Isolation and characterization of the gene and cDNA encoding human mitochondrial creatine kinase

Creatine kinase (CK; EC 2.7.3.2) isoenzymes play prominent roles in energy metabolism. Nuclear genes encode three known CK subunits: cytoplasmic muscle (MCK), cytoplasmic brain (BCK), and mitochondrial (MtCK). We have isolated the gene and cDNA encoding human placental MtCK. By using a dog heart MCK cDNA-derived probe, the 7.0-kb EcoRI fragment from one cross-hybridizing genomic clone was isolated and its complete nucleotide sequence determined. A region of this clone encoded predicted amino acid sequence identical to residues 15-26 of the human heart MtCK NH2-terminal protein sequence. The human placental MtCK cDNA was isolated by hybridization to a genomic fragment encoding this region. The human placental MtCK gene contains 9 exons encoding 416 amino acids, including a 38-amino acid transit peptide, presumably essential for mitochondrial import. Residues 1-14 of human placental MtCK cDNA-derived NH2-terminal sequence differ from the human heart MtCK protein sequence, suggesting that tissue-specific MtCK mRNAs are derived from multiple MtCK genes. RNA blot analysis demonstrated abundant MtCK mRNA in adult human ventricle and skeletal muscle, low amounts in placenta and small intestine, and a dramatic increase during in vitro differentiation induced by serum-deprivation in the non-fusing mouse smooth muscle cell line, BC3H1. These findings demonstrate coordinate regulation of MtCK and cytosolic CK gene expression and support the phosphocreatine shuttle hypothesis.     

C-terminal lysines determine phospholipid interaction of sarcomeric mitochondrial creatine kinase

High affinity interaction between octameric mitochondrial creatine kinase (MtCK) and the phospholipid cardiolipin in the inner mitochondrial membrane plays an important role in metabolite channeling between MtCK and inner membrane adenylate translocator, which itself is tightly bound to cardiolipin. Three C-terminal basic residues revealed as putative cardiolipin anchors in the x-ray structures of MtCK and corresponding to lysines in human sarcomeric MtCK (sMtCK) were exchanged by in vitro mutagenesis (K369A/E, K379Q/A/E, K380Q/A/E) to yield double and triple mutants. sMtCK proteins were bacterially expressed, purified to homogeneity, and verified for structural integrity by enzymatic activity, gel filtration chromatography, and CD spectroscopy. Interaction with cardiolipin and other acidic phospholipids was quantitatively analyzed by light scattering, surface plasmon resonance, and fluorescence spectroscopy. All mutant sMtCKs showed a strong decrease in vesicle cross-linking, membrane affinity, binding capacity, membrane ordering capability, and binding-induced changes in protein structure as compared with wild type. These effects did not depend on the nature of the replacing amino acid but on the number of exchanged lysines. They were moderate for Lys-379/Lys-380 double mutants but pronounced for triple mutants, with a 30-fold lower membrane affinity and an entire lack of alterations in protein structure compared with wild-type sMtCK. However, even triple mutants partially maintained an increased order of cardiolipin-containing membranes. Thus, the three C-terminal lysines determine high affinity sMtCK/cardiolipin interaction and its effects on MtCK structure, whereas low level binding and some effect on membrane fluidity depend on other structural components. These results are discussed in regard to MtCK microcompartments and evolution.     

Divergent enzyme kinetics and structural properties of the two human mitochondrial creatine kinase isoenzymes

