ABSTRACT
Thermal adaptation at the biochemical level has been demonstrated in previous
studies of bony fishes and other vertebrates. One criterion that has been used to
assess the possible presence of thermal adaptation is the Michaelis-Menten constant,
or KK values have been shown to be conserved among teleosts and some other
vertebrates within their physiological temperature ranges. One possible mechanism
by which homologous proteins may conserve K at extreme temperatures is by
changes in primary structure. The relationship between temperature adaptation and
changes in primary structure has been explored in teleost fishes, but has yet to be
studied in elasmobranchs. In this study, the variation of pyruvate Kp with
temperature and the deduced amino acid sequences of four elasmobranch Aj-LDH
orthologs were compared. Both the subtropical and the cold temperate species of
sharks were found to display conservation of pyruvate Kp over physiological
temperature ranges, while the subtropical and the Antarctic species of rays did not.
The deduced amino acid sequences of the shark enzymes exhibited different regions
of variability than the LDH's of teleosts previously studied, while the sequences of
the ray enzymes exhibited similar regions of variability to those of teleost orthologs.
It was concluded that there may a limited number of sites at which temperature
adaptation in AA-LDH occurs, and that even closely related organisms do not
necessarily use the same sequence changes to adapt to temperature.
INTRODUCTION
Life occurs at a wide range of temperatures, from below zero to above 100 degrees
Celsius. Because life processes including development, mitosis, and membrane
function are affected by the thermal environment in which they occur, organisms
must adjust to their physiological temperature ranges. This adjustment must occur
at all levels, including the function of proteins. For example, because glycolysis is a
nearly universal biological process, glycolytic enzymes must function similarly in a
broad range of thermal environments. In affecting the chemical environment in
which a protein functions, temperature influences the assembly of subunits, global
protein stability, substrate binding and conformational changes during catalysis.
In order to compensate for the perturbing effects of temperature, proteins may
undergo changes in primary structure through evolution. These changes do not
have to be dramatic; substitutions are found outside the active site, in regions that
do not change the chemistry of catalysis, but affect the movement of catalytic
domains (Holland et al. 1997). Moreover, differences in stability and flexibility may
only consist of a glycine/alanine substitution (Fields and Somero, 1997). Such
changes have been explored in teleost fishes (Fields and Somero, 1997; Fields and
Somero in prep.), and the study of elasmobranchs may help us discover whether
evolutionarily distinct organisms use similar sequence changes in their enzymes to
adapt to their thermal environments.
Elasmobranchs provide an interesting study system not only because of their
evolutionary distance from bony fishes but also because of their unique
biochemistry. Elasmobranchs such as sharks and rays concentrate urea and
trimethylamine oxide (TMAÖ) in their body fluids in order to maintain osmotic
balance with the surrounding seawater. Although urea is a protein denaturant,
elasmobranch enzymes clearly function at physiological urea concentrations. They
may even require the presence of urea to exhibit enzyme kinetics comparable to
other organisms (Yancey and Somero 1978). However, it has also been demonstrated
that TMAÖ may counteract the effects of urea in some organisms (Yancey and
Somero 1980). The combination of elasmobranchs’ novel biochemistry and
evolutionary distance from teleosts makes the study of their temperature adaptation
at the macromolecular level of interest to biochemists, evolutionary biologists, and
comparative biologists.
Iused lactate dehydrogenase-A (Az-LDH) to explore the mechanism by which
homologous proteins might adapt to different thermal environments. Aj-LDH is
the enzyme that converts pyruvate and NADH to lactate and NADt during
anaerobic glycolysis. In order to describe temperature adaptation, I compared the
PYR)
sequences and apparent Michaelis-Menten kinetic constants of pyruvate (Kp
among four elasmobranch Az-LDH orthologs. Kn values are typically very sensitive
to temperature, usually rising as measurement temperature is increased. However,
in previous studies comparing various organisms' pyruvate Kp's over a range of
temperatures, it has been found that enzymes tend to conserve Kp at the
temperatures at which they function (Somero 1995, Holland et al. 1997). In this
study, changes in primary structure were compared with the different ways in which
PrR varied with temperature among the four species' LDH orthologs.
the Km
Knowledge of LDH 3-D structure (Abad-Zapatero et al. 1987) and conformational
changes during catalysis (Dunn et al. 1991) were used to interpret correlations
between kinetics and sequence variation, and to compare these correlations to
biochemical temperature adaptation in teleosts.
