©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lanterns of the Firefly Photinus pyralis Contain Abundant Diadenosine 5`,5```-P,P-Tetraphosphate Pyrophosphohydrolase Activity (*)

(Received for publication, November 7, 1994)

Alexander G. McLennan (§) Elaine Mayers Ian Walker-Smith Haijuan Chen (¶)

From the Cellular and Metabolic Regulation Group, Department of Biochemistry, University of Liverpool, P. O. Box 147, Liverpool L69 3BX, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The enzyme diadenosine 5`,5‴-P^1,P^4-tetraphosphate (Ap(4)A) pyrophosphohydrolase has been purified to homogeneity from firefly lanterns. It is a single polypeptide of M(r) 16,000 with a K for Ap(4)A of 1.9 µM and k = 3.6 s. It is inhibited competitively by adenosine 5`-tetraphosphate (K = 7.5 nM) and non-competitively by fluoride ions (K = 50 µM). 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 Ap(4)A and related adenine-containing dinucleoside tetraphosphates in vitro. The high activity of Ap(4)A hydrolase in lanterns may be related to this ability and could be relevant to the use of the luciferase gene as a reporter gene.


INTRODUCTION

Adenine-containing dinucleoside polyphosphates, such as diadenosine 5`,5‴-P^1,P^4-tetraphosphate (Ap(4)A), (^1)Ap(3)A, Ap(4)G, etc. are synthesized in all cells by aminoacyl-tRNA synthetases(1, 2, 3) . The adenylate moiety of an aminoacyladenylate is joined to an acceptor nucleotide such as ATP, ADP, or GTP to form the corresponding ApN. Ap(4)N compounds, and particularly Ap(4)A, have been implicated in a number of intracellular processes including DNA replication and responses to metabolic stress(4, 5, 6, 7) . Recently, firefly luciferase has been shown to synthesize ApN (n geq 4) in vitro in an analogous reaction involving the formation of an enzyme-bound acyladenylate (luciferyl-AMP) and transfer of the AMP to an appropriate acceptor nucleotide(8, 9, 10) . Conditions in vitro (e.g. inclusion of inorganic pyrophosphatase) can be adjusted such that quantitative conversion of, for example, ATP to Ap(4)A is achieved.

The intracellular level of Ap(4)A and related Ap(4)N compounds is almost certainly regulated both at the level of synthesis (11) and degradation by specific enzymes(12) . Higher eukaryotes (plants and animals) contain a small, 17-21 kDa (asymmetrical) Ap(4)A hydrolase (Ap(4)A AMP + ATP; EC 3.6.1.17)(13, 14) , while lower eukaryotes have either an (asymmetrical) Ap(4)A hydrolase, e.g.Schizosaccharomyces pombe(15) , a (symmetrical) Ap(4)A hydrolase (Ap(4)A 2ADP; EC 3.6.1.41), e.g.Physarum polycephalum(16) , a reversible Ap(4)A phosphorylase (Ap(4)A + P(i) ATP + ADP; EC 2.7.7.53), e.g.Saccharomyces cerevisiae(17) , or a hydrolase and a phosphorylase, e.g.Scenedesmus obliquus(18) . Although generally described as Ap(4)A-metabolizing enzymes, all these enzymes will all act upon Ap(4)N compounds.

In view of the availability of a possible additional mechanism for Ap(4)N synthesis in firefly lanterns, it is clearly of interest to know whether the rate of Ap(4)N synthesis in vivo is higher in this tissue than in others and whether Ap(4)N accumulates in lanterns. This is relevant not only to the physiological function of Ap(4)N in general, but also to the control of luciferase-catalyzed light production in fireflies. Here, we report that firefly lanterns contain an apparently normal level of Ap(4)N but an unusually high concentration of (asymmetrical) Ap(4)A hydrolase.


EXPERIMENTAL PROCEDURES

Materials

Desiccated whole fireflies (Photinus pyralis) and lanterns, phenylmethylsulfonyl fluoride, benzamidine, E-64, and all nucleotides were from Sigma. Ultrogel AcA44 was from Réactifs IBF. ATP Monitoring Reagent (which contains firefly luciferase, luciferin, and Mg ions) was from BioOrbit. Procion Red P-3BN-Sepharose (19) and dihydroxyboryl-Biorex 70 (20) were prepared as described. Breakage buffer was 30 mM HEPES-KOH, pH 7.8, 150 mM sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 µM E-64.