The mitochondrial isoenzymes of creatine kinase (MtCK), ubiquitous uMtCK and sarcomeric sMtCK, are key enzymes of oxidative cellular energy metabolism and play an important role in human health and disease. Very little is known about uMtCK in general, or about sMtCK of human origin. Here we have heterologously expressed and purified both human MtCK isoenzymes to perform a biochemical, kinetic and structural characterization. Both isoenzymes occurred as octamers, which can dissociate into dimers. Distinct Stokes' radii of uMtCK and sMtCK in solution were indicative for conformational differences between these equally sized proteins. Both human MtCKs formed 2D-crystals on cardiolipin layers, which revealed further subtle differences in octamer structure and stability. Octameric human sMtCK displayed p4 symmetry with lattice parameters of 145 A, indicating a 'flattening' of the octamer on the phospholipid layer. pH optima and enzyme kinetic constants of the two human isoenzymes were significantly different. A pronounced substrate binding synergism (Kd > Km) was observed for all substrates, but was most pronounced in the forward reaction (PCr production) of uMtCK and led to a significantly lower Km for creatine (1.01 mM) and ATP (0.11 mM) as compared to sMtCK (creatine, 7.31 mM; ATP, 0.68 mM).     

Purification and characterization of human mitochondrial creatine kinase. A single enzyme form

Purification of human mitochondrial creatine kinase has been difficult and procedures that were highly successful in purifying canine enzyme failed for human mitochondrial creatine kinase. In the present study, we employed ultracentrifugation to remove the lipid, urea to prevent aggregation, followed by a final step of chromatofocusing which yielded a preparation of human mitochondrial creatine kinase with a specific enzyme activity of greater than 400 IU/mg. Biochemical and immunological characterization showed the preparation to be highly pure and free of even trace amounts of other creatine kinase isoenzymes. Antiserum specific for mitochondrial creatine kinase was developed which exhibited no cross-reactivity to cytosolic creatine kinase and mitochondrial creatine kinase did not cross-react with antiserum to the cytosolic forms. Marked differences were noted, both biochemically and immunologically, between mitochondrial creatine kinase and the cytosolic forms. Human mitochondrial creatine kinase was shown to have a molecular weight of around 82,000 and to be composed of two subunits of equal molecular weights around 41,000. Aggregates of mitochondrial creatine kinase were observed with molecular weights of around 200,000 in the absence of urea or if isolated from material after having undergone proteolysis. Isolation from fresh material or in the presence of urea inhibited aggregate formation for both canine and human mitochondrial creatine kinase. Despite claims of several investigators that mitochondrial creatine kinase exhibits two to three forms with varying molecular weights, our data indicate a single enzyme form made up of a subunit with a molecular weight of 41,000 and the high molecular weight aggregates appear to be induced artifacts. A radioimmunoassay was developed for human mitochondrial creatine kinase which, with appropriate modifications, should detect mitochondrial creatine kinase in human plasma.     

Purification of mitochondrial creatine kinase. Biochemical and immunological characterization

Mitochondrial creatine kinase was purified from canine myocardium. The preparation exhibited a positively charged isoenzyme free of other creatine kinase isoenzymes and on sodium dodecyl sulfate gel exhibited a single protein band. Amino acid composition showed mitochondrial creatine kinase to be different from that of MM or BB creatine kinase and did not hybridize with the M or B subunits of the cytosolic forms. Antiserum was developed to mitochondrial creatine kinase which did not cross-react with cytosolic creatine kinases. Antiserum to cytosolic creatine kinase exhibited no reaction to mitochondrial creatine kinase. Utilizing the specific antiserum, a radioimmunoassay was developed for the specific detection of mitochondrial creatine kinase. Thus, mitochondrial creatine kinase was purified and shown to be comprised of a unique subunit which is biochemically and immunologically distinct from the cytosolic creatine kinases.     

Crystal structure of human ubiquitous mitochondrial creatine kinase

Creatine kinase (CK), catalyzing the reversible trans-phosphorylation between ATP and creatine, plays a key role in the energy metabolism of cells with high and fluctuating energy requirements. We have solved the X-ray structure of octameric human ubiquitous mitochondrial CK (uMtCK) at 2.7 A resolution, representing the first human CK structure. The structure is very similar to the previously determined structure of sarcomeric mitochondrial CK (sMtCK). The cuboidal octamer has 422 point group symmetry with four dimers arranged along the fourfold axis and a central channel of approximately 20 A diameter, which extends through the whole octamer. Structural differences with respect to sMtCK are found in isoform-specific regions important for octamer formation and membrane binding. Octameric uMtCK is stabilized by numerous additional polar interactions between the N-termini of neighboring dimers, which extend into the central channel and form clamp-like structures, and by a pair of salt bridges in the hydrophobic interaction patch. The five C-terminal residues of uMtCK, carrying positive charges likely to be involved in phospholipid-binding, are poorly defined by electron density, indicating a more flexible region than the corresponding one in sMtCK. The structural differences between uMtCK and sMtCK are consistent with biochemical studies on octamer stability and membrane binding of the two isoforms.     

