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PubMed1999

Purification and characterization of an alpha-galactosyltransferase from Trypanosoma brucei.

A membrane-associated galactosyltransferase from Trypanosoma brucei was purified 34000-fold by affinity chromatography on UDP-hexanolamine-Sepharosetrade mark. Using SDS/PAGE under reducing conditions, the isolated enzyme ran as a relatively broad band with apparent molecular masses of 53 kDa and 52 kDa, indicative of glycosylation and the existence of two isoforms. N-Glycosylation of the enzyme was subsequently confirmed using Western blotting and either specific binding of concanavalin A or peptide-N4-(N-acetylglucosaminyl)asparagine amidase digestion. The de-N-glycosylated enzyme ran with apparent molecular masses of 51 kDa and 50 kDa, indicative of a single N-glycosylation site. The galactosyltransferase exhibited a pH optimum at 7.2 and had a pronounced requirement for Mn2+ ions (KM=2.5 mM) for its action. The transferase activity was independent of the concentration of Triton X-100. The enzyme was capable of transferring galactose from UDP-galactose to a variety of galactose-based acceptors in alpha-glycosidic linkages. The apparent KM values for UDP-galactose and for the preferred acceptor substrate N-acetyl-lactosamine are 46 microM and 4.5 mM respectively. From these results we would like to suggest that the galactosyltransferase functions in the processing of terminal N-acetyl-lactosamine structures of trypanosomal glycoproteins.

Animals↗
PubMed2000

In vitro interactions between epithelial cells and Gyrodactylus derjavini.

Skin responses of fish to various parasites have been shown to involve various immunologically competent cells producing factors which guide the reactions of epithelial cells. However, the present study has demonstrated that a monoculture of epithelial cells has the ability to encapsulate and partially degrade ectoparasites without involvement of leukocytes. The ectoparasitic monogeneanGyrodactylus derjavini was kept on a monolayer of Epithelioma Papulosum Cyprini (EPC) cells in 24-well multidishes supplied with tissue culture medium. Gyrodactylus derjavini did not reproduce but survived an incubation period of up to139h in the system. Due to sterile conditions, dead gyrodactylids were not subjected to microbial degradation and remained intact for several weeks. However, at 40 days G. derjavini was overgrown by EPC-cells and became partly degraded during the following 15 days. Analysis of enzyme reactivity in EPC-cells showed reactions for ten enzymes including esterases, amidases, phosphatases and phosphohydrolases. No marked differences for the ten enzymes between cell cultures with and without the ectoparasites were found but it cannot be excluded that some of these enzymes took part in parasite degradation. The study showed the in vitro capability of epithelial cells to interact, encapsulate and degrade G. derjavini without the involvement of leukocytes. This response probably is non-specific and will not exclude that various immunocompetent cells and their products normally optimize and accelerate elimination of invading parasites in vivo.

Animals↗
PubMed2000

Intracellular glucosaminidase of the bacterium Xanthomonas campestris IBPM B-124: purification and properties.

A system of intracellular autolytic enzymes of the bacterium Xanthomonas campestris IBPM B-124 was found to include enzymes with muramidase and glucosaminidase activities, while a system of extracellular bacteriolytic enzymes of the same bacterium includes muramidase, muramoylalanine amidase, and endopeptidase. Using a purification technique including fractional precipitation with ammonium sulfate, gel-filtration on Toyopearl HW-55F, and FPLC ion-exchange chromatography on Mono Q, a preparation of intracellular glucosaminidase was purified 435-fold with 16% yield (SDS-PAGE data indicated the presence of minor protein contaminants). Some physicochemical properties of the purified enzyme were determined: molecular mass 26 kD, Km = 5.6 x 10(-4) M with p-nitrophenyl-2-acetamido-2-deoxy-beta-D-glucopyranoside as the substrate, and pH optimum 8.0-8.5. The enzyme is active over a wide range of Tris-HCl buffer concentrations (0.01-0.5 M) and has temperature optimum at 37-40 degrees C. The glucosaminidase activity is sensitive to p-chloromercuribenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), and the disodium salt of ethylenediamine tetraacetic acid (EDTA). The properties of this glucosaminidase markedly differ from those of all extracellular bacteriolytic enzymes of Xanthomonas campestris. These findings indicate that the system of autolytic enzymes of this bacterium functions independently and is not connected with the system of extracellular bacteriolytic enzymes.

