Fluxes through cytosolic and mitochondrial creatine kinase, measured by P-31 NMR

The kinetic properties of the cytoplasmic and the mitochondrial iso-enzymes of creatine kinase from striated muscle were studied in vitro and in vivo. The creatine kinase (CK) iso-enzyme family has a multi-faceted role in cellular energy metabolism and is characterized by a complex pattern of tissue-specific expression and subcellular distribution. In mammalian tissues, there is always co-expression of at least two different CK isoforms. As a result, previous studies into the role of CK in energy metabolism have not been able to directly differentiate between the individual CK species. Here, we describe experiments which were directed at achieving this goal. First, we studied the kinetic properties of the muscle-specific cytoplasmic and mitochondrial CK isoforms in purified form under in vitro conditions, using a combination of P-31 NMR and spectrophotometry. Secondly, P-31 NMR measurements of the flux through the CK reaction were carried out on intact skeletal and heart muscle from wild-type mice and from transgenic mice, homozygous for a complete deficiency of the muscle-type cytoplasmic CK isoform. Skeletal muscle and heart were compared because they differ strongly in the relative abundance of the CK isoforms. The present data indicate that the kinetic properties of cytoplasmic and mitochondrial CK are substantially different, both in vitro and in vivo. This finding particularly has implications for the interpretation of in vivo studies with P-31 NMR. (Mol Cell Biochem 174: 33–42, 1997)     

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.     

Enzyme kinetics of a highly purified mitochondrial creatine kinase in comparison with cytosolic forms

Summary Mitochondrial creatine kinase (CK) purified from canine myocardium showed a single protein band on SDS-PAGE and was free of MMCK. Its amino acid composition was different than MMCK or BBCK and did not react to antiserum to MMCK or BBCK. Using purified mitochondrial, MM and BBCK, the velocity of reaction (V) was estimated for creatine phosphate (CP), creatine (C), adenosine triphosphate (ATP) and adenosine diphosphate (ADP) over a wide range of concentrations including those at V_max. The values for K_m (mM/L) derived from Lineweaver-Burke plots are shown: The affinity of mitochondrial CK for C is much greater than MMCK which is compatible with the energy shuttle hypothesis, namely ATP is converted by mitochondrial CK to CP, and then diffuses to the myofibril for conversion to ATP for utilization.     

The influence of the cytosolic oncotic pressure on the permeability of the mitochondrial outer membrane for ADP: implications for the kinetic properties of mitochondrial creatine kinase and for ADP channelling into the intermembrane space

Summary Cytosolic proteins as components of the physiological mitochondrial environment were substituted by dextrans added to media normally used for incubation of isolated mitochondria. Under these conditions the volume of the intermembrane space decreases and the contact sites between the both mitochondrial membranes increase drastically. These morphological changes are accompanied by a reduced permeability of the mitochondrial outer compartment for adenine nucleotides as it was shown by extensive kinetic studies of mitochondrial enzymes (oxidative phosphorylation, mi-creatine kinase, mi-adenylate kinase). The decreased permeability of the mitochondrial outer membrane causes increased rate dependent concentration gradients in the micromolar range for adenine nucleotides between the intermembrane space and the extramitochondrial space. Although all metabolites crossing the outer membrane exhibit the same concentration gradients, considerable compartmentations are detectable for ADP only due to its low extramitochondrial concentration. The consequences of ADP-compartmentation in the mitochondrial intermembrane space for ADP-channelling into the mitochondria are discussed.     

