International Journal of

Molecular Sciences

Article
Enzymatic Kinetic Properties of the Lactate
Dehydrogenase Isoenzyme C4 of the Plateau
Pika (Ochotona curzoniae)
Yang Wang, Lian Wei, Dengbang Wei *, Xiao Li, Lina Xu and Linna Wei
Received: 7 September 2015; Accepted: 21 December 2015; Published: 7 January 2016
Academic Editors: Christo Z. Christov and Mihai V. Putz

Research Center for High Altitude Medicine, Qinghai University, Xining 810016, China;
yangwangmd@163.com (Y.W.); weilian5318@163.com (L.W.); lixiao2234566@163.com (X.L.);
xu020916@163.com (L.X.); weilinnanana@163.com (L.W.)
* Correspondence: weidengbang@163.com; Tel./Fax: +86-971-531-0695

Abstract: Testis-specific lactate dehydrogenase (LDH-C4 ) is one of the lactate dehydrogenase (LDH)
isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C4
in mammals was previously thought to be expressed only in spermatozoa and testis and not in other
tissues. Plateau pika (Ochotona curzoniae) belongs to the genus Ochotona of the Ochotonidea family. It is
a hypoxia-tolerant species living in remote mountain areas at altitudes of 30005000 m above sea level
on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also
in somatic tissues of plateau pika. To shed light on the function of LDH-C4 in somatic cells, Ldh-a,
Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes
of Lactate Dehydrogenase A4 (LDH-A4 ), Lactate Dehydrogenase B4 (LDH-B4 ), and LDH-C4 were
prepared by a series of expression and purification processes, and the three enzymes were identified
by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native
polyacrylamide gel electrophoresis (PAGE). The enzymatic kinetics properties of these enzymes were
studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km)
of LDH-C4 for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate
90 times higher affinity of LDH-C4 for pyruvate than for lactate. At relatively high concentrations of
lactate, the inhibition constant (Ki) of the LDH isoenzymes varied: LDH-A4 (Ki = 26.900 mmol/L),
LDH-B4 (Ki = 23.800 mmol/L), and LDH-C4 (Ki = 65.500 mmol/L). These data suggest that inhibition
of lactate by LDH-A4 and LDH-B4 were stronger than LDH-C4 . In light of the enzymatic kinetics
properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its
adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C4 .

Keywords: testis-specific lactate dehydrogenase (LDH-C4 ); enzymatic kinetics; plateau pika

(Ochotona curzoniae); hypoxia; Qinghai-Tibet Plateau

1. Introduction
The Qinghai-Tibet Plateau, with an average elevation of over 3000 m above sea level, is the highest
and also largest plateau on earth and is characterized by extremely harsh environmental conditions
such as hypoxia, strong ultraviolet radiation, and cold temperature which expectedly have profound
effects on human and animal survival. Over long-term adaption and evolution, both plateau-native
humans and animals have developed a series of strategies to adapt to the plateau environment. Also,
inhabitants have developed some unique features to avoid disadvantageous environmental aspects
and effectively obtain material or energy from their survival environment as to ensure their normal
growth and breeding.

Int. J. Mol. Sci. 2016, 17, 39; doi:10.3390/ijms17010039 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2016, 17, 39 2 of 14

Plateau pika (Ochotona curzoniae), which is a member of the genus Ochotona of the Ochotonidae
family, is a small, non-hibernating rodent that lives in remote mountain areas at an elevation of
30005000 m on the Qinghai-Tibet Plateau. The pika plays an important role in biodiversity of
the ecosystem on the plateau and is regarded as a key species since ancient times [1,2]. Fossil
samples of pika that are nearly 37 million years old were found by archaeologistson the north
edge of the Qinghai-Tibetan plateau [3]. During long-term evolution, the pikas evolved a series
of physiological adaptations that allow them to thrive in the harsh environment and become a highly
advanced hypoxia-tolerant mammal. Specifically, the pika obtained oxygen effectively from the
hypoxic environment by larger superficial pulmonary alveoli and higher capillary density [4], thin
walled pulmonary arterioles and blunted hypoxic pulmonary vasoconstriction (HPV) [5], an increase
in erythrocyte count [6], reduction in the mean corpuscular volume [7], changes in hemoglobin
(Hb) [8], and 2,3-diphosphoglycerate concentrations [5], and an increase in the oxygen affinity to
Hb [8]; Secondly, a pika has a strong cardiac pumping function due to its larger heart and smaller
weights of right-to-left ventricular plus septum [9]; Thirdly, a pika has a high oxygen utilization
ratio by increasing the densities of capillary and mitochondrial [10], and myoglobin concentration
in tissues [6,9]. In addition to these physiological mechanisms, a pika reduces dependence on
oxygen by increasing anaerobic glycolysis in its skeletal muscle [11] and gluconeogenesis in liver [12].
The molecular mechanisms of these adaptations in pika have occurred due to a series of changes
such as genetic evolution, the expression of tissue-specific proteins, and changes related to high
altitude, including many functional cytokines as vascular endothelial growth factor (VEGF) [13,14],
hemoglobin [3], HIF-1 [15,16], LDH-C4 [17], pyruvate carboxylase [12], myoglobin [9], inducible
nitric oxide synthase (iNOS) [18], leptin [19,20] and cytochromec oxidase [21].
It is well known that LDH family enzymes catalyze the inter-conversion of pyruvate and lactate
with the concomitant oxidation/reduction of the reduced form of nicotinamide adenine dinucleotide
hydrogen (NADH) to nicotinamide adenine dinucleotide (NAD+ ) in the reactions [22]. Different forms
of Lactate dehydrogenase (LDH) isozymes are comprised of expression products of three typical genes:
Ldh-a, Ldh-b, and Ldh-c, which encode LDHA, LDHB and LDHC subunits, respectively [23,24]. LDH
consists of A and B subunits that assemble into homo- or hetero- tetramers. These A and B subunits are
distributed in various combinations in the body reflecting various energy metabolic requirements of
different tissues, and are consistent with the catalytic properties of each isozyme [25,26]. However, the
homo-tetramer LDH-C4 was previously detected only in testis and spermatozoa of mammals [2730].
Surprisingly, our previous studies identified the expression of Ldh-c not only in testis and sperm, but
also in somatic cells of plateau pika [17].
LDH-C4 has high thermostability [17], and its homolog from different species showed activity
against longer carbon chain with -hydroxy and -keto acids than those of pyruvate and lactate [3141],
indicating that LDH-C4 has unique structural and functional properties [31,4042]. In the present
study, we compared the enzymatic kinetic characteristics among LDH-A4 , LDH-B4 , and LDH-C4 of
plateau pika with the aim of exploring the pikas adaptation mechanism to the hypoxic environment
of the Qinghai-Tibet Plateau.

