHEA shows good SEC properties, although some nonideal size exclusion behavior suggests it has a low cationic character. On the other hand, the separation of peptides is affected by the sample salt concentration. The technique was applied to the analysis of several casein hydrolysates, and the mean chain length ofpeptides in the collected fractions indicates a good separation of peptides on the basis of their size

Identification of transformation sensitive proteins recorded in human two-dimensional gel protein databases by mass spectrometric peptide mapping alone and in combination with microsequencing.

A comprehensive human keratinocyte two-dimensional (2-D) gel protein database has been established to study the expression levels and properties of the thousands of proteins that orchestrate various keratinocyte functions both in health and disease, cancer included. A major task in establishing such a database is to identify known proteins in the 2-D gel patterns as well as to reveal hitherto unknown proteins. To date, protein identification has been performed by one or a combination of the following methods: (i) comigration with known proteins, (ii) Western blotting using specific antibodies, (iii) microsequencing and (iv) vaccinia virus expression of full length cDNAs. Recently, the systematic identification of proteins has gained a new dimension with the advent of computer programs for searching peptide molecular mass databases with experimentally obtained peptide mass maps. Here we investigate this approach to identify proteins that are highly up- or down-regulated in simian virus SV40 transformed human keratinocytes (K14). Peptide mass maps of several proteins, including keratins 7, 8, 18 and 19 were obtained either by plasma desorption mass spectrometry (PDMS) analysis of high performance liquid chromatography (HPLC) purified peptides or by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of total digests. The results demonstrated that peptide mass maps can be used for a rapid and sensitive protein identification allowing fast screening of proteins recorded in 2-D gel databases. The mass spectrometric approach when combined with microsequencing strengthened identification, and added the possibility of full characterization of post-translational modifications and sequence variations.

A cDNA clone encoding Chlamydomonas reinhardtii preferredoxin.

Fds are ubiquitous low mo1 wt proteins containing iron sulfur center(s) involved in numerous electron transfer reactions. In plants, one of the major functions of Fd is to provide the reducing power for chloroplastic NADP photoreduction. In addition, Fd is also needed for nitrite, ammonia, and sulfite assimilation as well as for pseudocyclic electron flow and fatty acid metabolism and for the light regulation of chloroplastic enzymes (Orme-Johnson, 1973). This protein is encoded by nuclear genes and is, therefore, produced as a precursor that is subsequently cleaved inside the chloroplast (Smeekens et al., 1985). We have previously purified Chlamydomonas reinhardtii Fd and determined its primary structure by direct amino acid sequencing (Schmitter et al., 1988). Based on this sequence and using the polymerase chain reaction, we have isolated a nucleotidic sequence coding for the mature portion of C. reinhardtii Fd and shown that the deduced amino acid sequence was identical with only one substitution Thr7 to Ser (Table I). In addition, we demonstrated that Escherichia coli cells were able to direct the synthesis of C. reinhardtii Fd polypeptide and to reassemble it together with an iron-sulfur center (Rogers et al., 1992). In this paper we report the sequence of a cDNA encoding C. reinhardtii preferredoxin. A Xgtll cDNA was sequenced (527 bp) and found to encode a 126-amino acid precursor with a molecular mass of 13,250 D, compared to the mature form, which contains 94 amino acids and has a molecular mass of 9,908 D. The deduced amino acid sequence was completely homologous to the sequence reported by Schmitter et al. (1988). In addition, the sequence revealed the structure of the 32-amino acid transit peptide (molecular mass 3342 D), which is as follows: MAMAMRSTFAARVGAKPAVRGARPASRMSCMA. Severa1 lines of evidence indicate that the first ATG is the initiation codon. First, it is preceded by TCA and AAA, triplets theoretically coding for Ser and Lys but rarely if ever used for nuclear genes in C. reinhardtii. Second, the flanking sequence surrounding the initiation codon (AAAAATGGC) fits well the eukaryotic translation initiation consensus (Wedel et al., 1992). Finally, the beginning of the transit peptide (MAMAM) is highly similar to the MAQM sequence reported by Wedel et al. (1992) and to the MAMAT of Hoffmann et al. (1988), and the dipeptide MA is overwhelm-