METHODS
Study Organisms
The four species of elasmobranchs used in this study were the nurse shark
Ginglymostoma cirratum (subtropical), the file tail cat shark Parmaturus xaniurus
(cold temperate), the cow nosed ray Rhinoptera bonasus (subtropical), and the
Antarctic bat skate Bathyraja eatonii.G. cirratum was collected in the Florida Keys,
and shipped on dry ice to Hopkins Marine Station by Elizabeth Clarke of the
University of Miami. P. xaniurus was collected from Monterey Bay Canyon and
donated to this study by the Monterey Bay Aquarium. R. bonasus was collected from
Tampa Bay, Florida, and sent on dry ice for the study by Joseph Torres of the
University of Southern Florida. B. eatonii was collected near Palmer Station in
Antarctica by Peter Fields of Hopkins Marine Station. All species were stored at -80°C
until use.
Determination of Km-PYR of A4-LDH
The Az-LDH of each species was purified using an agarose-oxamate affinity column
(Yancey and Somero 1978). A homogenate of the white muscle of each species was
allowed to pass down the oxamate affinity column, with NADH present in excess to
ensure efficient binding of the LDH. The LDH was eluted with pyruvate, which
binds the protein with a greater affinity than oxamate, and collected in fractions of
approximately 3ml each. The fractions were tested for LDH activity, and the active
fractions were pooled. Äfter dialysis against 50 mmol 11 potassium phosphate
buffer, pH 6.8, the purified protein was frozen at -80°C.
-PYR) was then determined
The apparent Michaelis-Menten constant of pyruvate (Km
at 0, 10, 20, and 30 degrees C for each species. The pyruvate Ky's were determined
using a Perkin Elmer Lambda 3B spectrophotometer equipped with a temperature-
controlled cell attached to a Lauda RM6 recirculating water bath. The temperature of
each cuvette was maintained at +/- 0.2°C. The reaction solutions were prepared
using an imidazole chloride buffer (160mM imidazole chloride, 300uM NADH; pH
7.0 at 20°C), which has been shown to exhibit similar pH to intracellular conditions
over a wide range of temperatures. For each Kp eight concentrations of pyruvate
were used: 0.5, 0.4, 0.3, 0.2 0.15 0.1 0.075, and 0.05 mmol 1-1. The spectrophotometer
was set to record the absorption of light with wavelength 340 nm, the wavelength at
which NADH absorbs most strongly. The decrease of [NADH] which followed the
addition of 25 ul of purified Az-LDH to the imidazole/NADH/pyruvate solution in
each cuvette was plotted against time. Conversion of absorbance to concentration
using Beer's law then yielded a series of velocities in units of change in
concentration of NADH (and thus pyruvate, as they decrease together in a 1:1 ratio)
per minute. Pyruvate concentration v. velocity data points were entered into the
Wilman computer program (1986) to calculate Kp using weighted linear regression
as outlined by Wilkinson (1961).
Sequencing of LDH-A CDNA
Messenger RNA (mRNA) was purified from each elasmobranch species using
microscopic magnetized beads (Dynabeads; Dynal, Inc.). 25-mers of poly¬
deoxythymidylate (poly-T) attached to the beads bound to the poly-adenylate tails of
the mRNA in a white muscle homogenate from each species. The beads were then
washed and the isolated mRNA eluted and stored at -80°C. Complementary DNA
(CDNA) was synthesized and the Az-LDH gene amplified using a Reverse
Transcriptase-Polymerase Chain Reaction (RT-PCR) procedure (Promega). LDH-A¬
specific primers were first designed using regions of agreement in barracuda (genus
Sphyraena; Holland et al. 1997) and dogfish (genus Squalus; Abad-Zapatero et al.
1987) LDH-A sequences. The 3' ends of the A4-LDH CDNA sequence were
synthesized and amplified using the 3'RACE procedure (GibcoBRL). AII CDNA
products were visualized using an ethidium-bromide stained agarose gel, and
purified using a gel extraction procedure (Qiagen). The CDNA sequence was obtained
by amplification of the purified CDNA products using a dye-labeled terminating
nucleotide protocol (Perkin-Elmer Corporation), and the amplification products
were visualized on the Applied Biosystems model 373A automated sequencer. The
resulting sequences were aligned and translated using the GCG software program
(Oxford Molecular Group).
