Baclofen and γ-hydroxybutyrate: similar effects on cerebral dopamine neurones

Baclofen (20 mg/kg) caused an increase in the content of homovanillic acid (HVA) and dopamine (DA) in rat brain 2–3 h after drug injection without appreciable changes in the level of other monoamines and their main metabolites. Six and eight hours after baclofen, the content of HVA but not that of DA was reduced. Moreover, baclofen initially (20 min after injection) reduced, but later (105 min post drug) enhanced the accumulation of HVA induced by probenecid. The shortlasting (20 min) initial reduction of HVA elevation in probenecid-pretreated animals as well as the longlasting (6–8 h) decrease of HVA levels in rats injected with baclofen alone are interpreted to be due to a decreased release and metabolism of DA, probably as a consequence of the blockade of impulse flow in mesolimbic and nigro-striatal DA neurones. The increase in HVA and DA seen during the first few hours is thought to result from enhanced DA synthesis similar to that known for γ-hydroxybutyrate (GHB). This initial rise in HVA due to synthesis stimulation probably masked a reduction of HVA to be expected immediately after baclofen injection. The similarity between baclofen and GHB is stressed by the finding that baclofen counteracted the increase of HVA occuring after chlorpromazine and D-amphetamine but not that induced by the benzoquinolizine derivative, Ro 4-1284.     

Use of an inhibitor to identify members of the hormone-sensitive lipase family

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids from the triacylglycerols stored in adipocytes, which provide the main source of energy in mammals. On the basis of amino acid sequence alignments and three-dimensional structures, this enzyme was previously found to be a suitable template for defining a family of serine carboxylester hydrolases. In this study, the HSL family members are characterized rather on the basis of their inhibition by 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one (compound 7600). This compound inhibits mammalian HSL as well as other HSL family members, such as EST2 from the thermophilic eubacterium Alicyclobacillus acidocaldarius and AFEST from the hyperthermophilic archaeon Archaeoglobus fulgidus. Various carboxylester hydrolases that are not members of the HSL family were found not to be inhibited by compound 7600 under the same experimental conditions. These include nonlipolytic hydrolases such as Torpedo californica acetylcholinesterase and pig liver esterase, as well as lipolytic hydrolases such as human pancreatic lipase, dog gastric lipase, Thermomyces lanuginosus lipase, and Bacillus subtilis LipA. When vinyl esters were used as substrates, the residual activity of HSL, AFEST, and EST2 decreased with an increase in compound 7600 concentration in the incubation mixture. The inhibitor concentration at which the enzyme activity decreased to 50% after incubation for 5 min was 70, 20, and 15 nM with HSL, AFEST, and EST2, respectively. Treating EST2 and AFEST with the inhibitor resulted in an increase in the molecular mass, as established by performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. This increase in the molecular mass, which corresponds approximately to the molecular mass of the inhibitor, indicates that a covalent enzyme-inhibitor complex has been formed. Surface-enhanced laser desorption ionization time-of-flight mass spectrometry analysis of a trypsin digest of AFEST treated with the inhibitor or not treated showed the occurrence of an increase in the molecular masses of the "GESAGG"-containing peptide, which is compatible with the formation of a covalent complex with the inhibitor.     

The α-<Emphasis Type="SmallCaps">l</Emphasis>-fucosidase from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>

Glycoside hydrolases form hyperthermophilic archaea are interesting model systems for the study of catalysis at high temperatures and, at the moment, their detailed enzymological characterization is the only approach to define their role in vivo. Family 29 of glycoside hydrolases classification groups α-l-fucosidases involved in a variety of biological events in Bacteria and Eukarya. In Archaea the first α-l-fucosidase was identified in Sulfolobus solfataricus as interrupted gene expressed by programmed −1 frameshifting. In this review, we describe the identification of the catalytic residues of the archaeal enzyme, by means of the chemical rescue strategy. The intrinsic stability of the hyperthermophilic enzyme allowed the use of this method, which resulted of general applicability for β and α glycoside hydrolases. In addition, the presence in the active site of the archaeal enzyme of a triad of catalytic residues is a rather uncommon feature among the glycoside hydrolases and suggested that in family 29 slightly different catalytic machineries coexist.     

Identification and characterization of a novel bacterial sulfite oxidase with no heme binding domain from Deinococcus radiodurans

An open reading frame (draSO) encoding a putative sulfite oxidase (SO) was identified in the sequence of chromosome II of Deinococcus radiodurans; the predicted gene product showed significant amino acid sequence homology to several bacterial and eukaryotic SOs, such as the biochemically and structurally characterized enzyme from Arabidopsis thaliana. Cloning of the Deinococcus SO gene was performed by PCR amplification from the bacterial genomic DNA, and heterologous gene expression of a histidine-tagged polypeptide was obtained in a molybdopterin-overproducing strain of Escherichia coli. The recombinant protein was purified to homogeneity by nickel chelating affinity chromatography, and its main kinetic and chemical physical parameters were determined. Northern blot and enzyme activity analyses indicated that draSO gene expression is constitutive in D. radiodurans and that there is no increase upon exposure to thiosulfate and/or molybdenum(II).     

