(Received for publication, November 7, 1994)
From the
The enzyme diadenosine
5`,5‴-P,P
-tetraphosphate
(Ap
A) pyrophosphohydrolase has been purified to homogeneity
from firefly lanterns. It is a single polypeptide of M
16,000 with a K
for Ap
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
A and related adenine-containing
dinucleoside tetraphosphates in vitro. The high activity of
Ap
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.
Adenine-containing dinucleoside polyphosphates, such as
diadenosine
5`,5‴-P,P
-tetraphosphate
(Ap
A), (
)Ap
A, Ap
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 Ap
N.
Ap
N compounds, and particularly Ap
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
Ap
N (n
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
A is achieved.
The intracellular level of
ApA and related Ap
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
A hydrolase (Ap
A
AMP + ATP; EC
3.6.1.17)(13, 14) , while lower eukaryotes have either
an (asymmetrical) Ap
A hydrolase, e.g.Schizosaccharomyces pombe(15) , a (symmetrical) Ap
A hydrolase (Ap
A
2ADP; EC 3.6.1.41), e.g.Physarum
polycephalum(16) , a reversible Ap
A
phosphorylase (Ap
A + P
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
A-metabolizing enzymes, all these
enzymes will all act upon Ap
N compounds.
In view of the
availability of a possible additional mechanism for ApN
synthesis in firefly lanterns, it is clearly of interest to know
whether the rate of Ap
N synthesis in vivo is
higher in this tissue than in others and whether Ap
N
accumulates in lanterns. This is relevant not only to the physiological
function of Ap
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
N but an unusually high concentration of
(asymmetrical) Ap
A hydrolase.
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 (). The column was calibrated with the
following molecular mass standards (
): 1, conalbumin (77
kDa); 2, bovine serum albumin (67 kDa); 3, ovalbumin
(45 kDa); 4,
-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
A(19) , was sufficient to yield homogeneous enzyme (Table 1). A single polypeptide of M
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
A concentration
from 10 to 20 µM(19) were required to ensure that
the final preparation was free of contaminating adenylate kinase.
Firefly luciferase catalyzes the formation of ApN
from luciferyl-AMP and NTP in vitro. One may speculate,
therefore, that it may also do so in vivo. Ap
N has
indeed been detected in firefly lanterns; however, because of the
practical difficulties in measuring Ap
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 ApN 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
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
N in higher
eukaryotes are reported as picomoles/milligram protein or
picomoles/10
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
cells(28) . Drosophila cells
contain 1 pmol of Ap
A + Ap
G/10
cells(29) . Given that 1 pmol of Ap
N/10
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 ApA in vivo led us to investigate the
level of Ap
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
A, and substrate/product
specificity, including the inability to degrade Ap
A and
Ap
A, the firefly enzyme is a typical higher eukaryotic,
asymmetrically cleaving Ap
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
for
Ap
A of 4 µM and a K
for
p
A of 10 nM(31) .
The most notable
feature of the firefly lantern ApA 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(
), and 0.09 milliunits/mg
for lupin meal(26) . Extracts of Artemia cysts, which
contain an abundant store of the alternative substrate Gp
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.
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 (
)when extracted and assayed under our
standard conditions. Thus, we conclude that firefly lanterns display
10-100 times more Ap
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 ApN 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
N
synthesis.
In vivo, luciferase is located in the
peroxisomes(34) . However, ApN compounds (M
=
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
=
768)(35) . Maintenance of the normal steady state level of
Ap
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
A synthesis.