Assay of Ap(4)A Hydrolase

The luminometric assay of column fractions was carried out as before (14) with the concentrations and volumes as described(18) . One unit of enzyme activity is the amount that hydrolyzes 1 µmol of Ap(4)A/min at 25 °C. In some experiments, the properties of the purified enzyme were determined using an anion-exchange high performance liquid chromatography assay(18) .

Purification of Firefly Ap(4)A Hydrolase

All operations were performed at 4 °C unless otherwise stated.

Step 1: Preparation of Crude Extract

Firefly tails (1 g) were pulverized by hand in a mortar. Breakage buffer (60 ml) was added and the powder allowed to hydrate for 45 min. After freezing and thawing once, the suspension was homogenized in a Potter homogenizer with 40 strokes of a loose-fitting pestle. After centrifugation at 125,000 times g for 1 h, the supernatant was concentrated to 25 ml by dialysis against solid sucrose.

Step 2: Ultrogel AcA44 Chromatography

The concentrated sample was applied at 60 ml/h to a 950 times 50-mm column of Ultrogel AcA44 equilibrated in 50 mM potassium phosphate, pH 6.8, 1 mM 2-mercaptoethanol, 10% glycerol and eluted with the same buffer. Fractions (13 ml) were collected and assayed for Ap(4)A hydrolase activity (luminometric assay).

Step 3: Procion Red P-3BN-Sepharose Chromatography

Pooled active fractions from Step 2 (182 ml) were applied at 7.3 ml/h to a 15 times 60-mm column of Procion Red P-3BN-Sepharose (19) equilibrated in 50 mM potassium phosphate, pH 6.8, 1 mM 2-mercaptoethanol, 10% glycerol, 0.5 mM EDTA. After elution of unbound protein, homogeneous Ap(4)A hydrolase was eluted at 3 ml/h with the same buffer containing 5 mM MgCl(2) and 20 µM p(4)A. Fractions (1 ml) were collected and assayed for Ap(4)A hydrolase activity (luminometric assay).

Determination of Firefly Tissue Ap(4)N

The endogenous Ap(4)N content of desiccated lanterns and separated non-lantern tissues was measured as follows. Tissues (0.5 g) were added to 16 ml of breakage buffer, and the mixture was shaken for 3 times 7.5 min periods with glass beads (0.45-0.50 mm) at 1700 revolutions/min in a Braun Mikro-Dismembrator U. Samples of homogenate (1.25 ml) were added to 2.5 ml 10% trichloroacetic acid, left on ice for 30 min, then centrifuged (7,000 times g, 5 min). The Ap(4)N content of a 3.3-ml sample of the supernatant was determined by chromatography on dihydroxyboryl-Biorex 70 and luminometry as described previously(20, 21) . The protein concentration in the initial homogenate was also measured(22) .

Other Methods

SDS-polyacrylamide gels were run according to Laemmli(23) . Protein concentrations were determined by the Bradford method using bovine -globulin as standard(22) .


RESULTS

Endogenous Ap(4)N Content of Lanterns and Non-lantern Tissues

Lanterns were separated from non-lantern tissues by dissection. Total Ap(4)N was extracted from each source, separated from ATP and other nucleotides on dihydroxyborylBioRex, and estimated by luminometry to be 6.4 and 5.0 nmol Ap(4)N/g dry weight, respectively. Given that 1 g dry weight approximates to 2.5 g wet weight, these values are equivalent to 2.6 and 2.0 nmol Ap(4)N/g wet weight tissue. Relative to soluble protein, the corresponding values were 6.6 and 5.6 pmol Ap(4)N/mg protein for lanterns and non-lantern tissue, respectively.

Presence of Ap(4)A Hydrolase in Firefly Lantern Extracts

In order to determine the specific activity of Ap(4)A hydrolase in crude lantern extracts, a 0.5-ml sample was applied to a calibrated HiLoad 16/60 Superdex 75 column equilibrated in 30 mM HEPES-KOH, pH 8.0, 50 mM KCl, 1 mM 2-mercaptoethanol, 0.1 mM EDTA. The column was eluted with the same buffer at 1 ml/min and 1-ml fractions collected and assayed immediately for hydrolase activity by luminometry. This procedure rapidly removes endogenous luciferase and ATPase activities that would interfere with the assay. A single peak of activity was eluted with an apparent M(r) of 14,500 (Fig. 1). Assuming 100% recovery of applied hydrolase activity, the specific activity of the crude extract was estimated to be at least 20 milliunits/mg protein. The same result was obtained with three different batches of lanterns.


