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P Roepstorff

Publications and source records attributed to P Roepstorff.

At least 145 records · Page 8Linked to original sources
PubMed1995

[Serratia marcescens nucleases. I. Comparison of native and recombinant nucleases using electrospray mass spectrometry].

Isoforms of the natural and recombinant nucleases of Serratia marcescens were characterized by their molecular mass, which was determined by electrospray mass spectrometry. The natural nuclease was isolated from the S. marcescens B10M1 culture, whereas the recombinant nuclease was obtained from Escherichia coli MT102 cells carrying plasmid p403-SD2 with the nuclease gene nuc. The primary structure for each of the isoforms isolated from the nuclease preparations was determined by comparing its molecular mass with that of known amino acid sequence, which was determined from the nucleotide sequence of the nuc gene. Both preparations included identical nuclease variants with N-terminal amino acid residues removed. The number of isoforms in the natural nuclease was, however, significantly greater than in the recombinant nuclease. The structures of some of the isoforms were confirmed by N-terminal analysis.

Chromatography, DEAE-Cellulose↗
PubMed1995

[Serratia marcescens nucleases. II. Primary structure analysis by peptide mapping combined with plasma-desorption mass spectrometry].

The primary structures of nucleases Sm1, Sm2, and Sm3 produced by Serratia marcescens were completely characterized using plasma desorption mass spectrometry (PDMS) of proteolytic peptides isolated by reverse-phase HPLC. The isoforms were separated by anion-exchange chromatography on DEAE cellulose and subjected to hydrolysis by the lysine-specific endoproteinase Lys-C. Comparative analysis of the peptides identified by PDMS showed that all three nucleases are N-terminal variants of the same protein: Sm2 represents a "mature" protein form, whereas Sm1 and Sm3 lack three and one N-terminal amino acid residues, respectively.

Amino Acid Sequence↗
PubMed1995

[Nuclease isoforms of natural and recombinant strains of Serratia marcescens. Comparative characteristics of plasma desorption mass spectrometry].

The primary structure of isoforms of natural nuclease secreted by Serratia marcescens as well as of recombinant nuclease produced by Escherichia coli has been characterized by plasma desorption mass spectrometry. The isoforms were isolated from the purified nuclease on a DEAE-cellulose anion-exchange column and digested with endoproteinase Lys-C. The peptides generated were isolated by reversed phase IIPLC and their molecular masses determined by plasma desorption mass spectrometry. Among the nuclease isoforms secreted by parent cells, a new isoform, Sm3, was found. Comparison of the peptides yielded by nuclease Sm2 and two isoenzymes, Sm1 and Sm3, demonstrated their distinction only in the N-termini; three amino acids are lacking in Sm1 and one residue in Sm3. Identity was demonstrated between nucleases Sm1 and Sm2 produced by S. marcescens and the isoforms rSm1 and rSm2 secreted by the recombinant E. coli strain.

Amino Acid Sequence↗
PubMed1995

[Characteristics of isoforms of Serratia marcescens from electrospray mass spectrometry].

Electrospray mass spectrometry was used to determine the molecular masses of nuclease isoforms isolated and purified from the cultural fluid of Serratia marcescens B10M1. The primary structure of each of the isoforms isolated from the nuclease preparations was established by comparing their masses with the known amino acid sequence encoded by the nuc-gene. The structure of some of these isoforms was verified by N-terminal sequencing, that of nearly all isoforms-by isoelectric focusing. All nuclease isoforms were found to be variants of native nuclease split at the molecular N-termini.

Chromatography, DEAE-Cellulose↗
PubMed1994

Identification of a substance, previously shown to enhance mitogenesis of human lymphocytes, as the acetamide of p-aminobenzoic acid.

We characterize here an arachidonic acid (AA)-derived metabolite previously found to have an adjuvant effect in phytohemagglutinin-induced mitogenesis of lymphocytes from mothers of newborn babies and from immunodeficient infants. We named the metabolite 'compound 4' due to its position in a thin-layer chromatography system developed for isolation of eicosanoids. The compound was originally found to be produced by peripheral blood mononuclear leukocytes and the T cell leukemia line Jurcat after long-term (18-24 h) incubation with [1-14C]AA. Compound 4 is also produced by lymphocytes, monocytes, platelets, thrombocytes, cultured fibroblasts and various types of malignant cell lines. We purified this metabolite by means of high pressure liquid chromatography with synchronous detection of radioactivity and measurement of ultraviolet-light absorption at 278 nm. Proton nuclear magnetic resonance spectroscopy and mass spectrometry with electron impact techniques demonstrated that compound 4 is not an eicosanoid, but is identical to p-acetamidobenzoic acid (PACBA). The cells synthesize PACBA from p-aminobenzoic acid and a two-carbon residue from AA.

