The use of caged substrates to assess the activity of 6-phosphogluconate dehydrogenase in living sea urchin eggs.
As part of our inquiries into the regulation of the hexose monophosphate shunt in the early development of sea urchin eggs and embryos, we have developed a novel method to assess the in vivo activity of the enzyme 6-phosphogluconate dehydrogenase (6PGDH) before and after fertilization. Our measurements show that the intracellular level of 6-phosphogluconate (6PG) in eggs decreases 60% after fertilization, which is consistent with the increase in the activity of 6PGDH previously reported using irreversibly permeabilized cell assays (Swezey and Epel, Proc. Natl. Acad. Sci USA 85, 812-816, 1988). The in vivo turnover of the 6PG pool was assessed using a new radioisotopic technique. 1-14C-labeled 6PG was chemically modified such that it was not metabolized by cellular 6PGDH and could be rapidly converted back to 6PG by photolysis. This "caged" 6PG was introduced into unfertilized sea urchin eggs using a transient permeabilization procedure, and then the oxidation of [1-14C]6PG in vivo upon irradiation was followed. Oxidation of 6PG was complete within 7-11 s of irradiation, indicating an extremely rapid turnover of this pool in sea urchin eggs. Based on the 6PG pool sizes and the kinetic properties of 6PGDH, determined here, along with the activity levels seen in permeabilized cells, the half-time for the label in the 6PG pool in sea urchin eggs is calculated to be 26 s. This is inconsistent with the in vivo turnover rates seen in these studies, indicating that the permeabilized cell assays overestimate the degree of inhibition of 6PGDH before fertilization. These results suggest that caution should be exercised in extrapolating data obtained from permeabilized cells to the situation in vivo.
Swezey
R R
RR
Department of Biological Sciences, Stanford University, Pacific Grove, California 93950.
Epel
D
D
D000818:Animals; D002250:Carbon Radioisotopes; D005306:Fertilization; D005942:Gluconates / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008027:Light; D010063:Ovum; D010734:Phosphogluconate Dehydrogenase / Q000032:analysis*; D012617:Sea Urchins
D002250:Carbon Radioisotopes; D005942:Gluconates; D010734:Phosphogluconate Dehydrogenase; C008884:6-phosphogluconic acid