RESULTS
Apparent Kp-PYR. Upon comparison of the two shark species, it was found that the
„PTR of the cold temperate species, P. xaniurus, was higher than that of the
Km
subtropical species, G. cirratum, at every temperature (Fig 1A). This trend is
consistent with earlier studies of temperature effects on Km in organisms adapted to
widely differing thermal environments (Yancey and Somero, 1978; Holland et al.
1997; Fields and Somero, 1997; Fields and Somero in prep.). Upon comparison of the
two ray species, however, R. bonasus, the warm-adapted species, exhibited higher
Knvalues at every temperature above 0°C when compared to the Antarctic species,
B. eatonii (Fig 1B). At 0°C, B. eatonii displayed a Km value slightly greater than that
of R. bonasus.
Amino acid sequences. There were fourteen differences among the 180 residues of
amino acid sequence obtained from the two species of rays. Of those fourteen
differences, four were non-conservative. (A non-conservative change is defined as
one in which the two residues were of different character, one being polar and one
hydrophobic, for example.) There were twenty-eight differences among the 220
residues of sequence obtained from both of the shark species, twelve of which were
non-conservative. The differences were distributed non-randomly throughout the
four A4-LDH sequences; regions of high conservation among all species include
residues 96-110, 155-180, 190-210, and 245-254 (Fig. 2). These regions include the
catalytic loop (aaf 96-113, Abad-Zapatero et al., 1987) and substrate and cofactor
binding residues (e.g., Arg107, Asp167, Arg170, His194, Thr249; Abad-Zapatero et al.,
1987; Sakowicz et al., 1993). [Please note that the residue numbers in this paper
correspond to the sequences I obtained; they are not identical to the sequence
numbering systems used in previous studies.
DISCUSSION
In analyzing these data on Kp and deduced amino acid sequences, I am attempting
to answer three questions: 1) Do the four elasmobranch Az-LDH orthologs exhibit
temperature-adaptive changes? 2) When compared to teleosts, are these
elasmobranch species using similar primary structure changes in their AA-LDH
sequences to adapt to temperature? 3) How might the primary structure changes
lead to the observed kinetic differences between the elasmobranch AA-LDH
orthologs studied?
Temperature adaptation as assessed by kinetic data. At every temperature of
measurement, the shark LDH ortholog of P. xaniurus, which experiences habitat
temperatures from 4° to 10°C (G. Somero, pers. comm.), exhibited higher Kp values
than the G. cirratum ortholog, which is found at temperatures ranging from 20° to
32°C (J. Torres, University of Southern Florida, pers. comm.). This pattern is
consistent with earlier studies which show that organisms tend to share comparable
Kn values within their physiological temperature ranges (Fields and Somero 1997,
Yancey and Somero 1978, Holland et al. 1997, Fields and Somero in prep.). Upon
comparison of the shark K patterns, it is apparent that the Kp values for each
shark within its temperature range are very similar (Fig. 1A).
The rays, in contrast, do not exhibit Kp conservation in this study. For each
temperature of measurement above zero, the LDH-A ortholog of B. eatonii, which
experiences habitat temperatures from -1.86° to 1°C (Eastman 1993), displays Km
values below those of the R. bonasus Az-LDH, which is found at temperatures
ranging from 20° to 32°C (. Torres, University of Southern Florida, pers. comm.)
(Fig. 1B). The lower Kp values, and corresponding higher pyruvate affinities of the
Antarctic species' Az-LDH homologue are puzzling. Because affinity rises (and Km
drops) as temperature decreases, one would expect that enzymes functioning at
colder temperatures would compensate by exhibiting lower intrinsic affinity (and
higher Kp) at any given temperature. One possible explanation for the rays' kinetic
data is that the presence of organic solutes such as urea and TMAO within the
organisms under physiological conditions alter the kinetics of the enzymes. It is
possible that the ray A4-LDH homologues display affinity (Kp) conservation under
physiological conditions.