2. Results

2.1. Plasmid Construction and Recombinant Protein Expression

To study the enzyme kinetics of LDH-A4 , LDH-B4 , and LDH-C4 of plateau pika, the expression
plasmids pCold-SUMO-Ldh-a, pET-30a-Ldh-b, and pET-30a-Ldh-c, respectively encoding the full-length
pika LDHA, LDHB, and LDHC were constructed. In order to verify that the vectors contained the
sequences of Ldh-a, Ldh-b, and Ldh-c and their correct expression, Polymerase Chain Reaction (PCR) of
the plasmids and sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) analysis of
the expressed proteins were performed. Figure 1AC show the agarose gel electrophoresis results of
PCR products of Ldh-a, Ldh-b, and Ldh-c, respectively. Lane 1 in Figure 1AC displayed clear and bright
Int. J. Mol. Sci. 2016, 17, 39 3 of 13
Int. J. Mol. Sci. 2016, 17, 39 3 of 14

sequences
Int. J. Mol. Sci.of Ldh-a,
2016, 17, 39Ldh-b
and Ldh-c in the final plasmid constructs were determined to be 999 3 ofbp,
13
1005bands
bp and at approximately
999 bp in length1000 and
bp, matching
identicaltheto same
thosenumber
in the of base pairs
Genbank of Ldh-a (999HQ704677
(HQ704676, bp), Ldh-b and
sequences
(1005 of
bp), Ldh-a,
and Ldh-b
Ldh-c
HQ704678, respectively). (999 and
bp) asLdh-c
our in the
previous final
study plasmid
[17]. By constructs
sequencing were
and determined
alignment, the to be 999
sequences of bp,
1005Ldh-a,
bp andLdh-b999
andbp in in
Ldh-c length and
the final identical
plasmid to those
constructs wereindetermined
the Genbank (HQ704676,
to be 999 bp, 1005 bpHQ704677
and 999 bp and
in length and identical
HQ704678, respectively). to those in the Genbank (HQ704676, HQ704677 and HQ704678, respectively).

Figure 1. Amplification of plateau pika Ldh-a, Ldh-b and Ldh-c. Lane 1: PCR products of (A) Ldh-a;
(B) Ldh-b; and (C) Ldh-c. M: DNA marker. The PCR fragments of Ldh-a, Ldh-b, and Ldh-c were 999 bp,
Figure bp1.and
1005Figure Amplification
1.999 bp. The of
Amplification plateau
sequence
of ofpika
plateau pikaLdh-a,
Ldh-a, Ldh-b
transcripts andLdh-c.
Ldh-c.
was identical
Ldh-b and Lane1:
toLane
that in1:PCR
PCR
the products
GenBank
products of
for(A)
of (A)
Ldh-a, Ldh-a;
Ldh-b,
Ldh-a;
(B)
andLdh-b;
Ldh-c, and
(B) Ldh-b; (C) (C)
and Ldh-c.
suggesting M:the
Ldh-c.
that DNA
M: DNA marker. The
ThePCR
marker.plasmids
expression fragments
fragments ofofLdh-a,
PCRpCold-SUMO-Ldh-a, Ldh-a, Ldh-b,
Ldh-b, andand Ldh-c
Ldh-c
pET-30a-Ldh-b, werewere
and999 999 bp,
bp,
pET-30a-
1005 bpencoding
Ldh-c1005 and 999999
bp and bp. The
bp.
the Thesequence of transcripts
sequencepika
full-length transcripts
LDHA, was was identical
identical
LDHB to that
andtoLDHC,
that inGenBank
the GenBank forLdh-b,
for Ldh-a,
in therespectively, were Ldh-a, Ldh-b,
and
successfully
and Ldh-c, suggesting that the expression plasmids pCold-SUMO-Ldh-a, pET-30a-Ldh-b,
Ldh-c, suggesting that the expression plasmids pCold-SUMO-Ldh-a, pET-30a-Ldh-b, and pET-30a-
constructed. and pET-30a-Ldh-c
Ldh-cencoding
encoding the full-length pika LDHA,
the full-length pika LDHB
LDHA, andLDHB
LDHC,andrespectively,
LDHC, were successfully
respectively, constructed.
were successfully
constructed.
The SDS-PAGE results of cell-free extracts (shown in Figure 2AC) displayed the protein in the
The SDS-PAGE results of cell-free extracts (shown in Figure 2AC) displayed the protein in
supernatant and precipitate fractions with dark bands around 4050 kD. The recombinant LDH
the supernatant
The SDS-PAGE and precipitate
results ofwith fractions
cell-free with dark
extracts bands around
(shown 4050 kD. The recombinant LDH
isoenzymes were expressed His-tag (LDHA with inHis-tag
Figureand 2AC) displayed
SUMO-tag) inthetheprotein in the
C-terminus,
isoenzymes
supernatant were expressed
and precipitate with His-tag
fractionsThese (LDHA
withresults with
dark bands His-tag and
around similar SUMO-tag)
4050 kD. in the C-terminus,
The recombinant LDH
which was favorable to purification. demonstrated molecular
which was favorable to purification. These results demonstrated similar molecular weights to those
weights to those
isoenzymes
of LDHA, were
LDHB, expressed
and LDHC, with His-tag (LDHA with His-tag and SUMO-tag) in the C-terminus,
of LDHA, LDHB, and LDHC,asaswe wepreviously reported[17],
previously reported [17],indicating
indicating that
that expression
expression plasmids
plasmids
which was favorable
pCold-SUMO-Ldh-a, to purification.
pET-30a-Ldh-b These results demonstrated similar molecular weights to those
pCold-SUMO-Ldh-a, pET-30a-Ldh-b and pET-30a-Ldh-c
and pET-30a-Ldh-c werewere successfully
successfully prepared,
prepared, and expressed
and expressed the
of
theLDHA,
proteinsLDHB,
proteins andcorrectly.
of interest
of interest LDHC,
correctly. as we previously reported [17], indicating that expression plasmids
pCold-SUMO-Ldh-a, pET-30a-Ldh-b and pET-30a-Ldh-c were successfully prepared, and expressed
the proteins of interest correctly.

