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Biochemical characterisation of dihydrolipoamide dehydrogenase from potato.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Biochemical characterisation of dihydrolipoamide dehydrogenase from potato./
作者:	Fullerton, Ruth Elisabeth.
面頁冊數:	1 online resource (195 pages)
附註:	Source: Dissertations Abstracts International, Volume: 80-12, Section: C.
Contained By:	Dissertations Abstracts International80-12C.
標題:	Plant sciences. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=10391317click for full text (PQDT)
ISBN:	9781369585438

Biochemical characterisation of dihydrolipoamide dehydrogenase from potato.
Fullerton, Ruth Elisabeth.

Biochemical characterisation of dihydrolipoamide dehydrogenase from potato. - 1 online resource (195 pages)

Source: Dissertations Abstracts International, Volume: 80-12, Section: C.

Thesis (Ph.D.)--University of Glasgow (United Kingdom), 1995.

Includes bibliographical references

Dihydrolipoamide dehydrogenase (E3), a member of the group of Flavin-containing pyridine nucleotide-disulphide oxidoreductases, exists as a homodimer containing 1 Flavin adenine dinucleotide (FAD) per subunit and a redox-active disulphide. Dihydrolipoamide dehydrogenase is also an integral component of the 2-oxoacid dehydrogenase complexes and the glycine decarboxylase complex, and is generally believed to be the identical gene product in these multi-enzyme complexes to those from mammalian sources. However, there is increasing evidence for the existence of isoforms of E3 from mammalian and bacterial sources. Preliminary immunological analyses of chloroplasts and mitochondria from pea provided the first evidence for the existence of organelle specific isoforms of E3 in plants, a novel affinity column developed specifically for the one-step purification of E3 from plant sources was successfully used in the purification of E3 from potato tuber mitochondria. Neither pea chloroplastic or mitochondrial E3 would bind effectively to the affinity matrix, but, however, their differing affinities for the column matrix did provide supplementary evidence for the existence of organelle specific forms of E3 from pea. Both potato and pea E3 activities also varied in their sensitivity to salt further confirming the presence of E3 organelle specific isoforms being present in plants. The affinity purified potato tuber mitochondrial E3 was observed to be composed of two distinct polypeptides α and β with Mr values of 58,000 and 56,000 respectively, which both cross-reacted strongly with anti-E3 IgG raised to the porcine heart enzyme. Further analysis of this E3 involving the use of anion exchange chromatography revealed the presence of three discrete peaks of E3 activity. On SDS-PAGE peak 1 was observed to contain a single band with an Mr value of 58,000, peak 3 a band with an Mr of 56,000, and peak 2 by a combination of the two. All three peaks were observed to cross-react strongly with E3 antiserum, and peaks 1 and 3 to have identical N-terminal sequences. Mr analysis of native E3 preparations on gel filtration were consistent with the presence of a dimeric organisation for these enzymes. Thus, peak 1 and peak 3 appear to be α2 and β2 homodimers respectively, and peak 2 an αβ heterodimer. All three isoforms behave as conventional dihydrolipoamide dehydrogenases containing a redox-active disulphide at their active sites, with basic kinetic characterisation providing evidence that they vary in their enzymatic properties, lending weight to the possible conclusion that they interact selectively or exclusively with individual mitochondrial complexes. The three isoforms were also observed to have differing affinities for the inner mitochondrial membrane, providing indirect evidence that the three isoforms appear to be selective. Tissue specific expression of the three isoforms was also observed, with the α2 isoform observed to be the predominant E3 isoform in potato leaves. Reconstitution experiments were performed employing bovine heart PDC and OGDC due to the inherent problems involved in the purification of intact multi-enzyme complexes from plants, which ideally would have been more suitable for this purpose. All three potato E3 isoforms were observed to recognise and bind to the E2/X-PDC and E1/E2-OGDC sub complexes but with lower affinity than their mammalian counterparts. The three potato isoforms were found to reconstitute approx. 6% of overall PDC activity compared to 35% of reconstituted activity by parent bovine heart E3, but they failed to reconstitute OGDC activity. No direct comparisons could be made between the three isoforms due to the low levels of PDC reconstitution achieved by the plant enzymes. It is thought that the three peaks of E3 activity present in potato mitochondria may represent complex specific isoforms which may function preferentially or exclusively with one of the three multi-enzyme complexes located in plant mitochondria (PDC, OGDC and GDC). To date, it is unknown whether this is a unique situation that occurs only in potato.

