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J Biol Chem, Vol. 273, Issue 50, 33774-33780, December 11, 1998
From the In vertebrates, the creatine kinase isoenzyme
family consists of four types of isoforms: cytosolic muscle type
(M-CK), cytosolic brain type (B-CK), mitochondrial ubiquitous, acidic
type (Miu-CK), and mitochondrial sarcomeric, basic type (Mis-CK). Until
recently, the existence of more than one subisoform of CK isoenzyme has been demonstrated only in fishes by starch gel electrophoresis. We
report herein the isolation of three full-length cDNAs that correspond to three closely related creatine kinase M-CK genes from
common carp (Cyprinus carpio), designated the M1-CK, M2-CK, and M3-CK genes. Using oligonucleotide probes that correspond to the
same region but with the most variable sequences, different restricted
genomic hybridization patterns have been obtained. These Southern blot
results indicate that the three cDNAs come from different genes.
Northern blot analysis using probes that correspond to the
3'-untranslated regions further show that all three subisoforms are
expressed specifically in carp muscle. The deduced amino acid sequences
of these three subisoforms of carp M-CK show about 85% identity to
mammalian M-CK isoenzyme. Finally, the three cDNAs have been
expressed in Escherichia coli with a molecular mass of
approximately 43,000 Da, and these recombinant proteins exhibit
creatine kinase activity. All of these data suggest that the M-CK
isoenzymes have at least three subisoforms in carp.
All living organisms require energy to survive and carry out the
many tasks that characterize biological activity. Cellular energy
demand and supply are generally balanced and tightly regulated for
economic and efficient energy use. The enzyme creatine kinase (CK1; EC 2.7.3.2) plays a key
role in the energy metabolism of cells that have fluctuating energy
requirements (for a review, see Ref. 1). Cells contain a number of
different CK isoenzymes, which are, in part, compartmentalized
specifically at places where energy is produced or utilized, such as in
mitochondria, skeletal and cardiac muscle fibers, neurons,
electrocytes, photoreceptors, and spermatozoa (for a review, see Ref.
2). Two fundamental types of CKs can be found in vertebrates: cytosolic
and mitochondrial CKs (3).
So far, there are four different CK isoforms known in vertebrates; two
are found in cytosol, and two are found in mitochondria. The cytosolic
forms are called M-CK (muscle) and B-CK (brain) and can dimerize with
each other (4-6). They are found in soluble form in the cytosol, but
fractions are also associated with the M-line of the sarcomeres, the
sarcoplasmic Ca2+-ATPase, or the spermatozoan tail (7-9).
MM creatine kinase purified from tissue exists in a single form but
upon release into the plasma exhibits three forms in both dogs
(MM1, MM2, and MM3) and humans
(MM-A, MM-B, and MM-C) (10, 11). The hydrolytic cleavage of a basic
amino acid, presumably by carboxypeptidase N, is responsible for
conversion of muscle tissue MM1 to MM2 and
MM3 (10). In humans, these isoforms are formed by the
successive removal of the COOH-terminal lysine residue from one M
subunit at a time, resulting in the conversion of MM-A to isoforms MM-B
and MM-C (11). In mammals, there is just one isoform of B-CK, and the two B-CK isoforms of chicken are derived from a single gene by alternative splicing of the second exon (12, 13). Additional heterogeneity of B-CK has been shown to be due to alternative initiation of translation or post-translation phosphorylation (14-17).
The four CK isoenzymes of teleost fish are termed CK-A to CK-D and are
all of cytoplasmic origin. CK-A, CK-C, and CK-D are expressed
predominantly in striated muscle, stomach, and testis, respectively,
while CK-B is expressed ubiquitously or is confined to neural tissue
(18). In trout, a cDNA encoding for a CK named TCK-1 has been
demonstrated to show enhanced testicular expression, and an s-CK
protein has been purified from sperm (19). Since Torpedo
electrocytes have been shown by isoenzyme and two-dimensional electrophoresis to contain the same major CK isoforms as muscle, the
two Torpedo CKs very likely represent the CK-A isoenzyme
(20-22). Based on a comparison of the tissue specificity of expression of the various isoenzymes, it has been hypothesized that CK-II of frogs
and CK-A of fish correspond to M-CK of mammals and birds, while CK-IV
and CK-C correspond to B-CK (23, 24).