Figure 2. Analysis
Figure 2. Analysisof the cell-free
of the cell-freeextracts
extractsrecombinant proteinby
recombinant protein bySDS-PAGE.
SDS-PAGE. (A)(A) LDHA;
LDHA; (B) LDHB;
(B) LDHB;
and and
(C) (C)
LDHC.
LDHC.For (AC)Lane
For (AC) Lane 1 Lane
1 and and 2Lane 2 represent
represent the total
the total protein protein of
of supernatant andsupernatant
precipitation, and
Figure 2. Analysis
respectively,
precipitation, of E.
in the thecoli
respectively, cell-free
BL21
in the extracts
(DE3)
E. cells;
coli recombinant
M:
BL21 molecular protein
weight
(DE3) cells; by SDS-PAGE.
standards.
M: molecular weight (A)
SDS-PAGE LDHA;
analysis
standards. (B) LDHB;
indicates
SDS-PAGE
and (C) indicates
LDHA,
analysis LDHC.
LDHB For and
LDHA, (AC)
LDHC Lane
expression
LDHB and1 LDHC
and Lane 2 represent
in supernatant and in
expression the total
precipitate protein
fractions.
supernatant and of supernatant
precipitate fractions. and
precipitation, respectively, in the E. coli BL21 (DE3) cells; M: molecular weight standards. SDS-PAGE
2.2.
2.2. LDH-ALDH-A
analysis 4 , LDH-B
4, indicates
LDH-B 4 and
4 LDHA,
and LDH-C
LDH-CLDHB Purification
4and LDHC and
4 Purification
andActivity
Activity
expression Measurement
inMeasurement
supernatant and precipitate fractions.
The native polyacrylamide gel electropheresis (native PAGE) results shown in Figure 3P indicate1 Figure 3P1
The native
2.2. LDH-A polyacrylamide gel electropheresis (native PAGE) results shown in
4, LDH-B4 and LDH-C4 Purification and Activity Measurement
that LDH-A 4 was observed in 100, 250 and 500 mmol/L imidazole buffer elutions with LDH activity,
indicate that LDH-A4 was observed in 100, 250 and 500 mmol/L imidazole buffer elutions with LDH
and
TheLDH-B
native 4 and LDH-C4 were observed
polyacrylamide in 250 and 500 (native
gel electropheresis mmol/LPAGE)
imidazole buffersshown
results elutions in
with LDH 3P1
Figure
activity, and LDH-B 4 and LDH-C4 were observed in 250 and 500 mmol/L imidazole buffers elutions
activity. LDH-A4 , LDH-B4 and LDH-C4 bound so completely to Ni-NTA that no active protein was
indicate thatactivity.
withassayed
LDH LDH-ALDH-A4 was observed in 100, 250 and 500 mmol/L imidazole buffer elutions with LDH
4, LDH-B4 and LDH-C4 bound so completely to Ni-NTA that no active
in the remainder of the purified supernatant. As shown in Figure 3P2 , the purified LDH-A4 ,
activity,
protein and LDH-B
was assayed 4 and LDH-C4 were observed in 250 and 500 mmol/L imidazole buffers elutions
in the remainder of with
the purified
LDH-B4 , and LDH-C4 , were represented only one supernatant.
band as observedAs by
shown in Figure
SDS-PAGE, and 3P
the2, the
with LDH
purified
molecular
activity.
LDH-A weights
LDH-A
4, LDH-B
4, LDH-B4 and LDH-C4 bound so completely to Ni-NTA that no active
were4,consistent
and LDH-C 4, LDHA,
with were represented with only
LDHB, and LDHC, one bandFigure
respectively. as observed by SDS-
3P3 presents
protein
PAGE, was
the and
LDHthe assayed in
molecular
activity the remainder
weightsLDH-A
of the purified of the purified
were consistent
4 , LDH-B4 , with
supernatant.
LDHA,
and LDH-C As
LDHB,
4 as
shown in
and LDHC,
determined Figure
by native 3P2, the
respectively.
PAGE.
purified
Figure 3PLDH-A 4, LDH-B4, and LDH-C4, were represented with only one band as observed by SDS-
3 presents the LDH activity of the purified LDH-A4, LDH-B4, and LDH-C4 as determined by
PAGE, and the
native PAGE. These molecular weights
results were
indicate thatconsistent
LDH-A4,with LDHA,
LDH-B LDHB, and LDHC, respectively.
4, and LDH-C4 of plateau pika were
Figure 3P3 presents
successfully purified.theAsLDH
shownactivity of the4,purified
in Figure LDH-Aenzyme
the respective 4, LDH-B4, and LDH-C4 as determined by
activity (n = 5) of LDH-A4, LDH-
native PAGE. These results indicate that LDH-A 4, LDH-B4, and LDH-C4 of plateau pika were
B4, and LDH-C4 was 2241 9.3 U/g protein, 3918 37.9 U/g protein, and 10,741 80.9 U/g protein,
successfully purified. As shown in Figure 4, the respective enzyme activity (n = 5) of LDH-A4, LDH-
 Int. J. Mol. Sci. 2016, 17, 39 4 of 14

These results indicate that LDH-A4 , LDH-B4 , and LDH-C4 of plateau pika were successfully purified.
As shown in Figure 4, the respective enzyme activity (n = 5) of LDH-A4 , LDH-B4 , and LDH-C4 was
2241 9.3 U/g protein, 3918 37.9 U/g protein, and 10,741 80.9 U/g protein, and that of LDH-C4
was significantly higher than that of LDH-A4 and LDH-B4 (p < 0.01).
Int.J.J.Mol.
Int. Mol.Sci.
Sci.2016,
2016,17,
17,39
39 44of
of13
13

Figure 3.3. Purification

Figure Purification and identification
identification of of LDH-A
LDH-A4, LDH-B LDH-B44, , and and LDH-C
LDH-C44. . (P (P11)) Native
Native PAGEPAGE of of
Figure 3. Purificationand and identification of LDH-A44,, LDH-B 4 , and LDH-C4 . (P1 ) Native PAGE of
recombinant
recombinant
recombinantproteinprotein washed
proteinwashed
washedby byimidazole
by imidazoleat
imidazole atdifferent
at differentconcentrations.
different concentrations.
concentrations. Lane
Lane
Lane 1:1:1: the
the
the supernatant
supernatant
supernatant before
before
before
purification,
purification, Lane 27: collected elutions from Ni-NTA resins eluted
purification, Lane 27: collected elutions from Ni-NTA resins eluted with 10, 20, 50, 100, 250 and500
Lane 27: collected elutions from Ni-NTA resins eluted with
with 10,
10, 20,
20, 50,
50, 100,
100, 250
250 and
and 500
mmol/L
mmol/L
500 mmol/L imidazole
imidazole buffers,
buffers,
imidazole respectively.
respectively.
buffers, Lane8:8:Lane
Lane
respectively. theremainder
the remainder
8: the remainder inthe
in thepurified
purified supernatant;
supernatant;
in the purified (P22))SDS-
(P
supernatant; SDS-
PAGE
PAGE analysis of
analysis
(P2 ) SDS-PAGE ofanalysis
the purified
the purified LDH-A
of theLDH-A
purified44, , LDH-B
LDH-B444, ,, and
LDH-A and
LDH-B LDH-C
LDH-C
4 , and44. . Lane
Lane
LDH-C 13
13
4 . represented
represented
Lane 13 theLDH-A
theLDH-A
represented 44, ,

LDH-B
LDH-B , and LDH-C
4, and4 ,LDH-C
theLDH-A4 , respectively;
LDH-B44, respectively;
4 and LDH-C4 ,M: M: marker;
marker; (PM:
respectively; (P ) Identification
3) Identification
3 of purified
of purified LDH-A
marker; (P3 ) Identification LDH-A , LDH-B
4, LDH-B
of purified4 LDH-A ,
4, 4
4 and
and
,
LDH-C
LDH-B
LDH-C 44. .4Lane
Lane
, and11LDH-C
represents
represents 4 . Lane(A)LDH-A
(A) 1LDH-A 44; ;(B)
represents (B)(A)
LDH-B
LDH-BLDH-A 4,4,and
and (C)LDH-B
4 ; (B)
(C) LDH-C
LDH-C byNative
44,4by
and Native
(C) LDH-C polyacrylamide
4 by Nativegel
polyacrylamide gel
polyacrylamide
electrophoresis
electrophoresis gel electrophoresis
(PAGE),
(PAGE), respectively.
respectively. (PAGE),
M:marker
M: respectively.
marker wasprepared
was M: marker
prepared bymixed
by was prepared
mixed kidneyand
kidney by mixed
and skeletal
skeletal kidney
muscle
muscle
and skeletal
tissues
tissues ofplateau
of muscle
plateau tissues of plateau pika.
pika.
pika.