Figure 1: Chromatography of crude firefly lantern extract on Superdex 75. Crude extract (0.5 ml) was applied to a HiLoad 16/60 Superdex 75 column in 30 mM HEPES-KOH, pH 8.0, 50 mM KCl, 1 mM 2-mercaptoethanol, 0.1 mM EDTA and eluted at 1 ml/min. Fractions (1 ml) were assayed immediately for hydrolase activity (bullet). The column was calibrated with the following molecular mass standards (circle): 1, conalbumin (77 kDa); 2, bovine serum albumin (67 kDa); 3, ovalbumin (45 kDa); 4, beta-lactoglobulin (36.8 kDa); 5, carbonic anhydrase (29 kDa); 6, chymotrypsinogen A (25 kDa); 7, soybean trypsin inhibitor (20.1 kDa); 8, myoglobin (17 kDa); and 9, cytochrome c (12.4 kDa). The elution volumes of these standards are indicated by the appropriate numbers. Absorbance at 280 nm (-).



In order to determine whether this high value is due to an unusually high k or to the presence of a large amount of enzyme, the hydrolase was purified to homogeneity and some of its properties examined. A two-step purification procedure, including biospecific elution from Procion Red-P-3BN-Sepharose with the competitive inhibitor p(4)A(19) , was sufficient to yield homogeneous enzyme (Table 1). A single polypeptide of M(r) 16,000 was observed after silver staining (not shown). Careful pooling of the AcA44 fractions and rapid elution of the hydrolase from the Procion Red-Sepharose column by increasing the p(4)A concentration from 10 to 20 µM(19) were required to ensure that the final preparation was free of contaminating adenylate kinase.



Properties of Firefly Ap(4)A Hydrolase

The firefly enzyme is a typical higher eukaryotic (asymmetrical) Ap(4)A hydrolase according to several criteria(12) : (i) its molecular mass is typical of this class of enzyme; (ii) it hydrolyzes Ap(n)A with n geq 4 and always produces ATP as one of the products (Table 2) (prolonged incubation did not alter the products, thus demonstrating the absence of adenylate kinase, phosphatase, and nonspecific phosphodiesterase activities); (iii) p(4)A is a potent competitive inhibitor (24) with a K(i) of 7.5 nM while fluoride is a non-competitive inhibitor (25) with a K(i) of 50 µM (not shown). The enzyme displayed Michaelis-Menten kinetics with K(m) = 1.9 µM and k = 3.6 s. These values are typical of those reported for other Ap(4)A hydrolases(12, 14, 18, 26) . Thus, the high Ap(4)A hydrolase activity of firefly lanterns must be due to the presence of an unusually high concentration of the enzyme. The data in Table 1would suggest that the hydrolase comprises 0.15% of the soluble lantern protein. This compares, for example, to figures of approximately 0.01% and 0.002% estimated for the enzymes from Artemia(14) and human placenta(19) , respectively.




DISCUSSION

Firefly luciferase catalyzes the formation of Ap(4)N from luciferyl-AMP and NTP in vitro. One may speculate, therefore, that it may also do so in vivo. Ap(4)N has indeed been detected in firefly lanterns; however, because of the practical difficulties in measuring Ap(4)N synthesis directly in lanterns, it is not possible to say whether this has arisen in whole or in part from luciferyl-AMP rather than the more usual aminoacyl-AMP.

The level of Ap(4)N detected in lanterns is not significantly higher than in the combined non-lantern tissues and is similar to levels detected in other tissues. For example, in normal rat liver, the level of Ap(4)N has been reported to be 1.0-1.4 nmol/g wet weight, rising to 5.0 nmol/g wet weight 24 h after partial hepatectomy (27) . The values determined for lanterns (2.6 nmol/g wet weight) and non-lantern tissues (2.0 nmol/g wet weight) fall in the middle of this range. Most of the numerous other literature values for intracellular Ap(4)N in higher eukaryotes are reported as picomoles/milligram protein or picomoles/10^6 cells(28) . Typical ranges in non-storage cells (i.e. excluding platelets and chromaffin cells) are 0.6-7.5 pmol/mg protein and 0.3-3.0 pmol/10^6 cells(28) . Drosophila cells contain 1 pmol of Ap(4)A + Ap(4)G/10^6 cells(29) . Given that 1 pmol of Ap(4)N/10^6 cells is approximately equivalent to 3-4 pmol/mg protein(30) , the values measured in lanterns and non-lantern tissues of 6.6 and 5.6 pmol/mg protein, respectively, fall again within the normal ranges.

