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PubMed1999

A new biotyping method for Streptococcus mutans with the API ZYM system.

OBJECTIVE: To test a new system for the biotyping of Streptococcus mutans, based on the measurement of enzyme activity, and to investigate the relationship between biotype and in vitro susceptibility to seven clinically useful antibiotics. METHODS: In total, 160 oral isolates of S. mutans were classified into different biotypes with the APIZYM test for enzyme activity, excluding results that were positive or negative in >80% of the strains. The susceptibility of all 160 strains to amoxycillin, cefazolin, erythromycin, clindamycin, vancomycin, teicoplanin and imipenem was tested by dilution in a solid medium. Statistical analysis of susceptibility (mean minimum inhibitory concentrations (MICs)) was based on chi-squared tests. RESULTS: Eight different biotypes (1-8) were identified on the basis of three kinds of enzyme activity: valine aryl amidase, acid phosphatase and alpha-galactosidase. Biotype 5 was found to be the most common. The mean MIC values showed strains belonging to biotype 4 to be the most susceptible to amoxycillin, cefazolin and erythromycin, whereas biotype 1 was the least susceptible to teicoplanin. CONCLUSIONS: The proposed biotyping method, which is relatively fast and simple to perform, provided reproducible results, and may contribute to clinically effective treatment of S. mutans infections.

Journal Article↗

PubMed1982

Substance P hydrolysis by human serum cholinesterase.

Highly purified human serum cholinesterase (EC 3.1.1.8, also known as pseudocholinesterase and butyrylcholinesterase) had peptidase activity toward substance P. Digestion of substance P was monitored by high performance liquid chromatography, which separated three product peptides. The cleavages occurred sequentially. The first peptide to appear as Arg1-Pro2. The Km for this hydrolysis was 0.3 mM; maximum activity was 7.9 nmol min-1 mg-1 of protein, which corresponded to a turnover number of 0.6 min-1. A second cleavage yielded Lys3-Pro4. A third cleavage occurred at the C-terminal, where the amide was removed from Met11 to yield a peptide containing residues 5-11. Both the peptidase and esterase activities of the enzyme were completely inhibited by the anticholinesterase agent, diisopropylfluorophosphate. Substance P inhibited the hydrolysis of benzoylcholine (a good ester substrate) with a KI of 0.17 mM, indicating that substance P interacted with cholinesterase rather than with a trace contaminant. Peptidase and amidase activities for serum cholinesterase are novel activities for this enzyme. It was demonstrated previously that the related enzyme acetylcholinesterase (EC 3.1.1.7) catalyzed the hydrolysis of substance P, but at entirely different cleavage sites from those reported in the present work. Since butyrylcholinesterase is present in brain and muscle, as well as in serum, it may be involved in the physiological regulation of substance P.

Amino Acids↗

PubMed1988

Slow penetration of thyrotropin-releasing hormone across the blood-brain barrier of an in situ perfused guinea pig brain.

Transport of 3H-labelled thyrotropin-releasing hormone (TRH) across the blood-brain barrier was studied in the ipsilateral perfused in situ guinea pig forebrain. The unidirectional transfer constant (Kin) calculated from the multiple time brain uptake analysis ranged from 1.14 X 10(-3) to 1.22 X 10(-3) ml min-1 g-1, in the parietal cortex, caudate nucleus, and hippocampus. Regional Kin values for [3H]TRH were significantly reduced by 43-48% in the presence of an aminopeptidase and amidase inhibitor, 2 mM bacitracin, suggesting an enzymatic degradation of tripeptide during interaction with the blood-brain barrier. In the presence of unlabelled 1 mM TRH and 2 mM bacitracin together, a reduction of [3H]TRH regional Kin values similar to that obtained with 2 mM bacitracin alone was obtained . L-Prolinamide, the N-terminal residue of tripeptide, at a 10 mM level had no effect on the kinetics of entry of [3H]TRH into the brain. The data indicate an absence of a specific saturable transport mechanism for TRH presented to the luminal side of the blood-brain barrier. It is concluded that intact TRH molecule may slowly penetrate the blood-brain barrier, the rate of transfer being some three times higher than that of D-mannitol.

