Lanterns of the Firefly Photinus pyralis Contain Abundant Diadenosine 5′,5“′-P1,P4-Tetraphosphate Pyrophosphohydrolase Activity

Abstract

The enzyme diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) pyrophosphohydrolase has been purified to homogeneity from firefly lanterns. It is a single polypeptide of M(r) 16,000 with a Km for Ap4A of 1.9 microM and kcat = 3.6 s-1. It is inhibited competitively by adenosine 5'-tetraphosphate (Ki = 7.5 nM) and non-competitively by fluoride ions (Ki = 50 microM). The specific activity of the enzyme in crude extracts of at least 20 milliunits/mg protein is 10-100 times higher than in any other eukaryote so far examined. Interestingly, firefly luciferase is known to synthesize Ap4A and related adenine-containing dinucleoside tetraphosphates in vitro. The high activity of Ap4A hydrolase in lanterns may be related to this ability and could be relevant to the use of the luciferase gene as a reporter gene.

Diadenosine oligophosphates (ApnA), a novel class of signalling molecules?

“…In vivo overexpression of mammalian TrpRS correlates with the elevated levels of Ap 3 A, not Ap 4 A [38]. Possible participation of the luciferase in the synthesis of Ap 4 A in vivo is indirectly supported by the unusually high level of Ap 4 A hydrolase activity in firefly lanterns [68].…”

pH opposite effects on synthesis of dinucleoside polyphosphates and on oxidation reactions catalyzed by firefly luciferase

“…These conditions, although far away from the ones currently used in experiments of light emission, are presumably not very different from those found in nature. Besides, Ap 4 A has been found in the lanterns of P. pyralis , indicating that their synthesis could be more than a simple artifact [13].…”

Methylene analogues of adenosine 5′‐tetraphosphate

“…The K i values estimated for the human enzyme, 1.6 and 2.3 nm, respectively, were over 30-and 20-fold lower than the K i estimated for the same enzyme for p 4 A (50 nm). Moreover, these values are five and three times smaller than the lowest K i estimated yet reported (7.5 nm) for the reaction of Ap 4 A hydrolysis catalysed by the firefly enzyme [52]. Significantly, the analogue, pppCH 2 pA, with its methylene bridge in the position most distant from the reaction site, is 100-fold less potent an inhibitor than ppCH 2 ppA.…”

“…Adenosine tetraphosphate itself has been known for a long time as an effective competitive inhibitor of the (asymmetrical) Ap 4 A hydrolases. Examples of the reported inhibition constants are 48 nm for the rat liver enzyme [50], 30 nm for the enzyme from Ehrlich ascites tumour cells [51] and 7.5 nm, the lowest value reported to date, for the enzyme from firefly lanterns [52]. Owing to such low K i values, this nucleotide has been used for the elution of the (asymmetrical) Ap 4 A hydrolases adsorbed to dye-ligand affinity columns as homogeneous proteins [53,54].…”