In this study, we report the cloning, sequence analysis, and expression
in Escherichia coli of three carp M-CK cDNAs. Although the existence of more than one subisoform of each CK isoenzyme has been
demonstrated in fish by starch gel electrophoresis, this is the first
time that the cloning of more than one M-CK subisoform cDNA in any
organism has been reported (25). We have explored the possible
existence of CK gene families in teleost fish, and so far, we have
cloned three M-CK subisoforms and four B-CK subisoforms. Here, we
report the nucleotide sequences; the predicted amino acid sequences of
the three subisoforms of M-CK along with data on their tissue
distribution; and the E. coli-expressed recombinant M-CK
protein catalytic activities. Also, since the common carp is a
poikilothermic fish, we have focused our attention on the catalytic
activity of the E. coli-expressed recombinant M-CK proteins of carp at various temperatures.
Experimental Animals--
Common carp (Cyprinus
carpio) were maintained in tanks of circulating aerated water at
25 °C for 3 months under a 12-h day length and were fed a commercial diet.
Isolation of cDNA Clones--
Poly(A)+ RNA was
prepared from striated muscle of the common carp by an established
procedure (26). The double-stranded cDNA was supplied with
EcoRI and XhoI linkers and was inserted into the
Sequence Analysis and Computer Homology Search--
The
nucleotide sequences were determined by doubled-stranded sequencing
according to the dideoxy chain termination method using an ABI PRISM
dye terminator cycle sequencing kit (Applied Biosystems). The
nucleotide sequences were then analyzed with an Applied Biosystems 377A
automated DNA sequencer. A sequence homology search and comparison were
performed with computer programs of the GCG sequence analysis software
package (Genetic Computer Group, Madison, WI). The homologous sequences
searched included GenBankTM accession numbers M11660 (dog
M-CK), X03233 (mouse M-CK), M10140 (rat M-CK), M14780 (human M-CK),
M10012 (chicken M-CK), M11508 (Torpedo M-CK), M14400 (rat
B-CK), X59736 (rat Mis-CK), and X57937 (rat Miu-CK).
Southern Hybridization Analysis--
Genomic DNA was prepared
from carp muscle tissue with the proteinase K method (27). Southern
blotting with nitrocellulose membrane (Hybond-C super,
Amersham Pharmacia Biotech) was carried out following standard
procedures from Maniatis et al. (27). Restriction digestion
of genomic and plasmid DNA was carried out overnight according to the
manufacturer's specifications (New England Biolabs).
Oligodeoxynucleotide probes were designed to cover the region 27 nucleotides after the stop codon of the three CK cDNAs that
contained the most variable sequences (for M1-CK, 5'-AGC GGG GAG CCC
TTC CAT TTT TTT CTA-3'; for M2-CK, 5'-AGC GGG AGC CCT TCC TCT TTT TTC
CTC-3'; and for M3-CK, 5'-AAT GGC AGA AGT GCT TTT CTT TTT TTA-3'). Each
probe was 5'-end-labeled with [ Northern Hybridization Analysis--
Total RNA was isolated by
the acid guanidinium isothiocyanate/phenol/chloroform method using
TRIzol reagent (Life Technologies, Inc.), electrophoresed on 1.2%
agarose-formaldehyde gels, and then transferred onto nitrocellulose
membranes (Hybond-C super, Amersham Pharmacia Biotech)
(28). Probes of different M-CK subisoforms were prepared with the
entire 3'-UTRs as templates and labeled by the rediPrime
labeling system (Amersham Pharmacia Biotech) with
[ Expression Construction, Preparation, and Isolation of
Recombinant Protein--
The three full-length carp M-CK cDNA
coding regions were amplified by PCR. To facilitate directional
subcloning into the expression vector, forward and reverse primers were
designed to contain restriction sites for NdeI and
EcoRI at the 5'- and 3'-termini, respectively (BamHI for the 5'-terminal of M2-CK). Both restriction sites
were absent in the M-CK cDNAs. The cDNA products were ligated
into the pET-28a(+) expression vector (Novagen), in which prokaryotic gene expression is driven by the T7 lac promoter. Nucleotide
sequencing was carried out to ensure the absence of PCR-induced
mutation. Strain BL21 (DE3) pLysS bacteria were transformed with the
resulting cDNA constructs and screened for positive clones. The
bacterial cultures were grown in LB broth until
A600 = 0.3 and then were induced with isopropyl
Immunoblot Analysis--
Polyclonal goat anti-human creatine
kinase MM isoenzyme was purchased from BIODESIGN. SDS-polyacrylamide
gel electrophoresis was performed according to the method of Laemmli
(29). After electrophoresis, the gel was stained with Coomassie Blue
(Sigma) and then electrotransferred to a polyvinylidene difluoride
membrane filter (Millipore Corp.) (28). The blotted filter was
incubated in 20 mM Tris, pH 7.6, 137 mM NaCl,
and 3% bovine serum albumin (fraction V, Sigma) for 1 h. The
filter was incubated with a 1:1000 dilution of anti-M-CK serum,
followed by interaction with anti-goat IgG alkaline
phosphatase-conjugated antibody as a secondary antibody (Zymed Laboratories Inc.) and staining with
5-bromo-4-chloro-3-indolylphosphate (Pierce) and nitro blue tetrazolium (Pierce).
