| Abstract: | The highly purified respiratory chain NADH dehydrogenase (EC 1.6.99.3) of Escherichia coli is inactive in the absence of detergent or phospholipid. Triton X-100 is the detergent that gives optimal activity, but the Triton X-100-activated enzyme is stimulated an additional 2-fold by E. coli phospholipids. Phosphatidylglycerol and diphosphatidylglycerol are the most effective lipid activators. The activated complex prepared with diphosphatidylglycerol is stable, whereas that with phosphatidylglycerol loses activity rapidly. Maximum activation by phospholipids occurs after preincubation at 0 degrees C and at pH 7. Triton X-100 is required at low concentrations for lipid activation, but high concentrations interfere with the activation. When the enzyme is optimally activated by phospholipids, it may be additionally activated 2-fold by spermidine, but not by magnesium. In contrast, the Triton X-100-activated form of the enzyme is stimulated by several divalent cations, without specificity. Thus, the most stable, active form of the purified NADH dehydrogenase is generated in the presence of diphosphatidylglycerol and spermidine. |
