Department of Agronomy, University of Illinois, Urbana, Illinois 61801.
Plant Physiol. 1986 Mar;80(3):707-10. doi: 10.1104/pp.80.3.707.
The exchange properties of the activator CO(2) of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO(2) on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO(2). The exchange kinetics of the activator CO(2) were monitored by activation of the enzyme to steady state in the presence of (12)CO(2), followed by addition of (14)CO(2) and determination of the amount of labeled CO(2) trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants (K(obs)) for exchange with both the purified enzyme (0.45 min(-1)) and in illuminated chloroplasts (0.18 min(-1)) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO(2) was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation.
菠菜核酮糖-1,5-二磷酸羧化酶/加氧酶的激活剂 CO(2)的交换性质在体外使用纯化酶和在分离的叶绿体中进行了体内研究。核酮糖-1,5-二磷酸阿拉伯糖醇-1,5-二磷酸，一种酶羧化酶活性的拟反应中间体类似物，用于在体外和体内捕获酶上的激活剂 CO(2)。在光/暗循环期间完整叶绿体中核酮糖-1,5-二磷酸羧化酶/加氧酶活性的调节与核酮糖-1,5-二磷酸阿拉伯糖醇-1,5-二磷酸捕获的 CO(2)的类似调节有关。通过在存在 (12)CO(2) 的情况下将酶激活至稳定状态，然后添加 (14)CO(2) 并通过核酮糖-1,5-二磷酸阿拉伯糖醇-1,5-二磷酸测定酶上标记 CO(2)的量来监测激活剂 CO(2)的交换动力学。与纯化酶（0.45 min(-1)) 和光照叶绿体（0.18 min(-1)) 相比，交换的速率常数 (K(obs)) 与两种条件下酶激活的观察到的速率常数相当。在黑暗中的叶绿体中未观察到激活剂 CO(2)的类似交换。对纯化酶交换性质的动力学分析与在稳态激活过程中酶的活性和非活性形式之间的平衡一致。