Isoenzyme Activity Assay--
Protein concentration
determination was performed using the Bradford method (30), using
bovine serum albumin as the protein standard (Pierce). Creatine kinase
activities were determined by the direct enzyme activity assay, which
measured the creatine (Cr) concentration formed in the reverse
reaction, PCr + ADP Enzyme Kinetic Analysis--
The same direct enzyme assay was
used in the kinetic experiments. Reaction conditions for each
individual kinetic experiment are described under "Results." The
influence of PCr and ADP concentrations on creatine kinase activity was
studied at 25 °C in Trizma (Tris base) buffer (with Mg2+
ion) (Sigma). Km for PCr was determined with various PCr (Sigma) concentrations (from 0.5 to 15 mM) and a fixed
ADP (Sigma) concentration (4 mM). Km for
ADP was determined with various ADP concentrations (from 0.1 to 10 mM) and a fixed PCr concentration (15 mM). For
the determination of all kinetic parameters, initial velocity data were
analyzed using the EZ-FIT program package by Perrella (33). Values of
Km for PCr and ADP and of
Vmax were calculated by a Lineweaver-Burk plot of three series of independent measurements. S.E. values for calculated Km and Vmax values are given.
Molecular Cloning of Carp M1-CK, M2-CK, and M3-CK
cDNAs--
The
The lengths for these three cDNA inserts, excluding the poly(A),
were 1559, 1580, and 1508 bp for M1-CK, M2-CK, and M3-CK, respectively
(Fig. 1). The presumptive polyadenylation
signal, ATAAA, was located at nucleotides 1528-1532 and 1541-1545 for M1-CK, nucleotides 1549-1553 and 1562-1566 for M2-CK, and nucleotides 1516-1520 for M3-CK. The lengths of the open reading frame were 1146, 1146, and 1143 bp for M1-CK, M2-CK, and M3-CK, respectively. The
homology of the open reading frame nucleotide sequences of M1-CK,
M2-CK, and M3-CK was 80% identity to dog and human M-CKs, and this
result indicates that these three cDNA inserts represent the muscle
form creatine kinase cDNAs (Table I).
Nucleotide homology comparison of these carp cDNAs among themselves
and with the M-CK, B-CK, Miu-CK, and Mis-CK cDNAs of other
organisms is shown in Table I. Nucleotide homology comparison of these
carp cDNAs among themselves in the 5'- and 3'-untranslated regions
and the open reading frames is shown in Table
II.
Amino Acid Primary Structure of Carp M1-CK, M2-CK, and M3-CK
Subisoforms--
The open reading frame sequences could be translated
into proteins of 381 amino acids for both M1-CK and M2-CK and 380 amino acids for M3-CK (Fig. 2). Computer
alignment and search were carried out using the programs Pileup and
Pretty of the GCG software package and protein sequence data banks. The
identity between M1-CK and M2-CK proteins is as high as 96% (Fig. 2,
Table I). For the M3-CK protein, the identity with the homologous M1-CK
and M2-CK subisoforms is somewhat lower, at approximately 87%, due to
differences within sequences of different domains, especially at the N
terminus (Fig. 2). Analysis of the deduced amino acid sequences of the
three carp muscle subisoforms revealed 83-87% identity with human and other species' creatine kinase muscle isoenzyme (Table I). In mammals
and birds, the amino acid identities within each isoenzyme class range
from 88 to 99% (24). Evidently, a higher degree of homology can be
observed between the B- and the M-CKs (77-82%), while the homology
between the cytosolic and the mitochondrial CK isoforms is lower
(60-65%) (Table I) (24).
