Thesis

Studies of isozymes of glucose-6-phosphate dehydrogenase in sea urchin eggs

In sea urchin eggs the hexose monophosphate shunt is the main glycolytic pathway before and after fertilization, and glucose-6-phosphate dehydrogenase (G6PD) is the main regulatory enzyme of the pathway. Activity of the enzyme increases with fertilization, thought to occur as a result of activation of NAD kinase and glycogen phosphorylase and simultaneously increased titers of NADP and glucose-6-phosphate (G6P). Part of the enzyme activity is associated with the egg surface membrane complex. Experiments have been done to determine the effects of extraction pH upon isozymes in unfertilized eggs, and what effects dithiothreitol (DTT, a sulfhydrl reducer), papain (a well known thiol protease), NADP, NAD, G6P, and other substrates have when added exogenously, upon the formation of isozymes and the mechanics of isozyme change as a part of the program of fertilization. Control extracts of pellet and gel fractions of unfertilized egg homogenates of the species Strongylocentrotus purpuratus show three isozyme bands when extracted using 0.01M MgCl2; these bands are designated B1, B2, and B3, the slowest to fastest migrating respectively with electrophoresis. Extraction with Lubrol WX yields an even slower moving band, BL. Fertilized eggs are shown to lack a B3 band. Extraction pH has been found to have no effect upon formation of isozymes between pH 6.4 and 7.9, whether pellet or gel extract. Unfertilized and fertilized egg pH 7.9 extracts with DTT added show the apparent extinction of activity of all but the B2 isozyme band, thought to be due to sulfhydryl inhibition of the binding of NADP to cysteine residues of the G6PD isozymes inactivated by DTT. Papain added to unfertilized egg extracts has no visible effect upon the isozymes, while fertilized egg extracts take on somewhat the appearance of unfertilized eggs, gaining the B3 band and partially losing B1, indicating a relationship between B1 and B3. Addition of NADP and G6P together causes the apparent disappearance of the B3 isozyme in unfertilized egg extracts and slightly increased mobility in both fertilized and unfertilized cases. With the experimental results in mind, it is proposed that increased NADP titers cause a release of lower molecular weight isozymes from membranes to soluble phases of the cell, and increased concentrations of substrate and specific cofactor complete formation of a larger G6PD isozyme, with the greater enzyme activity available to supply the energy for the metabolism of the newly formed sea urchin zygote.

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