Figure
Figure4.4.4.The
Figure The enzyme
Theenzyme activities of
enzymeactivities
activities ofLDH-A
of LDH-A4,,4,LDH-B
LDH-A LDH-B44and
LDH-B andLDH-C
and LDH-C.44.The
LDH-C . The
Theenzyme
enzyme
enzyme activity
activity
activity of LDH-A
of of LDH-A
LDH-A 44, ,
4 4 4 4,
LDH-B
LDH-B ,
4 , and
and LDH-C
LDH-C 4 was
was 2241
2241 9.3
9.3 U/g
U/g protein,
protein, 3918
3918 37.9
37.9 U/g
U/g protein,
protein, 10741
10741
LDH-B4 , and LDH-C4 was 2241 9.3 U/g protein, 3918 37.9 U/g protein, 10741 80.9 U/g protein,
4 4 80.9
80.9 U/g
U/g protein,
protein,
each
each
each sample
sample
samplesizesize was
was5,5,
sizewas **ppp<<< 0.01.The
5, **
** 0.01.The
0.01. enzymeactivity
The enzyme
enzyme activityof
activity ofLDH-C
of LDH-C
LDH-C was
4 was
4 4was
significantly
significantly
significantly higher
higher
higher that
that
that of
of of
LDH-A
LDH-A
LDH-A and
44and
4 andLDH-B
LDH-B
LDH-B 44 4(p
(p <
(p< 0.01).
0.01).
< 0.01).

2.3.
2.3. Enzyme
Enzyme
2.3. Kinetics
Kinetics
Enzyme Properties
Propertiesofof
KineticsProperties LDH-A444,,,LDH-B
ofLDH-A
LDH-A LDH-B444,,,and
LDH-B andLDH-C
and LDH-C444
LDH-C
The
The Km
TheKm values
Kmvalues for LDH-A
values for LDH-A444,,, LDH-B
LDH-A LDH-B444 andandLDH-C
and LDH-C
LDH-C weredetermined
were
444were determinedfrom
determined from
from Lineweaver-Burk
Lineweaver-Burk
Lineweaver-Burk
double-reciprocal
double-reciprocal plots.
plots. The
The activities
activities
double-reciprocal plots. The activities of
of the
the LDH
LDH isoenzymes
isoenzymes inin forward
forward reaction
reaction (pyruvate
(pyruvate
LDH isoenzymes in forward reaction (pyruvate ++NADH
NADH
+ NADH
++HH
++ lactate
lactate++NAD
NAD))increased
++ increasedwithwithincreasing
increasingconcentration
concentrationof ofthe
thesubstrate
substratepyruvate,
pyruvate,as asshown
shown
in Figure 5. The kinetic parameters of the forward reaction for LDH-A
in Figure 5. The kinetic parameters of the forward reaction for LDH-A4, LDH-B4, and LDH-C44,,
4 , LDH-B 4 , and LDH-C
calculatedfrom
calculated fromthe
theLineweaver-Burk
Lineweaver-Burkdouble-reciprocal
double-reciprocalplots
plotsof
ofreciprocal
reciprocalpyruvate
pyruvateconcentration
concentrationvs. vs.
reciprocal velocity, had Km values of 0.260, 0.172, and 0.052 mmol/L, respectively.
reciprocal velocity, had Km values of 0.260, 0.172, and 0.052 mmol/L, respectively. The activities of The activities of
LDH isoenzymes
LDH isoenzymesin in reverse
reverse reaction
reaction (lactate NAD+
(lactate++ NAD + pyruvate
pyruvate ++ NADH
NADH ++ H H+)) also
+ also increased
increased with
with
Int. J. Mol. Sci. 2016, 17, 39 5 of 14

+ H+ lactate + NAD+ ) increased with increasing concentration of the substrate pyruvate, as shown
in Figure 5. The kinetic parameters of the forward reaction for LDH-A4 , LDH-B4 , and LDH-C4 ,
calculated from the Lineweaver-Burk double-reciprocal plots of reciprocal pyruvate concentration
vs. reciprocal velocity, had Km values of 0.260, 0.172, and 0.052 mmol/L, respectively. The activities
of LDH isoenzymes in reverse reaction (lactate + NAD+ pyruvate + NADH + H+ ) also increased
with increasing concentration of the substrate lactate, as shown in Figure 6. The kinetic parameters
of the reverse reaction for LDH-A4 , LDH-B4 , and LDH-C4 , calculated from the Lineweaver-Burk
double-reciprocal plots of reciprocal lactate concentration vs. reciprocal velocity, had Km values of
19.968,
Int. J. Mol.8.980 and17,
Sci. 2016, 4.934
39 mmol/L, respectively. 5 of 13

5.Double
Figure 5. Doublereciprocal
reciprocal plots
plots by
by pyruvate
pyruvate as as substrate of plateau pika LDH LDH isozymes.
isozymes. Double
reciprocal plots
plots of
of(A)
(A)LDH-A
LDH-A4;4(B)
; (B)LDH-B
LDH-B 4 ; and
4; and (C)(C) LDH-C
LDH-C 4 , respectively.
4, respectively. Reciprocal
Reciprocal velocity
velocity (V)
(V) was
was calculated
calculated withwith
the the reciprocal
reciprocal 340
340 nm/min,
nm/min, plots
plots werepreformed
were preformedby bycalculating
calculating the the reciprocal
reaction velocity vs. reciprocal
reciprocal pyruvate
pyruvate concentration
concentration at at a constant concentration of NADH. The
concentrations
concentrations of pyruvate used were 0.05,
0.05, 0.1, 0.2, 0.4, 0.6
0.1, 0.2, 0.4, 0.6 and
and0.8 0.8mmol/L,
mmol/L, respectively.
respectively. NADH
concentration was maintained
maintained at at0.15
0.15mmol/L.
mmol/L.The Theresulting
resulting Km of LDH-A
Km of LDH-A4, LDH-B
4 , LDH-B 4 , and
4, and LDH-C
LDH-C 4 for
4
for pyruvate
pyruvate werewere 0.260,
0.260, 0.172
0.172 andand 0.052
0.052 mmol/L,
mmol/L, respectively.
respectively.

The double reciprocal plots of initial velocities at different pyruvate concentrations and the
inhibitory effect of lactate at different concentrations on pika LDH isozymes are shown in Figure 7.
In all cases, the competitive inhibition was observed. Initial velocities were determined in the presence
and absence of lactate of different concentrations. The Ki values calculated from the Lineweaver-Burk
double-reciprocal plots of reciprocal pyruvate concentration vs. reciprocal velocity. Reciprocals of initial
velocity with substrate concentrations below the above-mentioned concentrations gave linear plots
with these substrates. The results showed that the Ki of lactate for LDH-C4 (Ki = 65.500 mmol/L) was almost
three times higher than that for LDH-A4 (Ki = 26.900 mmol/L) and LDH-B4 (Ki = 23.800 mmol/L).
The inhibitory effects of lactate at different concentrations on the enzyme reaction velocity of LDH
isozymes are shown in Figure 8. Under the experimental conditions employed in the present study,
lactate at higher concentrations exhibited a strong inhibition of LDH-A4 and LDH-B4 activity, but
only slightly inhibited LDH-C4 activity. The results indicated that LDH-C4 was less inhibited by
lactate, which was likely beneficial in catalyzing the conversion of pyruvate to lactate even at high
concentrations of lactate.

