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At least 847 records · Page 47Linked to original sources
PubMed1992

Use of resorufin-labelled N-glycopeptide in a high-performance liquid chromatography assay to monitor endoglycosidase activities during cultivation of Flavobacterium meningosepticum.

Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F (PNGase F) and endo-beta-N-acetyl glucosaminidase F (Endo F) activities were monitored during cultivation of Flavobacterium meningosepticum using a new fluorescence-HPLC procedure based on a commercially available substrate. The PNGase F activity reached a maximum level at the end of the log phase and remained constant during the stationary phase, while Endo F continuously increased until late stationary phase. PNGase F obtained at the end of the log phase was less contaminated by other proteins compared with late stationary phase.

Amidohydrolases↗
PubMed2004

Total enzymatic synthesis of cholecystokinin CCK-5.

This paper describes the enzymatic synthesis of the C-terminal fragment H-Gly-Trp-Met-Asp-Phe-NH2 of cholecystokinin. Immobilized enzymes were used for the formation of all peptide bonds except thermolysin. Beginning the synthesis with phenylacetyl (PhAc) glycine carboxamidomethyl ester (OCam) and H-Trp-OMe by using immobilized papain as biocatalyst in buffered ethyl acetate, the dipeptide methyl ester was then coupled directly with Met-OEt.HCl by alpha-chymotrypsin/Celite 545 in a solvent free system. For the 3+2 coupling PhAc-Gly-Trp-Met-OEt had to be converted into its OCam ester. The other fragment H-Asp(OMe)-Phe-NH2 resulted from the coupling of Cbo-Asp(OMe)-OH with H-Phe-NH2.HCl and thermolysin as catalyst, followed by catalytic hydrogenation. Finally PhAc-Gly-Trp-Met-Asp-Phe-NH2 was obtained in a smooth reaction from PhAc-Gly-Trp-Met-OCam and H-Asp(OMe)-Phe-NH2 with alpha-chymotrypsin/Celite 545 in acetonitrile, followed by basic hydrolysis of the beta-methyl ester. The PhAc-group is removed with penicillin G amidase and CCK-5 is obtained in an overall isolated yield of 19.6%.

Acetates↗
PubMed2007

Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast Pichia pastoris.

Human chorionic gonadotropin (hCG) is a heterodimeric, placental glycoprotein hormone involved in the maintenance of the corpus luteum during the first trimester of pregnancy. Biologically active hCG has been successfully expressed in the yeast Pichia pastoris (phCG). In the context of structural studies and therapeutic applications of phCG, detailed information about its glycosylation pattern is a prerequisite. To this end N-glycans were released with peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated via anion-exchange chromatography (Resource Q) yielding both neutral (80%) and charged, phosphate-containing (20%) high-mannose-type structures. Subfractionations were carried out via normal phase (Lichrosorb-NH(2)) and high-pH anion-exchange (CarboPac PA-1) chromatography. Structural analyses of the released N-glycans were carried out by using HPLC profiling of fluorescent 2-aminobenzamide derivatives, MALDI-TOF mass spectrometry, and 500-MHz(1)H-NMR spectroscopy. Detailed neutral oligosaccharide structures, in the range of Man(8)GlcNAc(2) to Man(11)GlcNAc(2) including molecular isomers, could be established, and structures up to Man(15)GlcNAc(2) were indicated. Phosphate-containing oligosaccharides ranged from Man(9)PGlcNAc(2) to Man(13)PGlcNAc(2). Mannosyl O-glycans were not detected. Profiling studies carried out on different production batches showed that the oligosaccharide structures are similar, but their relative amounts varied with the culturing media.

Acetylglucosamine↗
PubMed1986

Characterization of cellular oligosaccharides from normal and cystic fibrotic fibroblasts using sequential endoglycosidase digestions.

A method was developed for obtaining detailed oligosaccharide profiles from [2-3H]mannose- or [6-3H]fucose-labeled cellular glycoproteins. The oligosaccharides were segregated first according to class, using endo-beta-N-acetylglucosaminidase H (Endo H) to release the high mannose species, and then with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F), which provided a complete array of complex oligosaccharide chains. The high mannose and complex oligosaccharides were fractionated subsequently according to net negative charge on QAE-Sephadex. High resolution gel filtration on TSK HW-40(S) resolved the neutral high mannose population into species of the type Man9-5 N-acetylglucosamine. Desialylation of the complex chains with neuraminidase allowed resolution of these oligosaccharides into their corresponding asialo bi-, tri-, and tetraantennary species. Fibroblasts from normal and cystic fibrosis cells were analyzed for differences in their glycosylation patterns using these techniques. Over 95% of the [2-3H]mannose-labeled glycoproteins were susceptible to the combined glycosidase digestions, but no difference in either the high mannose or complex oligosaccharides were observed. Nonetheless, the methodology developed in this study provides an important new approach for investigating oligosaccharides of different cell types and variants of the same type. Metabolic changes induced in cellular glycoproteins, as illustrated by use of the processing inhibitor swainsonine, demonstrated the versatility of this procedure for investigating questions relating to glycoprotein structure and enzyme specificity. Thus, by employing a variation of this method, it was possible to confirm the location of fucose in the core of PNGase F-released hybrid oligosaccharides by the subsequent release with Endo H of the disaccharide, fucosyl-N-acetylglucosamine.