A previously defined "CK framework," consisting of the six most
conserved sequence blocks and "diagnostic boxes," which are characteristic for any creatine kinase isoenzyme and which may serve to
distinguish this isoenzyme from all others, could be observed in the
protein sequences of the three carp M-CKs (24). The six highly
conserved blocks are boxed in the carp M-CK consensus sequences in Fig. 2. These conserved sequences are flanked by regions
that are less conserved, among them the N and C termini (Fig. 2). The
absolutely conserved Cys-283, alkylation of which is always paralleled
by a very pronounced or even complete loss of enzymatic activity, is
also present (41). A putative adenine nucleotide binding motif
(glycine-rich loop) LGXGXXGXV and the absolutely conserved seven-amino acid sequence
Cys-Pro-Ser-Asn-Leu-Gly-Thr, referred to as the active site, are also
observed (Fig. 2) (42, 43).
Analysis of Carp M-CK Genomic DNA Variants--
Detection of the
carp M-CK genes was by Southern hybridization. The results of the
hybridization of the three restricted M-CK genes are shown in Fig.
3. The oligonucleotide probes specific for each of the three genes hybridized strongly to their respective target DNAs. The respective gene-specific probes hybridized to a 6.5-kb
HindIII fragment, a 3.8-kb PstI fragment, and a
4.7-kb PvuII fragment for the carp M1-CK gene; a 5.2-kb
HindIII fragment, a 2.2-kb PstI fragment, and a
3.4-kb PvuII fragment for the carp M2-CK gene; and a 16.2-kb
HindIII fragment, a 5.8-kb PstI fragment, and a
3.5-kb PvuII fragment for the carp M3-CK gene (Fig. 3). In
the standard plasmid DNA lanes that carried the different cDNAs and
the restricted genomic lanes, only slight cross-hybridization could be
observed between M1-CK and M2-CK and between M2-CK and M3-CK (Fig. 3).
These results demonstrate that under the hybridization conditions used,
M1-CK, M2-CK, and M3-CK genes could be detected by Southern analysis
with these gene-specific oligonucleotide probes. Therefore, there are
at least three M-CK genes in the carp genome.
Tissue Distribution of Carp M1-CK, M2-CK, and M3-CK
mRNAs--
Tissue distribution of the three carp M1-CK, M2-CK, and
M3-CK mRNAs was characterized by Northern blot analysis (Fig.
4). These three subisoform mRNAs
(about 1.6 kb) in carp tissues were abundantly expressed in red and
white muscles, whereas faint signals were observed in heart and no
transcripts were detected in brain, kidney, gill, or liver. This
distribution is similar to those of other vertebrate M-CK isoenzymes,
which were found to express abundantly in striated muscle but with no
expression in the brain (44). These results indicate that carp M1-CK,
M2-CK, and M3-CK are subisoforms of M-CK isoenzymes.
Expression of the Three Carp M-CK Recombinant Subisoforms--
To
express and purify the recombinant carp M-CK proteins, a 1-L culture of
BL21 (DE3) pLysS cells harboring the expression plasmid pETM1, pETM2,
or pETM3 was grown and harvested. The SDS-polyacrylamide gel depicting
the purified recombinant proteins is shown in Fig. 5A. Western analysis showed
that they were all immunoreactive with anti-human M-CK antibody (Fig.
5B).
Different Enzyme Catalytic Activities and Kinetic Parameters of
Carp M-CK Subisoforms--
The specific activities of carp M1-CK,
M2-CK, and M3-CK recombinant proteins were 45.1, 54.4, and 20.7 units/mg, respectively (Table III).
Specific activities were determined under standard conditions of 20 mM phosphocreatine, 4 mM ADP, and 4 mM MgCl2. Kinetic parameters
Km and Vmax were calculated
from three sets of independent experiments, and in each set of
experiments, three independent measurements were taken, with the
phosphocreatine concentrations being varied between 0.5 and 15 mM while with constant concentrations of ADP at 4 mM and of MgCl2 at 4 mM. The enzyme amount in our assay was 0.04 µg, and the enzyme activity was linear up to 0.04 µg of enzyme. Also, the enzyme activity was linear up to
30 min under the assay conditions; therefore, the end point creatine
value was taken at 30 min for this reaction (data not shown).
