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Human liver slices: An in vitro system for determination of N-acetylation and acetylator statusGunawardhana, Lhanoo, 1959- January 1989 (has links)
An in vitro system has been developed to study N-acetyltransferase (NAT) activity using human liver slices in dynamic organ culture. Acetylation of para-aminobenzoic acid (PABA) and sulfamethazine (SMZ) in the presence of human liver slices was monitored by measuring the disappearance of the parent amine from the incubation medium using the colorimetric procedure of Bratton & Marshall. Presence of the acetyl conjugate was confirmed using HPLC. PABA acetylation rates varied from 0.72-2.52 nmoles/hr/mg protein (n = 8). This small variation (4 fold) is consistent with the classification of PABA as a monomorphic substrate. The variation in the rate of SMZ acetylation was greater than 20 fold (0.144-3.68 nmoles/hr/mg protein; n = 9). This larger variation is characteristic of SMZ as a polymorphic substrate. The results obtained indicate that human liver slices in dynamic organ culture can be used for the determination of hepatic NAT activity and acetylator status of individual human livers.
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Functions of the unique N-terminus of a GCN5 histone acetylase in toxoplasma gondii /Bhatti, Micah M. January 2007 (has links)
Thesis (M.A.)--Indiana University, 2007. / Title from screen (viewed on May 23, 2007) Department of Pharmacology & Toxicology, Indiana University-Purdue University Indianapolis (IUPUI) Includes vita. Includes bibliographical references (leaves 204-226)
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Towards the Characterization of Enzymes Involved in the Metabolism of Tyrosine and Tyrosine DerivativesMehere, Prajwalini V. 30 December 2010 (has links)
Tyrosine is involved in many biological processes including protein synthesis. This dissertation is focused on two different aspects: tyrosine catabolism and tyrosine derivative metabolism. Tyrosine undergoes degradation via tyrosine aminotransferase (TAT). Deficiency of TAT leads to some disease conditions or tyrosinemia type II. TAT has been characterized in several species, including humans. Mouse tyrosine aminotransferase was used as a model protein for the tyrosine catabolism portion of this study. Characterization of TAT included its expression in a bacterial expression system, purification using various chromatographic techniques, crystallization under different conditions, and its kinetic analysis, and molecular dynamics simulations. Based on sequence, structure, and kinetic data we have shown that mouse TAT behaves like human TAT. Our crystallization studies added new insights into the mechanism of TAT by shedding light on involvement of a disulfide bond in the regulation of mTAT. Molecular dynamics analysis provided perspective on the differences (preferences) in the substrate specificities of mouse and Trypanosome cruzi TAT.
Tyrosine is a precursor of several key neurotransmitters. These neurotransmitters must be regulated in order to function properly. The hypothetical N-acetyltransferases from Aedes aegypti were used as model proteins for investigation of tyrosine derivative metabolism. We found nine potential arylalkylamine N-acetyltransferase (AANAT) genes in Ae. aegypti. Phylogenetic analysis suggests that these Ae. aegypti AANATs (AeAANATs) can be further divided into three clusters. Phylogenetic analysis suggests that insect AANATs may have different functions as compared with the mammalian AANATs, for which function is specific to circadian rhythm regulation. PCR amplification indicates that eight of the nine putative AeAANATs are expressed in the mosquito. Expression of the eight putative AeAANATs and substrate screening of their recombinant proteins against dopamine, octopamine, tyramine, epinephrine, tryptamine, 5-hydroxytryptamine, and methoxytryptamine established that five of the eight putative AeAANATs are true AANATs.
The discontinuous expression profiles of AeAANAT genes were studied in detail. Six of the AeAANATs were expressed in the head before and after blood feeding, suggesting their potential role in neurotransmission inactivation. Down-regulation of these genes after blood feeding suggests that blood feeding or factors related to blood feeding impact on the regulation of these genes. Kinetic studies determined that two AeAANAT proteins are highly efficient in mediating the acetylation of dopamine and 5-hydroxytryptamine. Substrate analysis of AeAANATs supports the notion that acetylation of arylalkylamines is vital to the biology of mosquito species, and that these genes emerged in response to specific pressures related to necessities for biogenic amine acetylation. / Ph. D.
