Search results for "Edman degradation"
showing 10 items of 12 documents
Sequencing and analysis of the gene encoding the α-toxin of Clostridium novyi proves its homology to toxins A and B of Clostridium difficile
1995
A library of total Clostridium novyi DNA was established and screened for the alpha-toxin gene (tcn alpha) by hybridization with oligonucleotides derived from a partial N-terminal sequence and by using specific antisera. Overlapping subgenic tcn alpha fragments were isolated and subsequently the total sequence of tcn alpha was determined. The 6534 nucleotide open reading frame encodes a polypeptide of M(r) 250,166 and pI 5.9. The N-terminal alpha-toxin (Tcn alpha) sequence MLITREQLMKIASIP determined by Edman degradation confirmed the identity of the reading frame and the assignment of the translation start point. The toxin is not modified posttranslationally at its N-terminus nor does it co…
Direct identification of the agonist binding site in the human brain cholecystokinin-B receptor
1999
In investigating the agonist binding site of the human brain cholecystokininB receptor (CCKBR), we employed the direct protein chemical approach using a photoreactive tritiated analogue of sulfated cholecystokinin octapeptide, which contains the p-benzoylbenzoyl moiety at the N-terminus, followed by purification of the affinity-labeled receptor to homogeneity. This probe bound specifically, saturably, and with high affinity (KD = 1.2 nM) to the CCKBR and has full agonistic activity. As the starting material for receptor purification, we used stably transfected HEK 293 cells overexpressing functional CCKBR. Covalent labeling of the WGA-lectin-enriched receptor revealed a 70-80 kDa glycoprote…
Molecular characterization of a male-specific glycosyl hydrolase, Lma-p72, secreted on to the abdominal surface of the Madeira cockroach Leucophaea m…
2003
0264-6021 (Print) Journal Article Research Support, Non-U.S. Gov't; The epicuticular surface protein Lma-p72 is specific to the abdominal secretions of Leucophaea maderae (Madeira cockroach) adult males. Natural Lma-p72 was purified and the complete cDNA sequence determined by reverse-transcription PCR using primers based on Edman degradation fragments. Northern blot and in situ hybridization analyses showed that Lma-p72 was expressed in the tergal and sternal glands. Sequence alignment indicates that Lma-p72 is closely related to the family 1 glycosyl hydrolases (EC 3.2.1). Native Lma-p72 was proved to be active in the abdominal secretions and exhibit a beta-galactosidase-like activity. Ho…
Proteomic Analyses Reveal an Acidic Prime Side Specificity for the Astacin Metalloprotease Family Reflected by Physiological Substrates
2011
Astacins are secreted and membrane-bound metalloproteases with clear associations to many important pathological and physiological processes. Yet with only a few substrates described their biological roles are enigmatic. Moreover, the lack of knowledge of astacin cleavage site specificities hampers assay and drug development. Using PICS (proteomic identification of protease cleavage site specificity) and TAILS (terminal amine isotopic labeling of substrates) degradomics approaches >3000 cleavage sites were proteomically identified for five different astacins. Such broad coverage enables family-wide determination of specificities N- and C-terminal to the scissile peptide bond. Remarkably, me…
Identification of major histocompatibility complex class II-associated peptides derived from freshly prepared rat Langerhans cells using MALDI-PSD an…
2000
The isolation and identification is described of MHC class II-bound peptides derived from Langerhans cells. A combination of preparative micro-HPLC, MALDI-MS, Edman degradation was used for determining the amino acid sequence of MHC-associated peptides. Sample handling was crucial because fractions containing trace amounts of material require immediate storage at −80 °C to prevent peptide losses.
Molecular characterization of Lma-p54, a new epicuticular surface protein in the cockroach Leucophaea maderae (Dictyoptera, oxyhaloinae)
2002
0965-1748 (Print) Journal Article Research Support, Non-U.S. Gov't; The epicuticular surface protein Lma-p54 is imbedded in the "cuticular waxes" which cover the abdominal surface of the adult Leucophaea maderae. Natural Lma-p54 was purified and the complete cDNA sequence was determined by RT-PCR using primers based on Edman degradation fragments. Northern blot and in situ hybridization analyses showed that Lma-p54 was expressed in the adult abdominal epidermis and in the chemical sense organs of both sexes. Sequence alignment indicates that Lma-p54 is closely related to aspartic proteases (EC 3.4.23). However, there are critical amino acid substitutions at the level of the active site and,…
Differential Infectivity of Two Pseudomonas Species and the Immune Response in the Milkweed Bug, Oncopeltus fasciatus (Insecta: Hemiptera)
2002
Pseudomonas aeruginosa and Pseudomonas putida show a profound differential infectivity after inoculation in Oncopeltus fasciatus. Whereas P. putida has no significant impact on nymphs, P. aeruginosa kills all experimental animals within 48 h. Both Pseudomonas species, however, induce the same four hemolymph peptides in O. fasciatus. Also injection of saline solution and injury induced these peptides. In general peptide induction was stronger in nymphs than in adult males. A significantly higher number of nymphs survived a challenge with P. aeruginosa when an immunization with P. putida preceded. The antibacterial properties of the hemolymph were demonstrated in inhibition experiments with P…
Molecular characterization of a new adult male putative calycin specific to tergal aphrodisiac secretion in the cockroach Leucophaea maderae
2001
0014-5793 (Print) Journal Article Research Support, Non-U.S. Gov't; Lma-p18 is an epicuticular surface protein specific to the tergal gland aphrodisiac secretion of Leucophaea maderae adult males. Native Lma-p18 was purified and the complete cDNA sequence was determined by RT-PCR using primers based on Edman degradation fragments. Northern blot and in situ hybridization analyses showed that Lma-p18 is expressed exclusively in the anterior part of male tergal gland, which is exposed only during sexual behavior. Sequence analysis indicated that Lma-p18 belongs to the calycin superfamily and is very similar to Lma-p22, the first known male-specific tergal protein in L. maderae. Lma-p18 and Lma…
Automated multi-dimensional liquid chromatography
2004
A comprehensive on-line sample clean-up with an integrated two-dimensional HPLC system was developed for the analysis of natural peptides. Samples comprised of endogenous peptides with molecular weights up to 20 kDa were generated from human hemofiltrate (HF) obtained from patients with chronic renal failure. The (poly-)peptides were separated using novel silica-based restricted access materials with strong cation-exchange functionalities (SCX-RAM). The size-selective sample fractionation step is followed by cation-exchange chromatography as the first dimension. The subsequent second dimension of separation is based on hydrophobic interaction using four parallel short reversed-phase (RP) co…
Porifera Lectins: diversity, physiological roles and biotechnological potential
2015
An overview on the diversity of 39 lectins from the phylum Porifera is presented, including 38 lectins, which were identified from the class of demosponges, and one lectin from the class of hexactinellida. Their purification from crude extracts was mainly performed by using affinity chromatography and gel filtration techniques. Other protocols were also developed in order to collect and study sponge lectins, including screening of sponge genomes and expression in heterologous bacterial systems. The characterization of the lectins was performed by Edman degradation or mass spectrometry. Regarding their physiological roles, sponge lectins showed to be involved in morphogenesis and cell intera…