Swezey R R, Epel D
Department of Biological Sciences, Stanford University, Hopkins Marine Station, Pacific Grove, CA 93950.
Proc Natl Acad Sci U S A. 1988 Feb;85(3):812-6. doi: 10.1073/pnas.85.3.812.
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.
海胆卵和胚胎在模拟细胞内环境的介质中受到高压放电作用后，仍能保持其结构完整性并保持通透性，使底物能够进入细胞质，从而可以测定酶活性。在底物饱和浓度下，所检测的六种酶中有五种在透化胚胎中的活性比透化卵中的活性更高（高三到十五倍），但从这些相同的透化细胞制备的匀浆中未观察到与受精相关的差异。此外，匀浆中的酶活性总是超过透化细胞悬液中的酶活性。未受精卵和受精卵之间酶反应速率的这种差异并非由于底物向透化细胞扩散的差异所致。对透化细胞中葡萄糖 - 6 - 磷酸脱氢酶（D - 葡萄糖 - 6 - 磷酸：NADP + 1 - 氧化还原酶，EC 1.1.1.49）的活性进行了更详细的研究，其具有以下特征。（i）在早期发育过程中活性调节持续存在。（ii）这种调节不是由可扩散的变构剂介导的。（iii）受精时的刺激由细胞内钙的升高引发，并通过细胞质碱化进一步促进。（iv）该酶在细胞内所经历的微环境与匀浆中的酶不同，这可从明显不同的pH与活性曲线得到证明。这些结果表明酶的调节状态在电透化细胞中得以保留，并表明这种调节取决于匀浆时被破坏的某些细胞结构特征。
