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PubMed1991

Rapid screening of colonies from Listeria selective agar.

A rapid method ('Liststrip', Lab M) of screening aesculin-positive colonies from Oxford selective agar was evaluated. This method relies on the ability of all Listeria spp. to hydrolyse aesculin and to have a rapid phosphatase but neither a pyroglutamic acid beta-naphthylamide amidase nor produce indole. Of 198 Listeria spp., all gave identical results (as above) in 10 min (except four isolates of L. murrayi), whereas of 112 aesculin-positive colonies from Oxford agar that were not Listeria only one gave a similar result. The method was compared with Gram stain and oxidase as a screening procedure. This strip test method is simple enough to be used by relatively unskilled staff and provides a rapid and reliable method to screen colonies from Oxford selective agar.

Bacteriological Techniques↗

PubMed1988

Sialate O-acetylesterases: key enzymes in sialic acid catabolism.

Sialate 9(4)-O-acetylesterases (EC 3.1.1.53) have been isolated from equine liver, bovine brain and influenza C virus. In this latter case, the esterase represents the receptor-destroying enzyme of the virus. The kinetic properties of these enzymes were determined with Neu5,9Ac2 and in part with 4-methylumbelliferyl acetate and Neu5,9Ac2-lactose. The Km values vary between 0.13 and 24 mM and the Vmax values from 0.55 to 11 U/mg of protein. The pH optima are in the range of 7.4-8.5, the molecular masses at 56,500 and 88,000 Da. In addition to a fast hydrolysis found for aromatic acetates, such as 4-methylumbelliferyl acetate or 4-nitrophenyl acetate, N-acetyl-9-O-acetylneuraminic acid is de-O-acetylated at the highest relative rate. Other substituents at the 9-position, such as lactoyl residues, or acetyl groups at other positions within the side chain are not hydrolyzed. Neu4,5Ac2, however, is a substrate for all 3 enzymes. The hydrolysis rates of this ester function, which renders sialic acids resistant to the action of sialidases, vary from 3 to 100% relative to Neu5,9Ac2. Whereas Neu5,9Ac2-lactose is hydrolyzed by the bovine and viral esterases, other O-acetylated sialic acids in glycoconjugates are only attacked by the enzyme from influenza C virus and not by that from bovine brain. The esterase from horse liver also releases 4-O-acetyl groups from equine submandibular gland mucin. By incubation with appropriate substrates and inhibition studies, carboxylesterase, amidase and choline esterase activities were excluded, as well as the cleavage of other acyls, e.g., butyryl groups. Thus, the enzymes investigated belong to the acetylesterases.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylesterase↗

PubMed1980

Enzymic formation of TRH-OH from TRH by rat hypothalamus.

Specific radioimmunoassays for thyrotrophin-releasing hormone (TRH) and deamido-TRH (TRH-OH) were used to investigate the inactivation of TRH by subcellular fractions from rat hypothalamus. TRH-OH was rapidly formed from TRH by an amidase present only in the supernatant fraction at an optimum pH of 7.38; although TRH was degraded by the particulate fraction, no TRH-OH formation was detected. Of several brain areas investigated, the cortex had the highest rate of TRH-OH production and the pituitary the lowest. Once formed in the supernatant fractions, it was found that TRH-OH was stable and did not undergo further degradation. Formation of TRH-OH from TRH appears to be an important step in the tripeptide's enzymic degradation in the brain, so the combined use of specific radioimmunoassays for TRH and TRH-OH may provide a suitable means for studies of the enzymes involved, since these enzymes may contribute to the mechanisms regulating the secretion and duration of action of TRH.

Amidohydrolases↗

PubMed1980

The heparin-accelerated neutralisation of bovine alpha and beta thrombins by antithrombin III.

The heparin-accelerated neutralisation of bovine alpha and beta thrombins has been examined using a peptide substrate H-D-phenylalanyl-pipecolyl-arginine-paranitroanilide-HCl to measure thrombin amidase activity. Alpha and beta thrombins were both neutralised by antithrombin III and this neutralisation was further accelerated by the presence of small amounts of heparin. Low and high molecular weight heparin and heparins fractionated by their affinity for antithrombin III were all able to accelerate the neutralisation of alpha and beta thrombin. This work is therefore unable to confirm reports that alpha and beta thrombins have different heparin sensitivities.

Animals↗

PubMed1989

Biotinidase and lipoamidase in guinea pig livers.