Figure 6.Double reciprocal plots by lactate as substrate of plateau pika LDH isozymes. Double
 Figure 5.Double reciprocal plots by pyruvate as substrate of plateau pika LDH isozymes. Double
reciprocal plots of (A) LDH-A4; (B) LDH-B4; and (C) LDH-C4, respectively. Reciprocal velocity (V) was
calculated with the reciprocal 340 nm/min, plots were preformed by calculating the reciprocal
reaction velocity vs. reciprocal pyruvate concentration at a constant concentration of NADH. The
concentrations of pyruvate used were 0.05, 0.1, 0.2, 0.4, 0.6 and 0.8 mmol/L, respectively. NADH
Int. J. Mol. Sci. 2016, 17, 39 was maintained at 0.15 mmol/L. The resulting Km of LDH-A4, LDH-B4, and LDH-C4 for
concentration 6 of 14
pyruvate were 0.260, 0.172 and 0.052 mmol/L, respectively.

Int. J. Mol. Sci. 2016, 17, 39 6 of 13

The double reciprocal plots of initial velocities at different pyruvate concentrations and the
inhibitory effect of lactate at different concentrations on pika LDH isozymes are shown in Figure 7.
In all cases, the competitive inhibition was observed. Initial velocities were determined in the
presence and absence of lactate of different concentrations. The Ki values calculated from the
Lineweaver-Burk double-reciprocal plots of reciprocal pyruvate concentration vs. reciprocal velocity.
FigureFigure 6.Double
6. Double reciprocalplots
reciprocal plotsbybylactate
lactate as as substrate
substrate of of plateau
plateaupika pikaLDHLDH isozymes.
isozymes.Double
Double
Reciprocals of initial velocity with substrate concentrations below the above-mentioned
reciprocal
reciprocal plotsplots
of of (A)
(A) LDH-A;4;(B)
LDH-A (B) LDH-B
LDH-B4; and
; and (C)(C)
LDH-C
LDH-C 4, respectively. Reciprocal velocity (V) was
, respectively.
concentrations gave linear plots 4 with these4 substrates. The results 4 showed that the Ki of lactate for (V)
Reciprocal velocity
calculated with
was LDH-C
calculated with the
the reciprocal
reciprocal 340
340 nm/min, plots were preformed by calculating the the
reciprocal
4 (Ki = 65.500 mmol/L) was almost three times higher than thatby
nm/min, plots were preformed forcalculating
LDH-A4 (Ki = reciprocal
26.900
reaction velocity vs. reciprocal pyruvate concentration at a constant concentration of NAD + .. The
+
mmol/L) and LDH-B (Ki = 23.800 mmol/L). The inhibitory effects
reaction velocity vs. reciprocal pyruvate concentration at a constant concentration
4 of lactate at different concentrations
of NAD
The concentrations
on the enzyme of
reaction lactate
velocityused were 2, 5, 10, 20, 30 and 40 mmol/L. NAD + concentration was
concentrations of lactate used were 2,of5,LDH isozymes
10, 20, 30 and 40 aremmol/L.
shown inNAD Figure 8. Under the experimental
+ concentration was maintained
maintainedemployed
conditions at 0.5 mmol/L.in Thepresent
the resultingstudy,
Km of lactate
LDH-Aat 4, LDH-B4, and LDH-C4 for lactate were 19.968,
higher concentrations exhibited a strong
at 0.58.980,
mmol/L.
4.934
The resulting Km of LDH-A 4 , LDH-B 4 , and LDH-C 4 for lactate were 19.968, 8.980,
inhibition of mmol/L,
LDH-A4 respectively.
and LDH-B4 activity, but only slightly inhibited LDH-C4 activity. The results
4.934 mmol/L, respectively.
indicated that LDH-C4 was less inhibited by lactate, which was likely beneficial in catalyzing the
conversion of pyruvate to lactate even at high concentrations of lactate.

Figure 7.Effect of pyruvate on the inhibitory activity of lactate on plateau pika LDH isozymes. Double
Effect ofplots
Figure 7.reciprocal pyruvate on the inhibitory activity of lactate on plateau pika LDH isozymes. Double
of (A) LDH-A4; (B) LDH-B4; and (C) LDH-C4, respectively. Reciprocal velocity (V) was
reciprocalcalculated
plots of with
(A) LDH-A 4 ; (B)340
the reciprocal LDH-B 4 ; and
nm/min, (C)were
plots LDH-C 4 , respectively.
preformed Reciprocal
by calculating velocity
the reciprocal value (V) was
calculatedof with thevelocity
reaction reciprocal 340 nm/min,
vs. reciprocal value ofplots wereconcentration
pyruvate preformed at byacalculating the reciprocal
constant concentration of value
NADH.
of reaction Thevs.
velocity concentrations
reciprocal valueof pyruvate used concentration
of pyruvate were 0.1 0.2, 0.4,
at a0.6constant
and 0.8concentration
mmol/L. NADH of NADH.
concentration was maintained at 0.15 mmol/L. Upper left: determination of Ki from plots of slope
The concentrations of pyruvate used were 0.1 0.2, 0.4, 0.6 and 0.8 mmol/L. NADH concentration was
values against lactate concentrations. The resulting Ki for pika LDH-A4, LDH-B4, LDH-C4 by lactate
maintained at 0.15 mmol/L. Upper left: determination of Ki from plots of slope values against lactate
were 25.9, 22.2 and 66.5 mmol/L, respectively.
concentrations. The resulting Ki for pika LDH-A4 , LDH-B4 , LDH-C4 by lactate were 25.9, 22.2 and
66.5 mmol/L, respectively.
Int. J. Mol. Sci. 2016, 17, 39 7 of 14
Int. J. Mol. Sci. 2016, 17, 39 7 of 13

Figure8.8.Effect
Figure Effectofofincreasing
increasingconcentration
concentrationof oflactate
lactateon
onplateau
plateaupika
pikaLDH
LDHisozymes.
isozymes.Pyruvate
Pyruvatewas
was
usedas
used assubstrate
substrateandandlactate
lactateas
asananinhibitor.
inhibitor.Velocities
Velocitieswere
werecalculated
calculatedtaking
takingthethemaximum
maximumactivity
activity
withoutthe
without theinhibitor
inhibitorlactate
lactateasas100%.
100%.Lactate
Lactatewas
wasadded
addedatatthe
theconcentration
concentrationofof10,10,20
20and
and40
40mmol/L.
mmol/L.
The enzyme reaction velocity of LDH-C was less affected than that of LDH-A and
The enzyme reaction velocity of LDH-C4 was less affected than that of LDH-A4 and LDH-B4 when the
4 4 LDH-B 4 when the
lactateconcentrations
lactate concentrations were
were at
at 040
040 mmol/L.
mmol/L.