Ammonium Sulfate↗
PubMedDate not supplied

[The respiratory activity of Rhodococcus rhodochrous M8 cells producing nitrile-hydrolyzing enzymes].

The respiratory activity of Rhodococcus rhodochrous M8 cells containing nitrile hydratase and amidase was studied in the presence of nitriles and amides of carbonic acids. Culturing of cells with acrylonitrile and acrylamide yielding their maximum respiratory activity was studied. The optimum conditions for measurements and maintenance of respiratory activity were found. Curves for the linear concentration dependence of cell respiratory activity on 0.01-0.5 mM acrylonitrile, 0.025-1.0 mM acetonitrile, and 0.01-0.1 mM acrylamide were plotted. The selectivity of cell respiratory activity for some substrates was analyzed.

Acrylamides↗
PubMed1978

Induction of bacteriolytic enzyme from pyocinogenic Pseudomonas aeruginosa and its enzymatic properties.

Mitomycin C induced a pyocinogenic Pseudomonas aeruginosa P15 to produce a bacteriolytic enzyme, PR1-lysozyme, together with pyocin R1. No significant accumulation of the enzyme was observed inside the induced cells. The enzyme was partially purified by acrinol treatment and Amberlie CG-50 column chromatography. The mode of action of the enzyme on the host bacterial cells as well as on Micrococcus lysodeikticus cells or peptidoglycan isolated from Salmonella typhimurium, was compared with that of hen egg-white lysozyme or phage lambda-lysozyme. It is suggested that PR1-lysozyme should be classified as a glycosidase, rather than an amidase or an endopeptidase.

Bacteriolysis↗
PubMed2002

[Study on production of acrylamide by microbial method (I)--Culture of bacterium cells and expression of high activity of nitrile hydratase].

The cultural conditions for the growth of Norcardia cell were studied in this paper. Controlling pH value, adding nutrient and optimizing the quantity of inducer during cultivation, the activity of nitrile hydratase reached 6567 u/mL (culture medium), which was the highest value appeared in native journals. In the farther hydratase experiments, no by-product, crylic acid, was detected. It showed that the activity of amidase was not promoted obviously while the activity of nitrile hydratase was increased greatly. The results set a strong foundation for the industrial application and the research on new technology.

Acrylamides↗
PubMedDate not supplied

[Characterization of urokinase type plasminogen activator modified by phenylglyoxal].

A chemical modification of single-chain urokinase-type plasminogen activator (scu-PA) with phenylglyoxal under mild conditions resulted in the scu-PA derivatives with various numbers of the modified Arg residues. The study of properties of the resulting derivatives demonstrated that the modification of 4-12 Arg residues did not cause any loss of the activator, fibrinolytic, and potential amidase activities of the activator. The scu-PA with four modified Arg residues was found to be the most stable derivative in human blood plasma; it causes a more efficient lysis of plasma clots than the native activator. Three of four modified Arg residues are supposed to be within the 178RRHRGGS184 cluster, which was localized in the superficial loop of the scu-PA globule and was shown to interact with the complementary series of negatively charged residues in the molecule of the main plasma inhibitor PAI-1. The neutralization of positively charged Arg residues in this cluster decreases the affinity of scu-PA and the double chain urokinase-type plasminogen activator for PAI-1, which results in an enhancement of the stability in plasma and the fibrinolytic efficiency of the activator. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

Arginine↗
PubMed2002

[Cloming, sequence analysis of imidase gene from Alcaligenes eatrophus and its expression in E. coli].