Guinea pig biotinidase and lipoamidase were mostly located in the liver microsomal fraction. Approx. 80% of the total activities of both enzymes were associated with the membranes subfractions of liver. The subunit molecular masses of these enzymes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of microsomal biotinidase and lipoamidase were 70 and 60 kDa, respectively. Sephadex G-200 gel-permeation chromatography in the presence of 0.1% Nonidet P-40 indicated that the native-state molecular masses of microsomal biotinidase and lipoamidase were 68 and 120 kDa, respectively. The isoelectric points of microsomal biotinidase and lipoamidase were 6.3 and 5.7, respectively. Linear sucrose density centrifugation analysis indicated that both enzymes exist in the rough endoplasmic reticulum. Comparison of amino acid analyses indicated a higher content of leucine and lower content of serine in lipoamidase than in biotinidase. Microsomal biotinidase and lipoamidase were classified as being thiol-type and serine-type amidases, respectively.

Amidohydrolases↗

PubMed1991

Enkephalin hydrolysis by human serum biotinidase.

Purified human serum biotinidase exhibited amino-exo-peptidase activity. Enkephalins and dynorphin A (less than 10-mer) seemed to be the most appropriate substrates among various physiological peptides in terms of the kcat/Km values. Similar kcat/Km values were obtained for both biocytin (biotinyllysine) and these opioid-neuropeptides. Neuro-oligo-peptides ranging from 2-mer to 18-mer were hydrolyzed. The presence of amino group at the carboxyl terminal position increased the kcat/Km value by decreasing the Km value. The results of inhibition studies using various kinds of antibiotic inhibitors, metals, and chelating agents indicated that enkephalin hydrolysis was mediated by the peptide-hydrolyzing center probably containing Zn ions. This aminopeptidase activity was uniquely inhibited by a vitamin of biocytin. The reason for the high content of biotinidase activity in serum may be related to the binary function of this enzyme; i.e., biocytin hydrolyzing amidase and enkephalin hydrolyzing aminopeptidase functions.

Amidohydrolases↗

PubMed1982

Characteristics of two trypsin type isozymes isolated from the arctic fish capelin (Mallotus villosus).

1. Two trypsin-like enzymes, assayed by their amidase activity with N-alpha-benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA) as the substrate, were isolated from the gut of the arctic fish capelin (Mallotus villosus). 2. Purification involved affinity chromatography (Benzamidine-CH-Sepharose 4B) of the 30 to 70% (NH4)2SO4 precipitation fraction of a crude extract of the gut, followed by DEAE-Sephadex chromatography, yielding two enzymes, designated Enzyme I and II. 3. Both enzymes had MW of about 28,000 as determined by SDS-electrophoresis. Their isoelectric points were 5.6-5.9 (Enzyme I) and 5.1-5.3 (Enzyme II) and they had similar amino acid composition. 4. Both enzymes were inhibited by standard trypsin inhibitors including the serine protease inhibitor phenylmethyl sulphonyl fluoride (PMSF), but not by the chymotrypsin inhibitor L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK). 5. The enzymes had a pH optimum of 8-9 and their stability was not affected by CaCl2. Low pH (2.3) caused an initial rapid loss of enzyme activity, followed by relatively slow decomposition of the activity remaining after 1 hr at 4 degrees C. 6. The enzymes had an apparent temperature optimum of 42 degrees C, resulting from rapid self digestion at higher temperatures.

Amino Acids↗

PubMed1984

Acid proteinase activity in fish--I. Comparative study of extraction of cathepsins B and D from Mujil auratus.

Acid catheptic activity was measured in crude extracts of muscle, liver, heart, spleen and gonads from the fishes Mujil auratus, Sparus aurata and Lightonatus mormyrus. The spleen was the organ which showed the highest activity. A comparative study of the seven most commonly used extraction methods was made. Some were modified to account for the characteristics of the fish organs and the activity extracted from them. The Siebert method resulted as the best extraction method only if 1 mM EDTA was present in the medium. The activity from Mujil auratus muscle was strongly inhibited by iodoacetate, N-ethylmaleimide, p-hydroxy mercuribenzoate, and diazo-acetyl-DL-norleucine methyl ester. The results indicated the presence of a carboxyl-proteinase and a thiol-proteinase. According to inhibition studies, the levels of proteinase and amidase activities shown by different organs of Mujil auratus were re-examined. The spleen extract showed the maximum activity for both cathepsins, but muscle extract accounted for more than 95% of total catheptic activity.

