Characterization of the interaction of P 1 ,P 4 -diadenosine 5 0 -tetraphosphate with luciferase q Luminita H. Pojoga, a,1 Jana E. Moose, a and Richard H. Hilderman a,b, * a Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634-0324, USA b South Carolina Experiment Station, Clemson University, Clemson, SC 29634-0324, USA Received 8 January 2004 Abstract Adenylated dinucleotides (Ap n A) are regulatory molecules that control various cellular processes. A very likely intracellular target for Ap 4 A are enzymes that require ATP as either substrate or modulator. We report the results of new biochemical studies aimed at characterizing the Ap 4 A interaction with firefly luciferase, by using the luminometric and thin layer chromatography techniques. The data presented herein demonstrate that Ap 4 A is a noncompetitive inhibitor for the ATP-induced luminescence. These results together with our previous findings that Ap 4 A is a luciferase substrate [Nucleosides Nucleotides Nucleic Acids 23 (2004) in press.] support the notion that, similar to its interaction with P 2 receptors, Ap 4 A also has a dual interaction with luciferase. Other Ap n As (n ¼ 2, 5, and 6) also inhibited the ATP–luciferase interaction. Since Ap n As may have similar interactions with other intracellular ATP-requiring enzymes, the study presented herein validates ulterior investigations of the Ap n A interaction with such enzymes, and opens the way to a better understanding of their intracellular roles. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Ap 4 A; Adenine dinucleotides; Alarmone; Firefly luciferase Diadenylated dinucleotides represent a diverse and interesting group of regulatory molecules that act as both intracellular and extracellular signal molecules, as recently reviewed [2–4]. These molecules, consisting of two adenosine moieties linked via their 5 0 positions by a phosphate bridge of variable length (Ap n A; n ¼ 2–7), are structurally similar to adenine mononucleotides such as ATP and can interact with various mononucleotide- specific targets [5–10]. The most abundant and best characterized Ap n A (Ap 4 A) was shown to interact, on the cell surface, with both dinucleotide-specific and mononucleotide-specific receptors [8–12]. However, the intracellular role of Ap 4 A is less understood in terms of its interaction with targets inside the cells. Due to its structural resemblance to ATP, the most likely target for Ap 4 A inside the cells consists of enzymes with an ATP- binding domain; therefore, our goal is to characterize the effects of Ap 4 A on such enzymes. Among the intracellular enzymes that exhibit an ATP-binding domain, several also display sequence and structural homology and form the “acyl-adenylate/thi- oester-forming” superfamily [13–18]. Many members of this superfamily are involved in essential biochemical pathways, which makes the class they belong to an in- teresting and worthy candidate for studying the Ap n A interaction with intracellular enzymes. This large family includes enzymes that catalyze reactions of ATP with carboxylate substrates to form acyl-adenylates, such as a variety of luciferases [17], acyl:CoA ligases, acyl–CoA synthetases [13], as well as the acyl-adenylate-forming domains of enzyme complexes involved in the nonrib- osomal synthesis of peptides [18]. We chose to approach our longterm objective (the understanding of Ap 4 A in- tracellular roles) by initially studying its interaction with the firefly luciferase, not only for the enzyme’s sequence q Abbreviations: ADP, adenosine 5 0 -diphosphate; AMP, adenosine 5 0 -monophosphate; Ap n A, diadenosine polyphosphates; ATP, adeno- sine 5 0 -triphosphate; BAEC, bovine aortic endothelial cells; LH 2 , luciferin; oxyluc, oxyluciferin; TLC, thin layer chromatography. * Corresponding author. Fax: 1-864-656-6879. E-mail address: hilderr@clemson.edu (R.H. Hilderman). 1 On leave from the Institute of Cellular Biology and Pathology “Nicolae Simionescu,” 8 B.P. Hasdeu Street, Bucharest 79691, Romania. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.01.121 Biochemical and Biophysical Research Communications 315 (2004) 756–762 BBRC www.elsevier.com/locate/ybbrc