Theoretical modelling of some spatial and temporal aspects of the mitochondrion/creatine kinase/ myofibril system in muscle

After discussing approaches to the modelling of mitochondrial regulation in muscle, we describe a model that takes account, in a simplified way, of some aspects of the metabolic and physical structure of the energy production/usage system. In this model, high-energy phosphates (ATP and phosphocreatine) and low energy metabolites (ADP and creatine) diffuse between the mitochondrion and the myofibrillar ATPase, and can be exchanged at any point by creatine kinase. Creatine kinase is not assumed to be at equilibrium, so explicit account can be taken of substantial changes in its activity of the sort that can now be achieved by transgenic technology in vivo. The ATPase rate is the input function. Oxidative ATP synthesis is controlled by juxtamitochondrial ADP concentration. To allow for possible functional coupling between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase, we define parameters and that set the fraction of the total flux carried by ATP rather than phosphocreatine out of the mitochondrial unit and into the ATPase unit, respectively. This simplification is justified by a detailed analysis of the interplay between the mitochondrial outer membrane porin proteins, mitochondrial creatine kinase and the adenine nucleotide translocase. As both processes of possible coupling are incorporated into the model as quantitative parameters, their effect on the energetics of the whole cell model can be explicitly assessed. The main findings are as follows: (1) At high creatine kinase activity, the hyperbolic relationship of oxidative ATP synthesis rate to spatially averaged ADP concentration at steady state implies also a near-linear relationship to creatine concentration, and a sigmoid relation to free energy of ATP hydrolysis. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on these relationships. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, and this is exaggerated when or is near unity (i.e. little coupling). (2) At high creatine kinase activity, the fraction of flow at steady state carried in the middle of the model by ATP is small, unaffected by the degree of functional coupling, but increases with ADP concentration and rate of ATP turnover. Lowering the creatine kinase activity raises this fraction, and this is exaggerated when or is near unity. (3) Both creatine and ADP concentrations show small gradients decreasing towards the mitochondrion (in the direction of their net flux), while ATP and phosphocreatine concentration show small gradients decreasing towards the myosin ATPase. Unless = 0 (i.e. complete coupling), there is a gradient of net creatine kinase flux that results from the need to transform some of the adenine nucleotide flux at the ends of the model into creatine flux in the middle; the overall net flux is small, but only zero if = . A reduction in cytosolic creatine kinase activity decreases ADP concentration at the mitochondrial end and increases it at the ATPase end. (4) During work-jump transitions, spatial average responses exhibit exponential kinetics similar to those of models of mitochondrial control that assume equilibrium conditions for creatine kinase. (5) In response to a step increase in ATPase activity, concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. This simplified model embodies many important features of muscle in vivo, and accommodates a range of current theories as special cases. We end by discussing its relationship to other approaches to mitochondrial regulation in muscle, and some possible extensions of the model.     

Interaction of creatine kinase and hexokinase with the mitochondrial membranes,and self-association of creatine kinase: crosslinking studies

Summary Covalent coupling of protein by crosslinking reagents have been used to study the interaction of mitochondrial creatine kinase (CKm) and hexokinase (HK) with the mitochondrial membranes.The effects of crosslinkers were studied either by following the inhibition of solubilization of enzymatic activities or by modification of the electrophoretic patterns of proteins solubilized from mitochondria after treatment with different crosslinkers.Dimethylsuberimidate (DMS) efficiently reduced the amount of HK activity solubilized by various agents but it did not modify solubilization of CKm from mitochondria. The effect of DMS on HK solubilization did not result from non specific crosslinking since it did not impede the solubilization of adenylate kinase.Bissuccinimidyl another class of crosslinker has been tested. Ethyleneglycol bis (succinimidyl succinate)(EGS) efficiently reduced HK solubilization, but in addition it induced osmotic stabilization of mitochondria and thus impeded release of soluble or solubilized proteins from the intermembrane space. Furthermore this agent drastically inhibited CKm activity and thus, in a second set of experiments the effect of crosslinkers have been studied by the disappearance of protein bands in the electrophoretic pattern of soluble fractions obtained from mitochondria, the outer membranes of which have been ruptured to allow free release of soluble proteins. Results of these experiments showed that succinimidyl reagents and Cu⁺⁺-Phenanthroline substantially reduced the amount of CKm released from mitochondria and confirmed that bisimidates were ineffective in inhibiting CKm solubilization.In addition crosslinking reagents have been used to study subunits interactions in purified CKm. Our results showed, in contrast with control experiments with a non oligomeric protein (ovalbumin) which did not give rise to polymers, that in the same conditions electrophoresis of crosslinked CKm resolved a set of species with molecular weights roughly equal to integral multiples of the protomer. These results proved that the polymeric form of CKm was an octamer.Abbreviations AK Adenylate Kinase (EC 2.7.4.3) - CKm Mitochondrial Isoenzyme of Creatine Kinase (EC 2.7.3.2) - DMS Dimethyl Suberimidate - DTT Dithiothreitol - EGS Ethylene Glycol bis (succinimidyl succinate) - EGTA Ethylene Glycol bis (aminoethyl ether) - N,N,N,N Tetraacetic acid - G6P Glucose 6 Phosphate, Hepes - N-2 Hydroxyethyl Piperazine N-2 Ethane Sulfonic Acid - HK Hexokinase (EC 2.7.1.1) - MABI methyl 4-Azido Benzoimidate - NaPi Sodium Phosphate - SANPAH N-Succinimidyl 6(4 azido 2 nitrophenylamino) Hexanoate - SDS Sodium Dodecyl Sulfate (sodium lauryl sulfate) - Tris Tris (hydroxymethyl) Aminomethane     