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2023

Mode of access: World Wide Web

ISBN: 9781369585438Subjects--Topical Terms:

3173832
Plant sciences.
Subjects--Index Terms:

DehydrogenasesIndex Terms--Genre/Form:

542853
Electronic books.

Biochemical characterisation of dihydrolipoamide dehydrogenase from potato.
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245 1 0 $a Biochemical characterisation of dihydrolipoamide dehydrogenase from potato.
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500 $a Source: Dissertations Abstracts International, Volume: 80-12, Section: C.
500 $a Publisher info.: Dissertation/Thesis.
500 $a Advisor: Lindsay, J. G.
502 $a Thesis (Ph.D.)--University of Glasgow (United Kingdom), 1995.
504 $a Includes bibliographical references
520 $a Dihydrolipoamide dehydrogenase (E3), a member of the group of Flavin-containing pyridine nucleotide-disulphide oxidoreductases, exists as a homodimer containing 1 Flavin adenine dinucleotide (FAD) per subunit and a redox-active disulphide. Dihydrolipoamide dehydrogenase is also an integral component of the 2-oxoacid dehydrogenase complexes and the glycine decarboxylase complex, and is generally believed to be the identical gene product in these multi-enzyme complexes to those from mammalian sources. However, there is increasing evidence for the existence of isoforms of E3 from mammalian and bacterial sources. Preliminary immunological analyses of chloroplasts and mitochondria from pea provided the first evidence for the existence of organelle specific isoforms of E3 in plants, a novel affinity column developed specifically for the one-step purification of E3 from plant sources was successfully used in the purification of E3 from potato tuber mitochondria. Neither pea chloroplastic or mitochondrial E3 would bind effectively to the affinity matrix, but, however, their differing affinities for the column matrix did provide supplementary evidence for the existence of organelle specific forms of E3 from pea. Both potato and pea E3 activities also varied in their sensitivity to salt further confirming the presence of E3 organelle specific isoforms being present in plants. The affinity purified potato tuber mitochondrial E3 was observed to be composed of two distinct polypeptides α and β with Mr values of 58,000 and 56,000 respectively, which both cross-reacted strongly with anti-E3 IgG raised to the porcine heart enzyme. Further analysis of this E3 involving the use of anion exchange chromatography revealed the presence of three discrete peaks of E3 activity. On SDS-PAGE peak 1 was observed to contain a single band with an Mr value of 58,000, peak 3 a band with an Mr of 56,000, and peak 2 by a combination of the two. All three peaks were observed to cross-react strongly with E3 antiserum, and peaks 1 and 3 to have identical N-terminal sequences. Mr analysis of native E3 preparations on gel filtration were consistent with the presence of a dimeric organisation for these enzymes. Thus, peak 1 and peak 3 appear to be α2 and β2 homodimers respectively, and peak 2 an αβ heterodimer. All three isoforms behave as conventional dihydrolipoamide dehydrogenases containing a redox-active disulphide at their active sites, with basic kinetic characterisation providing evidence that they vary in their enzymatic properties, lending weight to the possible conclusion that they interact selectively or exclusively with individual mitochondrial complexes. The three isoforms were also observed to have differing affinities for the inner mitochondrial membrane, providing indirect evidence that the three isoforms appear to be selective. Tissue specific expression of the three isoforms was also observed, with the α2 isoform observed to be the predominant E3 isoform in potato leaves. Reconstitution experiments were performed employing bovine heart PDC and OGDC due to the inherent problems involved in the purification of intact multi-enzyme complexes from plants, which ideally would have been more suitable for this purpose. All three potato E3 isoforms were observed to recognise and bind to the E2/X-PDC and E1/E2-OGDC sub complexes but with lower affinity than their mammalian counterparts. The three potato isoforms were found to reconstitute approx. 6% of overall PDC activity compared to 35% of reconstituted activity by parent bovine heart E3, but they failed to reconstitute OGDC activity. No direct comparisons could be made between the three isoforms due to the low levels of PDC reconstitution achieved by the plant enzymes. It is thought that the three peaks of E3 activity present in potato mitochondria may represent complex specific isoforms which may function preferentially or exclusively with one of the three multi-enzyme complexes located in plant mitochondria (PDC, OGDC and GDC). To date, it is unknown whether this is a unique situation that occurs only in potato.
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538 $a Mode of access: World Wide Web
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650 4 $a Adenosine. $3 1362513
650 4 $a Plant mitochondria. $3 1004384
650 4 $a Vitamin B. $3 3561720
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650 4 $a Immunology. $3 611031
650 4 $a Bacteria. $3 550366
650 4 $a Symmetry. $3 536815
650 4 $a Urine. $3 3681463
650 4 $a Amino acids. $3 558768
650 4 $a Mitochondria. $3 592563
650 4 $a E coli. $3 3556834
650 4 $a Metabolism. $3 541349
650 4 $a Chromatography. $3 1073639
650 4 $a Dehydrogenases. $3 3563094
650 4 $a Alzheimers disease. $3 3560855
650 4 $a Proteins. $3 558769
650 4 $a Velocity. $3 3560495
650 4 $a Mammals. $3 531989
650 4 $a Molecular weight. $3 3683783
650 4 $a Heart. $3 578987
650 4 $a Polyethylene glycol. $3 3686750
650 4 $a Enzymes. $3 520899
650 4 $a Metabolites. $3 683644
650 4 $a Polypeptides. $3 1085124
653 $a Dehydrogenases
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856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=10391317 $z click for full text (PQDT)

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