A novel alpha-D-galactosynthase from Thermotoga maritima converts beta-D-galactopyranosyl azide to alpha-galacto-oligosaccharides

The large-scale production of oligosaccharides is a daunting task, hampering the study of the role of glycans in vivo and the testing of the efficacy of novel glycan-based drugs. Glycosynthases, mutated glycosidases that synthesize oligosaccharides in high yields, are becoming important chemo-enzymatic tools for the production of oligosaccharides. However, while β-glycosynthase can be produced with a rather well-established technology, examples of α-glycosynthases are thus far limited only to enzymes from glycoside hydrolase 29 (GH29), GH31 and GH95 families. α-L-Fucosynthases from GH29 use convenient glycosyl azide derivatives as a strategic alternative to glycosyl fluoride donors. However, the general applicability of this method to other α-glycosynthases is not trivial and remains to be confirmed. Here, β-D-galactopyranosyl azide was converted to α-galacto-oligosaccharides with good yields and high regioselectivity, catalyzed by a novel α-galactosynthase based on the GH36 α-galactosidase from the hyperthermophilic bacterium Thermotoga maritima. These results open a new avenue to the practical synthesis of biologically interesting α-galacto-oligosaccharides and demonstrate more widespread use of β-glycosyl-azide as donors, confirming their utility to expand the repertoire of glycosynthases.     

Selective inhibition of acylpeptide hydrolase in SAOS-2 osteosarcoma cells: is this enzyme a viable anticancer target?

Molecular Biology Reports - Serine hydrolases play crucial roles in many physiological and pathophysiological processes and a panel of these enzymes are targets of approved drugs. Despite this,...     

The extremo-α-carbonic anhydrase (CA) from Sulfurihydrogenibium azorense,the fastest CA known,is highly activated by amino acids and amines

The α-carbonic anhydrase (CA, EC 4.2.1.1) from the extremophilic bacterium Sulfurihydrogenibium azorense (SazCA) was recently shown to be the fastest CA known. Here we investigated this enzyme for its activation with a series of amino acids and amines. The best SazCA activators were d-Phe, l-DOPA, l- and d-Trp, dopamine and serotonin, which showed activation constants in the range of 3–23 nM. l- and d-His, l-Phe, l-Tyr, 2-pyridyl-methylamine and L-adrenaline were also effective activators (K_As in the range of 62–90 nM), whereas d-Dopa, d-Tyr and several heterocyclic amines showed activity in the micromolar range. The good thermal stability, robustness, very high catalytic activity and propensity to be activated by simple amino acids and amines, make SazCA a very interesting candidate for biomimetic CO₂ capture processes.     

A xylan-degrading strain of<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis>: isolation and characterization of the xylanase activity

Two strains (O and X₂) of the hyperthermophilic crenarchaeon Sulfolobus solfataricus strain MT4 were selected and isolated for their ability to grow on xylan. O and X₂, grown on media containing oat spelt xylan and birchwood xylan as the sole nutrient source, respectively, produced the same thermostable xylanase that was demonstrated to be inducible in xylan cultures. In an oat spelt medium, S. solfataricus O underwent interesting morphological changes in the cell envelope, exhibiting mobile appendages not present in the typical coccal shape. The enzyme was prevalently membrane associated and showed a molecular mass of approximately 57.0 kDa. It was also highly thermostable, with a half-life of 47 min at 100°C, and exhibited an optimal temperature and pH of 90°C and 7.0, respectively. Xylo-oligosaccharides were the enzymatic products of xylan hydrolysis, and the smallest degradation product was xylobiose, thus indicating that the enzyme was an endoxylanase. The enzyme was able to bind weakly to crystalline cellulose (Avicel) and more strongly to insoluble xylan in a substrate amount-and temperature-dependent manner.Communicated by G. Antranikian     

Biochemical characterization of a CDC6-like protein from the crenarchaeon Sulfolobus solfataricus

Cdc6 proteins play an essential role in the initiation of chromosomal DNA replication in Eukarya. Genes coding for putative homologs of Cdc6 have been also identified in the genomic sequence of Archaea, but the properties of the corresponding proteins have been poorly investigated so far. Herein, we report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. Gel filtration and glycerol gradient ultracentrifugation experiments indicated that this protein behaves as a monomer in solution (molecular mass of about 45 kDa). We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 --> Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function.