3.3. Discussion
Discussion
LDH isis the
LDH theterminal
terminal enzymeenzyme of of glycolysis
glycolysis with with the
the role
role of
ofreducing
reducingpyruvate pyruvate to tolactate
lactate with
with
limiting oxygen [43]. LDH-C 4 catalyzes the terminal reaction in the glycolytic pathway and displays
limiting oxygen [43]. LDH-C4 catalyzes the terminal reaction in the glycolytic pathway and displays a
a unique
unique structure
structure and and functional
functional properties
properties [43,44].
[43,44].
Ultimately,the
Ultimately, thebiochemical
biochemicalproperties
propertiesthat thatdistinguish
distinguishLDH-CLDH-C4 from from the theother
otherLDH LDHisoforms
isoforms
4
may be attributed to its high glycolytic rate. Studies have shown
may be attributed to its high glycolytic rate. Studies have shown that since the same chemical that since the same chemical reaction
is being is
reaction catalyzed, in spite ofindifferent
being catalyzed, spite of kinetic
different constants, the equilibrium
kinetic constants, constant (Kconstant
the equilibrium eq) is the same for
(Keq ) is
all LDH isoenzymes, as stated in the Haldane equation [45,46].
the same for all LDH isoenzymes, as stated in the Haldane equation [45,46]. The complex Haldane The complex Haldane kinetics
mechanism may be reduced to the Michaelis-Menten mechanism
kinetics mechanism may be reduced to the Michaelis-Menten mechanism more tractably under special more tractably under special
conditions of
conditions of substrate-enzyme
substrate-enzyme interactions
interactions [46]. [46].Also,
Also,the
thebasic
basicMichaelis-Menten
Michaelis-Menten description
description can
canbe
modified in a logistical manner to fit the reaction parameters with
be modified in a logistical manner to fit the reaction parameters with in vitro conditions for thein vitro conditions for the enzyme-
catalyzed reactionsreactions
enzyme-catalyzed [47]. In the [47].sense
In theit issense
evenithyperbolic to logistictokinetics
is even hyperbolic logisticthat kinetic
kinetics thatconstants
kinetic
may be considered
constants may be considered as being the same,
as being however
the same, however with thethe
with amendment
amendmentthey theywould respectively
would respectively
correspondto
correspond tovarious
variousfractions
fractionsfrom frommaximum
maximumvelocity velocityof ofouttake
outtake[45,47].
[45,47]. Based
Based on on this
this theory,
theory,thethe
LDHisoenzyme
LDH isoenzyme pattern pattern in in itself
itself may
maynot notchange
changethe theequilibrium
equilibrium of ofthetheLDH LDHreaction
reactionor orinfluence
influence
the tissue lactate concentration [45]. However, the situation could be quite
the tissue lactate concentration [45]. However, the situation could be quite different during transition different during transition
inenergy
in energymetabolism
metabolismwhere whereespecially
especiallythe theglycolytic
glycolyticfluxfluxcancanundergo
undergogreat greatrapid
rapidchanges.
changes.During During
fastmetabolic
fast metabolictransitions
transitionsinvolving
involvingsignificant
significantpyruvate
pyruvateconcentrations
concentrationschanges, changes,primarily
primarilyresulting
resulting
from major changes glycolytic rate, the LDH isoenzyme pattern
from major changes glycolytic rate, the LDH isoenzyme pattern may be of great importance may be of great importance in the
in
compound metabolic response to the altered energy metabolism
the compound metabolic response to the altered energy metabolism [45].Therefore, we presumed [45].Therefore, we presumed that
ATPATP
that was wasproducedproducedrapidly through
rapidly throughanaerobic glycolysis
anaerobic by LDH-C
glycolysis 4 in plateau pika, which stirs up
by LDH-C 4 in plateau pika, which
the metabolic response to the changed energy metabolism,
stirs up the metabolic response to the changed energy metabolism, enhancing enhancing its adaptation to the hypoxic
its adaptation to the
environment.
hypoxic environment.
Previousstudies
Previous studieshavehaveinvestigated
investigatedinindetail detailthetheenzymatic
enzymatickinetics
kineticsproperties
propertiesofofLDH-C LDH-C4in inother
other
4
species [31,48,49]. The biochemical properties distinguishing LDH-C
species [31,48,49]. The biochemical properties distinguishing LDH-C4 from the other LDH isoforms 4 from the other LDH isoforms

may be
may be conducible
conducible to to the
the high
high glycolytic
glycolyticrate. rate.Compared
Comparedwith withLDH-A
LDH-A 4, pika LDH-C4 has a high Km
4 , pika LDH-C4 has a high
for lactate (~2.0 mmol/L) and a low Km for pyruvate (~0.030
Km for lactate (~2.0 mmol/L) and a low Km for pyruvate (~0.030 mmol/L) [31,4850]. mmol/L) [31,4850]. This finding Thissuggests
finding
that LDH-C 4 has a 60-fold higher affinity for pyruvate than that for lactate. The finding also implies
suggests that LDH-C4 has a 60-fold higher affinity for pyruvate than that for lactate. The finding also
that pyruvate
implies conversion
that pyruvate to lactate
conversion may may
to lactate occuroccurevenevenat high concentrations
at high concentrations of of
extracellular
extracellular or
endogenous lactate [31,4850]. This theory has been supported
or endogenous lactate [31,4850]. This theory has been supported by an experiment that addition by an experiment that addition of
excess lactate in the environment (50-fold excess in relation to pyruvate) did not affect adenosine
triphosphate (ATP) production in capacitating spermatozoa [51]. In the present study, to investigate
Int. J. Mol. Sci. 2016, 17, 39 8 of 14