Animals↗

PubMedDate not supplied

Purification and characterization of two chymotrypsin-like proteases from the pyloric caeca of rainbow trout (Oncorhynchus mykiss).

1. Two chymotrypsins, called chymotrypsin I and II, were purified from the pyloric caeca of rainbow trout, by (NH4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). 2. The approximate molecular weights of chymotrypsin I and II were 28,200 (+/- 1200) and 28,800 (+/- 900), respectively, as determined by SDS-PAGE and their isoelectric points were about 5. 3. The pH optima of the enzymes were centered around nine, when assayed for succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-NA) as substrate and both enzymes were unstable at pH values below 5. 4. The amidase activity of both enzymes increased with temperature up to about 55 degrees C. Chymotrypsin I was found to be more heat stable than chymotrypsin II, an effect most likely explained by stronger calcium binding of the former. 5. The trout chymotrypsins were significantly more active than bovine alpha-chymotrypsin when assayed against Suc-AAPF-NA at 25 degrees C and casein at low temperatures (10-20 degrees C), indicating an adaptation of the activities of the trout chymotrypsins to the habitation temperatures of the fish.

Amino Acid Sequence↗

PubMed1989

Influence of cationic peptides on the activity of the autolytic endo-beta-N-acetylglucosaminidase of Staphylococcus simulans 22.

The peptidoglycan hydrolyzing endo-beta-N-acetylglucosaminidase of Staphylococcus simulans 22 is not able to attack intact cell walls of S. simulans 22, but hydrolyzes cell walls of Micrococcus luteus and soluble peptidoglycan chains of S. simulans 22. Hydrolysis of cell walls of M. luteus is activated in presence of organic cations such as poly-L-lysine (n = 17) and the peptide antibiotics Pep 5 and nisin, whereas hydrolysis of soluble peptidoglycan chains is not influenced. High concentrations of inorganic cations inhibit enzyme activity. These effects are discussed with respect to the cationic nature of the enzyme (pI greater than 9.5) and the regulation of the concerted action of the N-acetylmuramoyl-L-alanine amidase and the glucosaminidase during S. simulans 22 autolysis in vivo.

Acetylglucosaminidase↗

PubMed1991

Purification and characterization of a cell wall hydrolase encoded by the cwlA gene of Bacillus subtilis.

A cell wall hydrolase of Bacillus subtilis was prepared from Escherichia coli cells harboring a plasmid containing the B. subtilis cwlA gene and purified by hydroxyapatite column chromatography and HPLC through TSK-gel G3000SWXL. In contrast to the molecular mass of 29,919 Da deduced from its nucleotide sequence, the purified CWLA is a 23 kDa protein. Characterization of the specific substrate bond cleaved by CWLA indicated the enzyme is an N-acetylmuramyl-L-alanine amidase. A 32-kDa precursor protein was detected on zymography of a crude cell homogenate. Some of the enzymatic properties of CWLA are also described.

Bacillus subtilis↗

PubMed1996

High-performance liquid chromatographic study of the aromatic nitrile metabolism in soil bacteria.

Simultaneous HPLC determination of bromoxynil, ioxynil and dichlobenil, three arylnitrile herbicides, and their metabolic products in soil extracts and microbiological media is described. Limits of detection (LODs) ranged from 0.56 to 3.97 ppb. Slight modification of the mobile phase composition allowed determination of 13 other aromatic nitriles. Assay of aromatic nitrile hydratase, amidase or nitrilase activities is possible by the method developed.

Benzamides↗

PubMed1992

Xenobiotic-metabolizing enzyme systems in test fish--IV. Comparative studies of liver microsomal and cytosolic hydrolases.

1. The initial slopes of the substrate-activity curves of several hydrolases were determined in the microsomal and cytosolic fractions of the liver of several fish recommended by OECD for the regulatory testing of chemicals. 2. Inter-species differences ranged within a factor of 7-17 for the esterases and reached a factor of 60 for the amidase. Guppy and carp appeared endowed with hydrolase activities which, overall, are much higher than zebra fish, trout and golden orfe. 3. The comparison with the rat liver microsomal hydrolases strongly suggests that fish are endowed with similar or higher levels of A-esterase and with much less B-esterase/amidase activities.

Animals↗

PubMed1992

Glycoprotein metabolism in neuronal ceroid lipofuscinosis fibroblasts.

