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J Biol Chem, Vol. 274, Issue 42, 29858-29861, October 15, 1999
Muscle as the Primary Site of Urea Cycle Enzyme Activity in an
Alkaline Lake-adapted Tilapia, Oreochromis alcalicus
grahami*
Timothy E.
Lindley ,
Cary L.
Scheiderer ,
Patrick J.
Walsh§,
Chris M.
Wood¶,
Harold L.
Bergman ,
Annie L.
Bergman ,
Pierre
Laurent¶,
Paul
Wilson¶, and
Paul
M.
Anderson **
From the Department of Biochemistry and Molecular
Biology, University of Minnesota-Duluth, Duluth, Minnesota 55812, the § Rosenstiel School of Marine and Atmospheric Science,
University of Miami, Miami, Florida 33149-1098, the
¶ Department of Biology, McMaster University, Hamilton, Ontario
L8S4K1, Canada, and the Department of Zoology and Physiology,
University of Wyoming, Laramie, Wyoming 82071
 |
ABSTRACT |
The tilapia fish Oreochromis alcalicus
grahami from Kenya has adapted to living in waters at pH 10.5 by
excreting the end product of nitrogen metabolism as urea rather than as
ammonia directly across the gills as occurs in most fish. The level of activity in liver of the first enzyme in the urea cycle pathway, carbamoyl-phosphate synthetase III (CPSase III), is too low to account
for the observed high rates of urea excretion. We report here the
surprising finding that CPSase III and all other urea cycle enzyme
activities are present in muscle of this species at levels more than
sufficient to account for the rate of urea excretion; in addition, the
basic kinetic properties of the CPSase III appear to be different from
those of other known type III CPSases. The sequence of the CPSase III
cDNA is reported as well as the finding that glutamine synthetase
activity is present in liver but not in muscle. This unusual form of
adaptation may have occurred because of the apparent impossibility of
packaging the needed amount of urea cycle enzymes in liver.
 |
INTRODUCTION |
Most teleostean fishes are ammonotelic, excreting nitrogen wastes
across their gills as ammonia (1, 2). An exception is a tilapia
(Oreochromis alcalicus grahami) from Lake Magadi in Kenya,
which has adapted to conditions of high pH (10-10.5) by using
ureotelism as a means of nitrogen excretion (3-5). However, the level
of activity in liver of the first enzyme in the urea cycle pathway,
carbamoyl-phosphate synthetase III (CPSase
III),1 is too low to account
for the observed high rates of urea excretion (3-5). We report here
the surprising finding that CPSase III and all other urea cycle enzyme
activities are present in muscle of this alkaline lake-adapted tilapia
at levels more than sufficient to account for the rate of urea
excretion; in addition, the basic kinetic properties of the CPSase III
appear to be different from those of other known type III CPSases. The
sequence of the CPSase III cDNA is reported as well as the finding
that GSase activity is present in liver but not in muscle.
In ureotelic terrestrial vertebrates the complete urea cycle pathway is
confined to liver and the first step is catalyzed by mitochondrial
CPSase I, which requires the allosteric effector AGA for activity and
utilizes only ammonia as the nitrogen-donating substrate; CPSase I is
thought to have evolved from CPSase III (6). CPSase III, found in
invertebrates and some fish, differs from CPSase I by the fact that it
can utilize glutamine as the nitrogen-donating substrate (6). In liver
of the two species of teleost fishes where expression of ureotelism has
been well documented, CPSase III is localized exclusively in the
mitochondrial matrix, as occurs in ureotelic terrestrial vertebrates,
but in contrast to ureotelic terrestrial vertebrates, GSase is also
localized entirely or partially in the mitochondria (7-11). CPSase III
and ornithine carbamoyltransferase, which catalyzes the second step of
the urea cycle, activities have recently been reported in muscle of
several different species of teleostean fishes (12-15), but the levels
of CPSase III activity are extremely low (e.g. 0.5-2 nmol/min/g of muscle), and the physiological significance, if any, has
not been established. These observations prompted us to examine muscle
as a site for urea cycle activity in the tilapia.
 |
EXPERIMENTAL PROCEDURES |
Lake Magadi tilapia (O. a. grahami) were captured by
seine net from Fish Springs Lagoon in Lake Magadi, Kenya in February 1997. Following a 20-min transport time to an on-site laboratory, fish
were anesthetized in metomidate, and tissues were rapidly dissected.