4-Aminobenzoic Acid↗
PubMed1994

Matrix assisted laser desorption/ionization mass spectrometry of enzymatically synthesized RNA up to 150 kDa.

Enzymatically synthesized RNA samples (in vitro transcripts) were analysed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Spectra of RNA up to 150 kDA (461 nucleotides) are shown. Polymerase generated sample heterogeneity and its contribution to mass resolution are discussed. A time course exonuclease digest of a 55 nt in vitro transcript was analyzed to investigate the performance of MALDI-MS on complex mixtures. Based on these data, the analysis by MALDI-MS of DNA sequencing reactions, produced by the action of an RNA polymerase, is discussed.

Base Sequence↗
PubMed1994

Yeast acyl-CoA-binding protein: acyl-CoA-binding affinity and effect on intracellular acyl-CoA pool size.

Acyl-CoA-binding protein (ACBP) is a 10 kDa protein characterized in vertebrates. We have isolated two ACBP homologues from the yeast Saccharomyces carlsbergensis, named yeast ACBP types 1 and 2. Both proteins contain 86 amino acid residues and are identical except for four conservative substitutions. In comparison with human ACBP, yeast ACBPs exhibit 48% (type 1) and 49% (type 2) conservation of amino acid residues. The amino acid sequence of S. carlsbergensis ACBP type 1 was found to be identical with the one ACBP present in Saccharomyces cerevisiae. A recombinant form of this protein was expressed in Escherichia coli and S. cerevisiae, purified, and its acyl-CoA-binding properties were characterized by isoelectric focusing and microcalorimetric analyses. The yeast ACBP was found to bind acyl-CoA esters with high affinity (Kd 0.55 x 10(-10) M). Overexpression of yeast ACBP in S. cerevisiae resulted in a significant expansion of the intracellular acyl-CoA pool. Finally, Southern-blotting analysis of the two genes encoding ACBP types 1 and 2 in S. carlsbergensis strongly indicated that this species is a hybrid between S. cerevisiae and Saccharomyces monacensis.

Acyl Coenzyme A↗
PubMed1994

The complete amino acid sequence and disulphide bond arrangement of oat alcohol-soluble avenin-3.

We have elucidated the complete amino acid sequence of one of the avenin components, avenin-3, isolated from oat (Avena sativa L.), variety Narymsky 943. The sequence of the protein was determined by sequencing of CNBr and trypsin-generated peptides in combination with mass spectrometry. The protein is a single polypeptide chain, consisting of 201 amino acid residues with M(r) 23,252.8. The N-terminal amino acid residue of the protein is blocked with 5-oxoproline (pyroglutamic acid). All eight cysteine residues in avenin-3 are involved in disulphide bonds. The positions of these bonds were established by identification of a CNBr cleavage product of the intact avenin containing all the disulfide bonds (S-S core). Subsequent subdigestion of this S-S core allowed isolation of disulphide bonded peptides detected by differential reverse-phase HPLC before and after reduction. As a result, all four disulphides Cys50 Cys183, Cys58 Cys77, Cys84 Cys85 and Cys97 Cys191 were identified. Comparison of avenins with other prolamins demonstrates a high degree of similarity, which is especially pronounced around the cysteine residues. Avenins differ slightly from other prolamins in having unique N-terminal sequences and some differences in the repeated sequence motifs.

Amino Acid Sequence↗
PubMed1994

Differentiational regulation and phosphorylation of the fatty acid-binding protein from rat mammary epithelial cells.

From the soluble protein fraction of lactating rat mammary epithelial cells, fatty acid-binding protein (FABP) was isolated by immunoaffinity chromatography. After digestion with trypsin, peptides were characterized with time-of-flight mass spectrometry and revealed identity with corresponding peptides derived from the heart-type FABP isolated from rat heart. In addition, by electrospray mass spectrometry the molecular mass has been determined to 14683.9 +/- 3 Da, further corroborating the identity. The content of FABP in mammary glands from virgin, pregnant and lactating rats was evaluated using two-dimensional gel electrophoresis and a FABP-specific immunosorbent assay. In the two-dimensional gels FABP was the apparently most abundant cytosolic protein in mammary epithelial cells from rats in late pregnancy as well as from lactating rats. The content of FABP was 59 +/- 19 microgram/mg (n = 11) of soluble proteins from the fully differentiated lactating mammary gland as determined by ELISA. This value represented an 80-fold increase compared with the FABP content of mammary gland from virgin rats, and is comparable with the level found in rat heart. Upon stimulation with insulin a small fraction of FABP was phosphorylated in lactating mammary epithelial cells. In conclusion, these findings indicate that the FABPs from rat mammary gland and heart are identical and further suggest that in mammary gland this FABP may play a role in signal transduction downstream from the insulin receptor.