Animals↗

PubMed1992

Purification and partial structural and functional characterization of mouse myelin/oligodendrocyte glycoprotein.

The myelin/oligodendrocyte glycoprotein (MOG) is found exclusively in the CNS, where it is localized on the surface of myelin and oligodendrocyte cytoplasmic membranes. The monoclonal antibody 8-18C5 identifies MOG. Several studies have shown that anti-MOG antibodies can induce demyelination, thus inferring an important role in myelin stability. In this study, we demonstrate that MOG consists of two polypeptides, with molecular masses of 26 and 28 kDa. This doublet becomes a single 25-kDa band after deglycosylation with trifluoromethanesulfonic acid or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, indicating that there are no or few O-linked sugars and that the doublet band represents differential glycosylation. Partial trypsin cleavage, which also gave a doublet band of lower molecular weight, confirmed this idea. MOG was purified by polyacrylamide gel electrophoresis, followed by electroelution. Three N-terminal sequences of eight to 26 amino acids were obtained. By western blot analysis, no binding was found between MOG and cerebellar soluble lectin. MOG does not seem to belong to the signal-transducing GTP-binding proteins. Reduced MOG concentrations were observed in jimpy and quaking dysmyelinating mutant mice, giving further support to its localization in compact myelin of the CNS.

Amino Acid Sequence↗

PubMed2005

Occurrence and possible biological role of the endocannabinoid system in the sea squirt Ciona intestinalis.

A cannabinoid receptor orthologue (CiCBR) has been described in the sea squirt Ciona intestinalis. Here we report that CiCBR mRNA expression is highest in cerebral ganglion, branchial pharynx, heart and testis of C. intestinalis, and that this organism also contains cannabinoid receptor ligands and some of the enzymes for ligand biosynthesis and inactivation. Using liquid chromatography-mass spectrometry, the endocannabinoid anandamide was found in all tissues analysed (0.063-5.423 pmol/mg of lipid extract), with the highest concentrations being found in brain and heart. The endocannabinoid 2-arachidonoylglycerol (2-AG) was fivefold more abundant than anandamide, and was most abundant in stomach and intestine and least abundant in heart and ovaries (2.677-50.607 pmol/mg of lipid extract). Using phylogenomic analysis, we identified orthologues of several endocannabinoid synthesizing and degrading enzymes. In particular, we identified and partly sequenced a fatty acid amide hydrolase (FAAH) orthologue, showing 44% identity with human FAAH and containing nearly all the amino acids necessary for a functional FAAH enzyme. Ciona intestinalis also contained specific binding sites for cannabinoid receptor ligands, and an amidase enzyme with pH-dependency and subcellular/tissue distribution similar to mammalian FAAHs. Finally, a typical C. intestinalis behavioural response, siphon reopening after closure induced by mechanical stimulation, was inhibited by the cannabinoid receptor agonist HU-210, and this effect was significantly attenuated by mammalian cannabinoid receptor antagonists.

Amidohydrolases↗

PubMed1996

Utilization of acetonitrile and other aliphatic nitriles by a Candida famata strain.

A variant of a yeast strain identified as Candida famata isolated from gold mine effluent was able to grow on acetonitrile, acrylonitrile, butyronitrile, isobutyronitrile, methacrylnitrile, propionitrile, succinonitrile, valeronitrile, acetamide, isobutyamide, and succinamide as sole nitrogen source, after acclimatization. The yeast grew on acetonitrile and acetamide at concentrations up to 4%. The utilisation of acetonitrile and acetamide by the C. famata strain probably involves hydrolysis in a two-step reaction mediated by both inducible and intracellular nitrile hydratase and amidase.

Acetonitriles↗

PubMed2006

Lysis of staphylococcal mastitis pathogens by bacteriophage phi11 endolysin.

The Staphylococcus aureus bacteriophage phi11 endolysin has two peptidoglycan hydrolase domains (endopeptidase and amidase) and an SH3b cell wall-binding domain. In turbidity reduction assays, the purified protein can lyse untreated staphylococcal mastitis pathogens, Staphylococcus aureus and coagulase-negative staphylococci (Staphylococcus chronogenes, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus simulans, Staphylococcus warneri and Staphylococcus xylosus), making it a strong candidate protein antimicrobial. This lytic activity is maintained at the pH (6.7), and the "free" calcium concentration (3 mM) of milk. Truncated endolysin-derived proteins containing only the endopeptidase domain also lyse staphylococci in the absence of the SH3b-binding domain.

