Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Monoclonal antibodies (MAbs) to a Burkholderia (Pseudomonas) cepacia 36-kDa protease (PSCP) which neutralize PSCP and Pseudomonas aeruginosa elastase but not P. aeruginosa alkaline protease have been isolated (C. Kooi et al., Infect. Immun. 62:2811-2817, 1994). These MAbs, designated 36-6-6 and 36-6-8, react with N-chlorosuccinimide cleavage products of P. aeruginosa elastase, consistent with the recognition of a 13.9-kDa fragment which contains the active site. Overlapping 9-mer peptides that span this region were synthesized. Neutralizing MAbs to PSCP reacted strongly with two peptides (341HGFTEQNSG349 and 395RYM DQPSRD403). Peptide 341HGFTEQNSG349 overlaps the motif 337HEXXH341, which has been found in many zinc-dependent endopeptidases. Peptide 395RYMDQPSRD403 lies between E361, which binds a zinc atom, and H420, which acts as a proton donor at the active site. Polyclonal rabbit sera raised against these peptides reacted with elastase on Western immunoblots and by enzyme-linked immunosorbent assay. With hide powder azure as the substrate, antisera to either HGFTEQNG and RYMDQPSRD completely neutralized the activities of elastase, thermolysin, Vibrio cholerae hemagglutinin/protease, and PSCP but had no effect on P. aeruginosa alkaline protease or the Serratia marcescens major protease. These results suggest that the MAbs recognize two different epitopes on P. aeruginosa elastase and that antibodies raised against synthetic peptides corresponding to either of these epitopes neutralize proteolytic activity.
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PMID:Identification of neutralizing epitopes on Pseudomonas aeruginosa elastase and effects of cross-reactions on other thermolysin-like proteases. 900 99

Immunoglobulin A1 (IgA1) from normal human serum is known to have O-linked sugar chains, sialylated Galbeta1,3GalNAc, in the hinge portion. In order to reduce the microheterogenity of the sugar chain, the hinge glycopeptide prepared from IgA1 was sequentially treated with neuraminidase and beta-galactosidase. The asialo-, agalacto-hinge glycopeptide (HGP-SG) composed of a 33-mer peptide (HP33) and N-acetylgalactosamine (GalNAc) residues was obtained. The HGP-SG was separated into three major peaks, A, B and C, by high-performance liquid chromatography (HPLC). Each glycopeptide fraction was further separated by capillary electrophoresis (CE). Peaks A, B and C with HPLC abundantly contained HP33 bearing five and six N-acetylgalactosamine residues (HGP33-5,6GN), HGP33-4,5GN and HGP33-3,4GN, respectively. Among these glycopeptide peaks, only the HGP33-5GN peak was partly split into two peaks based on the CE analysis - HGP33-5GN-alpha and -beta. The glycopeptide, HGP25-5GN shortened by the thermolysin digest of HGP33-SG was also well separated into the alpha and beta forms by CE analysis. No differences in their mass and peptide portion were observed between HGP25-5GN-alpha and -beta. Therefore, the obtained result might indicate that HGP25-5GN-alpha was an isomer of HGP25-5GN-beta differing in its stereospecific structure of the peptide portion and/or the attachment site of the GalNAc residue.
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PMID:Mutual separation of hinge-glycopeptide isomers bearing five N-acetylgalactosamine residues from normal human serum immunoglobulin A1 by capillary electrophoresis. 1040 3

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.
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PMID:Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus. 1132 28