The in vivo rate of glucose-6-phosphate dehydrogenase activity in sea urchin eggs determined with a photolabile caged substrate.
The in vivo rate of glucose-6-phosphate dehydrogenase activity in sea urchin eggs determined with a photolabile caged substrate.
复制标题
海胆卵中葡萄糖-6-磷酸脱氢酶活性的体内速率用光不稳定的笼式底物测定。
DOI：
10.1006/dbio.1995.1183
复制
发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Epel,D
中科院分区：
文献类型：
--
作者：
Swezey,RR;Epel,D
海胆卵受精后最早的一些代谢变化围绕着磷酸戊糖分流的活动。我们在这里报告的葡萄糖-6-磷酸脱氢酶（G6 PDH），第一个酶的分流，在体内活性的光不稳定（笼）的类似物的基板，葡萄糖-6-磷酸（G6 P）进行测定。笼G6 P从放射性标记的（5- 3 H或1- 14 C）葡萄糖合成，并加载到未受精的海胆卵通过瞬时电穿孔。照射这些卵子（受精前或受精后）使笼状的G6 P光解，从而用3 H和14 C标记的G6 P脉冲细胞。从标记的G6 P氧化成3 H2O和14 CO2的速率计算G6 P进入糖酵解和戊糖分流的通量;由于6-磷酸葡萄糖酸脱氢酶对6-磷酸葡萄糖酸池的周转几乎是瞬时的（Swezey，R.R.，和Epel，D.（1092）实验Cell Res.201：366-372），戊糖旁路产生14 CO2的速率等于G6 P通过G6 PDH的通量。数据表明，G6 PDH活性在未受精卵中非常低，在受精后2分钟增加184至427倍，然后降低至未受精水平的74至209倍（未受精卵最高为0.005 × 10- 8个/卵，受精后2 min最高为2.14 × 10- 8个/卵，受精后20 min最高为1.05 × 10- 8个/卵）。尽管这种实质性的激活，酶的活性是相当抑制;与破碎的细胞提取物中的活性相比，G6 PDH在这些发育时间在体内分别以其潜在活性的0- 0.003%，0.52- 1.21%和0.21- 0.59%起作用。这些结果进行了讨论，在各种假设有关的G6 PDH活性的调制受精。这些活性测量值与体内活性的其他指标密切相关。受精后不久NADPH的主要用途是产生H2 O2，H2 O2被用作受精膜硬化的底物;我们的数据表明，戊糖分流活性产生的NADPH是受精后产生H2 O2所需的NADPH的30-70%。
Some of the earliest metabolic changes after fertilization of sea urchin eggs center around the activity of the pentose phosphate shunt. We here report on the in vivo activity of glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme of this shunt, as assayed with a photolabile (caged) analog of the substrate, glucose-6-phosphate (G6P). Caged G6P was synthesized from radiolabeled (5-3H or 1-14C) glucose and loaded into unfertilized sea urchin eggs by transient electroporation. Irradiation of these eggs (either before or after fertilization) photolyses the caged G6P, thereby pulsing the cell with3H- and14C-labeled G6P. The fluxes of G6P into glycolysis and the pentose shunt are calculated from the rates of oxidation of labeled G6P to3H2O and14CO2; since the turnover of the 6-phosphogluconate pool by 6-phosphogluconate dehydrogenase is nearly instantaneous (Swezey, R.R., and Epel, D. (1092) Exp. Cell Res. 201:366-372), the rate of14CO2production by the pentose shunt is equal to the flux of G6P through G6PDH. The data indicate that G6PDH activity is very low in unfertilized eggs, 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 (maximally 0.005 × 10-8units per egg in unfertilized eggs, 2.14 × 10-8units per egg by 2 min after fertilization, and 1.05 × 10-8units per egg by 20 min after fertilization). In spite of this substantial activation, the enzyme activity is considerably repressed; compared with activity in broken cell extracts, G6PDH at these developmental times operates in vivo at 0-0.003%, 0.52-1.21%, and 0.21-0.59%, respectively, of its potential activity. These results are discussed in terms of various hypotheses regarding the modulation of G6PDH activity by fertilization. These activity measurements relate well to other indices of in vivo activity. The major use of the NADPH shortly after fertilization is to produce H2O2, which is used as a substrate for fertilization membrane hardening; our data indicate that the NADPH that is produced by the pentose shunt activity is 30-70% of that required for this postfertilization generation of H2O2.