Km values of carp M1-CK, M2-CK, and M3-CK
recombinant proteins for PCr were 1.4 ± 0.2, 0.8 ± 0.1, and
3.0 ± 0.4 mM, respectively.
Vmax values of the M1-CK (103.2 ± 0.9 units/mg) and M2-CK (102.7 ± 0.5 units/mg) recombinant proteins
were similar, while that of the M3-CK (70.8 ± 0.8 units/mg)
recombinant protein was lower (Table III). Also, while the
Km values of ADP were determined, the values
fluctuated widely from experiment to experiment. This assay method, for
technical reasons, might not be suitable for accurate determination of
Km values of ADP of CKs, since these
Km values might be far below 100 µM
(45).
When specific activities were measured at different temperatures, the
M1-CK protein was found to exhibit its highest specific activity at
37 °C (64 unit/mg) and then to fall to around 56% at 20 °C, and
at 10 °C only 18% activity was retained (Fig.
6). The temperature dependence of the
M2-CK protein was very similar to that of human M-CK. The highest
specific activity appeared at 37 °C (63 unit/mg), was maintained at
85% activity from 30 to 25 °C, and then was reduced further to 66%
activity at 20 °C and finally to 28% activity at 10 °C (Fig. 6).
In contrast, the temperature dependence of the M3-CK protein-specific
activities was quite different. The highest activity was measured at
25 °C (21 units/mg), and at 37 and 10 °C, 41 and 28% activities
were measured, respectively (Fig. 6).
In this report, we describe the cloning, characterization, and
expression of three creatine kinase isoenzymes from common carp
striated muscle. According to the nucleotide and amino acid sequence
homology comparison, the three carp M-CKs should belong to cytosolic
M-CKs. Southern hybridization analysis using oligonucleotides specific
to individual cDNA as probes indicates that three carp M-CK
cDNAs are encoded by different genes. From Northern blot analysis,
mRNAs for M-CKs were detected in striated muscle (red, white, and
cardiac muscle), and the expression levels varied in each muscle.
However, with our probe design we could not tell whether there is any
quantitative difference in expression levels of each enzyme. The
Northern signal is faint in carp heart, and we speculate that there is
the possibility that a mixture of MB and BB forms exists in heart (25).
Another more interesting possibility is that another heart-specific
M-CK subisoform exists whose DNA sequence in the corresponding position
might be sufficiently different so that it only weakly cross-hybridized
with our probes. All of these results indicate that these three carp
CKs are muscle creatine kinase subisoforms.
In the experiments where the M-CK expression plasmids were transformed
and expressed in E. coli cells, we detected the expressed recombinant proteins by immunoblot analysis with human muscle creatine
kinase antibody. The three recombinant proteins exhibited somewhat
different Km values, despite the amino acid homology
between M1-CK and M2-CK being as high as 96%. As for Vmax values, M1-CK and M2-CK showed similar
values, while that of M3-CK was 30% lower. The Km
of carp M-CK has previously been found to be around 2.84 mM
for PCr, and in light of our present findings, it is possible that this
previous value was a measurement of a mixture of M-CKs (46). Finally,
in the temperature dependence-specific activity studies, the decreasing
trends of carp M1-CK and M2-CK enzyme activity when temperatures
decreased from 37 to 10 °C were somewhat similar. Yet, more
interestingly, the specific activities of M3-CK recombinant protein
showed a plateau between 30 and 20 °C. All of these results suggest
that these carp muscle creatine kinase subisoforms possess quite
different enzyme properties.
Taken together, the three carp M-CKs are a new combination of cytosolic
CK subisoforms that may have overlapping but not necessarily redundant
physiological enzyme functions. Whether their mRNA expression patterns are similar or different at different developmental stages or
at different environmental temperatures is an interesting question. Likewise, it is important to learn the different mechanisms or physiological roles played by the three carp M-CKs, if any, in energy
homeostasis during environmental adaptation in ectothermic animals.
Since there are multiple subisoforms of M-CK in carp, it would be
important to learn whether heterodimers of M-CKs exist in a single carp
muscle cell and whether there are differential subcellular
localizations of these subisoforms.
