Swezey R R, Epel D
Department of Biological Sciences, Stanford University, Pacific Grove, California 93950, USA.
Dev Biol. 1995 Jun;169(2):733-44. doi: 10.1006/dbio.1995.1183.
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 with 3H- and 14C-labeled G6P. The fluxes of G6P into glycolysis and the pentose shunt are calculated from the rates of oxidation of labeled G6P to 3H2O and 14CO2; since the turnover of the 6-phosphogluconate pool by 6-phosphogluconate dehydrogenase is nearly instantaneous (Swezey, R.R., and Epel, D. (1992) Exp. Cell Res. 201:366-372), the rate of 14CO2 production 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 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) units 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.
海胆卵受精后最早出现的一些代谢变化集中在磷酸戊糖途径的活性上。我们在此报告葡萄糖-6-磷酸脱氢酶（G6PDH）的体内活性，该酶是此途径的首个酶，通过底物葡萄糖-6-磷酸（G6P）的光不稳定（笼形）类似物进行测定。笼形G6P由放射性标记（5-³H或1-¹⁴C）的葡萄糖合成，并通过短暂电穿孔加载到未受精的海胆卵中。对这些卵（受精前或受精后）进行照射会使笼形G6P光解，从而用³H和¹⁴C标记的G6P对细胞进行脉冲处理。G6P进入糖酵解和戊糖途径的通量根据标记的G6P氧化为³H₂O和¹⁴CO₂的速率来计算；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间完成的（斯韦齐，R.R.，和埃佩尔，D.（1992年）《细胞实验研究》201:366 - 372），戊糖途径产生¹⁴CO₂的速率等于G6P通过G6PDH的通量。数据表明，未受精卵中的G6PDH活性非常低，受精后2分钟增加184至427倍，然后降至未受精水平的74至209倍（未受精卵中最大为0.005×10⁻⁸单位/卵，受精后2分钟为2.14×10⁻⁸单位/卵，受精后20分钟为1.05×10⁻⁸单位/卵）。尽管有这种显著的激活，但酶活性仍受到相当程度的抑制；与破碎细胞提取物中的活性相比，在这些发育阶段，G6PDH在体内的活性分别为其潜在活性的0 - 0.003%、0.52 - 1.21%和0.21 - 0.59%。根据关于受精对G6PDH活性调节的各种假说对这些结果进行了讨论。这些活性测量结果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H₂O₂，其用作受精膜硬化的底物；我们的数据表明，戊糖途径活性产生的NADPH占受精后产生H₂O₂所需量的30 - 70%。