Mechanisms of temperature adaptation in elasmobranchs and teleosts. I compared
the subtropical elasmobranch Az-LDH sequences to their cold habitat counterparts to
explore the primary structure differences that might occur in organisms adapted to
different temperatures. In the rays, non-conservative differences occurred with
higher frequency in two regions: the aH helix and the BH-alG loop (Fig. 2).
The first region of high variability between the two species of rays was the aH helix,
at the carboxy end of the monomer (aaf 311-327). Helix aH plays a significant part
during catalysis because aH, along with helix alG-a2G and the catalytic loop (aaft 97-
107) form the mouth of the molecule, moving toward one another during
formation of the internal vacuole during catalysis (Dunn et al. 1991) (Fig. 3).
The most dramatic sequence difference between the rays in the aH region is at the
amino end of the helix. A proline residue in the subtropical species is replaced by a
serine in the Antarctic species, a pattern that has also been shown to occur in
Antarctic teleosts (Fig. 2). Proline is a unique residue because its side chain is bonded
not only to the alpha carbon, but also to nitrogen, forming a five-membered ring.
This ring prevents proline from assuming a broad range of conformations.
Replacing a proline with a serine would therefore increase the flexibility of helix aH
relative to the rest of the molecule.
The second region of high variability between the rays was the BH-alG loop (aat
207-226). The BH-alG loop links part of the Aj-LDH active site (containing residues
His194, Asp167 and Arg172) to one part of the 'mouth' of the monomer, helix alG¬
a2G, which contains Tyr140, a residue required for enzyme activity. The ßH-alG
loop, which does not possess secondary structure, possesses a high temperature
factor (Abad-Zapatero et al. 1987) and may ease the movement of the alG-a2G helix
during conformational changes during catalysis (Fields and Somero in prep.) (Fig. 3).
Hence it is a possible source of adjustment for increased flexibility of catalytically
important regions in the enzyme.
In the ßH-alG loop, there are two non-conservative differences between the
subtropical and Antarctic ray species. In the first, a serine at position 211 in the
subtropical species (R. bonasus) is replaced by a glutamine in the Antarctic species (B.
eatonii). In the second, a glutamine at position 226 in the subtropical species is
replaced by an aspartate in the Antarctic species (Fig. 2). In both of these
substitutions, the Antarctic residue is more polar than the subtropical residue. This
increase in the polarity of the BH-alG loop has also been shown to occur in
Antarctic teleosts. Because ßH-alG forms hydrophobic interactions with the
neighboring subunit, increased polarity in this region might correspond to increased
flexibility of the monomer. This increased flexibility results when the interaction of
the loop with the neighboring monomer is reduced while interaction with the
surrounding polar solvent is increased. An increase in the flexibility of ßH-alG
might enable ease of movement of alG-a2G during catalysis. These changes should
favor a higher catalytic rate constant (kcat) for the enzyme of the Antarctic species, a
conjecture to be tested in future work with these enzymes.
In the sharks, the highest concentration of non-conservative amino acid
substitutions occurs from residue 9 to residue 21, near helix aA, in the amino
terminal arm of the Ag-LDH monomer (Fig. 3). This region of the monomer forms
polar inter-subunit interactions with the neighboring monomer (Abad-Zapatero et
al. 1987. In three of the four non-conservative changes I observed, a polar residue in
the subtropical shark was replaced by a charged residue in the cold temperate shark.
In the fourth non-conservative difference, a charged residue in the subtropical
species was replaced by a polar residue in the cold temperate species (Fig. 2). Because
I did not obtain the portion of sequence in G. cirratum which interacts with the
amino arm in neighboring monomers, it is not possible to analyze how these
changes in polarity of amino arm residues might affect the overall protein stability.
However, a single substitution at position 8 has been shown to lead to changes in
Km and thermal stability in barracuda A4-LDH (Holland et al. 1997). Therefore, it is
possible that the high variability within the shark LDH sequences in the amino
terminal arm contributes to their differences in kinetic data.
While analysis of the shark sequence differences is limited, it is noteworthy that the
shark species do not display high variability in the ßH-alG loop, as previously seen
in teleosts. It is also interesting that the two species of rays seem to use similar
primary sequence changes to the teleosts in thermal adaptation of their Aj-LDH
orthologs. Two conclusions may be drawn from these variability patterns: 1)There
may be a finite number of areas in the A4-LDH sequence that allow temperature
adaptation. 2)Elasmobranchs do not all use similar sequence changes to adapt to
different temperatures.