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Mapping the genome and characterization of an acetyltransferase of Trypanosoma cruzi /Ochaya, Stephen O. January 2006 (has links)
Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 2 uppsatser.
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Isolation, partial purification and characterization of an N-acetyltransferase from Streptomyces akiyoshiensis L-138Rodriguez-Juarez, Rocio C., University of Lethbridge. Faculty of Arts and Science January 2003 (has links)
A novel N-acetyltranferase (NAT) with high specificity for L-dopa was partially purified and characterized during this study. Streptomyces akiyoshiensis NAT was isolated from liquid cultures and the cell free extract was partially purified via two chromatographic approaches. The purest enzyme preparation (354.4-fold) was obtained through a combination of affinity (Affi-gel blue) and gel filtration chromatography. This preparation could not be stored due to poor stability. An alternate approach using affinity and anion-exchange column chromatogrpahy (macro-prep DEAE) provided a 125-fold purification. A second macro-prep DEAE showed the enrichment of a band at around 14.4 kDa. The enzyme activity was strongly inhibited by the presence of Cu2+ and Hg2+ salts suggesting the presence of critical histidine or cysteine redidues. In addition, NaC1 plays an important role as a stabilizing agent and/or slight activator of this enzyme. The Km and Vmax values for L-dopa determined at 30degreesC were 5.39 x 10-2 mM and 2.19 x 10-5 mM's, respectively. At 37 degreesC these kinetic constants were 0.11 mM and 2.57 x 10-4 mM's, respectively. The Km and Vmax values for AcCoA determined at 30 degreesC were 0.42 mM and 4.32 x 10-4 mM's, respectively. At 37 degreesC these values were 0.58 mM and 1.15 x 10-4 mM's, respectively. The optimal temperature and pH were 43 degreesC and 8.0 respectively. S. akiyoshiensis NAT acetylated arylalkylamine substrates but not arylamine substrates to any significant extent. These findings suggest that this enzyme but not arylamine subsrates to any significant extent. These findings suggest that this enzyme may be closely related to arylalkylamine-N-acetyltranferases (AANATs), however the certification of this postulate requires the knowledge of the amino acid sequence and ultimately 3D structure of S. akiyoshiensis NAT. / xvii, 128 leaves : ill. ; 28 cm.
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Protein-ligand interactions of arylamine N-acetyltransferase from Mycobacterium smegmatisBrooke, Edward W. January 2003 (has links)
Tuberculosis is the world's largest cause of death from an infectious agent. Treatment is by an extended period of combination chemotherapy. Drug resistance is an increasing problem in tuberculosis therapy, particularly to the frontline anti-tubercular drug isoniazid (INH). Recombinant arylamine N-acetyltransferase (NAT) of Mycobacterium tuberculosis N-acetylates INH using the cofactor Acetyl Coenzyme A. NAT from M. tuberculosis is a polymorphic enzyme and also acetylates INH in vivo. Acetylated INH is inactive therapeutically against M. tuberculosis both in vivo and in vitro. The acetylation of isoniazid in the mycobacterial cell may compete with the activation of INH by the catalase-peroxidase, katG, and hence contribute to INH resistance in clinical isolates. Inhibition of NAT in M. tuberculosis may thus increase the efficacy of INH therapy. A novel assay based around the detection of free Coenzyme A released during the acetylation reaction was used to determine the substrate specificity of recombinant NAT from the related Mycobacterium M. smegmatis (MSNAT). A relationship was observed between the lipophilicity of simple arylamine substrates and the rate of acetylation by MSNAT. Several MSNAT substrates possess antibacterial activity. The assay could also be used to screen compound libraries for MSNAT inhibitors. Synthesis of seventeen thiazolidinedione sultams in collaboration with Dr.Vickers (Dyson Perrins), identified as weak inhibitors of MSNAT, gave a minimum competitive inhibitory constant of 14μM. Screening a library of 5,074 drug-like compounds for inhibition of MSNAT identified thirteen compounds with semi-maximal inhibition constants (IC<sub>50</sub>) of below 10μM. Based on this, fifteen maleimides were synthesised and were irreversible inhibitors of MSNAT with submicromolar potency. Similarly, ninety-six aminothiazoles were synthesised by Dr. Vickers and were uncompetitive inhibitors of MSNAT with a minimum IC<sub>50</sub> of 1.5μM. The most potent aminothiazole showed no effect on the growth of M. smegmatis or M. bovis BCG or the sensitivity of the bacteria to isoniazid. However the aminothiazoles were shown not to penetrate the cells.