Amino Acid Sequence↗
PubMed1994

Amino acid sequence of beta-microseminoprotein from porcine seminal plasma.

beta-Microseminoprotein is a small mucus-associated protein of unknown function, previously characterized in man and the ape. The corresponding porcine protein was identified in tracheal mucus and seminal plasma by its cross-reaction with antibodies raised against the human protein. The porcine protein was isolated from seminal plasma, and its complete amino acid sequence determined. It is a single-chain protein of 91 amino acids with a molecular mass of 10,068 Da. Compared to primate beta-microseminoprotein, the porcine protein has a blocked N-terminus and a deletion of three amino acids in the N-terminal region, but otherwise manifests similarity in amino acid sequence including conservation of 10 cysteine residues. A proportion of individual pigs have two forms of the protein, differing in charge but not in size. The characterization of beta-microseminoprotein in a species suited for experiments should help determine the function of this protein.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of a cadmium-induced metallothionein from the shore crab Carcinus maenas (L.).

Two metallothionein variants were purified from the midgut gland of crabs (Carcinus maenas) exposed to a high cadmium concentration (2 p.p.m.). One of the variants was purified from crabs exposed to a low cadmium concentration (0.5 p.p.m.). The purification method involved acetone precipitation, gel filtration and reversed-phase h.p.l.c. The complete amino acid sequences of both variants have been elucidated by m.s. and automated sequence analysis on S-methylated proteins or fragments produced by cleavage of the S-methylated proteins with Staphylococcus aureus proteinase. The two variants from crabs exposed to the high cadmium concentration differed only by a single residue of methionine at the N-terminus. The single variant isolated from crabs exposed to the low cadmium concentration was the one without the N-terminal methionine, indicating that high cadmium concentrations either inhibit the processing enzymes and/or that the processing enzymes cannot keep pace with the increased metallothionein synthesis when cadmium availability is high. Cadmium-induced metallothionein from C. maenas shows a high degree of structural similarity to metallothioneins from the decapod crustaceans Scylla serrata and Homarus americanus.

Amino Acid Sequence↗
PubMed1994

Identification of proteins in polyacrylamide gels by mass spectrometric peptide mapping combined with database search.

Mass spectrometric peptide mapping of proteins separated by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis has been investigated. The best results are obtained after blotting of the proteins onto polyvinylidene difluoride membranes followed by enzymatic digestion of the protein on the membrane. The peptide maps were investigated in terms of completeness and applicability for protein identification using a previously developed database search program as well as for the possibility for full characterization of covalent modifications in the proteins. The most complete peptide maps were obtained when the proteins were reduced and alkylated on the membrane prior to enzymatic digestion followed by separation of the resulting mixture by high performance liquid chromatography prior to mass spectrometric analysis. Such peptide maps cover up to 98% of the sequence and consequently may allow complete characterization of post-translational modifications in proteins for which the amino acid sequence is known. The fastest and most sensitive procedure to obtain peptide maps sufficient for protein identification was direct analysis of the extracted peptide mixture by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The use of external and internal calibration of MALDI spectra for database searches is evaluated as well as the possibility of including a post-calibration routine within the search program.

Alkylation↗
PubMed1994

Localization of an O-glycosylated site in the recombinant barley alpha-amylase 1 produced in yeast and correction of the amino acid sequence using matrix-assisted laser desorption/ionization mass spectrometry of peptide mixtures.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of peptide mixtures was used to characterize recombinant barley alpha-amylase 1, produced in yeast. Three peptide mixtures were generated by cleavage with CNBr, digestion with endoproteinase Lys-C and Asp-N, respectively, and analyzed directly by MALDI-MS. Based on the three mass spectrometric peptide maps, an error in the sequence deduced from cDNA, resulting in a mass difference of 28 Da, was located to a sequence stretch of 5 amino acid residues; furthermore, a dihexose substituent was identified on Thr410. Subsequent Edman degradation of two selected peptides isolated from the endoproteinase Lys-C digest corrected the sequence to be Val instead of Ala in position 284 and confirmed the O-glycosylation. These results demonstrate that the direct peptide mixture analysis by MALDI-MS is a rapid and sensitive method for protein characterization and provides valuable information before further characterization.