A hydantoin-cleaving microorganism 112R4 is screened and identified to be Alcaligenes eutrophus. The resting cell of Alcaligenes eutrophus 112R4 can catalyze the hydrolysis of hydantoin, dihydropyrimidine and succinimide effectively, but not function to 5-monosubstituted hydantoins or 5,5'-disubstituted hydantoins. The microorganism can utilize succinimide as a sole carbon source and nitrogen source, which indicates the presence of a complete transformation pathway of succinimide, and a hydantoin-cleaving enzyme, imidase, is suggested to be contained in this metabolic pathway. A 6 kb EcoRI-EcoRI fragment isolated from the genome DNA of Alcaligenes eutrophus 112R4 is shown to be correlative with the transformation of succinimide. A 2 kb DNA fragment containing the gene of imidase is subcloned and sequenced. Deletion analysis verifies that one open reading frame of 876 nucleotides, which encodes a peptide of 291 amino acids, with a calculated molecular weight of 33688, is responsible for the encoding of imidase. This is the first report of the nucleotide and amino acid sequences of imidase (GenBank accession number: AF373287). A homology search performed in protein database reveals an identity of 14% with polysaccharide deacetylase conserved domain, an identity of 60% with N-terminal 20 amino acids of Blastobacter sp. A17p-4, but no apparent similarity with all known cyclic-amide-cleaving enzymes. This result suggested that the imidase should be classified as a new member of cyclic amidases. Under the control of lac promoter and IPTG induction, the imidase activity of transformed E. coli reached 3200 U/L, which is about 7-fold higher than that of gene donor strain.

Alcaligenes↗
PubMed2003

Endocannabinoids as autoregulatory signaling molecules: coupling to nitric oxide and a possible association with the relaxation response.

Endocannabinoid signaling processes are present in diverse organisms and in organisms 500 million years divergent in evolution. Cannabinoid receptor-1 expression (CB1), anandamide, and anandamide amidase have been found in invertebrates. Furthermore, this signaling system is coupled to constitutive nitric oxide synthase (cNOS)-derived nitric oxide (NO) release in both vertebrates and invertebrates, thereby regulating neural, immune, and vascular-like functions in these divergent organisms. In human endothelial cells from various blood vessels, CB1 immunoreactive components are present as is its coupling to anandamide-stimulated cNOS-derived NO production, which exerts an autoregulatory role on cNOS release. The modulation of vascular diameter and vascular tone represents a crucial point of interest in these pathways, and interactions between NO and the sympathetic nerve system are of importance, i.e, norepinephrine. Here, a possible association of NO and endocannabinoid signaling with the relaxation response, a physiological counterpart of the stress response, may exist.

Animals↗
PubMed1992

[Studies on metabolism of fungicide benzoic acid, 1,3-dithiolan-2-ylidenehydrazide in vitro].

The metabolism of fungicide benzoic acid, 1,3-dithiolan-2-ylidenehydrazied (Yekuling) was studied quantitatively in rat liver microsomes and liver soluble fractions pretreated with phenobarbital (PB) and 3-methylcholanthrene (3-MC) by high pressure liquid chromatography. The experimental results indicated that the major metabolic pathway of Yekuling in vitro was hydrolysis. PB can enhance amidase activity to increase formation of benzoic acid and 1,3-dithiolan-2-ylidenehydrazine. 3-MC treatment elevated rat liver microsomal cytochrome P-448, enhancing S-oxidation of Yekuling. On the other hand, S-oxidation of Yekuling by rat liver microsomal MFO was NADPH-dependent.

Animals↗
PubMed1992

Fish egg glycophosphoproteins have species-specific N-linked glycan units previously found in a storage pool of free glycan chains.

