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M.antonietta Di Bella - One of the best experts on this subject based on the ideXlab platform.
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Retinyl ester Hydrolases in retinal pigment epithelium.
Archives of biochemistry and biophysics, 1991Co-Authors: Maria Concetta Gueli, Concetta M. A. Nicotra, Anna Maria Pintaudi, Alessandra Paganini, Leonardo Pandolfo, Giacomo De Leo, M.antonietta Di BellaAbstract:In bovine retinal pigment epithelium membranes we have found three Hydrolases which were active against trans-retinyl palmitate. This was possible by assaying different subcellular fractions as a function of pH in the range 3-9. Detection of these activities has been favored by the use in the enzyme assay of Triton X-100, which has an activating effect up to a concentration of 0.03% at a detergent-protein ratio of about 1.5-3.0. Apparent kinetic parameters for the retinyl ester Hydrolases have been determined after a study of the optimization of assay conditions. Vmax values for Hydrolases acting at pH 4.5, 6.0, and 7.0 were, respectively, 156, 55, and 70 nmol/h/mg. To identify the subcellular site for these hydrolytic activities, assays of marker enzymes from various organelles in each subcellular preparation were carried out, demonstrating the lysosomal origin of the pH 4.5 retinyl ester hydrolase and the microsomal origin of the pH 6.0 retinyl ester hydrolase and suggesting that the pH 7.0 retinyl ester hydrolase originates from the Golgi complex.
Gary W. Black - One of the best experts on this subject based on the ideXlab platform.
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Functional analysis of a group A streptococcal glycoside hydrolase Spy1600 from family 84 reveals it is a β-N-acetylglucosaminidase and not a hyaluronidase
Biochemical Journal, 2006Co-Authors: William L. Sheldon, Matthew S. Macauley, Charlotte E. Robinson, Simon J. Charnock, Edward J Taylor, Gideon J. Davies, David J Vocadlo, Gary W. BlackAbstract:Group A streptococcus (Streptococcus pyogenes) is the causative agent of severe invasive infections such as necrotizing fasciitis (the so-called ‘flesh eating disease’) and toxic-shock syndrome. Spy1600, a glycoside hydrolase from family 84 of the large superfamily of glycoside Hydrolases, has been proposed to be a virulence factor. In the present study we show that Spy1600 has no activity toward galactosaminides or hyaluronan, but does remove β-O-linked N-acetylglucosamine from mammalian glycoproteins – an observation consistent with the inclusion of eukaryotic O-glycoprotein 2-acetamido-2-deoxy-β-D-glucopyranosidases within glycoside hydrolase family 84. Proton NMR studies, structure–reactivity studies for a series of fluorinated analogues and analysis of 1,2-dideoxy-2′-methyl-α-D-glucopyranoso-[2,1-d]-Δ2′-thiazoline as a competitive inhibitor reveals that Spy1600 uses a double-displacement mechanism involving substrate-assisted catalysis. Family 84 glycoside Hydrolases are therefore comprised of both prokaryotic and eukaryotic β-N-acetylglucosaminidases using a conserved catalytic mechanism involving substrate-assisted catalysis. Since these enzymes do not have detectable hyaluronidase activity, many family 84 glycoside Hydrolases are most likely incorrectly annotated as hyaluronidases.
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Functional analysis of a group A streptococcal glycoside hydrolase Spy1600 from family 84 reveals it is a {beta}-N-acetylglucosaminidase and not a hyaluronidase
Biochemical Journal, 2006Co-Authors: William L. Sheldon, Matthew S. Macauley, Charlotte E. Robinson, Simon J. Charnock, Edward J Taylor, Gideon J. Davies, David J Vocadlo, Gary W. BlackAbstract:Group A streptococcus (Streptococcus pyogenes) is the causative agent of severe invasive infections such as necrotizing fasciitis (the so-called "flesh eating disease") and toxic shock syndrome. Spy1600, a glycoside hydrolase from family 84 of the large superfamily of glycoside Hydrolases, has been proposed to be a virulence factor. Here we show that Spy1600 has no activity toward galactosaminides or hyaluronan, but does remove {beta}-O-linked N-acetylglucosamine from mammalian glycoproteins; an observation consistent with the inclusion of eukaryotic O-GlcNAcases within Glycoside Hydrolase family 84. Proton NMR studies, structure-reactivity studies for a series of fluorinated analogues, and analysis of NAG-thiazoline as a competitive inhibitor reveals that Spy1600 uses a double-displacement mechanism involving substrate-assisted catalysis. Family 84 glycoside Hydrolases are therefore comprised of both prokaryotic and eukaryotic {beta}-N-acetylglucosaminidases using a conserved catalytic mechanism involving substrate-assisted catalysis. Since these enzymes do not have detectable hyaluronidase activity, many family 84 glycoside Hydrolases are most likely incorrectly annotated as hyaluronidases.
