Effect of periodate-oxidized ATP and other nucleotides on firefly luciferase.
Addition of periodate-oxidized ATP (oATP) to firefly luciferase-containing reaction mixtures enhanced light production when the reaction mixture contained > approximately 8 microM ATP. The time course of light production was changed from a flash pattern to a constant light output during incubations of < approximately 10 min. During longer incubation, firefly luciferase was inactivated in a concentration-dependent fashion by oATP. Firefly luciferase has two different time courses of light production that depend on ATP concentration (DeLuca and McElroy, Biochem. Biophys. Res. Commun., 123, 764, 1984). The enhancement of light production occurred only when higher ATP concentrations (> 8 microM) were used. There is little effect of oATP on firefly luciferase activity at low ATP concentrations (< 2 microM) which gave steady production of light. ATP did not antagonize the inactivation of firefly luciferase by oATP. When the oATP was chemically reduced with sodium borohydride (giving or ATP), there was no inactivation of firefly luciferase on incubation. When or ATP was used in a short incubation the enhancement of light production and time course change were the same as those observed with oATP. The corresponding AMP and adenosine compounds (o and or) were slightly inhibitory to firefly luciferase activity. ADP was without effect but both oADP and or ADP enhanced light production. Of these periodate-oxidized ADP, AMP, and adenosine derivatives only oADP inactivated firefly luciferase. The activating effect can be explained by a change in the conformation of the enzyme-product complex so that the product is released faster. In addition there is an inactivation of the enzyme by certain periodate-oxidized nucleotides during longer incubations.
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078-0454.
S R
SR
Ford
K D
KD
Chenault
M S
MS
Hall
S J
SJ
Pangburn
F R
FR
Leach
D000227:Adenine Nucleotides / Q000494:pharmacology; D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology; D000818:Animals; D001517:Coleoptera / Q000201:enzymology; D004789:Enzyme Activation; D007700:Kinetics; D008027:Light; D008156:Luciferases / Q000378:metabolism*; D008163:Luminescent Measurements; D013997:Time Factors
D000227:Adenine Nucleotides; D000345:Affinity Labels; C017199:2',3'-dialdehyde ATP; D000255:Adenosine Triphosphate; D008156:Luciferases; D000241:Adenosine