The structural features of beef heart mitochondrial creatine kinase.

Two forms of mitochondrial creatine kinase (Mi-CK) having Mr 320 kDa and 240 kDa as determined by gel-filtration on Sephacryl S-300 in 0.1 M Tris-HCl pH 7.4 were investigated. The sedimentation coefficient values for these two forms were found to be identical and equal to 12.3 S. When studied by electron microscopy the main type of images for the 320 kDa and 240 kDa Mi-CK appeared as annular particles, 12-14 nm in diameter, with a well-detected subunit structure and a central hollow, 3-4nm in diameter filled with the dye. The results of the averaging of the main type of individual Mi-CK images and particles of the two-dimensional crystal layer point to the overall geometry of the Mi-CK molecule structure as containing eight subunits arranged by a 4-fold symmetry around the central hollow. It may be that the eight identical subunits of crystalline Mi-CK are arranged with a P422 symmetry. However in both cases the averaged main images do not show a mirror symmetry. The multiplicity of the observed projections close to annular one provides additional evidence in favour of the great lability and structural mobility of the Mi-CK subunits. It allows to assume that two forms (320 kDa and 240 kDa) are not the different oligomers but they are two functionally distinct conformational states of octameric molecule of Mi-CK.     

Native mitochondrial creatine kinase forms octameric structures. I. Isolation of two interconvertible mitochondrial creatine kinase forms, dimeric and octameric mitochondrial creatine kinase: characterization, localization, and structure-function relationships

The mitochondrial isoform of creatine kinase (Mi-CK, EC 2.7.3.2) purified to homogeneity from chicken cardiac muscle by the mild and efficient technique described in this article was greater than or equal to 99.5% pure and consisted of greater than or equal to 95% of a distinct, octameric Mi-CK protein species, with a Mr of 364,000 +/- 30,000 and an apparent subunit Mr of 42,000. The remaining 5% were dimeric Mi-CK with an apparent Mr of 86,000 +/- 8,000. Octamerization was not due to covalent linkages or intermolecular disulfide bonding. Upon dilution into buffers of low ionic strength and alkaline pH, octameric Mi-CK slowly dissociated in a time-dependent manner (weeks-months) into dimeric Mi-CK. However, the time scale of dimerization was reduced to minutes by the addition to diluted Mi-CK octamers of a mixture of Mg2+, ADP, creatine and nitrate known to induce a transition-state analogue complex (Milner-White, E.J., and Watts, D. C. (1971) Biochem. J. 122, 727-740). The conversion was fully reversible, and octamers were reformed by simple concentrations of Mi-CK dimer solutions to greater than or equal to 1 mg/ml at near neutral pH and physiological salt concentrations in the absence of adenine nucleotide. After separation of the two Mi-CK species by gel filtration, electron microscopic analysis revealed uniform square-shaped particles with a central negative-stain-filled cavity in the octamer fractions and "banana-shaped" structures in the dimer fractions. Mi-CK was localized inside the mitochondria by immunogold labeling with polyclonal antibodies. A dynamic model of the octamer-dimer equilibrium of Mi-CK and the preferential association of the octameric Mi-CK form with the inner mitochondrial membrane is discussed in the context of regulation of Mi-CK activity, mitochondrial respiration, and the CP shuttle.     