Flexibility and thermal adaptation of proteins. My analysis of the relationship
between proteins' primary sequence and temperature adaptation can be explained by
a theory developed by Fields and Somero (in prep.) as follows. At a given
temperature, a protein may exist in a number of different conformations. As the
temperature increases, the number of possible conformations and the rate at which
the protein changes between them increases. However, the amount of time spent in
any one conformation decreases as temperature increases. Thus the amount of time
spent in a conformation appropriate for binding decreases, and Kp increases.
Alternatively, increased flexibility of a protein due to primary structure changes can
also cause the protein to undergo conformational changes more rapidly. Thus
changes in the flexibility of a protein and changes in the temperature at which it
functions can cause similar (or counteracting) effects on the kinetic function of a
protein. Therefore, the perturbing effects of temperature may be compensated for by
varying the flexibility of the protein through changes in primary structure.
10
ACKNOWLEDGMENTS
Thank-you to George Somero and Peter Fields for their enduring patience and
willingness to teach. I would also like to thank Jonathon Stillman, Andy Gracey,
Lars Tomanek, and Rachel Ream for their kind tolerance.
LITERATURE CITED
Abad-Zapatero, C. et al. 1987. Refined Crystal Structure of Dogfish MA Apo-lactate
Dehydrogenase. J. Mol. Biol. 198:445-467.
Dunn, C. R. et al. 1991. Design and synthesis of new enzymes based on the lactate
dehydrogenase framework. Phil. Trans. R. Soc. Lond. 332:177-184.
Eastman, J. T. Antarctic Fish Biolo
y: Evolution in a Unique Environment. San
Diego: Academic Press Inc., 1993.
Fields, P. A. and Somero, G. N. 1997. Amino Acid Sequence Differences Cannot
Fully Explain Interspecific Variation in Thermal Sensitivities of Gobiid Fish A4
lactate Dehydrogenases (A-LDHs). J. exp. Biol. 200:1839-1850.
Holland, L. Z. et al. 1997. Evolution of Lactate Dehydrogenase-A Homologs of
Barracuda Fishes (Genus Sphyraena) from Different Thermal Environments:
Differences in Kinetic Properties and Thermal Stability Are Due to Amino Acid
Substitution Outside the Active Site. Biochemistry 36:3207-3215.
Sakowicz, R. 1993. Threonine 246 at the Active Site of the L-Lactate Dehydrogenase
of Bacillus stearothermophilus Is Important for Catalysis but Not for Substrate
Binding. Biochemistry. 32:12730-12735.
Somero, G. N. 1995. Proteins and Temperature. A. Rev. Physiol. 57:43-68.
Stock, D. W. and Powers, D. A. 1995. The CDNA sequence of the lactate
dehydrogenase-A of the spiny dogfish (Squalus acanthius): Corrections to the amino
acid sequence and an analysis of the phylogeny of vertebrate lactate dehydrogenases.
PYR) of A4-LDH from the shark
Fig. 1. (A) Apparent Michaelis-Menten constants (Kr
species G. cirratum and P. xaniurus. (B) Apparent Michaelis-Menten constants (Km¬
PYR) of A4-LDH from the ray species R. bonasus and B. eatonii.
Fig. 2. Deduced regions of amino acid sequences of LDH-A for the four
elasmobranch species studied, as well as the dogfish (S. acanthius) sequence (Stock
and Powers 1995). Residues in bold type indicate non-conservative differences
within the ray sequences or shark sequences. The text above the sequences indicates
the location of second degree structure (Abad-Zapatero et al. 1987).
Fig. 3. Cartoon of dogfish Az-LDH structure after Abad-Zapatero et al. (1987). One
monomer is shown with selected secondary structure labeled.