of excess lactate in the environment (50-fold excess in relation to pyruvate) did not affect adenosine
triphosphate (ATP) production in capacitating spermatozoa [51]. In the present study, to investigate
the enzyme kinetics of LDH-A4 , LDH-B4 , and LDH-C4 of plateau pika, the expression plasmids
pCold-SUMO-Ldh-a, pET-30a-Ldh-b, and pET-30a-Ldh-c, encoding the full-length of pika LDHA, LDHB,
and LDHC, were constructed and expressed. SDS-PAGE was used to determine the molecular weight
and confirm the recombinant proteins. Figure 2AC demonstrates that their molecular weights were
about 50 kD, 4045 kD and 4045 kD for LDHA, LDHB, and LDHC, respectively. According to our
previous paper [17], the open reading frames (ORF) of pika Ldh-a, Ldh-b and Ldh-c (accession numbers
HQ704676, HQ704677, and HQ704678 in GenBank) were 999 bp, 1005 bp and 999 bp, respectively,
encoding 332, 334 and 332 amino acids proteins, with their molecular weights of 36,557.5, 36,464.3 and
36,052.9 Da. Thus the molecular weight of the recombinant LDHA, LDHB, and LDHC in the current
study (Figure 2AC, Figure 3P2 ) was higher than the value calculated from their amino acid sequences
deduced from the encoding cDNA. The addition of the His-tag resulted in a 510 kD deviation in the
molecular weight of fusion proteins as shown in SDS-PAGE, and also indicated in other studies [5254].
In addition, fusion proteins with the SUMO-tag added 12 kD to its molecular weight [55].
The recombinant expression shuttles (pET-30a-Ldh-b, pET-30a-Ldh-c, pCold-SUMO-Ldh-a) were
transformed into E. coli BL21 cells and the enzymatic proteins (LDH-A4 , LDH-B4 , and LDH-C4 ) with
functional activity were purified from protein fluid extracts. Our results indicate that the enzyme
activity of the pika LDH-C4 was significantly higher than that of A4 and B4 , that LDH-C4 was less
sensitive to lactate inhibition than A4 and B4 (p < 0.01), and the Km values of LDH-C4 for pyruvate and
lactate were also higher than that of A4 and B4 . Compared with LDH-A4 and LDH-B4 , LDH-C4 had a
low Km for pyruvate (~0.052 mmol/L) and a high Km for lactate (~4.934 mmol/L); and the affinity of
LDH-C4 for pyruvate is 90-fold higher than that for lactate. These properties of pika LDH-C4 were
beneficial to catalyze the conversion of pyruvate to lactate even at high concentration of lactate.
Although the conversion mediated by LDH does not generate ATP, the reaction depends on
NADH as a cofactor that accompanies oxidation to NAD+ ; the concentration of NAD+ is a rate-limiting
factor in glycolysis and necessary for continued glycolysis [56]. Previous studies demonstrated that
knockout of Ldh-c or inhibition of LDH-C4 in sperm led to rapid decline in sperm ATP levels [56,57],
a decrease in progressive motility, and a failure to develop hyper-activated motility. It was revealed
that all consumed13 C-labeled pyruvate added in sperm culture medium was converted to lactate
rather than oxidized in the tricarboxylic acid (TCA) cycle in metabolic tracing experiments; in the
presence of exogenous pyruvate, the ATP concentration was increased by more than 50% [51]. When
carbonyl cyanide m-chlorophenylhydrazone (CCCP) and NaCN was applied to inhibit the oxidative
phosphorylation in mitochondria, the amount of ATP was kept at the equivalent level to that without
CCCP and the vigorous motility of sperm was maintained [51,58]. These results suggest that LDH-C4
is essential in sperm glycolysis which has an important role in providing abundant ATP for sperm
motility [56,58,59], and it is related to LDH-C4 enzymatic kinetics properties.
Plateau pika has a strong adaptability to the hypoxic plateau environment of the Qinghai-Tibet
Plateau, given the sperm-specific lactate dehydrogenase (Ldh-c) gene is also expressed in their somatic
cells. In light of the enzymatic kinetics properties, we propose that the pika could reduce dependence
on oxygen and enhance the adaptation to the hypoxic environments due to increased anaerobic
glycolysis by LDH-C4 .

4. Materials and Methods

4.1. Reagents and Animal Procedures

All reagents were from Sangon (Sangon, Shanghai, China) unless otherwise noted. Plateau pikas
were live-trapped from Haibei Alpine Meadow Ecosystem Research Station at an altitude of 3800 m in
Qinghai Province, China. All animals were first anesthetized with sodium pentobarbital (5%), and then
sacrificed by cervical dislocation immediately before dissection. Skeletal muscle, kidney, and testis
Int. J. Mol. Sci. 2016, 17, 39 9 of 14

were rapidly removed and frozen in liquid nitrogen for long-term storage. All procedures involved
in the handling and care of animals were approved by the China Zoological Society according to the
China Practice for the Care and Use of Laboratory Animals (permit number: GB 14923-2010).

4.2. Expression Plasmids Construction

In our previous study [17], Ldh-a, Ldh-b, and Ldh-c of plateau pika testis were cloned and deposited
in GenBank with the accession numbers HQ704676, HQ704677, and HQ704678, respectively. Three
lengths of BamHI/XhoI fragments (999 bp, 1005 bp, 999 bp representing Ldh-a, Ldh-b, and Ldh-c coding
sequences, respectively) were PCR amplified from plateau pika testis cDNA using Premix Ex Taq
Version Kit (Takara, Kyoto, Japan). The PCR primers for Ldh-a were 51 -CGGAATTCATGGCAGCTC
TCAAGGATCAG-31 (sense) and 51 -CCGCTCGAGGAACTGCAGCTCCTTCTGGAT-31 (antisense),
for Ldh-b were 51 -CGGAATTCATGGCAACCCTGAAGGAAAAACTCAT (sense) and 51 -CCGCTCG
AGCAGGTCCTTCAGGTCCTTCTGGA-31 (antisense), and for Ldh-c were 51 -CGGGATCCATGTCGAC
AGTCAAGGAGC-31 (sense) and 51 -CCGCTCGAGA AACACCAGGTC CTTCTGGAC-31 (antisense),
respectively. PCR conditions were 5 min at 95 C, 30 cycles of 45 s at 95 C, 45 s at 65 C, 1 min at
72 C, and a final elongation step at 72 C for 10 min. PCR products were determined with agarose
gel electrophoresis. Subsequently, the BamHI/XhoI fragment, Ldh-a was inserted into pCold-SUMO
expression vector (BPI, Beijing, China); the other two fragments, Ldh-b and Ldh-c were inserted into
pET-30a (+) expression vector (Novagen, Darmstadt, Germany). The final plasmid constructs of
pCold-SUMO-Ldh-a, pET-30a-Ldh-b, and pET-30a-Ldh-c had been sequenced in BGI (Beijing Genomics
Institute, Beijing, China).The recombinant expression shuttles (pCold-SUMO-Ldh-a, pET-30a-Ldh-b,
and pET-30a-Ldh-c) with six His-tags in the C-terminus were transformed into Escherichia coli (E. coli)
BL21 (DE3) cells.