Amino Acid Sequence↗
PubMed1994

Peptide sequence information derived by partial acid hydrolysis and matrix-assisted laser desorption/ionization mass spectrometry.

Partial acid hydrolysis of purified peptides followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the resulting peptide mixture is a fast and simple procedure to confirm the identity of a peptide tentatively identified by its determined molecular weight. Often partial amino acid sequence information can be obtained directly. The sensitivity of the technique is in the high-femtomole to low-picomole range.

Acids↗
PubMed1994

Enzyme IIBcellobiose of the phosphoenol-pyruvate-dependent phosphotransferase system of Escherichia coli: backbone assignment and secondary structure determined by three-dimensional NMR spectroscopy.

The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBcellobiose of the Escherichia coli cellobiose-specific phosphoenol-pyruvate-dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple resonance experiments, the HNCA and HN(CO)CA experiments, correlating backbone 1H, 15N, and 13C alpha resonances, and the HN(CA)CO and HNCO experiments, correlating backbone 1H,15N and 13CO resonances. Heteronuclear 1H-NOE 1H-15N single quantum coherence (15N-NOESY-HSQC) spectroscopy and heteronuclear 1H total correlation 1H-15N single quantum coherence (15N-TOCSY-HSQC) spectroscopy were used to resolve ambiguities arising from overlapping 13C alpha and 13CO frequencies and to check the assignments from the triple resonance experiments. This procedure, together with a 3-dimensional 1H alpha-13C alpha-13CO experiment (COCAH), yielded the assignment for all observed backbone resonances. The secondary structure was determined using information both from the deviation of observed 1H alpha and 13C alpha chemical shifts from their random coil values and 1H-NOE information from the 15N-NOESY-HSQC. These data show that enzyme IIBcellobiose consists of a 4-stranded parallel beta-sheet and 5 alpha-helices. In the wild-type enzyme IIBcellobiose, the catalytic residue appears to be located at the end of a beta-strand.

Amino Acid Sequence↗
PubMed1994

A hydroxyproline-containing class IV chitinase of sugar beet is glycosylated with xylose.

Two acidic chitinase isoforms, SP1 and SP2, have been purified to homogeneity from leaves of sugar beet (Beta vulgaris) infected with Cercospora beticola. SP1 and SP2 are extracellular proteins with an apparent molecular mass of 35 kDa and an approximate pI of 4.2. Since the only major difference was slightly diverging M(r)'s, only the SP2 chitinase was further characterized. Partial amino acid sequence data for SP2 was used to generate a polymerase chain reaction (PCR) clone employed for the isolation of a cDNA clone encoding SP2. SP2 exhibits significant structural identity with the class IV chitinases from sugar beet, rapeseed, bean and maize, but differs from the other members of this class in having a longer hinge region, comprising 22 amino acid residues, with a repeated 'TTP' motif. Western blotting analyses, using antibody raised against SP2, demonstrated an induction of SP protein during infection with C. beticola. The induction was very local, with high protein accumulation found close to the infection site only. Amino acid compositional analysis of SP2 revealed that five out of fourteen prolines are hydroxylated. No glucosamine or galactosamine residues are present. Evidence was obtained that SP2 is glycosylated with a limited number (< or = 7) of xylose residues: (1) SP2 was stained with the periodic acid-Schiff (PAS) reagent, (2) electrospray mass spectrometry on SP2 gave a series of M(r)'s with a consistent increase between two molecular masses of 132 Da, (3) SP2 was recognized by an antibody specific for beta-1,4-D-xylopyranose. The vacuolar class I chitinases A and B in tobacco have recently been shown to comprise a new class of hydroxyproline-containing proteins (Sticher et al., Science 257 (1992) 655-657). The SP2 chitinase differs from these in being glycosylated and, thus, represents a novel type of hydroxyproline-containing glycoproteins in plants.

Amino Acid Sequence↗
PubMed1994

The primary structure of an endocuticular protein from two locus species, Locusta migratoria and Schistocerca gregaria, determined by a combination of mass spectrometry and automatic Edman degradation.

The complete primary structures of two variants of a protein, Abd-5, isolated from the endocuticles of the migratory locust Locusta migratoria and the desert locust Schistocerca gregaria, have been determined. The proteins from the two species are N-terminally blocked with pyroglutamic acid. Their sequences differed only in two positions. Comparison of the sequences to those of other cuticular proteins shows that moderate homologies exist to 11 other cuticular proteins from insects representing four different orders. Amino acid residues in certain positions appear to be strictly conserved.

Amino Acid Sequence↗