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Control of isocitrate dehydrogenase in Escherichia coli strain ML308
Başlık:
Control of isocitrate dehydrogenase in Escherichia coli strain ML308
Yazar:
Borthwick, Andrew Cullen, author.
ISBN:
9780438054295
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (293 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Advisors: H. G. Nimmo.
Özet:
In most bacteria there is only one form of isocitrate dehydrogenase and in almost all cases it is dependent on the coenzyme NAIF for activity. The level of the activity of NADP-dependent isocitrate dehydrogenase (ICEH) that is present in Escherichia coli ML308 decreases when the organism adapts to growth on acetate as a sole source of carbon and energy. In these conditions the operation of the 'glyoxylate bypass' is essential to provide sufficient intermediates for biosynthesis. To allow the glyoxylate bypass enzyme isocitrate lyase to compete with ICDH for isocitrate the ICDH activity is lowered by covalent attachment of a phosphate group to each enzyme subunit. In this study two forms of ICEH have been isolated and characterised from E.coli ML308. An active form was purified from cells grown on glycerol using ammonium sulphate fractionation, ion exchange and also dye ligand chromatography. Using the same procedure a totally inactive form of ICEH, which is only present when the organism is adapted to growth on acetate, was separated from the active form during the dye ligand chromatography step. After a second ion exchange step and chromatofocusing the inactive form of ICEH was judged homogeneous by the criterion of poly aery lajnide gel electrophoresis. The physical and chemical characterisation of the two forms of ICEH indicates that they both exist as dimers of identical subunits with a subunit molecular weight of 45,000; both forms give the same 1 and 2-dimensional peptide "maps" as well as the same amino acid composition. However the specific activity of the inactive form is less than 1 unit/mg compared to 210 units/mg for the active form. The inactive form is also more mobile in conditions of non-denaturing polyacrylamide gel electrophoresis and it contains approximately 1 molecule of phosphate/subunit as compared to no phosphate/subunit in the active form. The analysis by non-denaturing gel electrophoresis of the inactivation and activation of ICEH catalysed vitro by ICDH kinase/phosphatase indicates clearly the interconversion of the two forms of ICEH tailing place. Furthermore when the inactivation/ phosphorylation of ICEH catalysed by ICEH kinase was carried out in the presence of (gamma-32P) ATP the decrease in the activity correlates with an increase in the phosphorylation of ICEH. Acid hydrolysis of this phosphorylated ICEH indicates that only phosphoserine is present. When the enzyme is digested with chymotrypsin a single major phosphorylated species is obtained in 2-dimensional ''mapping" of the peptides. This peptide is isolated by reverse phase high pressure liquid chromatography. The isolated phosphopeptide contains 22 amino acids and the sequence of the first residues is found to be, Thr-Thr-Pro-Val- Gly-Gly-Gly-Ile-Arg-(P-Ser)-Leu-Asn-Val-Ala. This sequence is the first example of a bacterial enzyme phosphorylation site to have been sequenced. Isolation of the dji vivo phosphorylated ICEH indicates that the same peptide is phosphorylated as in the in vitro labelled enzyme. An attempt is also made to qualititate directly the change in the level of ICEH activity relative to the change in the phosphorylation state of the enzyme is discussed. Finally the reasons for the evolution of such a powerful control mechanism in bacteria are discussed.
Notlar:
School code: 0547
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(684492.1) | 684492-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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