Primary structure of the major isomorph of the crustacean hyperglycemic hormone (CHH-I) from the sinus gland of the Mexican crayfish Procambarus bouvieri (Ortmann): Interspecies comparison

The amino acid sequence of this neuropeptide was elucidated by means of a combined approach of enzymatic digestions, manual and automatic Edman degradations, and mass spectrometry. It is a 72 residue peptide (molecular mass 8388 Da), with six cysteines forming three disulfide bridges connecting residues 7–43, 23–39, and 26–52, with blocked N- and C-termini, and lacking the amino acids histidine, methionine, and tryptophan. The CHH-I of Procambarus bouvieri is compared with the other known CHHs from Orconectes limosus (98.6% identity), Homarus americanus isomorph A (83.3% identity), Homarus americanus isomorph B (79.2% identity), and Carcinus maenas (61.1% identity).

Adsorption of heme-containing peptides on silicon surfaces

To elucidate the role of minor alteration of amino acid composition of protein in interfacial behavior, the adsorption of three heme peptides on hydrophilic and hydrophobic silicon surfaces was studied. It was found that all three peptides did not adsorb on the hydrophilic silicon surfaces and irreversibly adsorbed on the hydrophobic silica prepared by pretreatment with dimethyldichlorosilane. Rate constants of adsorption at pH = 7.2 for all the heme peptides were the same, indicating that at the initial stage of adsorption heme peptides interacted with the hydrophobic surface through identical segments. On the basis of plateau values obtained, calculation of surface areas occupied per heme peptide molecule showed that the higher the heme peptide molecular mass, the smaller is its occupied area on the hydrophobic silicon surface at pH = 7.2. This “illogical” observation was explored by electrostatic interaction of adsorbed heme peptide molecules, on the basis of their a bell-shaped pH-dependent adsorption and on the p K a values of amino acid residues in heme peptides.

Comparison of helium and argon as collision gases in the high energy collision-induced decomposition of MH + ions of peptides

Collision-induced decompositions (CID) of protonated peptides were studied using a four-sector mass spectrometer. The collision gases employed were helium and argon. The CID spectra of several peptides covering the molecular mass region of 905–2465 u were recorded. These investigations established several previously unrecognized differences between the CID spectra obtained with helium and argon as collision gases. These can be summarized as follows: (1) Structurally significant and specific side chain fragmentations (d n , w n , and v n ion types) are greatly reduced or completely missing in the CID spectra obtained with helium as a collision gas compared to those obtained with argon. (2) As the peptide molecular mass increases, argon, which is heavier than helium, is increasingly more efficient than helium for generating fragment ions.

Expression of Osmotin-Like Genes in the Halophyte Atriplex nummularia L.

A peptide (molecular mass 50 kilodaltons) that is immunologically related to tobacco osmotin was detected in cells of the halophyte Atriplex nummularia. This protein was constitutively expressed in both unadapted and NaCl-adapted cells. A predominant osmotin-like peptide (molecular mass 24 kilodaltons) was also found in culture media after cell growth. Two unique A. nummularia cDNA clones, pA8 and pA9, encoding osmotin-like proteins have been isolated. The pA8 and pA9 inserts are 952 and 792 base pairs and encode peptides of 222 and 224 amino acids, respectively. The peptide deduced from pA8 has a molecular mass of 23,808 daltons and theoretical isoelectric point of 8.31, whereas the peptide derived from pA9 has a molecular mass of 23,827 daltons and an isoelectric point of 6.88. Unique transcripts were detected by the inserts of the cDNA clones, two (1.2 and 1.0 kilobases) by pA8 and one (0.9 kilobase) by pA9. The pA8 transcripts were constitutively accumulated in unadapted and NaCl-adapted cells, whereas the mRNA levels were up-regulated by abscisic acid treatment. The level of pA9 mRNA was induced by NaCl treatment and increased in cells as a function of NaCl adaptation. Southern analysis of the genomic DNA indicated the presence of osmotin-like multigene families in A. nummularia.

Characterization of two forms of cationic peroxidase from cultured peanut cells.

