[The effect of substrates on stability of firefly luciferase, embedded in phosphatidylcholine liposomes]. / Vliianie substratov na stabil'nost' liutsiferazy svetliakov, vstroennoi v liposomy iz fosfatidilkholina.

The effects of substrates (ATP and luciferin) on stability of firefly luciferase embedded into phosphatidylcholine liposomes have been studied. Luciferin did not exert any appreciable influence on enzyme inactivation. Minor concentrations of adenosine 5'-triphosphate destabilized the enzyme; however, the increase in ATP concentration markedly stabilized the enzyme entrapped into liposomes. A kinetic scheme of ATP action on enzyme inactivation is proposed. According to this scheme, the enzyme has two ATP-binding sites.

New approaches to the preparation and application of firefly luciferase.

Allowing for the lipid nature of firefly luciferase we have developed a new method for obtaining high-activity and high-stability enzyme preparations for bioluminescent microassay. The method includes the step of differential centrifugation in presence of stabilizing additives which entails a partial purification of the enzyme and its essential stabilization likely due to the fact that luciferase retains its lipid environment which plays an important role in catalysis. The resultant luciferase preparation is stable in solution at 4 degrees C for 2-3 months and allows the detection of down to 10(-11) M ATP. A new method has been offered for luciferase immobilization on film carriers precoated with a phospholipid layer. By sorption of the enzyme on such carriers, the samples of immobilized luciferase have been obtained suitable for constructing chemiluminescent biosensors, in the form of luciferase-containing films. There are many-fold applications for detection of ATP micro-quantities.

Kinetics of the inactivation of the protein-lipid complex, firefly luciferase, by sodium deoxycholate and its reactivation by phosphatidylcholine.

Firefly luciferase has been shown to be a protein-lipid complex. Phospholipids and neutral lipids bound to luciferase have been identified. Sodium deoxycholate rapidly inactivated the enzyme, but an excess of phosphatidylcholine recovered luciferase activity. From the kinetics of inactivation and reactivation, a mechanism for interaction of the enzyme with detergents and phospholipids has been proposed. The substrates ATP and Mg2+ stabilized luciferase during delipidation.