Enzyme stimulation upon fertilization is revealed in electrically permeabilized sea urchin eggs.
Sea urchin eggs and embryos subjected to high-voltage electric discharge in a medium mimicking the intracellular milieu retain their structural integrity and remain permeable, permitting substrates to enter the cytoplasm and thus assay of enzyme activity. At saturating concentrations of substrates, five of six enzymes assayed for more active (three to fifteen times) in permeabilized embryos than in permeabilized eggs, but no fertilization-related differences are seen in homogenates prepared from these same permeabilized cells. Furthermore, enzyme activity in homogenates always exceeds that in the permeabilized cell suspensions. This difference in enzyme reaction rates between unfertilized eggs and fertilized eggs is not due to differences in the diffusibility of substrates into the permeabilized cells. The activity of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) in permeabilized cells was studied in greater detail and has the following characteristics. (i) Regulation of activity persists during early development. (ii) This regulation is not mediated by diffusible allosteric agents. (iii) Stimulation at fertilization is initiated by a rise in intracellular calcium and is further promoted by cytoplasmic alkalinization. (iv) The microenvironment experienced by this enzyme intracellularly differs from that of the enzyme in homogenates as evidenced by markedly different pH vs. activity profiles. These results indicate that the regulatory status of enzymes is preserved in electrically permeabilized cells and suggest that this regulation depends on some cell structural feature(s) that is (are) destroyed upon homogenization.
Department of Biological Sciences, Stanford University, Hopkins Marine Station, Pacific Grove, CA 93950.
R R
RR
Swezey
D
D
Epel
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability; D004558:Electric Stimulation; D004789:Enzyme Activation; D005306:Fertilization; D005954:Glucosephosphate Dehydrogenase / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D010063:Ovum / Q000201:enzymology* / Q000502:physiology; D012617:Sea Urchins / Q000196:embryology
D005954:Glucosephosphate Dehydrogenase; D002118:Calcium