Two forms of cationic peroxidase from peanut cells were differentiated by concanavalin A affinity chromatography. They differed in molecular mass as well as concanavalin A binding, leading to the initial suggestion that they represented two isozymes of peroxidase. However, similar values for the specific activity, Soret absorption, calcium content, and peptide molecular mass were observed for each of the forms. Therefore, the binding and nonbinding fractions most likely represent two molecular forms of cationic peanut peroxidase, rather than two distinct cationic isozymes. The difference between these two forms is discussed in terms of glycosylation. Through the amino acid sequence analysis of the formic acid treated peptide, the cationic isozyme has been shown to be identical in amino acid sequence to the cDNA clone PNC1.

Insect immunity. Isolation from a coleopteran insect of a novel inducible antibacterial peptide and of new members of the insect defensin family.

Injection of heat-killed bacteria into larvae of the large tenebrionid beetle Zophobas atratus (Insecta, Endopterygota, Coleoptera) results in the appearance in the hemolymph of a potent antibacterial activity as evidenced by a plate growth inhibition assay. We have isolated three peptides (A-C) from this immune hemolymph which probably account for most of this activity. Their primary structures were established by a combination of peptide sequencing and molecular mass determination by mass spectrometry. Peptide A, which is bactericidal against Gram-negative cells, is a 74-residue glycine-rich molecule with no sequence homology to known peptides. We propose the name coleoptericin for this novel inducible antibacterial peptide. Peptides B and C are isoforms of a 43-residue peptide which contains 6 cysteines and shows significant sequence homology to insect defensins, initially reported from dipteran insects. This peptide is active against Gram-positive bacteria. The results are discussed in connection with recent studies on inducible antibacterial peptides present in the three other major orders of the endopterygote clade of insects: the Lepidoptera, Diptera, and Hymenoptera.

Sucrose-phosphate synthase from wheat. Characterization of peptides by immunoblotting analysis

The structure of sucrose-phosphate synthase (SPS: EC 2.4.1.14) from wheat (Triticum aestivum L. cv. San Agustin was studied using antibodies prepared against the enzyme purified from wheat germ. The antibodies revealed the presence of 55 and 35 kDa polypeptides in wheat germ, endosperm, embryos and whole seed, while in whole wheat leaf, a 90 kDa was detected. It is not clear whether the 35 and 55 kDa polypeptide are truly subunits of SPS or they are the product of protease action, more active in non-photosynthetic tissues than in leaves. The antibodies from wheat germ clearly recognized polypeptides in leaf protein preparations from other plants (barley, soybean, maize) and, weakly in others (peanut, tobacco). It did not recognize any polypeptide in spinach and mustard leaf extracts. In the case of maize leaf, a peptide of higher molecular mass (116 kDa) than the wheat ones was revealed. The results may indicate the presence of different polypeptide compositions for sucrose-phosphate synthase, and suggest the existence of at least two types of this enzyme.

Degradation of Human Factor X by Human Polymorphonuclear Leucocyte Cathepsin G and Elastase

Cathepsin G and elastase from human polymorphonuclear leucocytes were used in vitro to digest human factor X. Clotting assays showed that both proteinases affected a rapid loss in the coagulant activity of factor X. Calcium ions almost totally protected the coagulant activity of factor X against the action of cathepsin G but not elastase. Polyacrylamide gel electrophoresis (in nonreducing conditions and in the presence of SDS) indicated that the proteolytic action of cathepsin G led to the removal of a peptide of low molecular mass (pX) with the consequent formation of a single stable high molecular mass product (PX). SDS electrophoresis (under reducing conditions and in the presence of SDS) indicated that a pX was derived from the light chain of factor X. The proteolytic action of elastase led to the formation of numerous degradation products. Analysis of the products generated by the action of cathepsin G indicated that cathepsin G cleaved position Phe40:Trp41 in the light chain of factor X. In the presence of citrated plasma, cathepsin G but not elastase, was responsible for a loss in coagulant activity.

