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dc.contributor.authorBlythe, Treena Jayne
dc.contributor.authorBlythe, Treena Jayne
dc.date.accessioned2011-08-01T02:32:37Z
dc.date.available2011-08-01T02:32:37Z
dc.date.issued1997
dc.identifier.urihttp://hdl.handle.net/10179/2574
dc.description.abstractThe primary biological role of the aldehyde dehydrogenase enzymes has long been a contentious issue. It was initially thought that the main function of these enzymes could be acetaldehyde metabolism; however, it seems unlikely that a large family of proteins evolved for this purpose. It has been suggested that an important function of aldehyde dehydrogenase enzymes may be in the metabolism of the vitamin A derivative, retinal. This thesis describes an investigation into the ability of human and sheep cytosolic aldehyde dehydrogenases to oxidise all-trans retinal, 9-cis retinal and CRBP-bound retinal under physiologically relevant conditions. A fluorescence-based assay following the production of NADH was employed, allowing the accurate measurement of low K m data. Firstly the ability of A1DH 1 to oxidise its putative biological ligands, free all-trans and 9-cis retinal, was demonstrated. It has been proposed that retinoids occur naturally as a 1:1 complex with the lipocalins cellular retinol binding protein (CRBP) and cellular retinoic acid binding protein (CRABP). If the sheep and human class 1 enzymes play a role in retinoid metabolism in vivo, it is likely that they will accept CRBP-bound retinal as a substrate. To investigate this possibility, recombinant CRBP was produced using an E.coli expression system. Using a spectrophotometric method, the purified recombinant CRBP was shown to bind all-trans but not 9-cis retinal, and using the same fluorescence-based assay as mentioned above, it was shown that both sheep and human A1DH 1 could accept CRBP-bound retinal as a substrate at physiologically relevant levels. In vivo studies into retinal oxidation were initiated using the retinoid-responsive human neuroblastoma cell-line SH-SY5Y. It was shown that A1DH 1 was expressed in this cell line by Western blotting, and that the cells were responsive to retinal in addition to retinoic acid, indicating that retinal was being converted to retinoic acid. In addition, a novel, putative alcohol dehydrogenase was isolated, purified and partially characterised. The protein was purified using the techniques of subcellular fractionation by centrifugation, PEG precipitation, ion-exchange chromatography, preparative isoelectric focusing, hydrophobic interaction chromatography and gel purification. Elucidated characteristics of this protein include: subunit molecular weight 42-45 kDa, native molecular weight 42-45 kDa, isoelectric point 8.3-8.5, and activity with ethanol and other longer chain alcohols, but not with glucose, sorbitol or methanol. The protein was blocked at the N-terminus, and cleavage and internal sequencing attempts yielded some sequence information. However, this information did not appear to match closely with any known protein sequence when submitted to a protein database, suggesting that the protein is novel. From all available information, we propose that in sheep and humans, the enzyme responsible for retinal oxidation is the major cytosolic class 1 aldehyde dehydrogenase, as opposed to the situation in rats and mice, where specific retinal-oxidising aldehyde dehydrogenases exist and the major class 1 enzymes play a more important role in acetaldehyde metabolism.en_US
dc.publisherMassey Universityen_US
dc.rightsThe Authoren_US
dc.subjectAldehyde dehydrogenaseen_US
dc.subjectEnzymesen_US
dc.titleStudies on proteins involved in retinoid and alcohol metabolism : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey Universityen_US
dc.typeThesisen_US
thesis.degree.disciplineBiochemistry
thesis.degree.grantorMassey University
thesis.degree.levelDoctoral
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophy (Ph.D.)


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