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PubMed1999

Cloning and expression of the N,N-dimethylformamidase gene from Alcaligenes sp. strain KUFA-1.

N,N-Dimethylformamidase (DMFase) from Alcaligenes sp. strain KUFA-1, a bacterium that can grow on N,N-dimethylformamide (DMF) as the sole carbon and nitrogen source, catalyzes the first step of the DMF degradation. The DMFase gene dmfA1A2 was cloned in Escherichia coli, and its nucleotides were sequenced. The deduced amino acid sequence of the enzyme consisted of two alpha- and two beta-subunits with 132 and 762 amino acids, respectively, and had little similarity to sequences in protein databases, including various amidases. The protein may be a new kind of amidase. DMFase activity was detected in E. coli cells transformed with an expression plasmid of the cloned DMFase gene. The properties of recombinant DMFase purified from E. coli were identical to those of Alcaligenes DMFase.

Alcaligenes↗

PubMed2001

Substrate recognition mechanism of carboxypeptidase Y.

To clarify the substrate-recognition mechanism of carboxypeptidase Y, Fmoc-(Glu)n Ala-OH (n = 1 to 6), Fmoc-(Glu)n Ala-NH2 (1 to 5), and Fmoc-Lys(Glu)3Ala-NH2 were synthesized, and kinetic parameters for these substrates were measured. Km for Fmoc-peptides significantly decreased as peptide length increased from n = 1 to n = 5 with only slight changes in kcat. Km for Fmoc-(Glu)(5,6)Ala-OH were almost the same as one for protein substrates described previously (Nakase et al., Bull. Chem. Soc. Jpn., 73, 2587-2590). These results show that the enzyme has six subsites (S1' and S1-S5). Each subsite affinity calculated from the Km revealed subsite properties, and from the differences of subsite affinity between pH 6.5 and 5.0, the residues in each subsite were predicted. For Fmoc-peptide amide substrates, the priorities of amidase and carboxamide peptidase activities were dependent on the substrate. It is likely that the interactions between side chains of peptide and subsites compensate for the lack of P1'-S1' interaction, so the amidase activity prevailed for Fmoc-(Glu)(3,5)Ala-NH2. These results suggest that these subsites contribute extensively to substrate recognition rather than a hydrogen bond network.

Amino Acid Sequence↗

PubMed2006

A supramolecular complex between proteinases and beta-cyclodextrin that preserves enzymatic activity: physicochemical characterization.

BACKGROUND: Cyclodextrins are suitable drug delivery systems because of their ability to subtly modify the physical, chemical, and biological properties of guest molecules through labile interactions by formation of inclusion and/or association complexes. Plant cysteine proteinases from Caricaceae and Bromeliaceae are the subject of therapeutic interest, because of their anti-inflammatory, antitumoral, immunogenic, and wound-healing properties. METHODS: In this study, we analyzed the association between beta-cyclodextrin (betaCD) and fraction P1G10 containing the bioactive proteinases from Carica candamarcensis, and described the physicochemical nature of the solid-state self-assembled complexes by Fourier transform infrared (FTIR) spectroscopy, thermogravimetry (TG), differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), and nuclear magnetic resonance (NMR), as well as in solution by circular dichroism (CD), isothermal titration calorimetry (ITC), and amidase activity. RESULTS AND DISCUSSION: The physicochemical analyses suggest the formation of a complex between P1G10 and betaCD. Higher secondary interactions, namely hydrophobic interactions, hydrogen bonding and van der Waals forces were observed at higher P1G10 : betaCD mass ratios. These results provide evidence of the occurrence of strong solid-state supramolecular non-covalent interactions between P1G10 and betaCD. Microcalorimetric analysis demonstrates that complexation results in a favorable enthalpic contribution, as has already been described during formation of similar betaCD inclusion compounds. The amidase activity of the complex shows that the enzyme activity is not readily available at 24 hours after dissolution of the complex in aqueous buffer; the proteinase becomes biologically active by the second day and remains stable until day 16, when a gradual decrease occurs, with basal activity attained by day 29. CONCLUSION: The reported results underscore the potential for betaCDs as candidates for complexing cysteine proteinases, resulting in supramolecular arrays with sustained proteolytic activity.

