Please use this identifier to cite or link to this item:
|Title:||Subunit structure and dual ATP effects of Na, K-ATPase|
|Presented at:||University of Leicester|
|Abstract:||The Na,-ATPase activity of membrane-bound sodium pump exhibits non-Michaelis kinetics with respect to ATP. The enzyme consists of promoters (one plus one -subunit) that may be organised into higher oligomers. Each -subunit is believed (although it has not been proven) to posses only one ATP binding site. It is not understood how the low-affinity ATP effect arises. Experiments thus far have not been able to convincingly distinguish between the following possibilities: 1) negative co-operatively between the ATP binding sites of the two halves of a dimeric enzyme, 2) the single ATP binding site of a protomeric enzyme exhibiting variable affinity and function around the reaction cycle, and 3) a second, uncharacterised, ATP binding site on the -subunit.;The experiments presented in this thesis investigate how the dual ATP effects are related to the subunit structure of the sodium pump. I solubilise Na,K-ATPase with dodecyl octaethyleneglycol monoether (C12E8). The aggregation state of the C12E8- solubilised enzyme is quantified by analytical ultracentrifugation and found to be predominantly protomeric. These soluble protomers retain dual responses towards ATP as determined from the substrate dependence curve of their Na,K-ATPase activity and their response to non-hydrolysable ATP analogs. Hence, the dual ATP responses are intrinsic to the protomer and do not arise from - interactions within an oligomeric enzyme. Furthermore, protomers that have their high-affinity ATP binding sites irreversibly blocked with fluorescein 5'-isothiocyanate can still bind 2'(3')-O-(2,4,6-trinitrophenyl)ADP indicating that each protomer possesses two nucleotide binding sites.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Cell Physiology and Pharmacology|
Items in LRA are protected by copyright, with all rights reserved, unless otherwise indicated.