The technique of ¹H spin echo n.m.r. has been used for the non-invasive study of enzyme catalysed ¹H/²H equilibrium isotope exchange at the C-2 position of lactate in suspensions of human erythroeytes. The intracellular environment of the enzymes involved in this exchange has been investigated by comparing the exchange properties of the enzymes in the intact cell with the properties they display in vitro. A study of the exchange of the lactate C-2 substituent with solvent, which is catalysed by a coupled system of four glycolytic enzymes, has teen used to examine the kinetic properties of the individual enzymes in vitro. Measurements of the exchange in the intact cell have been used to investigate the in situ kinetic properties of one of these enzymes, glyceraldehydephosphate dehydrogenase. Contrary to the conclusions of previous studies with the isolated enzyme in vitro, these measurements have shown that the enzyme is not rate determining for glycolytic flux in the human erythrocyte and that it is unlikely that it is bound to the cell membrane in situ. A study of ¹H/²H exchange between the C-2 positions of methyl labelled lactate molecules, catalysed by lactate dehydrogenase, has been used to investigate the in situ kinetic properties of this enzyme. Comparison of these properties with those it displays in vitro indicate that the free intracellular NAD(H) concentration in the erythrocyte is only approximately 10% of the total extractable concentration. A considerable fraction of the coenzyme must be bound, therefore, in the intact cell. This type of experiment should be widely applicable to a variety of tissues and possibly to different dehydrogenases. Theoretical aspects of bulk isotope exchange kinetics in multi-enzyme systems are examined and the effects of chemical flux, and of isotope effects, on the measurement of isotopic flux are considered. The advantages of the n.m.r. method over conventional radioactive tracer techniques are described. It is concluded that ¹H n.m.r. studies of ¹H/²H isotope exchange may be used to obtain information about the kinetic properties of enzymes in intact cellular systems. The technique should be a useful complement, therefore, to the currently more widely used n.m.r. methods employing the ³¹P and ¹³C nuclei and to other methods used for the non-invasive study of metabolism.

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains xiii, 109 p. : ill. Includes abstract. Includes bibliographical references (p. 102-109).

Thesis (M.S.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains ix, 72 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 40).

Thesis (Ph. D.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains ix, 165 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 118-122).

Thesis (Ph. D.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains xiii, 115 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 95-99).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xiii, 120 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 114-120).

A técnica de dupla ressonância unidimensional para alta resolução em líquidos constitui o objetivo deste trabalho. Para isso construiu-se uma sonda de dupla ressonância, apropriada para experiências heteronucleares do tipo 13C-{1H}. Empregam-se algumas sequências de pulsos para permitir as medidas do EON e do desacoplamento nuclear entre 1H e 13C. Emprega-se o EON com a finalidade de obter um aumento na intensidade do sinal de RMN do 13C; emprega-se o desacoplamento para eliminar a estrutura fina que surge devido ao acoplamento nuclear entre 1H e 13C. Inclui-se uma breve revisão sobre a teoria com a fmalidade de unificar o tratamento desta com as aplicações. As técnicas experimentais são discutidas e alguns espectros são mostrados para ilustrar os principais resultados / The one dimensional double resonance technique for high resolition in liquids is the main purpose of this work. For this we have built a double resonance probe, suitable for heteronuclear 13C-{1H} experiment. Some pulse sequences are employed to allow the NOE and decoupling between 1H e 13C to be measured. The NOE is employed in order to obtain a three-fold enhancement of the 13C NMR signal; decoupling is employed to eliminate the fme structW\'e arising from spin coupling between 1H e 13C. A short review about theory is included to provide unified treatment with applications. The experimental techniques are discussed and some spectra are shown to illustrate the main results.

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