Acetylglucosaminidase↗
PubMed1994

The role of N-linked oligosaccharides of MHC class II antigens in T cell stimulation.

A specific increase in T cell extracellular acidification rate has been demonstrated recently when complexes of purified MHC class II molecules and antigenic peptides interact with T cell receptors (TCRs) on cloned T cells. The present study shows that such measurements of an increase in extracellular acidification rate can be used to evaluate the functional role of various N-linked oligosaccharides of MHC class II antigens. Affinity-purified murine IAk and IAs were deglycosylated in the presence of aspargine-amidase enzyme and were characterized by SDS-polyacrylamide gel electrophoresis. The complete removal of all three N-linked oligosaccharides from the alpha/beta heterodimer was confirmed by four different lectin-linked Western blot analyses. Similar to the native heterodimer, both deglycosylated IAk and deglycosylated IAs were fully capable of binding synthetic antigenic peptides derived from myelin basic protein (MBP). When equivalent amount of glycosylated and deglycosylated class II-peptide complexes were exposed to restricted cloned T cells, identical increases in T cell extracellular acidification rates were observed. The specificity of such increases in extracellular acidification rate was demonstrated by exposing cloned T cells to irrelevant complexes of glycosylated and deglycosylated class II and antigenic peptides. These results show how measurement of extracellular acidification rate can be used to study structure-function correlations of ligand-receptor interactions, and support an earlier observation that N-linked oligosaccharides of murine MHC class II molecules are not involved in either antigenic peptide binding or T cell recognition.

Amidohydrolases↗
PubMed1987

Role of membrane N-linked oligosaccharides in host cell interaction with invasive forms of Trypanosoma cruzi.

The removal of N-linked oligosaccharides by peptide-N4-[N-acetyl-beta-glucoseaminyl]asparagine amidase (previously known as aspartoglycosylamine amidohydrolase and abbreviated N-glycanase) from the surface of blood or insect-transmissible forms of Trypanosoma cruzi markedly increased the capacity of these organisms to associate with (i.e., bind and penetrate) either mouse peritoneal macrophages or rat heart myoblasts. This effect was evidenced by a significant elevation in both the percentage of infected host cells and the average number of parasites per 100 cells. Conversely, N-glycanase treatment of either host cell markedly reduced both parameters to levels significantly below those obtained with cells mock treated with medium alone. The N-glycanase effect on the parasites was inhibited by heat inactivation of the enzyme or by the presence of fetuin, an N-glycanase substrate. The enhanced capacity of N-glycanase-treated T. cruzi to engage the host cells started to subside 2 h after the treatment, indicating the reversibility of the effect. The decreased reactivity of N-glycanase-treated macrophages or myoblasts with T. cruzi suggests that N-linked oligosaccharides on these host cells are involved in the initial phase of the cell infection process. Instead, because T. cruzi interacted more effectively with host cells after treatment with N-glycanase, parasite surface N-linked oligosaccharides would seem to interfere with the association.

Amidohydrolases↗
PubMed1995

Do de-N-glycosylation enzymes have an important role in plant cells?

In this review de-N-glycosylation was defined as the removal of the glycan(s) from a N-glycosylprotein, by means of enzymes acting on the di-N-acetylchitobiosyl part of the invariant pentasaccharide inner-core of N-glycosylproteins. Peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidases (PNGase) and endo-N-acetyl-beta-D-glucosaminidases (ENGase) were both considered as de-N-glycosylation enzymes. A detailed description of the characterization and the function of plant PNGases and ENGases is presented, together with a brief presentation on the occurrence and the current knowledge on the function of microbial and animal enzymes. De-N-glycosylation of plant glycoproteins was proposed as a possible mechanism for the release of oligosaccharides displaying biological activities and the removal of N-glycans could also explain the regulation of protein activity. Each enzyme seems to have a specific function during germination and post-germinative development. All the arguments concur that de-N-glycosylation enzymes have an important role in plant cells and confirm that the N-glycosylation/de-N-glycosylation system should occur more commonly than presently recognized in living organisms.

Amidohydrolases↗
PubMed1981

The metabolic fate of 14C-labeled immunoadjuvant peptidoglycan monomer. II. In vitro studies.

