Swezey R R, Epel D
Department of Biological Sciences, Stanford University, Pacific Grove, California 93950.
Exp Cell Res. 1992 Aug;201(2):366-72. doi: 10.1016/0014-4827(92)90285-g.
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.
作为我们对海胆卵和胚胎早期发育过程中磷酸己糖支路调控研究的一部分，我们开发了一种新方法，用于评估受精前后6 - 磷酸葡萄糖酸脱氢酶（6PGDH）的体内活性。我们的测量结果表明，受精后卵中6 - 磷酸葡萄糖酸（6PG）的细胞内水平降低了60%，这与之前使用不可逆通透细胞分析法报道的6PGDH活性增加一致（Swezey和Epel，《美国国家科学院院刊》85，812 - 816，1988）。使用一种新的放射性同位素技术评估了6PG库的体内周转率。1 - 14C标记的6PG经过化学修饰，使其不会被细胞中的6PGDH代谢，并且可以通过光解迅速转化回6PG。这种“笼化”的6PG通过瞬时通透化程序引入未受精的海胆卵中，然后追踪照射后[1 - 14C]6PG在体内的氧化情况。6PG的氧化在照射后7 - 11秒内完成，表明海胆卵中该库的周转率极快。根据这里测定的6PG库大小和6PGDH的动力学特性，以及通透细胞中观察到的活性水平，计算出海胆卵中6PG库中标记物的半衰期为26秒。这与这些研究中观察到的体内周转率不一致，表明通透细胞分析法高估了受精前6PGDH的抑制程度。这些结果表明，在将从通透细胞获得的数据外推到体内情况时应谨慎行事。