0.40 -
0.35
0.30
0.25
0.20
0.15
0.10
0.05 -
0.00
0.40
0.35
0.30
0.25 -
0.20
0.15
0.10 -
0.05
0.00
Fig. 1
—— Temp. V P. xaniurus
A
— Temp. v G. cirratum


5 10 15 20 25 30 35
Temperature (°C)
—0— Temp. V B. eatonii
+ Temp. V R. bonasus
B
I

5 10 15
20
25
30 35
Temperature (°C)
Fig. 2
-B—— — — — — — — — — ——
BA--
-A¬
-BB
SSS -SODKGS HNKXTIVGVG AVXMACAISI LMKDLADELA 50
P. xaniurus
S. acanthius
MATLKDKLIG HLATSQEPRS YNKITVVGVG AVGMACAISI LMKDLADEVA
S--G OLXTSOPGHS NNKITIVGVG AVGMACAISI LLKDLADEVA
G. cirratum
ssss sss  ssssss wewnnnnnwe
B. eatonii
wwwww
R. bonasus
—10 — — — — — — — — — — — — ———
8c---
pD---
P. X.
LVDVMEDKLK GEMMDLOHGS LFLOTSKIVS GKDYKVSAGS
KLVVITAGAR 100
S. a.
LVDVMEDKLK GEMMDLOHGS
LFLHTAKIVS GKDYSVSAGS KLVVITAGAR
G. c.
LVDVMEDKLK GEMMDLOHGS
LFLHTRKIVS GKDYSVSAGS KLVVVTAGAR
s ossssss sssssss ossssss ssssww
B. e.
Ph osssss ossssss osssss ssww
-(D----------GE------
-1F----
P. x. QOEGESRLNL VORNVNIFKH
IIPDIVKHSP NCIILVVSNP
VDILTYVAWK 150
QOEGESRLNI
S. a.
VORNVNIFKF IIPDIVKHSP DCIILVVSNI
VDVLTYVAWK
QQEGESRLNL VORNVNIFKH IIPDIVKNSP DCIILVVSNP
G. c.
VDXLTYVAWK
s sssss wosssss o TVK
B. e.
Wsssssss o  s wwwuwwwa
R. b.
-2F-----------
-BG---
P. X.
FRYLMGERLG
IHSSSCHAWV IGEHGDSSVP 200
LSGLPMHRVI GSGCNLDSAR
S. a.
IGEHGDSSVP
LSGLPMHRII
GSGCNLDSAR FRYLMGERLG
VHSSSCHGWV
IGEHGDSSVP
G. c
LSGLPMHRVI
GSGCNLDSAF
FRYLMGERLG IHSSSCHAWV
FRYLMGERLG IHSSSCHGWV IGEHGDSSVP
B. e.
LSGLPMHRVI
GSGCNLDSAR
R. b.
LSGFXMHRVI
GSGCNLDSAR
FRYLMGERLG IHSSSCHGWV IGEHGDSSVP
-a1G—----26—- ----
-83-
SLKEIHPDIG TEKDKENWKK VHKDVVDSAY EVIKLKGYTS 250
VWSGMNVAGV
P. X.
TDKDKENWKK LHKDVVDSAY EVIKLKGYTS
VWSGMNVAGV SLKELHPELG
TDHDKENWKA IHKE
G. c
VWSGMNVAGV NLKELHPEIC
TDKDKEDWKK VHRDVVDSAY EVIKLKGYTS
VWSGMNVAGV NLKELHPEIG
VWSGMNVAGV SLKELHPEIG TDKDKENWKK VHKDVVDSAY EVIKLKGYTS
-BK-
-B1-
-03G-
P. X. WAIGMSVADL
FHGIKNDVFL SLPCVLDNHG 300
GETIMKNLCK VHPISTMVKD
S. a.
WAIGLSVADL AETIMKNLCR VHPVSTMVKD FYGIKNDVFL SLPCVLDNHG

sssss sss wonnn
G. C.
WAIGMSVADM AETLMKNLRR VHPVSTMVKN
FYGITNDVFL SLPSVLDNHG
B. e.
WAIGMSVADL AETIMKNLRR VHPISTMVKT FYGITNEVFL SLPSVLDNNG
R. b.
-BM--
-H--
ITNVVKMVLK PEEENQLQOS ATTLWDIQKD L va 350
P. X.
ISNIVKMKLK PDEEQOLOKS ATTLWDIQKD LKF'TSPL'P SYAAVTSMII
s. a.
s wos
ISNVVKMTLK SEEEAKLOOS ATTLWNIQKD LKFS e
R. b. ISNIVKMTLK PDEEAKLOHS AT
O
L
(