Kirkman H N, Gaetani G F
J Biol Chem. 1986 Mar 25;261(9):4033-8.
Glucose-6-phosphate dehydrogenase catalyzes the initial and committed step of the pathway that is the principal source of NADPH in many cells. The intracellular rate of the enzyme in human erythrocytes was estimated from the rate at which the cells generated 14CO2 from 14C-labeled glucose in the presence of different amounts of methylene blue. This investigation differed from earlier studies in that: (a) accumulations of 6-phosphogluconate were considered in calculations of rate and (b) the cells were suspended in Krebs-Ringer bicarbonate buffer, which is the buffer system for erythrocytes in vivo. As with earlier studies, however, the intracellular enzyme was under unexplained inhibition or restraint relative to kinetic properties of the purified enzyme. Also, the intracellular enzyme exhibited sigmoid kinetics. In contrast, the isolated enzyme has been found to exhibit classical kinetics. In the course of dilution/ultrafiltration of the hemolysate a possible cause for the reduced activity of the enzyme was found: most of the NADP was bound to soluble macromolecules of the erythrocyte. The amount of NADP available to the enzyme was much less than the amount indicated by measurements of total (bound and unbound) NADP.
葡萄糖-6-磷酸脱氢酶催化该途径的起始和关键步骤，此途径是许多细胞中NADPH的主要来源。通过在不同量亚甲蓝存在下细胞从14C标记的葡萄糖生成14CO2的速率，估算人红细胞中该酶的细胞内速率。本研究与早期研究的不同之处在于：（a）在速率计算中考虑了6-磷酸葡萄糖酸的积累，以及（b）细胞悬浮于 Krebs-Ringer 碳酸氢盐缓冲液中，这是红细胞在体内的缓冲系统。然而，与早期研究一样，相对于纯化酶的动力学特性，细胞内酶受到无法解释的抑制或限制。此外，细胞内酶表现出S形动力学。相比之下，已发现分离出的酶表现出典型动力学。在溶血产物的稀释/超滤过程中，发现了酶活性降低的一个可能原因：大部分NADP与红细胞的可溶性大分子结合。酶可利用的NADP量远低于总（结合和未结合）NADP测量值所示的量。