Recent findings (Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H., and Inoue, Y. (1989) J. Biol. Chem. 264, 1623-1630; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K., and Inoue, Y. (1989) J. Biol. Chem. 264, 18520-18526) of a relatively large quantity of complex-type free sialo-oligosaccharides in the unfertilized eggs of freshwater fish, Plecoglossus altivelis and Tribolodon hakonensis, prompted us to search for their progenitor glycoproteins. First we demonstrated a third occurrence of free sialoglycans in the unfertilized eggs of Medaka fish (Oryzias latipes). Next, in all three species studied, a uniformly high level of glycophosphoproteins (GPP) was identified and found to possess N-linked glycan units. The carbohydrate structures of the GPP were determined to be identical with those of the free glycans isolated from the unfertilized eggs of the respective fish species. Thus, the most likely candidate for the progenitor of free sialoglycans appeared to be the oocyte GPPs. This implies that the liberation of the free glycans by a putative peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase may represent a necessary biochemical event during vitellogenesis or oogenesis. The present results may provide insight into a new concept of a "protein N-glycosylation/de-N-glycosylation system" recently proposed by us (Seko, A., Kitajima, K., Inoue, Y., and Inoue, S. (1991) J. Biol. Chem. 266, 22110-22114).

Amino Acids↗
PubMed1992

[Studies on metabolism of a fungicide yekuling in rat].

The metabolism of a fungicide Yekuling was studied in rat after oral administration. Four metabolites were isolated and purified by means of reverse-phase HPLC and TLC. They were identified to be acetic acid, 1,3-dithiolan-2-ylidenehydrazide; pyruvic acid, 1,3-dithiolan-2-ylidenehydrazide; benzoic acid and hippuric acid with UV and MS. The latter was further confirmed by chemical synthesis. The experimental results showed that Yekuling was metabolized extensively in rat. Yekuling was hydrolyzed by amidase to produce benzoic acid and 1,3-dithiolan-2-ylidenehydrazide. The latter was further acetylated by N-acetyltransferase to form acetic acid, 1,3-dithiolan-2-ylidenehydrazide, or condensed spontaneously to form pyruvic acid, 1,3-dithiolan-2-ylidenehydrazide. Benzoic acid was further conjugated with glycine to produce hippuric acid.

Acetates↗
PubMed1992

Isolation of promoter sequences from Brevibacterium sp. R312.

Promoter sequences recognized by Escherichia coli RNA polymerase were isolated from Brevibacterium sp. R312, a coryneform strain producing nitrile hydratase and amidase. Ten Escherichia coli clones containing promoter sequences were selected for their ability to grow with chloramphenicol concentrations of up to 1500 micrograms/ml. The strength of these promoter sequences was determined. We carried out a preliminary study of the strongest promoter having a chloramphenicol acetyl-transferase/beta-lactamase activities ratio of 18.4.

Amidohydrolases↗
PubMed1992

Development of genetic engineering in Bacillus megaterium.

