Hunter S C, Ohlrogge J B
Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan, 48824, USA.
Arch Biochem Biophys. 1998 Nov 15;359(2):170-8. doi: 10.1006/abbi.1998.0900.
We have investigated several factors which influence acetyl-CoA carboxylase (ACCase) activity in lysed spinach chloroplasts. (1) When assayed after rapid lysis of light-incubated chloroplasts, ACCase activity was 2-fold higher than activity from dark-incubated chloroplasts. Within 5 min after lysis, activity from dark-incubated chloroplasts increased, suggesting a transient inactivation or inhibition of ACCase in the dark. (2) When lysed chloroplast suspensions were incubated with 30 to 100 microM acetyl-CoA before starting assays, activity was 4-fold higher than if suspensions were not preincubated with acetyl-CoA. CoA, malonyl-CoA, propionyl-CoA, and butyryl-CoA also activated ACCase. Full acetyl-CoA activation required MgATP and was essentially complete after 8 min. ACCase activity decreased upon removal of acetyl-CoA by gel filtration and was partially restored by readdition of acetyl-CoA. Thus, ACCase activation by acetyl-CoA was reversible. (3) Dithiothreitol and thioredoxin stimulated ACCase activity, but only in preparations where ACCase activity was low. (4) ACCase was assayed in concentrations of ATP, ADP, NADPH, NADP+, Mg2+, and CO2/HCO-3, which are estimated to occur in the stroma of chloroplasts under illumination or darkness. ACCase activity from lysed chloroplast suspensions was 10-fold higher when illuminated conditions were used. However, this activity was still 5-fold to 10-fold lower than the rates required to sustain known in vivo rates of fatty acid synthesis and in vitro rates achieved under optimum assay conditions with saturating substrates.
我们研究了几种影响裂解菠菜叶绿体中乙酰辅酶A羧化酶（ACCase）活性的因素。（1）在对光照处理的叶绿体进行快速裂解后进行测定时，ACCase活性比黑暗处理的叶绿体高2倍。裂解后5分钟内，黑暗处理的叶绿体的活性增加，这表明ACCase在黑暗中存在短暂失活或抑制。（2）在开始测定前，将裂解的叶绿体悬浮液与30至100微摩尔的乙酰辅酶A一起孵育，其活性比未用乙酰辅酶A预孵育的悬浮液高4倍。辅酶A、丙二酰辅酶A、丙酰辅酶A和丁酰辅酶A也能激活ACCase。完全的乙酰辅酶A激活需要MgATP，8分钟后基本完成。通过凝胶过滤去除乙酰辅酶A后，ACCase活性降低，重新添加乙酰辅酶A后部分恢复。因此，乙酰辅酶A对ACCase的激活是可逆的。（3）二硫苏糖醇和硫氧还蛋白刺激ACCase活性，但仅在ACCase活性较低的制剂中。（4）在估计光照或黑暗条件下叶绿体基质中存在的ATP、ADP、NADPH、NADP +、Mg2 +和CO2/HCO - 3浓度下测定ACCase。使用光照条件时，裂解的叶绿体悬浮液的ACCase活性高10倍。然而，该活性仍比维持已知体内脂肪酸合成速率和在最佳测定条件下使用饱和底物所达到的体外速率所需的速率低5至10倍。
