Date of Award
Dissertation - Restricted
Doctor of Philosophy (PhD)
Samuel A. Morell
Paul M. Roll
Regulation of glucose metabolism in the human erythrocyte has been investigated at both the cellular and molecular levels. The red cell metabolizes glucose by only two pathways: glycolysis and the hexose monophosphate shunt provide about 75% and 25% of the energy requirements in the form to ATP and TPNH respectively. TPNH protects the cell against oxidation, the shunt being increased at the expense of glycolysis. From sutides on erythrocytes treated with peroxide, it has been proposed that the glutathione ratio, GSSG/GSH, functions as the primary regulator. However, in a recent study at the enzymatic level of control, TPN and TPNH have been proposed as allosteric effectors of glucose-6-phosphate dehydrogenase (G-6-PD), the "committed" step of the shunt, and thereby serve as the primary regulators. In this thesis, both of these hypotheses have been investigated using purified G-6-PD. The role of the red cell redox systems in regulating glucose metabolism at the G-6-PD level has also been studied.
Before kinetic studies could be undertaken, purification of red cell G-6-PD was necessary. A reproducible 12,000-fold purification was achieved. The absence of contaminating enzymes which might interfere with the proposed studies was established.
The two redox systems proposed as primary shunt regulators were tested. Neither form of glutathione nor any ratio of the two had any effect on the enzyme. Consequently the possibility of primary control by glutathione was discarded. Allosteric regulation et G-6-PD by TPN and TPNH has been confirmed and the hypothesis of primary regulation by the TPN/TPNH ratio verified. Since changes in this ratio are related to other redox systeas of the cell, their effects on G-6-PD were investigated. Of the systems tested, only the reduced pyridine nucleotides and ascorbic acid were found to inhibit the enzyme. Since a regulatory role tor the vitamin may be related to its redox properties, ascorbic acid was studied further.
Inhibition of erythrocyte G-6-PD by ascorbic acid was characterized as mixed-type. Degradation products of ascorbic acid formed in solution and other compounds exhibiting adjacent dicarbonyl or ene-diol structures were also found to be inhibitory. Most are members of redox pairs. The ascorbate inhibition could not be attributed to the following: a pH effect, a peroxide effect, a non-enzymatic reaction, a contaminating enzyme activity involving G-6-P, TPN, or TPNH. The ascorbate inhibition could be reversed by either sulfhydryl or disulfide compounds. This may be related to the observation that ascorbate inhibition of G-6-PD is associated with a change in the number of titratable sulfhydryl groups in the protein.
Comparative studies on G-6-PD from Torulopsis utilis showed that the yeast enzyme is not allosterically controlled by TPN/TPNH although TPNH is a competitive inhibitor. The yeast enzyme, like that of the red cell, is inhibited by ascorbic acid and similar dicarbonyl or ene-diol compounds. Ascorbate inhibition is also associated with changes in the number of titratable sulthydryl groups in the yeast enzyme. In contrast to the red cell enzyme, however, the yeast enzyme is inhibited by thiols and disulfides. Whereas either thiols or disulfides reverse the ascorbate inhibition of the red cell enzyme, ascorbate reverses the thiol or disulfide inhibition of the yeast enzyme. It appears that ascorbate inhibition of both enzymes may involve similar functional groups participating in oxidation-reduction reactions. A physiological role for control of glucose metabolism by ascorbic acid is proposed.