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Reference: Brindle, Kevin., (1982). Proton NMR studies of intact cells. DPhil. University of Oxford.Citable link to this page:

 

Proton NMR studies of intact cells

Abstract: ´╗┐The technique of 1H spin echo n.m.r. has been used for thenon-invasive study of enzyme catalysed 1H/2H equilibrium isotopeexchange at the C-2 position of lactate in suspensions of humanerythroeytes. The intracellular environment of the enzymes involved inthis exchange has been investigated by comparing the exchange propertiesof the enzymes in the intact cell with the properties they display invitro.A study of the exchange of the lactate C-2 substituent withsolvent, which is catalysed by a coupled system of four glycolyticenzymes, has teen used to examine the kinetic properties of theindividual enzymes in vitro. Measurements of the exchange in the intactcell have been used to investigate the in situ kinetic properties of oneof these enzymes, glyceraldehydephosphate dehydrogenase. Contrary tothe conclusions of previous studies with the isolated enzyme in vitro,these measurements have shown that the enzyme is not rate determiningfor glycolytic flux in the human erythrocyte and that it is unlikelythat it is bound to the cell membrane in situ.A study of 1H/2H exchange between the C-2 positions of methyllabelled lactate molecules, catalysed by lactate dehydrogenase, has beenused to investigate the in situ kinetic properties of this enzyme.Comparison of these properties with those it displays in vitro indicatethat the free intracellular NAD(H) concentration in the erythrocyte isonly approximately 10% of the total extractable concentration. Aconsiderable fraction of the coenzyme must be bound, therefore, in theintact cell. This type of experiment should be widely applicable to avariety of tissues and possibly to different dehydrogenases.Theoretical aspects of bulk isotope exchange kinetics inmulti-enzyme systems are examined and the effects of chemical flux, andof isotope effects, on the measurement of isotopic flux are considered.The advantages of the n.m.r. method over conventional radioactive tracertechniques are described.It is concluded that 1H n.m.r. studies of 1H/2H isotope exchangemay be used to obtain information about the kinetic properties ofenzymes in intact cellular systems. The technique should be a usefulcomplement, therefore, to the currently more widely used n.m.r. methodsemploying the 31P and 13C nuclei and to other methods used for thenon-invasive study of metabolism.

Type of Award:DPhil Level of Award:Doctoral Awarding Institution: University of Oxford Notes:The digital copy of this thesis has been made available thanks to the generosity of Dr Leonard Polonsky

Contributors

Campbell, Iain D.More by this contributor

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Dr. Iain CampbellMore by this contributor

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 Bibliographic Details

Issue Date: 1982Identifiers

Urn: uuid:f6fcac0c-0c31-470b-9e42-31ad25adf4f1

Source identifier: 603839283 Item Description

Type: Thesis;

Language: eng Subjects: Nuclear magnetic resonance Photon-echo nuclear double resonance Enzymes Erythrocytes Tiny URL: td:603839283

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Author: Brindle, Kevin. - institutionUniversity of Oxford facultyLife and Environmental Sciences Division - - - - Contributors Campbell,

Source: https://ora.ox.ac.uk/objects/uuid:f6fcac0c-0c31-470b-9e42-31ad25adf4f1



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