The opportunities for industrial genetic engineering in several species of Bacillus other than B. subtilis and B. thuringiensis are now becoming a reality. Many species have advantages for certain industrial applications, such as the lack of alkaline proteases, stable plasmid maintenance, and production of thermostable enzymes. It is now possible to increase production levels in many Bacillus strains that are already high producers of amylases, proteases, penicillinases, and penicillin amidases, by the introduction of such genes on high-copy-number plasmids. Possible problems in gene regulation, expression, limits on protein production, and secretion will be encountered, but recent reports on comparisons of expression of bacterial and eukaryotic genes in B. subtilis and B. megaterium (Shivakumar et al. 1989; Donovan et al. 1989a, 1989c; Ginsburgh et al. 1989) suggest that some species may prove to be better expression hosts for specific genes than others. What is needed is extensive comparative studies in promising species to better understand the parameters affecting cloning, gene expression, and protein secretion in the bacilli. In this chapter we have emphasized the development of genetic analysis and rDNA methods in B. megaterium. While the chromosomal map is still not a complete circle, many gene loci have been mapped, some with three-factor crosses, and have been characterized by enzyme assays (leu, trp, hem, cob, cbl) as well as complementation with B. subtilis genes (trp, dnaK, pur, met, ssp) E. coli genes (ATPase), hybridization (spoVG, abrB, sigK, ssp), and by growth on intermediates (trp, leu, thy, gua, ade, pur, pyr, his, arg). A mapping kit of 12 strains is available, and can facilitate the mapping of new mutations rapidly. This is of great advantage in strain construction since genes can be transferred at will by cotransduction. A partial physical map generated by pulse gel electrophoresis is also available (Muse 1990). It remains to be determined whether gene conversion occurs in B. megaterium as it does in B. subtilis. This is a powerful tool for rescuing genes and mutations of interest from the chromosome. There are now over 600 mutants available in our laboratory, over 100 in the laboratory of J.C. Vary, and several more that have been characterized in various laboratories throughout the world. Many of these are available in stock culture collections, although this source needs to be expanded. Mutants that should be useful for genetic engineering include recombination negative, protease negative, and plasmidless wild type, as well as Lac negative and several auxotrophs in the plasmidless background, and many sporulation negative mutants.

Bacillus megaterium↗
PubMedDate not supplied

[Isolation and various properties of alpha-aminocaprolactam hydrolase from Klebsiella aerogenes].

L-alpha-aminocaprolactam hydrolase possessing the L lysine amidase activity was isolated from Klebsiella aerogenes and purified. The procedure of enzymes purification included cell destruction on USDN-I, fractionation by ammonium sulfate, gel chromatography on G-200. The preparation of the purified enzyme possessed specific activity of 50 mumol of lysin per 1 mg of protein per hour. Km was 2.6 mM in case of phosphate buffer (ph 7.2) for I-alpha-aminocaprolactam. Besides L-alpha-aminocaprolactam the enzyme hydrolyses lysine amide, leucine amide tryptophanamide. Magnesium ions are necessary for manifestation of catalytic activity of the enzyme.

Amidohydrolases↗
PubMed2005

[Research on the protein C-activating property and influencial factors of protein C activator isolated from the venom of Agkistrodon halys].

OBJECTIVE: To investigate the protein C-activating property and experimental conditions that may affect the activities of protein C activator (PCA-SV). METHODS: PCA-SV's functional characteristic, possible anticoagulant and amidase activities were measured by method of APTT and that of specific chromogenic substrate respectively in various experimental conditions. RESULTS: PCA-SV can specifically act on protein C. The pH-optimum and temperature-optimum for the action of PCA-SV were 6.0 - 7.0 and 37 - 40 degrees C respectively. The anticoagulants (heparin, sodium citrate) had no effect on the activities. CONCLUSION: PCA-SV is a new protein C-activating reagent that has high potency and specificity.

Agkistrodon↗
PubMed1979

The interaction between heparin and plasmin on amidolysis.

The effect of the interaction between heparin and plasmin not only on fibrinolytic, caseinolytic and esterolytic activities but also on amidolytic activity, since plasmin has amidolytic or amidase activity, was investigated. Following were the results obtained from these investigations: 1. Heparin enhanced amidolytic activity of a fixed level of plasmin which was prepared by activating plasminogen with urokinase within the range from 2 to 64 units/ml of the final concentration of heparin. 2. Heparin also enhanced amidolytic activity of a fixed level of plasmin which was prepared by converting plasminogen with insolubilized urokinase. 3. Heparin did not enhance or inhibit fibrinolytic, caseinolytic and esterolytic activities of a fixed level of plasmin which was prepared by activating plasminogen with urokinase, in the fibrinolytic activity within the range from 0.032 to 125 units/ml, in the caseinolytic activity within the range from 0.0125 to 100 units/ml, and in the esterolytic activity within the range from 0.016 to 128 units/ml, of the final concentration of heparin respectively.

Amides↗