The possibility that firefly luciferase may synthesize Ap(4)A in vivo led us to investigate the level of Ap(4)A hydrolase activity in lanterns. Only one other insect hydrolase (from Drosophila) has been reported in the literature(31) . According to its size, sensitivity to F and p(4)A, and substrate/product specificity, including the inability to degrade Ap(2)A and Ap(3)A, the firefly enzyme is a typical higher eukaryotic, asymmetrically cleaving Ap(4)A hydrolase. In fact, it is more typical of this class than the partially purified Drosophila enzyme, which has an unusually high molecular mass of 26,000 and is stimulated preferentially by Co ions(31) . The Drosophila enzyme has a K(m) for Ap(4)A of 4 µM and a K(i) for p(4)A of 10 nM(31) .

The most notable feature of the firefly lantern Ap(4)A hydrolase is its unusually high concentration of 20 milliunits/mg protein (minimum value). Using a variety of assay procedures, values reported for other tissues are: 0.22 milliunits/mg for human placenta (19) , 0.15 milliunits/mg for human leukemic cells(32) , 0.13 milliunits/mg for human platelets and leukocytes(^2), and 0.09 milliunits/mg for lupin meal(26) . Extracts of Artemia cysts, which contain an abundant store of the alternative substrate Gp(4)G, have a specific activity of 2.7 milliunits/mg(14) . Values of 3.0-12.0 milliunits/mg have been reported for various rat tissues(13) ; however, using the extraction procedures and assay systems reported here, we have found the same rat extracts to have specific activities between 0.4 and 2.2 milliunits/mg.^2 Furthermore, in a survey of over 40 eukaryotic cells and tissues, we have found no other example with a specific activity above 2.7 milliunits/mg (^3)when extracted and assayed under our standard conditions. Thus, we conclude that firefly lanterns display 10-100 times more Ap(4)A hydrolase activity than other cells and tissues that have been examined.

Although we have no direct supporting evidence, it is possible that the reason for this abnormally high activity is to maintain the normal steady state levels of these compounds in a tissue which has an additional, luciferase-dependent capacity for Ap(4)N synthesis in vivo. Interestingly, firefly lanterns contain unusually high inorganic pyrophosphatase activity(33) ; pyrophosphatase would promote the back reaction of the luciferyl-AMP with ATP and other nucleotides in preference to PPi and so favor Ap(4)N synthesis.

In vivo, luciferase is located in the peroxisomes(34) . However, Ap(4)N compounds (M(r) = 800-850) synthesized by luciferase would have free access to the cytoplasm and nucleus since the peroxisomal membrane is non-selectively permeable to molecules at least as large as coenzyme A (M(r) = 768)(35) . Maintenance of the normal steady state level of Ap(4)N in the cytoplasm through increased hydrolase activity would be necessary to prevent the induction of responses normally associated with metabolic stress. If so, this could be of practical significance in view of the widespread use of luciferase expression in reporter gene assays in cellular systems that may not be equipped to deal with the consequences of enhanced Ap(4)A synthesis.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: +44 51 794 4369; Fax: +44 51 794 4349.

Supported by a grant from the Medical Research Council.

(^1)
The abbreviations used are: Ap(4)A, diadenosine 5`,5‴-P^1,P^4-tetraphosphate; Ap(2)A, diadenosine 5`,5‴-P^1,P^2-diphosphate; Ap(3)A, diadenosine 5`,5‴-P^1,P^3-triphosphate; Ap(5)A, diadenosine 5`,5‴-P^1,P^5-pentaphosphate; Ap(6)A, diadenosine 5`,5‴-P^1,P^6-hexaphosphate; Ap(4)G, adenosine 5`-guanosine 5`-P^1,P^4-tetraphosphate; Ap(4)N, adenosine 5`-nucleoside 5`-P^1,P^4-tetraphosphate; ApN, adenosine 5`-nucleoside 5`-P^1,P-polyphosphate; p(4)A, adenosine 5`-tetraphosphate; E-64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane.

(^2)
S. Hankin, N. M. H. Thorne, and A. G. McLennan, unpublished results.

(^3)
E. Mayers, K. Boyle, S. Jose, and A. G. McLennan, unpublished results.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.