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Functional characterization of the regulation of transcription factor MEF2C by histone acetyltransferase p300 and histone deacetylase 4 /Chan, Jonathan Ka Lok. January 2004 (has links)
Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 135-159). Also available in electronic version. Access restricted to campus users.
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Characterization of the chromosomal aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium /Draker, Kari-Ann. Wright, Gerard D. January 1900 (has links)
Thesis (Ph.D.)--McMaster University, 2004. / Advisor: G.D. Wright. Includes bibliographical references. Also available via World Wide Web.
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Investigating the endogenous role of human N-acetyltransferase 1, as potential breast cancer biomarker, using chemical biologyLaurieri, Nicola January 2012 (has links)
No description available.
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The regulation of Serotonin N-acetyltransferase in the rat pineal glandOlivieri, Gianfranco January 1993 (has links)
The synthesis of the pineal hormone, melatonin, is finely regulated by the pineal enzyme serotonin N-acetyltransferase (NAT). In the absence of light, the activity of NAT is markedly enhanced by the release of nor-adrenaline from sympathetic nerve endings in the pineal. Exposure of animals to light during darkness causes a sudden and dramatic reduction in the activity of NAT. The present study investigated a possible mechanism for this sudden decline in NAT activity. These investigations included the determination of the effects of S-adenosylmethionine (SAM), adenosine nucleotides and calcium on NAT activity. In vitro experiments using SAM showed that pineals pre-incubated with SAM prior to adrenergic stimulation did not significantly alter NAT activity or pineal indoleamine metabolism. However, measurement of pineal cyclic AMP showed that SAM exposure reduced the adrenergic-induced rise in pineal cyclic AMP. Experiments using adenosine 5'-monophosphate (5'-AMP) showed that this nucleotide enhanced both dark- and isoproterenol-induced NAT activity. Adenosine 5'-triphosphate (A TP), on the other hand, reduced NAT activity with a concomitant reduction in pineal indoleamine metabolism. Exposure of isoproterenol-stimulated pineals in organ culture to propranolol resulted in a marked rise in ATP and adenosine 5'-diphosphate (ADP) synthesis accompanied by a decline in 5'-AMP levels as compared with pineals treated with isoproterenol alone. This then implies that exposure of animals to light could cause a change in pineal nucleotide levels. Since nucleotide levels are also controlled by calcium, experiments were carried out to determine the effect of calcium on pineal NAT activity. These experiments showed that ethyleneglycol-bis-N,N,N,N,-tetraacetic acid (EGTA) enhanced NAT activity whilst calcium reduced the activity in pineal homogenates, implying that calcium may act directly on NAT to regulate its activity. Exposure of pineal glands in organ culture to the calmodulin antagonist R24571 caused a rise in pineal cyclic AMP levels with a concomitant decrease in cAMP-phosphodiesterase activity. This was, however, accompanied by a decline in Nacetyl serotonin and melatonin synthesis. These findings implicate a number of factors in the regulation of pineal NAT activity. A mechanism for the regulation of pineal NAT is proposed.
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