For studies of urea cycle enzymes, tissues were flash-frozen in liquid
nitrogen, transported to North America in a dry shipper, and kept at
70 °C until use.
Extracts for measuring units/gram of tissue of urea cycle enzymes in
frozen samples (Table I) were prepared and assayed as described
previously (7, 12), except tissue samples were smaller (0.2 g), and the
ATP-regenerating system was not used in the CPSase assay.
Subcellular localization studies (Table II) of GSase, glutaminase, and
arginase were conducted on-site using the isolation and assay methods
previously described (7), except homogenates were centrifuged at
4 °C at 82 × g for 5 min to pellet the debris fraction and the resulting supernatant at 13,800 × g
for 5 min to pellet the mitochondrial fraction. Glutaminase was assayed as described previously (16). lactate dehydrogenase and glutamate dehydrogenase were assayed as cytosolic and mitochondrial marker enzymes, respectively, as described previously (7).
Muscle CPSase III was partially purified and its molecular weight
estimated by gel filtration chromatography. Muscle tissue (0.6 g)
stored at 80 °C was homogenized with 2.5 ml of 0.1 M Hepes buffer, pH 7.5, containing 1 mM dithiothreitol, 0.1 M KCl, 0.1 mM EDTA, and 15% glycerol,
sonicated, and centrifuged as described previously (13). The
supernatant was applied to a Sephacryl S-300 column (0.8 × 25 cm)
equilibrated with the same buffer used to prepare extract and eluted at
4 °C in 1.2-ml fractions. The peak of enzyme activity (in three or
four fractions) was pooled and used for the kinetic studies. The
molecular size of the CPSase was estimated by comparison of the elution
volume with the elution volumes of cyanase and alcohol dehydrogenase
(molecular weights of 170,000 and 150,000, respectively), which had
been added to the extract before placing on the column.
Kinetic studies of the CPSase III (Table III) were conducted on enzyme
from muscle extracts that had been partially purified as noted above.
The reaction mixtures contained 10 mM glutamine or 10 mM NH4Cl, 25 mM MgCl2,
20 mM ATP, 5 mM
[14C]HCO3 (1 × 106 cpm), 50 mM Hepes buffer, pH 7.6, 50 mM KCl, and 0.8 mM AGA where indicated.
Km and Vmax values were
determined by measuring rates at different concentrations of ATP (5 mM excess MgCl2), AGA, NH4Cl, or
glutamine, respectively, and fitting the data to the Lineweaver-Burk
equation for double-reciprocal plots of rate versus
concentration with weighting to the fourth power; correlation coefficients were >0.97. Where the plots were not linear, the concentration that gave a half-maximal rate is cited for
Km.
SDS-PAGE was carried out essentially as described previously (17). For
sequencing, the protein bands were blotted to a polyvinylidene difluoride membrane and the N-terminal sequence of the tilapia muscle
band with the same mobility as rat CPSase I was determined by the Mayo
Protein Core Facility (Rochester, MN).