Animals↗

PubMed1986

Activation of factor XII in acetone-treated human plasma: significance of the functional state of plasma kallikrein for the extent of activation.

The kaolin-induced activation of factor XII (XII) to XIIa was studied in plasminogen-free human citrated plasma treated with acetone in the presence of benzamidine 7.5 mM. XIIa was assayed as prekallikrein (PK) activator. The significance of the concentrations of XII, PK and high molecular weight kininogen (HMrK) was examined using mixtures of normal plasma and plasma genetically deficient in these factors. At the high plasma dilution used (1 + 23 v/v in the kaolin incubate) a joint estimation of the factors was obtained. A reduction in amount of XII, PK or HMrK resulted in a correspondingly reduced yield of XIIa. Plasma kallikrein present was assayed as S-2302 amidase. The concentration of PK in XII-deficient plasma was normal, in HMrK-deficient plasma about 30% of normal. The activation of XII was studied in fresh plasma as well as in plasma stored for 3-6 months at -70 degrees, and the activation with acetone was carried out in the presence and in the absence of benzamidine, EDTA or purified HMrK. In previous work benzamidine was found to protect the cofactor function of purified HMrK in the assay system used, and EDTA was found to inhibit purified human plasma kallikrein assayed as plasminogen activator. The present results support the previous observations, and indicate that acetone treatment of fresh human plasma (benzamidine present) results in the activation of plasma kallikrein in a functional state that requires kinin-free, but otherwise native HMrK as a cofactor for the activation of XII.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetone↗

PubMed1986

Effects of intravenous radiographic contrast media on the blood pressure and on factors of the contact activation system in the rat.

Blood pressure (BP), plasma prekallikrein (PK), and the extent of activation of factor XII (XII-ACT) were studied after the intravenous injection into rats of dextran (Macrodex), the ionic radiographic contrast substance iodipamide (Biligrafin), or the non-ionic contrast substance iohexol (Omnipaque). After acetone activation plasma kallikrein was assayed as plasminogen activator, BAEe esterase or S-2302 amidase, and factor XIIa was assayed as kaolin-activated prekallikrein activator. Dextran induced a strong and lasting hypotension, preceded by significant lowerings in PK and XII-ACT. Iodipamide induced a rapid and dose dependent BP fall, no change in plasma PK, but a slightly reduced XII-ACT. Iohexol induced no significant alterations, neither in BP, nor in plasma parameters. Pretreatments of the rats with iodipamide abolished the dextran-induced reductions in PK and XII-ACT, and almost blocked the fall in BP. We conclude that the ionic contrast substance iodipamide is capable of blocking dextran shock in the rat by preventing an activation of the contact activating system in plasma.

Anaphylaxis↗

PubMed1987

Acetylcysteine in rats: inhibition of activation of prekallikrein and factor XII--protection against dextran-induced blood pressure fall.

Acetylcysteine (AC) injected intravenously into rats (200 mg/kg) had no effect on blood pressure, but significantly inhibited dextran-induced (40 mg/kg) blood pressure fall. Injection of AC also reversibly blocked the activation of prekallikrein (PK) normally obtained in plasma incubated with acetone. Kallikrein was assayed as plasminogen activator, S-2302 amidase and BAEe esterase. Also the activation of factor XII to factor XIIa, assayed as prekallikrein activator, was strongly inhibited in AC-treated rats. It is suggested that the partial blockade of dextran-induced shock is correlated with an inhibition of activation of PK and factor XII. Previous experiments had demonstrated an extensive, but reversible in vitro inhibition of human plasma kallikrein by AC. In view of such data it is concluded that the present results obtained with AC in rats are probably due to an inhibition of plasma kallikrein and its activation of factor XII.

Acetylcysteine↗

PubMed2005

A DmpA-homologous protein from Pseudomonas sp. is a dipeptidase specific for beta-alanyl dipeptides.