Macrocompartmentation of total creatine in cardiomyocytes revisited

Distribution of total creatine (free creatine + phosphocreatine) between two subcellular macrocompartments – mitochondrial matrix space and cytoplasm – in heart and skeletal muscle cells was reinvestigated by using a permeabilized cell technique. Isolated cardiomyocytes were treated with saponin (50 g/ml for 30 min or 600 g/ml for 1 min) to open the outer cellular membrane and release the metabolites from cytoplasm (cytoplasmic fraction, CF). All mitochondrial population in permeabilized cells remained intact: the outer membrane was impermeable for exogenous cytochrome c, the acceptor control index of respiration exceeded 10, the mitochondrial creatine kinase reaction was fully coupled to the adenine nucleotide translocator. Metabolites were released from mitochondrial fraction (MF) by 2–5% Triton X100. Total cellular pool of free creatine + phosphocreatine (69.6 ± 2.1 nmoles per mg of protein) was found exclusively in CF and was practically absent in MF. When fibers were prepared from perfused rat hearts, cellular distribution of creatine was not dependent on functional state of the heart and only slightly modified by ischemia. It is concluded that there is no stable pool of creatine or phosphocreatine in the mitochondrial matrix in the intact muscle cells, and the total creatine pool is localized in only one macrocompartment – cytoplasm.     

Creatine kinase and creatine kinase isoenzyme responses to heat stress

During this investigation the effects of heat acclimation and exercise on creatine kinase and creatine kinase BB isoenzyme responses in various tissues and serum of male Sprague-Dawley rats were ascertained. Forty rats were randomly divided into two groups of 20 rats each. One group was housed at 22+/-1 degrees C and the other at 33+/-1 degrees C. Each of the two groups were subdivided into two subgroups of ten rats each. One subgroup of each group was subjected to a programme of treadmill running of progressive intensity over a period of 6 weeks at the temperature at which it was housed while the other served as a resting control. At the end of the acclimation programme the rats were running at 23 m/min for 80 min. On the day of sacrifice all four subgroups were subjected to a discontinuous exercise protocol (10 min running alternated by a 2-min rest period; repeated three times) at 30+/-1 degrees C on a rodent treadmill at 23 m/min. The tissues investigated were kidney, heart and muscle. The rats were anaesthetized with pentobarbital sodium (6 mg/100 g body mass) injected intraperitoneally. The tissues were freeze-clamped and stored in liquid air until analysed. The body temperature of the four subgroups at the end of the experimental protocol were not significantly different. Acclimation at 33+/-1 degrees C resulted in significantly lower creatine kinase activity levels. Exercise at 30+/-1 degrees C also resulted in decreased creatine kinase activity levels in both acclimated groups. A similar trend was observed regarding creatine kinase BB isoenzyme activity levels, especially in kidney.     