Biosynthesis and processing of delta sleep‐inducing peptide‐like precursors in primary cultures of mouse anterior pituitary cells

The biosynthesis and processing of material resembling delta sleep-inducing peptide (DSIP) have been studied in mouse anterior pituitary primary cell cultures. Cells were pulse/chase incubated with 3H-labelled amino acids (Gly, Arg or Ala) and cell extracts were immunoprecipitated with DSIP antiserum. Labelled DSIP-related proteins were resolved by SDS/PAGE. Multiple forms of DSIP-immunoprecipitable material were observed, including three precursors of molecular mass 50-60 kDa which were processed to two major groups of intermediates of 35-45 kDa and 9-16.5 kDa. These intermediates appear to be processed to a DSIP-related peptide (molecular mass less than 3 kDa), which co-ran on reversed-phase HPLC with an endogenous form of DSIP in mouse anterior pituitary, but not with rabbit DSIP. This less than 3-kDa peptide incorporated [3H]Gly, but not [3H]Arg or [3H]Ala. In addition, it incorporated [3H]glucosamine, indicating that it was a glycopeptide. Secretion studies showed release of the less than 3-kDa DSIP-like glycopeptide and the 9-16.5-kDa group of intermediates into the medium. The present study demonstrates the biosynthesis of a small DSIP-like glycopeptide in mouse anterior pituitary cells, which is not identical with, but has similarities to, rabbit DSIP.

Electron microscopic immunolocalization of seminal vesicle-specific antigen in human seminal vesicle.

Seminal vesicle-specific antigen (SVSA) has been shown to be a polymorphic antigen represented by multiple immunoreactive peptides when fresh human semen is probed with monoclonal antibody (MHS-5) on Western blots. Semen samples collected directly into sodium dodecyl sulfate (SDS) demonstrate major immunoreactive peptide bands at 69-71 kDa and 58 kDa as well as a series of peptides of lower molecular mass. As semen liquefies, the higher molecular mass forms of SVSA are transformed into lower molecular mass bands, with 10-13 kDa immunoreactive peptides predominating after 8 h of liquefaction (McGee and Herr, Biol. Reprod. 37:431-439, 1987). In the present study, the 10-13 kDa form of SVSA was purified by preparative electrophoresis from SDS gels and a polyclonal antibody was generated in guinea pigs. Human seminal vesicle was fixed by immersion in combinations of glutaraldehyde and paraformaldehyde and embedded in Araldite or LR Gold. Both the guinea pig polyclonal antibody and the murine monoclonal antibody MHS-5 were employed to localize SVSA in human seminal vesicle by immunoelectron microscopy using Protein-A gold complexes. Gold particles were quantified in various subcellular compartments by a Videoplan computer. With either antibody probe, SVSA was found predominantly in the central electron-dense cores of secretory granules, with no staining evident over the electron lucent halo surrounding the granule core. With preimmune serum, the mean number of gold particles overlying secretory granules was 3/microns2; with polyclonal anti-SVSA, the mean number of particles observed over secretory granules was 182/microns2. This study represents, to our knowledge, the first fine-structural localization of a specific secretory protein to the electron-dense cores of secretory granules in principal cells of the human seminal vesicle.

Catfish plasma lipoproteins: In vivo studies of apoprotein synthesis and catabolism

AbstractThree distinct lipoprotein classes, very‐low‐density (VLDL), low‐density (LDL), and high‐density (HDL), were isolated from plasma of channel catfish, Ictalurus punctatus. HDL was the major lipoprotein class, accounting for 22–32% of the total plasma protein. The major peptide of VLDL and LDL was an apoB‐like protein (molecular mass 250,000 daltons). When a protease inhibitor was added to the plasma, there was no evidence of apoC‐ or apoE‐like proteins in catfish VLDL. The major peptide of HDL in male catfish was an apoAI‐like peptide (molecular mass 25,000 daltons). The major peptide of the HDL of female catfish undergoing vitellogensis had a molecular mass of 92,000 daltons. Plasma lipoprotein metabolism was investigated by measurement of plasma titers and the synthesis and catabolic rates of apoVLDL, apoLDL, and apoHDL. Apoprotein synthesis rates (ks) were measured in fed, 3‐day‐fasted, and 20‐day‐fasted channel catfish by continuous infusion of 14C‐tyrosine. In vivo synthesis rates of plasma apoproteins have not been previously measured. Analysis of data required the derivation of a series of equations that accounted for the kinetics of secretion of plasma proteins and their equilibration with an extravascular compartment. The plasma titer of apoVLDL was closely correlated to dietary intake, although apoVLDL synthesis rates did not significantly change with fasting. ApoLDL plasma titers significantly decreased after a 20‐day fast but not after a 3‐day fast. Fasting resulted in a significant increase in apoLDL synthesis. The apoHDL titer and synthesis rates were not significantly affected by fasting. Male and female fish have similar apoVLDL and apoLDL titers and synthesis rates. ApoHDL plasma titers and synthesis rates were significantly higher in females (14.0 ± 1.6 mg ml−1; ks = 34.9% ± 15.3% day−1) than in males (9.0 ± 1.6 mg ml−1; ks = 8.7% ± 2.6% day−1). The higher apoHDL titer and synthesis rate in female fish were due to the presence of a peptide (molecular mass 92,000 daltons) not present in males.