Human skin fibroblast lines of the infantile form of neuronal ceroid lipofuscinosis and control lines were cultured in the presence of [3H]glucosamine plus [3H]mannose and [35S]methionine. The labeled glycoconjugates were compared by quantitative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The infantile form of the disease showed a 75% decrease of four glycoprotein components of M(r) 120-140 kDa. These components appeared to be N-linked glycoproteins as peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase F) released 86-96% of the labeled carbohydrate from the labeled protein. These results suggest that the infantile form of this disease may be characterized by abnormalities in glycoconjugate metabolism leading to reduction of specific glycoproteins.

Adolescent↗

PubMedDate not supplied

Endotoxic activity in faeces of mice from different microbiological environments.

Chromogenic Limulus amoebocyte lysate (LAL) tests were evaluated for measuring endotoxic activity in faeces of mice from different microbiological environments. Faeces of BALB/c mice from germ-free (GF), specific-pathogen-free (SPF) and conventional (CV) colonies had no inhibitory activity on the test reaction. GF and SPF faeces had heat-labile non-specific amidase activity which gave false-positive reaction, while CV did not. GF faeces had a heat-stable non-endotoxic effect on the LAL system. Average endotoxin levels in faeces of GF, SPF and CV mice were 0.2, 0.8 and 17 to 24 micrograms/g wet weight, respectively. Results suggest that the microbiological status of mice influences not only the faecal endotoxin level but also the specificity of the test reaction.

Amidohydrolases↗

PubMedDate not supplied

The binding of novel phenolic derivatives of anandamide to brain cannabinoid receptors.

Arachidonylethanolamide (N-2-hydroxyethyl-arachidonamide) or 'anandamide' is a naturally occurring derivative of arachidonic acid that has been shown to bind and activate cannabinoid receptors in the brain. Since other potent ligands for the cannabinoid receptor have an aromatic hydroxyl group, we investigated the hypothesis that replacement of the ethanolamine hydroxyl with an aromatic hydroxyl will increase the binding affinity for the cannabinoid receptor. Two novel congeners of anandamide containing aromatic hydroxyl groups were synthesized: N-2-(4-hydroxyphenyl)ethyl arachidonamide (HEA) and N-2-hydroxyphenyl arachidonamide (HPA). The affinity of these congeners for the brain cannabinoid receptor was determined by competition with [3H]CP55940. HEA competed for [3H]CP55940 binding with a Ki of 600 nM; HPA had a Ki of 2200 nM. These results indicate that increased size in the amide portion of anandamide decreases affinity for the receptor. Phenylmethylsulfonyl fluoride (PMSF), an inhibitor of anandamide catabolism by brain membranes, had no effect on the binding of either HEA or HPA. We conclude that these congeners are not substrates for the amidase that catabolizes anandamide.

Animals↗

PubMed1994

Microbial hydrolysis of glutaronitrile derivatives with Brevibacterium sp. R 312.

The enantiomerically pure (S)-cyano acids 3 and 4 can be obtained by biotransformation with Brevibacterium sp. R 312 of the corresponding prochiral dinitriles 5 and 6, respectively. The hydrolysis is probably a two step process involving a nitrile hydratase and an amidase. In connection with these investigations a facile method for the synthesis of racemic 4-cyano-3-hydroxybutanoic acid derivatives was developed.

Amidohydrolases↗

PubMed2006

Analysis of plasma proteins that bind to glycosaminoglycans.

Glycosaminoglycan-binding proteins, with specific emphasis on dermatan sulfate, have been investigated in human plasma by affinity chromatography, mass spectrometry and Western blotting. Diluted plasma was applied to affinity columns and bound protein was eluted with 500 mM NaCl. Dermatan sulfate and heparan sulfate bound 7% of the total protein. Heparin bound 22% of the total protein, but chondroitin sulfate A bound only 0.23%. Mass spectrometric analysis identified 20 proteins as dermatan-sulfate-binding proteins, most of which were confirmed by Western blotting. Some of these binding proteins, such as fibrinogen, fibronectin, apolipoprotein B, LMW kininogen, inter-alpha-trypsin inhibitor, and factor H, were degraded to various extents during the chromatography step, but this degradation could be prevented by the inclusion of a serine protease inhibitor. The protein fraction binding to the dermatan sulfate column showed amidase activity, whereas that binding to the heparan sulfate and heparin columns showed 1/2 and 1/20, respectively, of the activity of the dermatan sulfate binding fraction. Dermatan sulfate was similar to heparan sulfate with respect to its capacity to bind plasma proteins and its activation of protease, but differed from chondroitin sulfate and heparin in these properties.

Amidohydrolases↗