The sequence of the CPSase III cDNA was determined following
previously described procedures for RNA isolation (from tilapia muscle), cDNA synthesis, analysis and isolation of PCR products, strategy for obtaining CPSase III-specific cDNA segments by PCR using consensus primers and sequence analysis of these segments, the
strategy for sequencing the remainder of the CPSase III cDNA by
primer walking, and sequence analysis (12, 13). Numbering of amino acid
residues of the deduced CPSase III amino acid sequence begins with the
initial methionine residue of the entire translated product,
i.e. including the mitochondrial targeting sequence.
 |
RESULTS |
As shown in Table I, all five urea
cycle enzymes are present in both liver and muscle, but except for
arginase the levels are higher in muscle; this is particularly true for
CPSase III, which appears to be rate-limiting on the basis of maximal
activity. The levels of activity in muscle are surprisingly high,
comparable with the levels of these enzymes in liver of ureoosmotic
elasmobranchs (18) and the ureogenic teleost gulf toadfish
(Opsanus beta) (7, 19). Liver and muscle represent 2 and
60% of total mass, respectively,2 so the total
units of CPSase III in muscle are 130 times that of liver. The rate
of urea excretion in tilapia has been reported to be about 0.07 µmol/min/g of fish (3-5). Assuming saturating concentrations of
substrate for CPSase III, this rate can be readily sustained by the
level of either ammonia- or glutamine-dependent CPSase III
( 0.11 µmol/min/g of fish), but the level of CPSase III in liver
(<0.001 µmol/min/g of fish) is not nearly sufficient to account for
this rate of urea excretion.
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Table I
Urea cycle enzymes in muscle and liver of O. a. grahami
Values are means ± S.E. (n = 3). OCTase,
ornithine carbomyltransferase; ASSase, argininosuccinate synthetase;
ASLase, argininosuccinate lyase.
|
|
GSase -glutamyltransferase activity is present in liver, but there
is very little in muscle where high levels of CPSase III are localized
(Table I). In addition, the GSase in liver is localized in the cytosol
(Table II), analogous to largemouth bass
(Micropterus salmoides) where the liver mitochondrial
glutamine-dependent CPSase III appears to have little or no
function (13). Since GSase biosynthetic activity is 5% of the
transferase activity (7, 20), the biosynthetic GSase activity in liver
and muscle together ( 0.02 µmol/min/g of fish) is not sufficient to
support urea synthesis if urea-related carbamoyl phosphate formation
required glutamine.
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Table II
Enzyme activities in different subcellular fractions of O. a. grahami
liver
Values are means ± S.E. (n = 3). LDHase, lactate
dehydrogenase; GDHase, glutamate dehydrogenase; GLNase, glutaminase.
|
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Based on these results, we examined the properties of tilapia CPSase
III to determine its ability to use ammonia directly as
nitrogen-donating substrate. The general kinetic properties of the
muscle CPSase III are summarized in Table
III. In other type III CPSases: 1) the
Vmax with ammonia as substrate is 10% or less
than the Vmax with glutamine as substrate, 2)
AGA under normal assay conditions is required for activity, 3) the
binding of glutamine and AGA are synergistic (i.e. as the
concentration of one is increased, the Km for the
other decreases), and 4) the Km for glutamine is
0.1-0.2 mM (21, 22). In contrast, for the tilapia CPSase
III assayed with 20 mM MgATP: 1) the
Vmax with ammonia as substrate is greater
than the Vmax with glutamine, 2) AGA is not
required for activity with either ammonia or glutamine as
nitrogen-donating substrate and the presence of AGA increases
Vmax only slightly, 3) AGA does not affect the apparent Km for glutamine (or ammonia), and 4) the
Km for glutamine is quite high. Similar results are
obtained at 1 mM MgATP, except that the
Km for ammonia in the absence of AGA is lower (1.8 mM). The Km values of 2-5
mM for ammonia are comparable with those observed for the
ammonia-dependent activity of other type III (21, 22) and
type I (23) CPSases. Like the ureoosmotic spiny dogfish shark