Amidohydrolases↗

PubMed1993

Dissection of the molecular pathology of aspartylglucosaminuria provides the basis for DNA diagnostics and future therapeutic interventions.

Aspartylglucosaminuria (AGU) is exceptional among lysosomal storage diseases since it represents the only known amidase deficiency in man, being caused by an inadequate function of aspartylglucosaminidase (AGA, E.C. 3.5.1.26.). This amidase is essential in one of the final steps in the ordered breakdown of glycoproteins since it cleaves Asn from the residual N-acetylglucosamines (for reviews see 1, 2). The deficiency of the enzyme activity results in the typical lysosomal accumulation of the abnormal degradation products (mainly aspartylglucosamine, 2-acetamido-1-beta-L-aspartamido-1,2-dideoxyglucose) in patients' cells and tissues. The diagnosis of AGU has so far been based on the detection of abnormal metabolites in urine and decreased enzyme activity in the cultured fibroblasts or isolated lymphocytes. Prenatal diagnosis has been possible by demonstrating the deficient enzyme activity of amniocytes or chorion villus biopsies. Identification of carriers has been difficult and unreliable due to the high individual variation in AGA activity and prerequisite for isolated blood lymphocytes. During the past few years we have purified the human enzyme into homogeneity, isolated the full length cDNA and characterized the majority of AGU mutations in this cDNA. This work facilitated the development of a reliable DNA diagnostic test suitable also for large scale carrier screening. The molecular pathology of the most common AGU mutation was unravelled, this being a prerequisite for the oncoming developments for therapy. Although AGU is a relatively rare disease, characterization of the AGU mutations and their cellular consequences have revealed highly interesting new phenomena in the biosynthesis of this lysosomal enzyme, some of which carry general biological significance.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylglucosamine↗

PubMed1996

In vitro metabolism of the nephrotoxicant N-(3,5-dichlorophenyl)succinimide in the Fischer 344 rat and New Zealand white rabbit.

1. The nephrotoxicant N-(3,5-dichlorophenyl)succinimide (NDPS) underwent nonenzymatic hydrolysis to N-(3,5-dichlorophenyl)succinamic acid (NDPSA) in buffer, rat liver and kidney homogenates, and rabbit liver homogenates. 2. In the presence of NADPH, rat liver homogenates converted NDPS to NDPSA and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA). 3. Using liver homogenates from the phenobarbital (PB)-pretreated rat, 2-NDHSA production was increased 5-fold, and the metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-3-hydroxysuccinamic acid (3NDHSA) were also detected. Formation of these latter metabolites was suppressed by CO or omission of NADPH. No hydroxylated metabolites were detected when NDPSA was incubated with PB-induced rat liver homogenates. 4. Oxidative metabolites were not produced when NDPS was incubated with kidney homogenates from the control or PB-pretreated rat. 5. NDHS underwent rapid hydrolysis in buffer to yield 2-NDHSA and 3-NDHSA. 6. Rabbit liver homogenates converted NDPS to NDPSA, 3,5-dichloroaniline (DCA), and succinic acid (SA). Production of DCA and SA was inhibited by the amidase inhibitor bis-p-nitrophenyl phosphate. Oxidative metabolism did not occur in rabbit tissue. 7. These experiments demonstrate that a PB-inducible form of rat liver P450 converts NDPS to NDHS, which then undergoes hydrolysis to 2-NDHSA and 3-NDHSA. An alternative route of production for 2-NDHSA and 3-NDHSA, via hydroxylation of NDPSA, does not occur. In rabbit liver NDPS metabolism was primarily amidase-mediated.

Animals↗

PubMedDate not supplied

On the latency of blood phenoloxidase of painted locust Poeciloceros pictus.

The blood phenoloxidase of Poeciloceros pictus exists as proenzyme. The amidase activity present in the haemocyte is responsible for the activation of haemocyte phenoloxidase. There is an amidase inhibitor in the plasma. Plasma phenoloxidase could be activated by various fatty acids, biological compounds like lipopolysaccharide zymosan and detergents. Electrophoretic studies suggest that activation of haemocyte phenoloxidase involves limited proteolysis. It is suggested that the natural activator of plasma phenoloxidase may be fatty acid and it does not undergo proteolysis during activation. The biological compounds like zymosan or LPS are easily recognised by the factor(s) from the membranes of haemocytes.