Heterogeneity of mitochondrial creatine kinase

The heterogeneity of cardiac sarcomeric mitochondrial creatine kinase (creatine N-phosphotransferase, EC 2.7.3.2, sMi-CK), namely, brain ubiquitous Mi-CK (uMi-CK) and an atypical Mi-CK detected in the serum of a patient with ovarian cancer, was studied by isoelectric focusing. These Mi-CKs were found to be slightly different from each other with respect to their pIs under the examined conditions. The atypical Mi-CK was found to be an atypically oxidized form of uMi-CK. Results suggest that these heterogeneities of Mi-CK are caused by the genotypes, structures, biological functions and metabolism/dissimilation of Mi-CKs in the mitochondria and intravascular circulation.     

Structure and expression of the human creatine kinase B gene

Various cDNAs for creatine kinase type B (CK-B) were isolated from human cDNA libraries using a 26-oligonucleotide guess-mer probe. One of the cDNAs appeared to be almost full-length and contained an open reading frame coding for the 381 amino acid residues of the human CK-B polypeptide. The nucleotide sequences of the translated region as well as the primary protein structure show a high degree of homology with known CK-B and CK-M sequences of other vertebrates. The level of CK-B RNA as a measure of CK-B gene activity was determined in various human tissues and cultured cells. Our results confirm that CK-B is expressed in a tissue-specific manner and give support to the previously proposed relation between CK-B gene activity and cell proliferation. Screening of genomic DNA with various cDNA regions as probes revealed that there is only one CK-B gene per haploid genome. Gene cloning and sequencing indicated that CK-B is coded for by a relatively small gene of 3.2 kb in size, which is partially overlapped by an HTF island (A. P. Bird (1986) Nature (London) 321, 557-558) with an extremely high G + C content at its 5' end.     

Expression of the mitochondrial creatine kinase genes

Mitochondrial Creatine Kinase (MtCK) is responsible for the transfer of high energy phosphate from mitochondria to the cytosolic carrier, creatine, and exists in mammals as two isoenzymes encoded by separate genes. In rats and humans, sarcomere-specific MtCK (sMtCK) is expressed only in skeletal and heart muscle, and has 87% nucleotide identity across the 1257 bp coding region. The ubiquitous isoenzyme of MtCK (uMtCK) is expressed in many tissues with highest levels in brain, gut, and kidney, and has 92% nucleotide identity between the 1254 bp coding regions of rat and human. Both genes are highly regulated developmentally in a tissue-specific manner. There is virtually no expression of sMtCK mRNA prior to birth. Unlike cytosolic muscle CK (MCK) and brain CK (BCK), there is no developmental isoenzyme switch between the MtCKs. Cell culture models representing the tissue-specific expression of either sMtCK or uMtCK are available, but there are no adequate developmental models to examine their regulation. Several animal models are available to examine the coordinate regulation of the CK gene family and include 1) Cardiac Stress by coarctation (sMtCK, BCK, and MCK), 2) Uterus and placenta during pregnancy (uMtCK and BCK), and 3) Diabetes and mitochondrial myopathy (sMtCK, BCK, and MCK). We report the details of these findings, and discuss the coordinate regulation of the genes necessary for high-energy transduction.     

Purification and immunological characterization of human myocardial MB creatine kinase

Elevated plasma MB creatine kinase (CK) is considered the most sensitive and specific diagnostic indicator of myocardial infarction. However, attempts to purify human MB CK have been unsuccessful. The need for purified human MB CK was further enhanced with the development of a radioimmunoassay for CK isoenzymes which would provide more prompt and specific detection of myocardial infarction. The major protein contaminant of MB CK is albumin which has been difficult to separate due to their similar electrophoretic mobility. Human hearts were obtained within 2 h postmortem and the tissue homogenized in 50 mm Tris-HCl (pH 7.4), 2 mm mercaptoethanol. The CK was recovered from the supernatant (31,000g) by ethanol extraction (50–70%). The resuspended pellet was fractionated on DEAE Sephadex A-50 with a salt gradient (50–500 mm, pH 8.0). The MB fraction contained about 90% albumin. The preparation was bound to an Affigel blue column and contaminating proteins other than albumin were eluted with 50 mm Tris-HCl (pH 8.0), 2 mm mercaptoethanol. MB CK was eluted with 250 mm NaCl, but the albumin remained bound. The MB fraction with a specific activity of 453 IU/mg represented an 80-fold increase in purity and exhibited a single protein band on polyacrylamide gels. Purified MB CK labeled with ¹²⁵I exhibited no binding to human albumin antiserum, but bound to MB CK antiserum, and unlabeled MB CK competitively inhibited binding of ¹²⁵I-MB CK in the radioimmunoassay system exhibiting a sensitivity for detection of plasma MB CK at the nanogram level.     