(Squalus acanthias, a representative ureoosmotic
elasmobranch) CPSase III (21) and type I CPSases (23), however, AGA
does affect MgATP binding and, therefore, activity at physiological
concentrations of ATP. Marked sigmoid kinetics (nonlinear
double-reciprocal plots of rate versus MgATP concentration)
are observed when AGA is absent (half-maximal rate obtained at 0.7 mM MgATP), but hyperbolic Michaelis-Menten kinetics (linear
double reciprocal plots of rate versus MgATP concentration)
with glutamine (Km = 0.2 mM) as
substrate or greatly reduced sigmoid kinetics with ammonia
(half-maximal rate obtained at 0.2 mM) as substrate are
observed when AGA is saturating. Other properties of the tilapia CPSase
III include: activities with glutamine and ammonia are not additive as
is observed for the ammonia- and glutamine-dependent CPSase
activities in liver extracts from an Indian catfish
(Heteroptneustes fossilis) (24), which has invited
speculation that CPSase I and CPSase III activities may both be present
in H. fossilis; like all other CPSases, K+ is
required for activity (Km = 0.01 M); pH
optimum is 7.8; Km for AGA is <0.01 mM;
activity is not inhibited by UTP, a negative allosteric effector for
the pyrimidine pathway-related CPSase II (6); asparagine cannot replace
glutamine as substrate. When subjected to gel filtration
chromatography, CPSase activity eluted between alcohol dehydrogenase
and cyanase, corresponding to a maximum molecular weight of 160,000.
This estimated value is typical of all type I and type III CPSases (6)
and is in close agreement with the molecular weight of 160,760 calculated from the amino acid sequence (without the mitochondrial
leader sequence), indicating that the CPSase III exists as a monomer under the described gel filtration chromatography conditions.
The amino acid sequence of the tilapia CPSase III is homologous to
other CPSases, e.g. 86, 71, and 50% identity with CPSase III from largemouth bass (13), CPSase I from rat (25), and shark CPSase
II (26), respectively. The sequence has the same domain structure as
other CPSases and the same highly conserved histidine residues that
have been shown to have an important mechanistic function (6). The open
reading frame codes for 1,535 amino acid residues, the first 37 of
which correspond to an expected mitochondrial signal sequence which,
when removed, would yield a mature protein with the N-terminal sequence
FSVKTQTAHL; the sequence also has the expected cysteine residues
required for activity with glutamine (Cys-298) and binding of AGA
(Cys-1352 and Cys-1362) (6).
CPSase III from shark and smallmouth bass have turnover rates of 0.2
µmol/min/mg of protein at 26 °C with glutamine as substrate (21,
22). If the turnover rate of the tilapia CPSase III is similar, the
presence of 0.2 unit of activity/g of muscle would correspond to 1
mg of CPSase/g of muscle, which should be detectable in crude extracts
by SDS-PAGE. As shown in Fig. 1, a
protein band with the same mobility as rat CPSase I can be readily
detected in tilapia, but not in trout, which has very little CPSase III activity (12). The identity of this band as CPSase III was confirmed by
showing that the sequence of the first 10 amino acid residues of the
protein in this protein band was identical to the N-terminal sequence
predicted for the mature protein after removal of the mitochondrial
signal sequence as noted above, which also indicates that the enzyme is
probably localized in the mitochondria. When aliquots of fractions
obtained after gel filtration chromatography were subjected to
SDS-PAGE, the intensity of this protein band correlated exactly with
the units of CPSase III activity. Quantitation of the intensities of
the two bands (rat CPSase I and tilapia CPSase III) indicated that
there is at least 0.7 mg of CPSase III/g of tilapia muscle if staining
of the tilapia CPSase III is the same as rat CPSase I.

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Fig. 1.
SDS-PAGE of purified rat CPSase I and of
trout and tilapia extracts of muscle. Lane 1, molecular
weight standards. The amount of protein applied was 0.34 mg of rat
CPSase I and that amount of trout and tilapia extract, which
corresponded to 0.5 mg of muscle. The arrow indicates the
bands for CPSase.