Amidohydrolases↗

PubMed1990

Chicken acrosin: extraction and purification.

The objective of the present research was to identify a procedure whereby chicken acrosin could be purified. Acrosin, as evidenced by amidase activity, was extracted with urea most efficiently at a concentration of 6 M. Extraction efficiency was enhanced by spermatozoal lysis prior to admixture with 6 M urea. Lysis was induced by passage of spermatozoal suspensions through a French pressure cell. Acrosin was purified by using gel filtration, chromatofocusing, and affinity chromatography. Based on amidase activity, a 19-fold purification was obtained with a 28% recovery. Native electrophoresis resolved two major protein bands with proteolytic activity. The methods described afford the procurement of milligram amounts of chicken acrosin.

Acrosin↗

PubMed1976

Isolation and purification of D-alanyl-meso-2, 6-diaminopimelic acid endopeptidase of Streptomyces L-3 enzyme using soluble substrates of known chemical structure from Lactobacillus plantarum cell wall digests.

D-alanyl-meso-2, 6-diaminopimelic acid (D-Alanyl-meso-A2pm) endopeptidase was isolated and purified from a crude Streptomyces L-3 enzyme preparation by ion exchange chromatography and isoelectric focusing in a density gradient. During its purification, its hydrolytic activity was assayed on cell walls of Lactobacillus plantarum ATCC 8014 and soluble glycopeptides and peptides, of known chemical structures, prepared enzymatically from these cell walls. A fraction with an isoelectric point of pH 7.9 cleaved the bond between the carboxyl group of the D-alanine residue at the C-terminal in one peptide subunit and one of the two amino groups of the A2pm residue in the neighboring peptide subunit. Unlike the crude enzyme, the endopeptidase in this fraction showed no N-acetylmuramyl-L-alanine amidase, A2pm carboxyamide amidase or proteinase(s) activity and it was immunologically homogeneous.

Alanine↗

PubMedDate not supplied

[Adaptation of acrylamide producer Rhodococcus rhodochrous M8 to change in ammonium concentration in medium].

The mechanism of adaptation of the acrylamide producing strain Rhodococcus rhodochrous M8 to changes in ammonium concentrations in the medium was studied. An increase in the content of ammonium in the medium changed the activity of glutamine synthetase (GS) (EC 6.3.1.2) and glutamine dehydrogenase (GD) (EC 1.4.1.4), the enzymes of ammonium assimilation, as well as the activities of enzymes responsible for nitrile utilization: nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4). This also caused inhibition of activation of GS induced by phosphodiesterase (EC 3.1.4.1). Increases in the activities of nitrile hydratase and amidase and resistance of these enzymes to ammonium were observed in mutant of R. rhodichrous resistant to phosphotricine, an inhibitor of GS. An important role of GS in the mechanism of adaptation is suggested.

Acrylamide↗

PubMed2006

[Characterization of two new pigmented mycobacteria isolates].

Two pigmented mycobacteria isolated from sputum specimens were described by biochemical tests, whole-cell fatty acid analyses by High Performance Liquid Chromatography (HPLC) and sequencing of 65-kDa heat shock protein gene and 16S rRNA gene. The hsp65 gene and 16S rRNA gene sequences of the Mycobacterium sp. G1368 and Mycobacterium sp. E498 were deposited in DDBJ/EMBL/GenBank under accession numbers AY553874, DQ324791 and AY379074, DQ324792, respectively. Mycobacterium sp. G1368 grew in about one week at 37 degrees C and 42 degrees C and produced smooth, yellow colonies. It reduced tellurite and hydrolyzed urea. Nitrate reduction, aryl sulfatase, pyrazin amidase, heat stable catalase and semiquantitative catalase tests were also positive, while Tween 80 hydrolysis was weakly positive. Mycobacterium sp. E498 grew in about 9 days at 37 degrees C and formed smooth, yellow colonies. It hydrolyzed Tween 80, possessed aryl sulfatase, pyrazin amidase and heat stable catalase, however, it did not possess urease and nitrate reductase. These data, in addition to their position in the phylogenetic tree, strongly support the status of novel species at least for Mycobacterium sp. G1368.

Bacterial Proteins↗

PubMed1977

A sensitive substrate for the clotting enzyme in horseshoe crab hemocytes.