Peptidoglycan monomer (GlcNAc-MurNAc-L-Ala-D-isoglutamine-meso-diaminopimelic acid-D-Ala-D-Ala), labeled with 14C both in the disaccharide and pentapeptide portions, was incubated with slices of mouse liver, kidney or spleen as well as with mouse and human blood, blood cells plasma and serum. Peptidoglycan monomer was isolated unchanged after incubations with mouse organs and blood cells. However, upon incubation with mouse or human blood, 10-50% of the peptidoglycan monomer underwent hydrolysis to the corresponding disaccharide and pentapeptide. After incubations with plasma and serum more than 90% of the [14C]peptidoglycan monomer was metabolized: about 50% of the administered radioactive dose was recovered in the disaccharide unit and about 35% in the pentapeptide part. These results suggest that in blood, plasma and serum of mouse and man, an N-acetylmuramoyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) exists which splits the amide bond between the lactyl carboxyl group of the muramyl residue and the amino group of the peptide moiety in the peptidoglycan molecule.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1993

Anomalies in cell wall turnover associated with the growth temperature of Bacillus subtilis.

Cell wall turnover appeared to be anomalously fast in Bacillus subtilis when the cells were grown at temperatures below 29 degrees C. Turnover rates k(generation-1), of exponential cultures at 25 degrees were approximately double those of cells grown at 37 degrees C. When autolysin levels were assayed in cell walls, it was found that the enzyme activities were constant between 25 degrees C and 40 degrees C, suggesting that there was no greater synthesis of autolysin at the lower temperature. Analyses of walls for individual components, extent of aminosugar substitution and extent of crosslinking, did not reveal significant differences between samples obtained from 25 degrees C or 37 degrees C cultures. The N-acetylmuramoyl-L-alanine amidase was stable over the temperature range studied. Lysis of cells, induced by carbonylcyanide-m-chlorophenylhydrazone, occurred at a faster rate for cells obtained at 25 degrees C than for cells obtained at 37 degrees C. In addition, the lysis of cells by hen egg white lysozyme was slightly faster when the cells were obtained from 25 degrees C cultures than from 37 degrees C cultures. It is possible the autolysin(s) responsible for cell wall turnover are cold-activated.

Bacillus subtilis↗
PubMed1990

Cloning and expression of gene fragments encoding the choline-binding domain of pneumococcal murein hydrolases.

The cloning in Escherichia coli of the 3' moieties of the lytA and cpl-1 genes is described, coding for the C-terminal regions of the lytic amidase of Streptococcus pneumoniae and the phage Cp-1 lysozyme, respectively. The truncated genes were overexpressed in E. coli and the purified polypeptides showed a great affinity for choline, although they were devoid of cell wall-degrading activity. Biochemical and circular dichroism analyses indicated that these are the domains responsible for the specific recognition of the choline-containing pneumococcal cell walls by the lytic enzymes. The data presented here suggested that these choline-binding domains can function independently of their catalytic domains.

Amidohydrolases↗
PubMed1990

Characterization of the transcription unit encoding the major pneumococcal autolysin.

The pneumococcal lytA gene coding for the major autolysin (amidase) can be expressed in Streptococcus pneumoniae and Escherichia coli using unchanged promoter and termination signals. A region containing several -10, -35 and -44 promoter elements, identical to other previously described prokaryotic promoter sequences, has been found upstream from the transcription start point. A transcription terminator consisting of a hairpin structure (-20.8 kcal/mol) typical of Rho-independent prokaryotic terminators was also localized. The lytA gene has a rather long (240-bp) leader sequence with a high A + T content (70%) that contrasts with the very short (2-bp) untranslated region of the polA gene [López et al., J. Biol. Chem. 264 (1989) 4255-4263], the unique pneumococcal transcription unit that had been characterized so far. Although two open reading frames have been found in the leader region it seems unlikely that these sequences can be translated due to the absence of appropriate ribosome-binding sites.

Amidohydrolases↗
PubMed1991

Sequence analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

The nucleotide (nt) sequence of a 2.0-kb NheI-XbaI DNA fragment containing a peptidoglycan hydrolase-encoding gene, lytA, tentatively identified as encoding an N-acetylmuramyl-L-alanine amidase, from Staphylococcus aureus, was determined. The nt sequencing revealed an open reading frame (ORF) of 1443 bp with a consensus ribosome-binding site located 7 nt upstream from the ATG start codon. The primary amino acid (aa) sequence deduced from the nt sequence revealed a putative protein of 481 aa residues with an Mr of 53815. Comparison of the aa sequence of the ORF with aa sequences in the GenBank data base (version 63, March 1990) revealed that the C-terminal sequence showed significant homology to the C-terminal sequence of lysostaphin from Staphylococcus simulans biovar staphylolyticus.