Cold is a major environmental problem for all living organisms.
Poikilothermic animals respond adaptively to chronic cold by a suite of
cellular responses that compensate to varying extents for the
rate-depressing effects of cooling. In ectothermic fish, body
temperature is totally dependent on ambient temperature, and yet, even
at low temperatures, carp have evolved strategies to maintain growth
and swimming ability. Various review articles concerning possible
acclimation mechanisms have been published (47, 48). Hazel and Prosser
(49) suggested that acclimation might involve a
temperature-dependent synthesis of new proteins, which
would require the expression of different sets of temperature-specific isoenzyme genes. Also, the nature and significance of changes in
enzymatic myosin ATPase activity and the recruitment of different muscle fiber types in relation to acclimation and environmental temperature in carp have been reviewed (50, 51). It has been reported
that there are different myosin heavy chain isoform genes that are
expressed at warm and cold environmental temperatures (52, 53).
Myofibrillar creatine kinase and myosin ATPase are associated with the
same microenvironment and provide and utilize ATP for muscle
contraction. Since M3-CK exhibited a specific activity peak at
25 °C, we could imagine that some cold-specific subisoform carp
M-CKs should exist to maintain normal muscle ability at lower environmental temperatures.
In conclusion, we have cloned three carp M-CK cDNAs encoded by
different genes. The three M-CK subisoforms are not due to alternative
initiation sites, alternative splicing, post-translational glycosylation, phosphorylation, or proteolytic modification. These results suggest that multiple genes give rise to creatine kinase heterogeneity in carp.
Hong-Yi Gong is gratefully acknowledged for
performing the Northern blot analysis. The technical contributions of
Jin-Ming Tsai for the cDNA library and Jun-Sin Chen for protein
expression are acknowledged. We appreciate the help of Dr. Gu-Gang
Chang in reviewing this manuscript. We thank Drs. Chi-Yao Chang,
Pung-Pung Hwang, and Sin-Che Lee for support and helpful discussions.
*
This work was supported by grants from Academia Sinica and
the Council of Agriculture, Taiwan, Republic of China.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) AF055288, AF055289, and AF055290 (for M1-CK,
M2-CK, and M3-CK cDNA, respectively).
¶
To whom correspondence may be addressed: Inst. of Zoology,
Academia Sinica, Nankang, Taipei 115, Taiwan, Republic of China. Tel.:
886-2-2789-9568; Fax: 886-2-2782-4595; E-mail:
ZOJLWU{at}ccvax.sinica.edu.tw.
The abbreviations used are:
CK, creatine kinase; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s); Cr, creatine; PCr, phosphocreatine.
Cloning, Characterization, and Expression in Escherichia
coli of Three Creatine Kinase Muscle Isoenzyme cDNAs from
Carp (Cyprinus carpio) Striated Muscle*
§,¶, and¶
Laboratory of Marine Molecular Biology and
Biotechnology, Institute of Zoology, Academia Sinica, Nankang, Taipei
115, Taiwan, Republic of China and the § Graduate Institute
of Life Sciences, National Defense Medical Center, Taipei 117, Taiwan,
Republic of China
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-ZAP II vector (Stratagene). The library was packaged in the
Gigapack II gold packaging extract (Stratagene) and was plated on
E. coli XL1-blue MRF' cells (27). The probe for screening the carp cDNA library was synthesized by PCR using carp striated muscle cDNA as a template with a sense oligonucleotide primer (CK-5', 5'-CAY AAY AAY CAY ATG GCN AA-3', alignment positions amino
acids 26-32 in the M-CK isoenzyme) and an antisense oligonucleotide primer (CK-3', 5'-CAT RTT NCC NCC YTT YTG CAT-3', alignment positions amino acids 239-246 in the M-CK isoenzyme). The amplified products were subcloned into pUC 19 (New England Biolabs) and transformed into
JM109 cells. Preliminary Northern blot analysis using the PCR product
(663 bp) as a probe revealed that it was expressed in carp striated
muscle. After in vitro packaging, 1 × 106
primary phages were amplified and plated at a density of ~50,000 plaques/plate (15 cm), with 12 plates being screened. The probe was
prepared by rediPrime labeling system (Amersham Pharmacia Biotech) with [
-32P]dATP (3000 Ci/mmol, Amersham
Pharmacia Biotech). Prehybridization and hybridization were carried out
in standard hybridization buffer at 42 °C for 16 h (27).
