Show simple item record

dc.contributor.advisorAposhian, H. Vaskenen_US
dc.contributor.authorWildfang, Eric Konrad
dc.creatorWildfang, Eric Konraden_US
dc.date.accessioned2013-04-11T08:35:53Z
dc.date.available2013-04-11T08:35:53Z
dc.date.issued2001en_US
dc.identifier.urihttp://hdl.handle.net/10150/279851
dc.description.abstractInorganic arsenic is an important environmental toxicant of both natural and anthropogenic sources. It is a human carcinogen for which appropriate animal models of most arsenic-induced cancers are lacking. Presently, 17 species of non-human primates were screened using an in vitro assay to determine their arsenic methylation ability as a predictive tool for better understanding the presence, and in some instances, deficiency of arsenic methyltransferase activity among animal species. Four of the 17 species investigated had arsenite methyltransferase activity, three of which were from the genus Macaca. This suggested a phylogenetic component to an animal having arsenic methyltransferase activity. That all of the hepatic tissues were viable was demonstrated by their all having arsenate reductase activity. These data suggested that arsenic methylation cannot be a detoxification mechanism for many non-human primates and that alternative methods of detoxifying inorganic arsenic must be considered and investigated. Biomethylation of inorganic arsenic has classically been considered its primary mechanism of detoxification, although results of recent investigations disagree. As arsenic methylation may represent a bioactivation process leading to cancer development, it now becomes critical to identify the methyltransferase protein, its endogenous function, as well as additional substrates and inhibitory molecules. Using a combination of chromatographic and isoelectric focusing protein separation methods, rabbit liver arsenite and methylarsonic acid methyltransferase activities were purified 19,000- and 604-fold, respectively. These activities co-purified during all steps. The arsenic methyltransferase protein had a molecular mass of approximately 46.5 kDa and a pl of 5.7 to 6.0 and is inhibited by Sadenosyl-L-homocysteine, sinefungin and trivalent antimony. The final purified fraction contained a heterogeneous protein mixture as assessed by SDS-PAGE analysis. Proteins in this fraction were identified using tandem and matrix-assisted laser desorption/ionization mass spectrometry techniques against databases of known proteins. Identified protein sequences were searched for putative S-adenosylmethionine binding motifs conserved among small molecule methyltransferases. Homologs of the identified proteins were obtained commercially, where available, and screened for arsenite methyltransferase activity. The arsenic methyltransferase was not identified among the obtained homologs and cloning efforts must be considered to screen the other proteins identified in the final purified fraction for arsenic methyltransferase functionality.
dc.language.isoen_USen_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.subjectHealth Sciences, Toxicology.en_US
dc.subjectChemistry, Biochemistry.en_US
dc.titlePurification, sequencing and characterization of rabbit liver arsenite and methylarsonic acid methyltransferaseen_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
thesis.degree.grantorUniversity of Arizonaen_US
thesis.degree.leveldoctoralen_US
dc.identifier.proquest3031355en_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplinePharmacology and Toxicologyen_US
thesis.degree.namePh.D.en_US
dc.identifier.bibrecord.b42283292en_US
refterms.dateFOA2018-06-16T16:04:51Z
html.description.abstractInorganic arsenic is an important environmental toxicant of both natural and anthropogenic sources. It is a human carcinogen for which appropriate animal models of most arsenic-induced cancers are lacking. Presently, 17 species of non-human primates were screened using an in vitro assay to determine their arsenic methylation ability as a predictive tool for better understanding the presence, and in some instances, deficiency of arsenic methyltransferase activity among animal species. Four of the 17 species investigated had arsenite methyltransferase activity, three of which were from the genus Macaca. This suggested a phylogenetic component to an animal having arsenic methyltransferase activity. That all of the hepatic tissues were viable was demonstrated by their all having arsenate reductase activity. These data suggested that arsenic methylation cannot be a detoxification mechanism for many non-human primates and that alternative methods of detoxifying inorganic arsenic must be considered and investigated. Biomethylation of inorganic arsenic has classically been considered its primary mechanism of detoxification, although results of recent investigations disagree. As arsenic methylation may represent a bioactivation process leading to cancer development, it now becomes critical to identify the methyltransferase protein, its endogenous function, as well as additional substrates and inhibitory molecules. Using a combination of chromatographic and isoelectric focusing protein separation methods, rabbit liver arsenite and methylarsonic acid methyltransferase activities were purified 19,000- and 604-fold, respectively. These activities co-purified during all steps. The arsenic methyltransferase protein had a molecular mass of approximately 46.5 kDa and a pl of 5.7 to 6.0 and is inhibited by Sadenosyl-L-homocysteine, sinefungin and trivalent antimony. The final purified fraction contained a heterogeneous protein mixture as assessed by SDS-PAGE analysis. Proteins in this fraction were identified using tandem and matrix-assisted laser desorption/ionization mass spectrometry techniques against databases of known proteins. Identified protein sequences were searched for putative S-adenosylmethionine binding motifs conserved among small molecule methyltransferases. Homologs of the identified proteins were obtained commercially, where available, and screened for arsenite methyltransferase activity. The arsenic methyltransferase was not identified among the obtained homologs and cloning efforts must be considered to screen the other proteins identified in the final purified fraction for arsenic methyltransferase functionality.


Files in this item

Thumbnail
Name:
azu_td_3031355_sip1_m.pdf
Size:
2.909Mb
Format:
PDF

This item appears in the following Collection(s)

Show simple item record