An endotoxin-activated hemocyte lysate from horseshoe crab (Tachypleus and Limulus) was found to hydrolyze specifically BZ-Ile-Glu-Gly-Arg-p-nitroanilide, which was recently introduced as the substrate for assay of the blood coagulation factor, Factor Xa. Further, this amidase activity increased by increasing the concentration of bacterial endotoxin (Salmonella minnesota R595) added to the lysate. Thus, the measurement of the amidase activity in the hemocyte lysate can be very useful to detect and determine the endotoxin.

Animals↗

PubMed1975

Protease II from Escherichia coli. Purification and characterization.

We have previously demonstrated the existence of two types of endopeptidase in Escherichia coli. A purification procedure is described for one of these, designated protease II. It has been purified about 13,500-fold with a recovery of 24%. The isolated enzyme appears homogeneous by electrophoresis and gel filtration. Its molecular weight is estimated by three different methods to be about 58,000. Its optimal pH is around 8. Protease II activity is unaffected by chelating agents and sulfhydryl reagents. Amidase and proteolytic activities are stimulated by calcium ion, which decreases the enzyme stability. Like pancreatic trypsin, this endopeptidase catalyses the hydrolysis of alpha-amino-substituted lysine and arginine esters. It appears distinct from the previously isolated protease I, which is a chymotrypsin-like enzyme. The apparent Michaelis constant for hydrolysis of N-benzoyl-L-arginine ethyl ester is 4.7 X 10(-4) M. The esterase activity is inhibited by diisopryopylphosphorofluoridate (Ki(app) equals 2.7 X 10(-3) M) and tosyl lysine chloromethyl ketone (Ki(app) equals 1.8 X 10(-5) M), indicating that serine and histidine residues may be present in the active site. However, protease II is insensitive to phenylmethanesulfonyl fluoride and several natural trypsin inhibitors. Its amidase and esterase activities are competitively inhibited by free arginine and aromatic amidines. The proteolytic activity measured on axocasein is very low. In contrast to trypsin, protease II is without effect on native beta-galactosidase. It easily degrades aspartokinase I and III. Nevertheless both enzymes are resistant to proteolysis in the presence of their respective allosteric effectors. These results provide further evidence that such differences in protease susceptibility can be related to the conformational state of the substrate. The possible implication of structural changes in the mechanism of preferential proteolysis in vivo, is discussed.

Binding, Competitive↗

PubMed1985

Management of inflammation: lipid peroxide as a parameter for a better understanding of inflammatory processes.

The management of inflammation, based on an overall, coordinated observation of the inflammatory system, requires the assessment of the validity and biological significance of the various components in inflammation, a procedure that is more reliable than the interpretation of individual measurements in isolation. Such an overall approach, here developed from the model of carrageenin-induced inflammation, also predicates that lysosomal enzymes, lipid peroxide and proamidase (related, respectively, to the inflammatory response in a narrow sense, to tissue damage and to tissue repair) are three basic parameters required when studying inflammatory processes. This overall approach, moreover, shows that the inflammatory process must be regarded as being diachronic, and that in the light of the observation of exudate fluid it can be divided into three main stages, namely an early stage of exudation related to lipid peroxide, an intermediate stage of exudate dilution related to granuloma amidase, and a later stage of exudate concentration related to granuloma amidase and plasma protein.

Amidohydrolases↗

PubMed1988

[Bacteriolytic enzymes produced by actinomycetes. I. The physicochemical properties of the enzymes and the spectrum of their lytic action].

This review is devoted to the bacteriolytic enzymes produced by many actinomycetes, mainly by Streptomyces genus. The bacteriolytic enzymes hydrolyse the specific bonds in bacterial peptidoglycans and cause the solubilization of the cellular walls and the disintegration of the bacterial cells. Many of the enzymes are purified to the electrophoretic homogeneity. The actinomycetes form the endo-N-acetylmuramidases more often, then the endopeptidases follow according to the frequency of occurrence, while the amidases and endo-N-acetylglucosaminidases are met rather seldom among the streptomycete-producers. The known amidases and exo-enzymes which are also produced by some species of actinomycetes are not related to the lytic enzymes proper. Almost all known endopeptidases from streptomyces hydrolyse the bridge peptide bonds in which the carboxyl group of terminal D-alanyl of peptide chain is involved. The bacteriolytic spectra of the different muramidases differ from each other and essentially differ from the spectrum of the egg-white lysozyme. Some endomuramidases from streptomyces are able to hydrolyse streptococci and some other important from the practical point of view microorganisms resistant to the action of lysozyme.

