THE IN-VIVO RATE OF GLUCOSE-6-PHOSPHATE-DEHYDROGENASE ACTIVITY IN SEA-URCHIN EGGS DETERMINED WITH A PHOTOLABILE CAGED SUBSTRATE

Some of the earliest metabolic changes after fertilization of sea urch in eggs center around the activity of the pentose phosphate shunt. We here report on the in vivo activity of glucose-6-phosphate dehydrogena se (G6PDH), the first enzyme of this shunt, as assayed with a photolab ile (caged) analog of the substrate, glucose-6-phosphate (G6P). Caged G6P was synthesized from radiolabeled (5-H-3 or 1-C-14) glucose and lo aded into unfertilized sea urchin eggs by transient electroporation. I rradiation of these eggs (either before or after fertilization) photol yses the caged G6P, thereby pulsing the cell with H-3- and C-14-labele d G6P. The fluxes of G6P into glycolysis and the pentose shunt are cal culated from the rates of oxidation of labeled G6P to (H2O)-H-3 and (C O2)-C-14; since the turnover of the 6-phosphogluconate pool by 6-phosp hogluconate dehydrogenase is nearly instantaneous (Swezey, R.R., and E pel, D. (1992) Exp. Cell Res. 201:366-372), the rate of (CO2)-C-14 pro duction by the pentose shunt is equal to the flux of G6P through G6PDH . The data indicate that G6PDH activity is very low in unfertilized eg gs, increases 184- to 427-fold by 2 min after fertilization, and then decreases to a value that is 74 to 209 times the unfertilized level (m aximally 0.005 X 10(-8) units per egg in unfertilized eggs, 2.14 X 10( -8) units per egg by 2 min after fertilization, and 1.05 X 10(-8) unit s per egg by 20 min after fertilization). In spite of this substantial activation, the enzyme activity is considerably repressed; compared w ith activity in broken cell extracts, G6PDH at these developmental tim es operates in vivo at 0-0.003%, 0.52-1.21%, and 0.21-0.59%, respectiv ely, of its potential activity. These results are discussed in terms o f various hypotheses regarding the modulation of G6PDH activity by fer tilization. These activity measurements relate well to other indices o f in vivo activity. The major use of the NADPH shortly after fertiliza tion is to produce H2O2, which is used as a substrate for fertilizatio n membrane hardening; our data indicate that the NADPH that is produce d by the pentose shunt activity is 30-70% of that required for this po stfertilization generation of H2O2. (C) 1995 Academic Press, Inc.