We have determined the nucleotide sequence of a DNA fragment covering the flanking region of the R-stereoselective amidase gene, ramA, from the Pseudomonas sp. MCI3434 genome and found an additional gene, bapA, coding for a protein showing sequence similarity to DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 (43% identity). The DmpA (called L-aminopeptidase D-Ala-esterase/amidase) hydrolyzes alanine-p-nitroanilide, alaninamide, and alanine methylester with a preference for the D-configuration of the alanine, whereas the enzyme acts as an L-stereoselective aminopeptidase on a tripeptide Ala-(Gly)2, indicating a reverse stereoselectivity [Fanuel L, Goffin C, Cheggour A, Devreese B, Van Driessche G, Joris B, Van Beeumen J & Frère J-M (1999) Biochem J341, 147-155]. A recombinant BapA exhibiting hydrolytic activity toward D-alanine-p-nitroanilide was purified from the cell-free extract of an Escherichia coli transformant overexpressing the bapA gene and characterized. The purified enzyme contained two polypeptides corresponding to residues 1-238 (alpha-peptide) and 239-366 (beta-peptide) of the precursor as observed for DmpA. On gel-filtration chromatography, BapA in the native form appeared to be a tetramer. It had maximal activity at 60 degrees C and pH 9.0-10.0, and was inactivated in the presence of p-chloromercuribenzoate, N-ethylmaleimide, dithiothreitol, Zn2+, Ag+, Cd2+ or Hg2+. The enzyme hydrolyzed D-alanine-p-nitroanilide more efficiently than L-alanine-p-nitroanilide the same as DmpA. Furthermore, BapA was found to hydrolyze peptide bonds of beta-alanyl dipeptides including beta-Ala-L-Ala, beta-Ala-Gly, beta-Ala-L-His (carnosine), beta-Ala-L-Leu, and (beta-Ala)2 with high efficiency compared to D-alanine-p-nitroanilide. Beta-alaninamide was also efficiently hydrolyzed, but the enzyme did not act on the peptides containing proteinogenic amino acids or their D-counterparts for N-terminal residues. Based on its unique substrate specificity, the enzyme should not be called L-aminopeptidase D-Ala-esterase/amidase but beta-Ala-Xaa dipeptidase.

Alanine↗

PubMed2006

Biochemical characterization of MelJ and MelK.

Melithiazol and myxothiazol are two myxobacterial metabolites that are highly efficient electron transport inhibitors of the respiratory chain. MelJ and MelK encoded in the melithiazol biosynthetic gene cluster were recently shown to be involved in the formation of the methyl ester from a hypothetical amide intermediate. In vivo studies suggest that the structurally highly similar amide myxothiazol A can be used as a substrate mimic of the hypothetical melithiazol amide to characterize the hydrolase MelJ. Both enzymes were produced in Escherichia coli as intein chitin fusion proteins and were purified using affinity chromatography. MelJ was found to catalyse the conversion of the amide myxothiazol to free myxothiazol acid. The formerly unknown myxothiazol acid was purified and used as a substrate for the methyl transferase MelK which methylates the compound using S-adenosyl-methionine as cosubstrate. Sequence analyses suggest that MelJ and MelK are members of the amidase signature family and of a new subclass of methyltransferases, respectively. Kinetic analyses point at a very high substrate specificity for both enzymes. Furthermore, the in vitro reconstitution of a unique mechanism of methyl ester formation found in myxobacteria is reported.

Acrylates↗

PubMed2003

Potentiation of anandamide effects in mesenteric beds isolated from endotoxemic rats.