Filters were finally washed with 0.1× SSC, 0.1% SDS for 1 h
twice at 65 °C. Positive clones were isolated, purified, and sequenced.
-32P]ATP (5000 Ci/mmol;
Amersham Pharmacia Biotech) to a specific activity of approximately
4 × 106 cpm/ml using bacteriophage T4 polynucleotide
kinase (Promega). The membranes were hybridized at a high stringency
temperature (TH) (27). Prehybridization and hybridization
were carried out in standard hybridization buffer at 65 °C for
16 h (27). The filters were finally washed with 6× SSC, 0.1% SDS
for 30 min at 52 °C. The membranes were exposed to a PhosphorImager
(Molecular Dynamics).
-32P]dCTP (3000 Ci/mmol; Amersham Pharmacia Biotech).
Hybridization was carried out at 42 °C overnight in standard
hybridization buffer with 50% formamide (28). After hybridization, the
membrane was finally washed twice at 68 °C for 15 min in 0.1× SSC,
0.1% SDS. The membrane was exposed to a PhosphorImager.
-D-thiogalactoside. The bacteria were pelleted by
centrifugation, resuspended in 20 mM Tris-HCl (pH 7.9)
containing 5 mM imidazole and 0.5 M NaCl, and
ultrasonicated. Purification of recombinant protein was achieved by
chromatography on His-Bind resin (Novagen) following the pET system
manual (Novagen). The eluted protein was dialyzed with 30 mM Tris-HCl (pH 7.0), 50 µM EDTA, and 5 mM 2-mercaptoethanol and then concentrated 10-fold by a
YM-10 filter (Amicom).
Cr + ATP, using the colorimetric detection
method (31, 32). Samples of the recombinant protein were diluted 1:100
in a buffer containing 30 mM Tris, pH 7.0, 50 µM EDTA, and 5 mM 2-mercaptoethanol. Reagents
were from the creatine kinase isoenzyme colorimetric detection kit
(Sigma Diagnostics) and were used according to the manufacturer's
instructions modified for optimum assay conditions. Reaction mixtures
containing 20 mM phosphocreatine (PCr) and 4 mM
ADP were incubated for 30 min at 25 °C. The reaction was stopped by
the addition of p-hydroxymercuribenzoate, and then
-naphthol solution and diacetyl solution were added and incubated
for 20 min at 37 °C. The sample was measured at
A520. CK activity was determined from the
creatine standard calibration curve. One enzyme unit corresponds to 1 µmol of creatine formed per min at pH 7.0 and 25 °C. Under this
condition, the analysis of the three subisoforms of carp M-CK
isoenzymes at different temperatures was carried out.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
ZAPII cDNA library constructed from poly(A)
mRNA of carp striated muscle was screened with a PCR-synthesized
nucleotide probe. After the first, second, and third screening, we
selected 12 clones for further analysis, and they could be grouped into three PstI restriction patterns. One clone from each group
of the cDNAs, designated M1-CK, M2-CK, and M3-CK was picked, and subjected to nucleotide sequence analysis.
View larger version (68K):
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Fig. 1.
cDNA sequences of carp M1-CK, M2-CK, and
M3-CK. Nucleotides are numbered in the 5' to 3' direction starting
with the first nucleotide. Dashed lines
below the M1-CK sequence denote DNA identical with the M1-CK
cDNA. Gaps in the nucleotide sequence alignment are indicated by
dots and are not taken into account for nucleotide
numbering. Numbers 1-70 and
1217-1568 in M1-CK, 1-86 and
1233-1580 in M2-CK, and 1-61 and
1205-1508 in M3-CK represent the 5'- and 3'-untranslated
regions, respectively. The start and stop codons are shaded.
Putative polyadenylation signals are underlined in the
3'-untranslated region, and the poly(A) tail is in boldface type. The
probe sequences for Southern hybridization are outlined with
boxes.
Sequence homology among CK isoenzymes within the coding region
Percentages of identity of different regions of the three carp creatine
kinase cDNAs
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Fig. 2.