Actinomycetales↗

PubMedDate not supplied

[Effect of the structure of polymeric materials on the properties of immobilized enzymes].

The relationship between the structure of triacetate cellulose fibres and films and properties of enzymes entrapped in these materials was investigated. Trypsin and penicillin amidase were entrapped in triacetate cellulose films and fibres during their formation. The effect of permeability of the films on the catalytic properties of entrapped trypsin was studied. The porous structure of triacetate cellulose fibres with entrapped penicillin amidase was studied, the fibres being produced by different methods. An increase of porosity and the specific surface of the fibre-biocatalyst reduced the Km value and the energy of activation of the hydrolytic decomposing of benzyl penicillin but increased the Vmax value.

Enzymes, Immobilized↗

PubMedDate not supplied

[Affinity chromatography of human gamma-thrombin on fibrin-agarose].

gamma-Thrombin was produced during autolysis or limited proteolysis of coagulant gamma-thrombin. This thrombin form loses its ability to coagulate fibrinogen but preserves the esterase and amidase activity on the low-molecular-weight synthetic substrates. This evidences for the integrity of the active site of gamma-thrombin and for the integrity break of the enzyme molecule region responsible for the binding with fibrinogen. gamma-Thrombin with the minimal coagulating activity, possessing high esterase and amidase activity is obtained. Fibrin-agarose possessing affinity to gamma-thrombin and specifically not binding gamma-thrombin was used to remove admixtures of the coagulant gamma-thrombin from the preparations of gamma-thrombin obtained during the enzyme autolysis.

Catalysis↗

PubMed1980

Formation and properties of L-glutaminase and L-asparaginase activities in Pichia polymorpha.

An ascogenous yeast with high potentialities for L-glutaminase and L-asparaginase formation was isolated from Egyptian soils by the application of the culture enrichment method. The organism, identified as Pichia polymorpha, was obtained through the enrichment of soil samples with a simple medium containing 0.5% L-glutaminase as a major carbon and nitrogen source at low pH values. The amidase activities were produced constitutively on a variety of media irrespective of the presence of their substrates in the growth medium. Assays of enzyme activity have revealed that optimum pH values for L-glutamine and L-asparagine hydrolysis are 6.0 and 6.7, respectively. The L-asparaginase activity of the cells was heat-stable for at least 10 minutes at 60 degrees C. The enzyme exhibited apparent Km of 1.37 x 10(-2) M and 1.95 x 10(-2) M for L-asparagine and L-glutamine, respectively. No metal requirement were detected for the amidase activities of the organism under study.

Ascomycota↗

PubMed1981

Immunoreactive forms of cationic trypsin in plasma and ascitic fluid of dogs in experimental pancreatitis.

A canine model of bile-induced pancreatitis has been employed to investigate time-dependent changes in the molecular forms of trypsin in blood and ascitic fluid in this disease. The distribution of immunoreactive trypsin as trypsinogen and trypsin bound to plasma inhibitors in ascitic fluid and plasma during the course of the disease has been investigated by means of a radioimmunoassay for canine pancreatic cationic trypsin. In addition, trypsinlike amidase activity was determined in plasma and ascitic fluid using Z-Gly-Gly-Arg-beta-Nap as substrate. Early plasma and ascitic fluid samples in four dogs that died contained primarily trypsinogen, while extensive activation of trypsinogen to alpha 2-macroglobulin and alpha 1-protease inhibitor-bound trypsin occurred in the course of the disease. A fifth dog survived and showed little activation of trypsinogen. In the four dogs that died, the levels of trypsinlike amidase activity in the ascitic fluid were substantial throughout the course of the disease. The plasma levels of trypsinlike activity in these animals were much lower, but increased during the disease process. The dog that survived had lower concentrations of trypsinlike activity in ascitic fluid and plasma. These results suggest that activation of trypsinogen resulting in inhibitor-bound forms of trypsin in ascitic fluid and plasma is important in the pathogenesis of acute pancreatitis.

Amidohydrolases↗