In vitro translation of canine mitochondrial creatine kinase messenger RNA

The cell-free translation products of mRNA from canine myocardium were immunoprecipitated using antiserum specific for either the MM or mitochondrial creatine kinase subunit. The two subunits were shown to be encoded by the nuclear genome and translated from separate mRNAs. The mitochondrial subunit was translated as a polypeptide with a molecular weight approximately 6,000 greater than the mature form of the enzyme. In contrast, the M-subunit was translated as a polypeptide having a molecular weight identical to that of the mature cytosolic M-subunit. It is assumed that the mitochondrial subunit precursor must be proteolytically processed during translocation from the cytoplasm into mitochondria.     

Facilitated diffusion of myoglobin and creatine kinase and reaction-diffusion constraints of aerobic metabolism under steady-state conditions in skeletal muscle

The roles of creatine kinase (CK) and myoglobin (Mb) on steady-state facilitated diffusion and temporal buffering of ATP and oxygen, respectively, are assessed within the context of a reaction-diffusion model of muscle energetics. Comparison of the reaction-diffusion model with experimental data from a wide range of muscle fibers shows that the experimentally observed skeletal muscle fibers are generally not limited by diffusion, and the model further indicates that while some muscle fibers operate near the edge of diffusion limitation, no detectable effects of Mb and CK on the effectiveness factor, a measure of diffusion constraints, are observed under steady-state conditions. However, CK had a significant effect on average ATP concentration over a wide range of rates and length scales within the reaction limited regime. The facilitated diffusion functions of Mb and CK become observable in the model for larger size cells with low mitochondrial volume fraction and for low boundary O(2) concentration and high ATP demand, where the fibers may be limited by diffusion. From the transient analysis it may be concluded that CK primarily functions to temporally buffer ATP as opposed to facilitating diffusion while Mb has a small temporal buffering effect on oxygen but does not play any significant role in steady-state facilitated diffusion in skeletal muscle fibers under most physiologically relevant regions.     

The structure of mitochondrial creatine kinase and its membrane binding properties

The biochemical and biophysical characterization of the mitochondrial creatine kinase (Mi-CK) from chicken cardiac muscle is reviewed with emphasis on the structure of the octameric oligomer by electron microscopy and on its membrane binding properties. Information about shape, molecular symmetry and dimensions of the Mi-CK octamer, as obtained by different sample preparation techniques in combination with image processing methods, are compared. The organization of the four dimeric subunits into the Mi-CK complex as apparent in the end-on projections is discussed and the consistently observed high binding affinity of the four-fold symmetric end-on faces towards many support films and towards each other is outlined. A study on the oligomeric state of the enzyme in solution and in intact mitochondria, using chemical crosslinking reagents, is presented together with the results of a search for a possible linkage of Mi-CK with the adenine nucleotide translocator (ANT). The nature of Mi-CK binding to model membranes, demonstrating that rather the octameric than the dimeric subspecies is involved in lipid interaction and membrane contact formation, is resumed and put into relation to our structural observations. The findings are discussed in light of a possiblein vivo function of the Mi-CK octamer bridging the gap between outer and inner mitochondrial membranes at the contact sites.     

Interfacial behavior of cytoplasmic and mitochondrial creatine kinase oligomeric states