Alexander G. Mclennan - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning of diadenosine tetraphosphatase from pig small intestinal mucosa and identification of sequence blocks common to diadenosine polyphosphate Hydrolases and phosphorylases
The International Journal of Biochemistry & Cell Biology, 1997Co-Authors: S Hankin, Katrine A Wintero, Alexander G. MclennanAbstract:Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) pyrophosphohydrolase is the enzyme responsible for reducing intracellular levels of the stress-responsive nucleotide diadenosine 5',5"'-P1,P4-tetraphosphate. In order to gain more information on the relationships between the enzymes hydrolysing diadenosine polyphosphates in different eukaryotes, the Ap4A hydrolase and a corresponding cDNA have been isolated from pig small intestinal mucosa by standard procedures. The enzyme is a typical mammalian Ap4A hydrolase (Km = 0.8 microM) being sensitive to inhibition by fluoride (Ki = 24 microM) and adenosine 5'-tetraphosphate (Ki = 10 nM) and yielding ATP and AMP as products. A low Km Ap4A hydrolase (Km = 0.3 microM) was also isolated from rabbit small intestinal mucosa. These enzymes differ from the rat intestinal mucosal hydrolase, which has much higher values of Km for Ap4A and Ki for adenosine 5'-tetraphosphate. A cDNA encoding the pig enzyme was isolated from a pig ileum cDNA library. The derived amino acid sequence of the 16.8 kDa gene product shows 88% identity and 96% similarity to that of the human enzyme. The sequence has the same modification of the MutT motif found in the human enzyme in which a threonine residue replaces a hydrophobic amino acid. Sequences comparisons among eukaryotic diadenosine polyphosphate Hydrolases and phosphorylases reveal two blocks of amino acid similarity, including a motif, Z[AD]Gx[ED]AGQ, which may be involved in polyphosphate binding by the Hydrolases, and an invariant histidine residue that may be involved in catalysis. These sequence similarities may have arisen by convergent evolution.
Benjamin F Cravatt - One of the best experts on this subject based on the ideXlab platform.
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competitive activity based protein profiling identifies aza β lactams as a versatile chemotype for serine hydrolase inhibition
Journal of the American Chemical Society, 2012Co-Authors: A Zuhl, Justin T Mohr, Daniel A Bachovchin, Sherry Niessen, Jacob M Berlin, Maximilian Dochnahl, Maria P Lopezalberca, Gregory C Fu, Benjamin F CravattAbstract:Serine Hydrolases are one of the largest and most diverse enzyme classes in Nature. Most serine Hydrolases lack selective inhibitors, which are valuable probes for assigning functions to these enzymes. We recently discovered a set of aza-β-lactams (ABLs) that act as potent and selective inhibitors of the mammalian serine hydrolase protein-phosphatase methylesterase-1 (PME-1). The ABLs inactivate PME-1 by covalent acylation of the enzyme’s serine nucleophile, suggesting that they could offer a general scaffold for serine hydrolase inhibitor discovery. Here, we have tested this hypothesis by screening ABLs more broadly against cell and tissue proteomes by competitive activity-based protein profiling (ABPP), leading to the discovery of lead inhibitors for several serine Hydrolases, including the uncharacterized enzyme α,β-hydrolase domain-containing 10 (ABHD10). ABPP-guided medicinal chemistry yielded a compound ABL303 that potently (IC50 ≈ 30 nM) and selectively inactivated ABHD10 in vitro and in living cel...
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a functional proteomic strategy to discover inhibitors for uncharacterized Hydrolases
Journal of the American Chemical Society, 2007Co-Authors: Jacqueline L Blankman, Benjamin F CravattAbstract:Hydrolytic enzymes constitute one of the largest and most diverse protein classes in Nature and play key roles in nearly all physiological and pathological processes. The mammalian serine hydrolase superfamily contains a remarkable number of uncharacterized members, with at least 40−50% of these enzymes lacking experimentally verified endogenous substrates and products. Assignment of metabolic and cellular functions to these enzymes requires the development of pharmacological tools to selectively perturb their activity. We describe herein a functional proteomic strategy to systematically develop potent and selective inhibitors for uncharacterized serine Hydrolases and its application to the brain-enriched enzyme α/β-hydrolase-6. We anticipate that the methods described herein will facilitate the development of selective chemical probes to annotate the metabolic and (patho)physiological functions of many of the uncharacterized serine Hydrolases that currently populate eukaryotic and prokaryotic proteomes.
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molecular characterization of an enzyme that degrades neuromodulatory fatty acid amides
Nature, 1996Co-Authors: Benjamin F Cravatt, Stephen P Mayfield, Dale L Boger, Dan K Giang, Richard A. Lerner, Norton B GilulaAbstract:ENDOGENOUS neuromodulatory molecules are commonly coupled to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolysed by acetylcholine esterase1 and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases2. Previously, we reported the structure and sleep-inducing properties of cis-9-octadecenamide, a lipid isolated from the cerebrospinal fluid of sleep-deprived cats3, cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems4 and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid3. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase5, from rat liver plasma mem-branes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor6, to arachidonic acid, indi-cating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling molecules, the fatty-acid amides6–8. Therefore we will hereafter refer to oleamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates.
Joseph L. Napoli - One of the best experts on this subject based on the ideXlab platform.
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[52] Bile salt-independent retinyl ester hydrolase activities associated with membranes of rat tissues
Methods in Enzymology, 1990Co-Authors: Earl H. Harrison, Joseph L. NapoliAbstract:Publisher Summary Recently, bile salt-independent retinyl ester Hydrolases has been detected in rat liver, kidney, lung, and testes. This chapter describes the properties of these bile salt-independent retinyl ester Hydrolases in liver and kidney—the two most studied tissues. The chapter presents information on the properties of the bile salt-independent retinyl ester hydrolase (REH) activity of rat liver. In particular, three areas are discussed that serve to distinguish the bile salt-independent hydrolase(s) from the bile salt-dependent enzyme(s). The cholate-dependent retinyl ester hydrolase activity in rat liver coelutes with cholesterol esterase and triacylglycerol lipase activities through at least three columns that separate by charge or hydrophobicity. The partially purified lipase(s) has higher specific activities assayed with cholesterol oleate and triolein than when assayed with retinyl palmitate. Fast protein liquid chromatography (FPLC) is useful for purification and resolution of retinyl ester hydrolase activity from other lipases.