Amino Acid Sequence↗
PubMed1992

Location, characterization and expression of lytic enzyme-encoding gene, lytA, of Lactococcus lactis bacteriophage phi US3.

Gene lytA, which encodes lytic enzyme (LytA), of the isometric Lactococcus lactis bacteriophage phi US3, was cloned and expressed in Escherichia coli. The lytA gene was located on the physical map of the phi US3 32-kb DNA that contains cohesive ends. Initial expression of lytA was detected by lysis of an overlay of cells of the phage-sensitive strain, L. lactis SK112. However, LytA appeared to have a broad spectrum and induced lysis in more than 30 different lactococcal strains. The nucleotide sequence of lytA showed a single open reading frame (ORF) of 774 bp encoding a protein of 258 amino acids (aa) with a calculated M(r) of 28,977. This is in agreement with the size of 29 kDa as determined for LytA produced in E. coli using a T7 expression system. The lytA gene is preceded by an ORF that may code for a hydrophobic peptide of 66 aa containing a putative secretion signal, and two putative transmembrane helices. The deduced aa sequence of the phage phi US3 LytA shows similarities to that of the autolysin of Streptococcus pneumoniae which is known to be an amidase.

Amino Acid Sequence↗
PubMedDate not supplied

Mode of action of Clostridium perfringens initiation protein (spore-lytic enzyme).

The extracellular initiation protein (IP; spore germination enzyme) produced by Clostridium perfringens was further purified and characterized. IP hydrolysed spore cortical fragments with the release of free amino groups. End group analysis of hydrolysed fragments indicated the presence of N-terminal alanine but no reducing sugars. Molecular weight analysis of IP- and lysozyme-treated fluorescamine-labelled cortical fragments indicated that IP acts only on peptidoglycan chains containing cross-linked peptide subunits. IP failed to hydrolyse a number of nitrophenyl-conjugated glucopyranosides and galactopyranosides. The results indicate that IP is an N-acetylmuramyl-L-alanine amidase.

Amidohydrolases↗
PubMed1992

Comparative efficacy of autolysin and pneumolysin as immunogens protecting mice against infection by Streptococcus pneumoniae.

Previous studies on Streptococcus pneumoniae have established that the pneumococcal proteins autolysin (N-acetylmuramyl-L-alanine amidase) and pneumolysin both contribute significantly to the virulence of the organism. In the present work, autolysin and a defined toxoid derivative of pneumolysin were tested, individually and in combination, for efficacy in a mouse model as antigens protecting against challenge with virulent, wild-type S. pneumoniae. While each antigen alone provided significant protection, the degree of protection was not increased when the antigens were administered together. In an additional experiment, mice were challenged with a genetically-modified mutant strain of pneumococcus unable to express active pneumolysin. Pre-immunization of such mice with autolysin failed to provide any significant protection against the challenge. The results of this study suggest that the most important contribution made by autolysin to the virulence of S. pneumoniae may be its role in mediating the release of pneumolysin from the pneumococcal cytoplasm during infection.

Animals↗
PubMed1990

Rapid protein purification using phenylbutylamine-Eupergit: a novel method for large-scale procedures.

Electrophoretic desorption was used to compare the protein binding capacities of some hydrophobic adsorbents [the phenylbutylamine (PBA) derivatives of Eupergit C and agarose and Phenyl-Sepharose] for low-pressure chromatography. The highest capacity was observed for the bifunctional adsorbent PBA-Eupergit. The hydrophobically adsorbed proteins can be selectively desorbed by decreasing the pH of the eluent due to electrostatic repulsion between positive charges on the adsorbed proteins and positively charged secondary amines on the adsorbent. This was used to purify 1500 U penicillin amidase from E. coli homogenates per gram wet weight of PBA-Eupergit in 50 adsorption-desorption cycles without organic solvents (greater than 90% yield, purification factor = 5.3).

Adsorption↗
PubMed2001

Enzymology of elongation and constriction of the murein sacculus of Escherichia coli.

Multiple deletions in murein hydrolases revealed that predominantly amidases are responsible for cleavage of the septum during cell division. Endopeptidases and lytic transglycosylases seem also be involved. In the absence of these enzymes E. coli grows normally but forms chains of adhering cells. Surprisingly, mutants lacking up to eight different murein hydrolases still grow with almost unaffected growth rate. Therefore it is speculated that general enlargement of the murein sacculus may differ from cell division by using transferases rather than the two sets of hydrolytic and synthetic enzymes as seems to be the case for the constriction process. A model is presented that describes growth of the murein of both Gram-positive and -negative bacteria by the activity of murein transferases. It is speculated that enzymes exist that catalyze a transpeptidation of the pre-existing murein onto murein precursors or nascent murein by using the chemical energy present in peptide cross-bridges. Such enzymes would at the same time cleave bonds in the murein net and insert new material into the growing sacculus.

Cell Wall↗