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|
As noted in Table II, liver arginase, which catalyzes the last step of
the urea cycle pathway and the subcellular localization of which is
quite variable in teleosts (2, 7, 27), appears to be localized
predominantly in the cytosol in this tilapia. The level of glutaminase,
which is localized in the mitochondria as expected, is significantly
higher ( 20 times) than in O. beta, which appears to
utilize glutamine as substrate for the liver mitochondrial CPSase III
(7).
 |
DISCUSSION |
This is the first report of the presence of all five urea cycle
enzymes in muscle of fish or muscle of any species. The high levels of
all urea cycle enzymes in tilapia muscle reported here identify muscle
as the likely major site for urea formation in this fish. This finding
highlights the possible significance of recent reports of low levels of
CPSase III and ornithine carbamoyltransferase activities in muscle of
several other species of fish (12-15). The presence in muscle of a
urea cycle pathway that is normally expressed at high levels only under
specific environmental (3-5, 7-11) or life cycle (12) circumstances
may be a common characteristic of fish.
The properties of the tilapia CPSase III differ from other type III
CPSases, most notably by the facts that the enzyme has substantial
activity in the absence of AGA and that ammonia appears to be as good a
nitrogen-donating substrate as glutamine. The latter would be
consistent with the very low level of GSase activity in muscle and
insufficient levels in liver to provide glutamine for the CPSase III in
muscle. Thus, adaptation may also include kinetic changes that favor
ammonia as the primary substrate.
At the observed urea excretion rate, a 100-g tilapia fish would require
25 mg of CPSase III/g of liver if CPSase III was located only in
liver. CPSase I in liver of ureotelic mammalian species accounts for
25% of the total protein in the mitochondrial matrix and this is
equivalent to only 5 mg of CPSase I/g of liver (28). If the CPSase III
is located in the muscle, however, then the 50 mg CPSase III would be
distributed in 60 g of tissue, or about 0.8 mg/g of muscle, close
to the value reported here. We speculate that it may be impossible to
achieve the observed high rate of urea excretion by packaging the
required amount of urea cycle enzymes in the mitochondrial matrix of
the small liver in this species. Because these fish require a laterally
compressed body plan for efficient swimming, it is unlikely that
additional ureogenesis through increased liver mass could be
accommodated by the visceral cavity. Thus, it appears that adaptation
to ureogenesis in this species includes a change in organ localization
of the urea cycle enzymes.
 |
ACKNOWLEDGEMENT |
We thank S. Powers-Lee for the gift of
purified rat CPSase I.
 |
FOOTNOTES |
*
This work was supported by National Science Foundation
Grants IBN-9727741 (to P. M. A.) and IBN-9507239 (to P. J. W.) and by grants from the National Sciences and Engineering
Research Council of Canada (to C. M. W.) and the University
of Wyoming and the Fulbright Foundation (to H. J. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF119250.
**
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, School of Medicine, University of Minnesota-Duluth, Duluth, MN 55812. Tel.: 218-726-7921; Fax:
218-726-8014; E-mail: panderso@d.umn.edu.
2
P. J. Walsh, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
CPSase, carbamoyl-phosphate synthetase;
GSase, glutamine synthetase;
AGA, N-acetyl-L-glutamate;
PAGE, polyacrylamide gel
electrophoresis.
 |
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March 8, 2002;
204(12):
2145 - 2154.
[Abstract]
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B. F. Terjesen, T. D. Chadwick, J. A. J. Verreth, I. Ronnestad, and P. A. Wright
Pathways for urea production during early life of an air-breathing teleost, the African catfish Clarias gariepinus Burchell
J. Exp. Biol.,
March 8, 2002;
204(12):
2155 - 2165.
[Abstract]
[Full Text]
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K Iwata, M Kajimura, and T Sakamoto
Functional ureogenesis in the gobiid fish Mugilogobius abei
J. Exp. Biol.,
January 12, 2000;
203(24):
3703 - 3715.
[Abstract]
[PDF]
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Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.
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