The aim of the present experiments was to study the effects of exogenously added anandamide on transient norepinephrine (NE)-induced contractions in mesenteric beds isolated from adult male Sprague-Dawley rats 6 h after the i.p. administration of 5 mg kg(-1) lipopolysaccharide (LPS). LPS treatment induced a 3-fold increase in total nitric-oxide synthase (NOS) activity without modifying either the systolic blood pressure or the vascular reactivity to NE of the isolated mesenteric bed. The endocannabinoid anandamide (0.01-10 microM) caused concentration-dependent reductions of the contractile responses to NE in the isolated mesenteric bed. This effect was significantly potentiated after LPS treatment compared with the controls. Anandamide-induced reductions of the contractile responses to NE in mesenteric beds isolated from LPS-treated rats were unmodified by endothelium removal but significantly diminished by either the anandamide amidase inhibitor phenylmethylsulfonyl fluoride (200 microM) or the vanilloid receptor antagonist capsazepine (1 microM). The vanilloid receptor agonist capsaicin (0.01-100 nM) also caused concentration-dependent relaxations that were potentiated in mesenteric beds from LPS-treated rats. Nevertheless, they were unmodified by 1 microM capsazepine. It is concluded that the potentiation of anandamide relaxations after LPS treatment, which are evident at early stages of endotoxic shock, could involve the participation of an anandamide metabolite and might be mediated, at least in part, through a vanilloid receptor.

Amidohydrolases↗

PubMed2006

Pharmacological characterization of hydrolysis-resistant analogs of oleoylethanolamide with potent anorexiant properties.

Oleoylethanolamide (OEA) is an endogenous lipid mediator that reduces food intake, promotes lipolysis, and decreases body weight gain in rodents by activating peroxisome proliferator-activated receptor-alpha (PPAR-alpha). The biological effects of OEA are terminated by two intracellular lipid hydrolase enzymes, fatty-acid amide hydrolase and N-acylethanolamine-hydrolyzing acid amidase. In the present study, we describe OEA analogs that resist enzymatic hydrolysis, activate PPAR-alpha with high potency in vitro, and persistently reduce feeding when administered in vivo either parenterally or orally. The most potent of these compounds, (Z)-(R)-9-octadecenamide,N-(2-hydroxyethyl,1-methyl) (KDS-5104), stimulates transcriptional activity of PPAR-alpha with a half-maximal effective concentration (EC50) of 100 +/- 21 nM (n = 11). Parenteral administration of KDS-5104 in rats produces persistent dose-dependent prolongation of feeding latency and postmeal interval (half-maximal effective dose, ED50 = 2.4 +/- 1.8 mg kg(-1) i.p.; n = 18), as well as increased and protracted tissue exposure compared with OEA. Oral administration of the compound also results in a significant tissue exposure and reduction of food intake in free-feeding rats. These results suggest that the endogenous high-affinity PPAR-alpha agonist OEA may provide a scaffold for the discovery of novel orally active PPAR-alpha ligands.

Animals↗

PubMed2006

Oxyhomologation of the amide bond potentiates neuroprotective effects of the endolipid N-palmitoylethanolamine.

The endolipid N-palmitoylethanolamine (PEA) shows a pleiotropic pattern of bioactivities, whose mechanistic characterization is still unclear and whose pharmacological potential is substantially limited by rapid metabolization by the amido hydrolyzing enzymes fatty acid amide hydrolases and N-acylethanolamine-hydrolyzing acid amidase. To overcome this problem, we have synthesized a new series of PEA homologs and characterized their activity on two in vitro models of neurodegeneration (oxidative stress, excitotoxicity). PEA partially prevented tert-butylhydroperoxide (t-BOOH; 100 microM; 3 h)-induced cell death (maximal effect, 26.3 +/- 7.5% in comparison with t-BOOH-untreated cells at 30 microM), whereas it was ineffective against the L-glutamate (1 mM; 24 h)-induced excitotoxicity at all concentrations tested (0.01-30 microM). Oxyhomologation of the amide bond, although leading to an increased enzymatic stability, also potentiated neuroprotective activity, especially for N-palmitoyl-N-(2-hydroxyethyl)hydroxylamine (EC(50) = 2.1 microM). These effects were not mediated by cannabinoid/vanilloid-dependent mechanisms but rather linked to a decreased t-BOOH-induced lipoperoxidation and reactive oxygen species formation and L-glutamate-induced intracellular Ca(2+) overload. The presence of the hydroxamic group and the absence of either redox active or radical scavenger moieties suggest that the improved neuroprotection is the result of increased metal-chelating properties that boost the antioxidant activity of these compounds.