Ehrlich chromogens, probable cross-links in elastin and collagen

(1) Proteolytic digests of tissue elastin contain material which reacts with dimethylaminobenzaldehyde in acid solution (Ehrlich's reagent) to give a cherry-pink colour. This Ehrlich chromogen(s) [EC(s)] is similar to but not identical with EC(s) previously demonstrated in tissue collagens [Scott, Hughes & Shuttleworth (1979) Biosci. Rep. 1, 611-618]. Both ECs react with diazonium salts in acid to give coloured products. (2) Diazobenzene linked via a phenolic ester to polyacrylamide beads (Biogel P10) has been used to absorb ECs specifically and almost quantitatively from proteolytic digests. The coupled deeply coloured azo-EC-peptides were then recovered after mild alkaline cleavage from the support and purified by gel chromatography. (3) Using 15N-labelled NaNO2, the collagen azo-EC-peptides were prepared, and 15N abundance measured therein. The molar absorption coefficient of the azo-EC group was calculated (18,700) based on the assumption that each azo-EC group contained one 15N atom. (4) Collagen azo-EC-peptides contained glucose and galactose, whereas elastin azo-EC peptides did not. The amino acid patterns of the two peptides were quite different, the former being rich in polar amino acids, the latter containing much alanine. The patterns were compatible with an origin from the cross-linking regions of collagen and elastin respectively. (5) Quantitative (molar) comparisons of the azo-EC group content with amino acid, amino end-group and sugar contents, and azo-EC peptide molecular mass, suggest that a structure is present in the collagen azo-EC-peptides containing two EC groups shared between four peptide chains. Three peptide chains probably meet at each (cross-linking) EC group. (6) Based on this structure, about 15% of adult bovine skin collagen contains EC groups.

Transition vesicle-enriched fractions isolated from rat liver exhibit a unique peptide pattern

Isolated fractions enriched in transition vesicles isolated by preparative free-flow electrophoresis from transition elements of endoplasmic reticulum incubated with ATP and a cytosol fraction in a cell free system, exhibited a polypeptide composition distinct from that of the original transitional endoplasmic reticulum fractions. The transition vesicles were deficient in peptides of apparent molecular mass between 200 and 270 kD, in the vicinity of 65 kD and less than 40 kD. Immunoblot analysis suggested a 140 kD protein to be concentrated in transition vesicles and low or absent from the endoplasmic reticulum fractions from which the transition vesicles were derived.

Protein phosphorylation in a myogenic cell line: effects of insulin, epinephrine and glucose.

Protein phosphorylation was studied in L6 cultured muscle cells by incubating cells with Na 32Pi and subsequently exposing them to external agents. L6 cells readily incorporated 32Pi into a number of peptides approaching steady-state incorporation by 2 h. Insulin stimulated the phosphorylation of one peptide of molecular mass 29,000 daltons by 37% with an ED50 of 3 mU/ml. This peptide was located in the high-speed pellet (105,000 g 60 min) which is consistent with an S6 ribosomal protein. Epinephrine (10(-5) M) led to only a modestly stimulated (less than 14%) phosphorylation of three peptides of molecular masses 39,000, 29,000 and 21,000 daltons. Glucose (5-50 mM) stimulated the phosphorylation of one peptide of molecular mass 19,000 daltons by 24%.