Characterization of isoforms of human mitochondrial creatine kinase by isoelectric focusing

High enzyme activity of mitochondrial creatine kinase (creatine-N-phosphotransferase, mCK, EC 2.7.3.2) was detected in serum from a patient with advanced carcinoma of the rectum and its isoforms were characterized by means of isoelectric focusing (IEF). Three forms of mCK, membrane-bound (pI 6.9–7.0), octameric (pI 7.0–7.9) and dimeric (pI 6.7, 6.8, 6.9 and 7.0), were detected in the fresh serum. These three forms of mCK were converted to five dimeric isoforms, and these were characterized as one reduced form (pI 7.0) and four oxidized (pI 6.6, 6.7, 6.8 and 6.9) forms upon treatment with urea, hydrogen peroxide or 2-mercaptoethanol (2-ME). The C-terminal of the mCKs was concluded to be a lysine residue because the mCKs treated with carboxypeptidase B migrated to positions closer to the anode than did those not treated with carboxypeptidase B. Therefore, four bands were concluded to represent one reduced-delysined isoform (pI 6.4) and three oxidized-delysined isoforms (pI 6.1, 6.2 and 6.3). The broad octameric mCK band disappeared and a narrow band focused at pI 6.8–6.9 appeared upon probable delysination of the mCKs. Thus, the number of lysine residues at the C-terminal of the octamer was concluded to be variable due to variable catalysis by carboxypeptidase N in the plasma. mCKs seemed to be inactivated during conversion from a membrane-bound form to dimeric oxidized-delysined forms via the octameric, dimeric reduced and oxidized forms.     

Over-expression, purification and characterization of the oligomerization dynamics of an invertebrate mitochondrial creatine kinase

Mitochondrial creatine kinase (MtCK) plays a central role in energy homeostasis within cells that display high and variable rates of ATP turnover. Vertebrate MtCKs exist primarily as octamers but readily dissociate into constituent dimers under a variety of circumstances. MtCK is an ancient protein that is also found in invertebrates including sponges, the most primitive of all multi-cellular animals. We have cloned, expressed, and purified one of these invertebrate MtCKs from a marine polychaete worm, Chaetopterus variopedatus (CVMtCK). Size exclusion chromatography and dynamic light scattering (DLS) were used to characterize oligomeric state in comparison with that of octameric chicken sarcomeric isoform (SarMtCK). At protein concentrations >1 mg/ml, CVMtCK was predominantly octameric (>90%). When diluted to 0.1 mg/ml, CVMtCK dissociated into dimers much more rapidly than SarMtCK when observed under identical conditions. The rate of dissociation for both MtCKs increased as temperature rose from 2 to 28 degrees C, and in CVMtCK, fell at higher incubation temperatures. The fraction of octameric CVMtCK at equilibrium increased with temperature and then fell. Temperature transition studies showed that octamers and dimers were rapidly interconvertible on a similar time scale. Importantly, when CVMtCK was converted to the transition state analog complex (TSAC), both size exclusion chromatography and DLS showed that there was minimal dissociation of octamers into dimers while SarMtCK octamers were highly unstable as the TSAC. These results clearly show distinct differences in octamer stability between CVMtCK and SarMtCK, which could impact function under physiological circumstances. Furthermore, the large yield of recombinant protein and high stability of CVMtCK in the TSAC suggest that this protein might be a good target for crystallization efforts.     