4.3. Protein Expression, Purification, Analysis and Enzyme Activity Measurement

E. coli BL21 (DE3) cells were cultured in media of Lysogeny broth (LB). E. coli clones with plasmids
(pCold-SUMO-Ldh-a, pET-30a-Ldh-b, and pET-30a-Ldh-c) were inoculated into a liter of medium with
100 g/mL ampicillin or 50 g/mL kanamycin and cultured at 37 C. When A600 value arrived at
0.6, isopropyl--D-thiogalactopyranoside (IPTG) reagent was added till the final concentration of
1 mmol/L, followed by further culturing at 25 C for 10 h to induce the expression of Ldh-a, Ldh-b,
and Ldh-c. To determine LDHA, LDHB and LDHC expression in the supernatant and precipitate of
E. coli cells, bacterial cells were collected by centrifugation and resuspended in 20 mmol/L phosphate
buffer saline (PBS) (pH 8.0), and disrupted by ultrasonication (36 5 s pulses with 5 s intervals).
The supernatant was then collected; the precipitate was resuspended and washed in 20 mmol/L PBS
(pH 8.0) with 8 mol/L urea. SDS-PAGE was performed to assay the collected supernatant and treated
precipitate with a steady current voltage electrophoresis instrument (Beijing Liuyi Instrument Factory,
Beijing, China) as follows: 6 L samples were loaded on a 12% (w/v) separating gel with a 5% (w/v)
stacking gel in Tris-glycine buffer (pH = 8.3). The electric voltage was 80 V in the stacking gel and
120 V in the separating gel. The gel was stained in with Coomassie staining solution (10% methyl
alcohol, 10% acetic acid, 0.2% R-250) for 4 h, and finally stored in the destaining solution (10% methyl
alcohol, 10% acetic acid).
To obtain the active enzyme protein in the supernatant of E. coli cells, bacterial cells were collected
with following procedures: 4000 r/min centrifugation (15 min), resuspension in 20 mmol/L PBS
(pH = 7.0), and disruption by repeated freeze-thaw cycles (in liquid nitrogen and 37 C water bath) for
10 times. In freeze-thaw cycles, Triton X-100 (final concentration of 1%), lysozyme (final concentration
of 1%) and DNA enzyme (final concentration of 0.2%) were added. The lysates were centrifuged at
15,000 r/min under a temperature of 4 C for 10 min, and the obtained supernatant was used for
purification. Ni-NTA resin (QIAGEN, Hilden, Germany) was used to purify the recombinant LDHs.
The resins were washed twice with increasing concentrations of imidazole at 10, 20, 50, 100, 250 and
500 mmol/L in PBS gradient (50 mmol/L NaH2 PO4 , 300 mmol/L NaCl, pH = 8.0). Each fraction was
Int. J. Mol. Sci. 2016, 17, 39 10 of 14

collected using individual collection tubes and detected by native PAGE. Elutions with LDH activity
were totally collected. Enzyme liquids were obtained from elutions after removing imidazole by
ultrafiltration (Ultrafiltration Tubes, 50 kD, 15 mL, Merck Millipore, Billerica, MA, USA). On average, a
final amount of 3 mg LDH isoenzymes were purified from every liter of bacterial culture following
this approach. LDH isoenzymes stocks were finally stored in PBS (pH = 8.0) at 4 C until following
steps. The LDH isoenzymes were prepared simultaneously under the same conditions [60].
SDS-PAGE was used to detect the protein purification and native PAGE was used to test
and verify the enzyme activity. Native PAGE experiment was also performed with a voltage
electrophoresis apparatus (Beijing Liuyi Instrument Factory, Beijing, China) with the following
conditions: 8% (w/v) separating gel with a 4% (w/v) stacking gel (non-denaturing polyacrylamide
gel) in Tris-glycine (pH = 8.3), and 6 L samples were loaded. LDH bands were stained at 37 C in
a specific staining reagent (a mixture of 0.5 mol/L phosphate buffer (pH = 7.5), 10 mL of 1 mg/mL
nitrobenzenethiocyanate chloride (NBT), 4 mL of 5 mg/mL NAD+ , 2.5 mL of 1 mol/L sodium lactate,
2.5 mL of 0.1 mol/L NaCl and 1 mL of 1 mg/mL phenazinemethosulfate (PMS)) for half an hour in the
dark. The stained gels were then rinsed with ddH2 O and stored in the storage liquid (10% glycerol
and 7% acetic acid). The reference marker for native PAGE was made with a mixture of pika kidney
and skeletal muscle, tissues were homogenized on ice as a 1:4 (w/v) dilution in 0.9% physiological
saline, and centrifuged at 4000 r/min at 4 C for 10 min, and finally the supernatant was collected.
Protein concentration was determined by Bradford assay (Bradford protein assay kit, Nanjing
Jiancheng Biotech Inc., Nanjing, China). Enzyme activity was measured by Bergmeyer assay (Lactate
dehydrogenase kit, Nanjing Jiancheng Biotech Inc., Nanjing, China).

4.4. LDH-A4 , LDH-B4 and LDH-C4 Kinetics Properties

The activity of LDH was measured using UV spectrophotometer (Unicon 2800, Shanghai, China)
by recording the change of 340 nm absorbance produced by NADH oxidation. Assays were kept
at consistent temperature of 37 C. Calculation of Km value for pyruvate and lactate was resulted
from Lineweaver-Burk plots. Ki value of lactate was determined from Km and Vmax, which was
obtained with or without inhibitor added to reaction buffer via various concentrations of pyruvate at a
constant inhibitor concentration, followed by plotting the slope (Km/Vmax value) against inhibitor
concentrations. The mixed reagent of reaction to from pyruvate lactate contained 0.15 mmol/L NADH,
50 mmol/L PBS (pH8.0) and the sodium pyruvate gradients as substrate were performed at 0.05,
0.1, 0.2, 0.4, 0.6 and 0.8 mmol/L for LDH-A4 , LDH-B4 , and LDH-C4 , and the enzyme persisted at a
constant inhibitor concentration. The reaction from lactate to pyruvate contained 0.5 mmol/L NAD+ ,
50 mmol/LPBS, (pH 8.0); sodium lactate gradients as substrate were performed at 2, 5, 10, 20, 30
and 40 mmol/L for LDH-A4 , LDH-B4 , and LDH-C4 . The concentration gradients of sodium lactate
as inhibitor were added as 10, 20, and 40 mmol/L, respectively; and the substrates, coenzymes, and
other buffers followed the method of the reaction from pyruvate to lactate as outlined above. A E340
value of 0.060.07 per minute was provided in the experiment. The enzyme preparation was diluted
with PBS (pH = 8.0) and assayed in a 1 cm light path. Before starting the reaction by addition of
substrate, the coenzyme was incubated with the buffer used in the assay for 10 min at 37 C. In total,
3 mL volumes of reagents were added in the process [61]. All chemicals were analytically pure from
Sangon (Sangon, Shanghai, China). All measurements in the study were repeated 5 times for the
average values.

4.5. Statistics
The data are presented as mean standard deviation (SD). Statistical analysis was performed
by one-way analysis of variance (ANOVA) followed by Duncans test using SPSS 22.0 (SPSS Inc.,
Chicago, IL, USA). Values of p < 0.01 was considered highly significant and p < 0.05 was considered
statistically significant.
Int. J. Mol. Sci. 2016, 17, 39 11 of 14

5. Conclusions
In light of the enzymatic kinetics properties, we suggest that the pika could reduce dependence on
oxygen and enhance adaptation to the hypoxic environment due to its increasing anaerobic glycolysis
to produce ATP rapidly by LDH-C4, which is often the role of LDH-A4 in most mammal species.

Acknowledgments: This work was supported by the National Natural Science Foundation of China (No. 31260512,
No. 30960054, and No. 31040011), the Key Project of Chinese Ministry of Education (209132) and the Natural
Science Foundation of Qinghai Province, China (No. 2012-Z-905 and No. 2014-ZJ-714).
Author Contributions: Dengbang Wei planned the experiments; Yang Wang and Lian Wei performed the
experiments; Yang Wang, Xiao Li, Linna Wei and Lina Xu analyzed the data; Yang Wang and Dengbang Wei wrote
the paper.
Conflicts of Interest: The authors declare no conflict of interest.

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