Deduced amino acid sequences of the three
carp M-CK isoenzymes with comparisons with chicken, human, and
Torpedo M-CK isoenzymes. The amino acid sequences of
the carp, chicken, human, and Torpedo were aligned and
arranged using the programs Pileup and Pretty of the GCG software
package. Amino acids that are identical to the carp M1-CK are
represented by hyphens in the corresponding sequences. Gaps
in the nucleotide sequence alignments are indicated by dots.
Boxed amino acid residues (numbered I
to VI) below the amino acid sequences mark the
regions with the most pronounced sequence conservation.
Shaded areas mark Cys-74, Thr-133, Lys-196,
Ser-239, Cys-283, Thr-322, and Asp-340. The underlined
regions (A to I) are either
isoform-specific or allow a clear cut distinction between mitochondrial
and cytosolic CK isoenzymes.
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Fig. 3.
Variant M-CK genes in the carp genome.
Each genomic DNA at 20 µg was digested with HindIII
(lane 1), PstI (lane
2), and PvuII (lane 3).
Each of the carp M1-CK (lane 4), M2-CK
(lane 5), and M3-CK (lane
6) cDNA clones at 500 pg was digested with
XhoI, and the fragments were resolved on an agarose gel and
transferred onto a nitrocellulose membrane. The three Southern blots
were hybridized separately to the three carp M-CK-specific probes, and
the sequences of these probes are shown in Fig. 1. The fragment sizes
of the marker DNA (phage DNA restricted with HindIII)
are indicated in kb. The hybridization patterns show that the three
carp M-CK cDNAs are encoded by different genes.
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Fig. 4.
Tissue distribution of the three carp M-CK
mRNAs. Tissue-specific expression of the mRNAs
corresponding to the three carp M-CKs was examined by high stringency
Northern analysis. Each lane contained approximately 10 µg of total
RNA. Blots were probed with random-primed 32P-labeled
probes derived from the 3'-untranslated region of carp M1-CK
(upper panel), M2-CK (middle
panel), and M3-CK (lower panel)
cDNA clones by PCR. Three M-CK mRNAs (1.6 kb) in carp tissues
were abundantly expressed in red and white muscle, whereas very faint
signals were observed in heart; and no transcripts were detected in the
brain, kidney, gill, or liver. Ethidium bromide-stained 18S rRNAs are
intended to show the loading differences in different lanes, and the
images have been converted artificially from white to black.
View larger version (77K):
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Fig. 5.
Immunoblot analysis of recombinant carp M-CK
isoenzymes. Lysates, from overnight bacterial cultures containing
1 mM isopropyl -D-thiogalactoside in the
last 2 h of culture, were subjected to purification by
chromatography on His-Bind resin. A, the Coomassie
Blue-stained SDS-polyacrylamide gel of crude bacterial lysates
(B.L.) and His-Bind affinity-purified M-CKs recombinant
protein (R.P.). Lane 1 contained
molecular size markers (NOVEX). Lanes 2,
4, and 6 contained approximately 20 µg of total
protein each of M1-CK, M2-CK, and M3-CK crude bacterial lysates, and
lanes 3, 5, and 7 contained
2 µg of the respective purified M-CK isoenzymes. B shows
Western blotting results of purified M-CK isoenzymes using antibodies
raised against human MM-CK isoenzyme. Lane 1 contained molecular size markers; and lanes 2-4
contained the recombinant proteins of approximately 2 µg each encoded
by carp M1-CK, M2-CK, and M3-CK cDNAs, respectively.
Lane 5 contained approximately 2 µg of purified
human M-CK as a control.
Comparison of specific activity and kinetic parameters of three carp
M-CK isoenzymes
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[in a new window]
Fig. 6.
Activity of three carp M-CK subisoforms and
human M-CK at different temperatures. This plot shows M-CK
specific activity with incubation at 37, 30, 25, 20, 15, and 10 °C.
CK activity measurements were performed at pH 7.0. [PCr] was 20 mM, and [ADP] was 4 mM. The concentration of
Mg2+ was the same as that of ADP. The values given
represent averages of three independent measurements for 0.04 µg of
recombinant M1-CK, M2-CK, and M3-CK proteins. The plotted data for
purified carp M1-CK ( 
), M2-CK (
), M3-CK (×), and human M-CK
(
), indicate different specific activities between the three M-CK
subisoforms at different temperatures. The highest specific activity of
each isoenzyme is defined as 100%.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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