Physiological role of the creatine kinase system and the problem of regulating the activity of mitochondrial creatine kinase

The review contains the analysis of present-day concepts on the physiological role of the creatine kinase system. A hypothesis on the buffering functions of the creatine kinase system which ensures a constant ATP level in cells and a hypothesis according to which phosphocreatine is a macroergic phosphate carrier from mitochondria to the sites of their utilization are considered. In connection with the creatine phosphate carrier hypothesis according to which the transport function of the creatine kinase system is provided for by an effective function of mitochondrial creatine kinase, feasible mechanisms of mitochondrial creatine kinase activity regulation are considered: as a result of creation of local concentration of nucleotide substrates as well as changes in the properties of creatine kinase itself which may result from the enzyme conversion from the membrane-bound to the free form or from the interconversion of oligomeric forms of the enzyme.     

Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes

Creatine kinase (EC 2.7.3.2) isoenzymes play a central role in energy transduction. Nuclear genes encode creatine kinase subunits from muscle, brain, and mitochondria (MtCK). We have recently isolated a cDNA clone encoding MtCK from a human placental library which is expressed in many human tissues (Haas, R. C., Korenfeld, C., Zhang, Z., Perryman, B., Roman, D., and Strauss, A. W. (1989) J. Biol. Chem. 264, 2890-2897). With nontranslated and coding region probes, we demonstrated by RNA blot analysis that the MtCK mRNA in sarcomeric muscle is distinct from this placenta-derived, ubiquitous MtCK cDNA. To compare these different mRNAs, a MtCK cDNA clone was isolated from a human heart library and characterized by complete nucleotide sequence analysis. The chemically determined NH2-terminal 26 residues of purified human heart MtCK protein are identical to those predicted from this sarcomeric MtCK cDNA. The human sarcomeric and ubiquitous cDNAs share 73% nucleotide and 80% predicted amino acid sequence identities, but have less than 66% identity with the cytosolic creatine kinases. The sarcomeric MtCK cDNA encodes a 419-amino acid protein which contains a 39-residue transit peptide essential for mitochondrial import. Primer extension analysis predicts a 348-base pair 5'-nontranslated region. RNA blot analysis demonstrates that heart-derived MtCK is sarcomere-specific, but the ubiquitous MtCK mRNA is expressed in most tissues. Thus, separate nuclear genes encode two closely related, tissue-specific isoenzymes of MtCK. Our finding that multiple genes encode different mitochondrial protein isoenzymes is rare.     

Association of chicken mitochondrial creatine kinase with the inner mitochondrial membrane

The stoichiometry and dissociation constant for the binding of homogeneous chicken heart mitochondrial creatine kinase (MiMi-CK) to mitoplasts was examined under a variety of conditions. Salts and substrates release MiMi-CK from mitoplasts in a manner that suggests an ionic interaction. The binding of MiMi-CK to mitoplasts is competitively inhibited by Adriamycin, suggesting that they compete for the same binding site. Fluorescence measurements also show that Adriamycin binds to MiMi-CK so that the effect of Adriamycin on the binding of MiMi-CK to mitoplasts is not simple. Titrating mitoplasts with homogeneous MiMi-CK at different pH values shows a pH-dependent equilibrium involving a group(s) on either the membrane or the enzyme with a pKa = 6. Extrapolating these titrations to infinite MiMi-CK concentration gives 14.6 IU bound/nmol cytochrome aa3 corresponding to 1.12 mol MiMi-CK/mol cytochrome aa3. Chicken heart mitochondria contain, after isolation, 2.86 +/- 0.42 IU/nmol cytochrome aa3. Titrating respiring mitoplasts with carboxyatractyloside gives at saturation 3.3 mol ADP/ATP translocase/mol cytochrome aa3. Therefore, chicken heart mitoplasts can maximally bind about 1 mol of MiMi-CK per 3 mol translocase; in normal chicken heart mitochondria about 1 mol of MiMi-CK is present per 13 mol translocase.