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Studies on the submitochondrial distribution and membrane topology of carnitine palmitoyltransferases
Başlık:
Studies on the submitochondrial distribution and membrane topology of carnitine palmitoyltransferases
Yazar:
Fraser, Fiona, author.
ISBN:
9780438057135
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (216 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Advisors: Victor Zammit.
Özet:
Liver mitochondrial overt carnitine palmitoyltransferase (L-CPT I) is an integral membrane protein. It has been suggested that the active site and malonyl-CoA binding site of the enzyme are on either side of the membrane. This hypothesis emerged primarily from data utilising proteolytic treatment of intact mitochondria. However, there are limitations in this technique and therefore this hypothesis is equivocal. It was therefore the aim of this thesis to resolve the topology of CPT I within the outer membrane. The work presented here establishes that CPT I is a polytopic protein with both its N- and C-terminus located in the cytosol and only a stretch of 27 amino acid residues resides within the inter-membrane space. In addition determinants for activity and sensitivity to inhibition by malonyl-CoA were also located cytosolically. This deduced topology was determined using a number of approaches. Intact mitochondria, in which only the cytosolic face of the outer membrane was exposed, or outer membrane ruptured mitochondria, in which both sides of the membrane were exposed, were treated with various proteases. The fragments of CPT I generated on proteolysis were identified using anti-peptide antibodies raised against synthetic peptides of CPT I and correlated with the changes in enzyme kinetics. The anti-peptide antibodies were also utilised in back titration experiments after incubation with mitochondria followed by e.l.i.s.a. In addition mitochondria were also treated with immobilised protease. This study also examined the possibility that CPT I and /or CPT II were located in contact-sites that exist between the inner and outer membranes of mitochondria. Inner- membranes, outer-membranes and contact sites were separated by sonication of mitochondria followed by sucrose density centrifugation. Specific marker enzymes within each fraction identified each micro-domain. Using anti-bodies to CPT I and CPT II it was found that both enzymes had a dual location, within their respective membranes and within the contact site fraction.
Notlar:
School code: 0547
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(684572.1) | 684572-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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