Amides↗

PubMed1988

Induction of an antibody that catalyzes the hydrolysis of an amide bond.

Catalysis of amide bond hydrolysis is of singular importance in enzymology. An antibody was induced to an analog of a high-energy intermediate anticipated along the reaction coordinate of amide hydrolysis. This antibody is an amidase with high specificity and a large rate enhancement (250,000) relative to the uncatalyzed reaction. This reaction represents the kinetically most difficult hydrolysis reaction yet catalyzed by an antibody.

Amidohydrolases↗

PubMed1981

Effect of penicillin G on release of peptidoglycan fragments by Neisseria gonorrhoeae: characterization of extracellular products.

The effect of penicillin G (penG) on the turnover and release of peptidoglycan (PG) by Neisseria gonorrhoeae RD5 was investigated. We previously showed that exponentially growing gonococci (labeled in the glycan moiety with glucosamine and muramic acid and in the peptide with diaminopimelic acid) turn over ca. 35% of their PG per generation. In current studies, addition of penG accelerated the rate of PG hydrolysis by more than twofold and resulted in a corresponding increase in the amount of soluble PG fragments found in the medium. This increase of soluble PG was accounted for mainly by the release of nonreducing (anhydro-muramyl-containing) disaccharide peptide dimers (molecular weight, about 2,000) and trimers that were composed of subunits, linked not by peptide cross-linking bonds, but probably only by glycosidic bonds. The enhanced release of these products suggested that penG, directly or indirectly, stimulates the activity of a glycan-splitting, gonococcal PG:PG-6 muramyl transferase (transglycosylase). PG monomers that were released in the presence of penG were identical, both qualitatively and quantitatively, to the monomers released as a result of turnover in the absence of penG and consisted of anhydro-muramyl-containing disaccharide tripeptides and tetrapeptides. PenG-treated bacteria consistently released slightly less free disaccharide and free peptides than did control cultures, implying a penG-associated depression in the activity of the gonococcal N-acetylmuramyl-L-alanine amidase. In addition to stimulating the release of PG fragments, penG was also associated with greatly enhanced release from cells of glucosamine-containing non-PG macromolecule(s).

Hydrolysis↗

PubMed1989

Reactions catalyzed by purified L-glutamine: keto-scyllo-inositol aminotransferase, an enzyme required for biosynthesis of aminocyclitol antibiotics.

Dialyzed extracts of the gentamicin producer Micromonospora purpurea catalyze reactions which represent transaminations proposed for 2-deoxystreptamine biosynthesis. To determine whether these transaminations were catalyzed by a single aminotransferase or by multiple enzymes, we purified and characterized an L-glutamine:keto-scyllo-inositol aminotransferase from M. purpurea. This enzyme was purified 130- to 150-fold from late-log-phase mycelia of both wild-type M. purpurea and a 2-deoxystreptamine-less idiotroph. The cofactor pyridoxal phosphate was found to be tightly bound to the enzyme, and spectral analysis demonstrated its participation in the transamination reactions of this enzyme. The major physiological amino donor for the enzyme appears to be L-glutamine; the keto acid product derived from glutamine was characterized as 2-ketoglutaramate, indicating that the alpha amino group of glutamine participates in the transamination. We found that crude extracts contained omega-amidase activity, which may render transaminations with glutamine irreversible in vivo. The substrate specificity of the aminotransferase was shown to be restricted to deoxycyclitols, monoaminocyclitols, and diaminocyclitols, glutamine, and 2-ketoglutaramate, which contrasts with the broader substrate specificity of mammalian glutamine aminotransferase. The appearance of the enzyme in late-log phase, coupled with its narrow substrate specificity, indicates that it participates predominantly in 2-deoxystreptamine biosynthesis rather than in general metabolism. The enzyme catalyzes reactions which represent both transamination steps of 2-deoxystreptamine biosynthesis. Although copurification of two aminotransferases cannot be ruled out, our data are consistent with the participation of a single aminotransferase in the formation of both amino groups of 2-deoxystreptamine during biosynthesis by M. purpurea. We propose that this aminotransferase participates in a key initial step in the biosynthesis of most aminocyclitol antibiotics.

Amidohydrolases↗