Adsorption to the air/water interface of isoenzymes of creatine kinase was investigated using surface pressure-area isotherms and Brewster angle microscopy (BAM) observations. Octameric mitochondrial creatine kinase (mtCK) exhibits a significant affinity for the air/water interface. Whatever the mode of formation of the interfacial film, i.e., injection of the protein in the subphase or spreading onto the buffer surface, the final arrangement and conformation adopted by mtCK molecules lead to a similar result. In contrast, the dimeric isoenzymes mtCK and cytosolic MMCK do not induce any surface pressure variation. However, when the subphase contains 0.3M NaCl, both isoenzymes adsorb to the interface. When treated with 0.8 or 3M GdnHCl, muscle creatine kinase (MMCK) becomes surface active and occupies a greater surface than mtCK. This result contrasts with previous observations, often derived from monomeric proteins, that their surface activity is increased upon unfolding. It underlines the possible influence exerted by the protein oligomeric state on its interfacial activity. At a subphase pH of 8.8, which corresponds to the pI of octameric mtCK, the profiles of the isotherms obtained with dimeric and octameric states and the resistance to compression of the protein monolayers are significantly affected when compared to those recorded at pH 7.4. These data suggest that the octamer is more hydrophobic than the dimer and may contribute to explaining why octamers bind to the inner mitochondrial membrane while dimers do not.     

Transgenic and tissue culture analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types

The muscle creatine kinase (MCK) gene is expressed at high levels only in differentiated skeletal and cardiac muscle. The activity of the cloned enhancer–promoter has previously been shown to be dependent on the Trex element which is specifically bound by a yet unidentified nuclear factor, TrexBF. We have further characterized the function of the Trex site by comparing wild-type and Trex-mutated MCK transgenes in five mouse skeletal muscles: quadriceps, extensor digitorum longus (EDL), soleus, diaphragm, and distal tongue, as well as in heart ventricular muscle. Several types of statistical analysis including analysis of variance (ANOVA) and rank sum tests were used to compare expression between muscle types and between constructs. Upon mutation of the Trex site, median transgene expression levels decreased 3- to 120-fold in the muscles examined, with statistically significant differences in all muscles except the EDL. Expression in the largely slow soleus muscle was more affected than in the EDL, and expression in the distal tongue and diaphragm muscles was affected more than in soleus. Median expression of the transgene in ventricle decreased about 18-fold upon Trex mutation. Transfections into neonatal rat myocardiocytes confirmed the importance of the Trex site for MCK enhancer activity in heart muscle, but the effect is larger in transgenic mice than in cultured cells.     

In situ measurements of creatine kinase flux by NMR. The lessons from bioengineered mice

P-31 nuclear magnetic resonance (NMR) is uniquely suited to measure the kinetics of the phosphoryl-exchange reaction catalyzed by creatine kinase in intact mammalian tissue, especially striated muscle. Recently developed transgenic mouse models of the creatine kinase iso-enzyme system open novel opportunities to assess the functional importance of the individual iso-enzymes and their relative contribution to the total in situ flux through the CK reaction. This chapter reviews the most recent findings from NMR flux measurements on such genetic models of CK function. Findings in intact mouse skeletal and cardiac muscle in vivo are compared to data from purified mitochondrial and cytosolic creatine kinase in vitro. The relevance of findings in transgenic animals for the function of CK in wild-type tissue is described and the perspectives of transgenic techniques in future quantitative studies on the creatine kinase iso-enzyme system are indicated.     

Mitochondrial creatine kinase functional development in post-natal rat skeletal muscle. A combined polarographic/31P NMR study

Mitochondrial creatine kinase (Mi-CK) function in viable mitochondria from developing rat skeletal muscle was assessed both by polarographic measurements of creatine-induced respiration and 31P NMR spectroscopy measurements of phosphocreatine (PCr) synthesis. Creatine-induced respiration was observed in very young rats and increased by 50% to 35 days of age. PCr synthesis was present in 7 day old animals and increased by 300% reaching levels measured in 35 day and adult muscle. Unlike reports showing Mi-CK enzymatic activities but no mitochondrial function in several situations, a concomitant progression of enzymatic activity and mitochondrial function was evidenced during the developmental stages of skeletal muscle Mi-CK in altricious animals. These results correlated with the progressive pattern of muscle differentiation during development of motricity in such animals. The observation that Mi-CK is functional in skeletal muscle mitochondria very early after birth, strongly favors the notion that adaptations in skeletal muscle of Mi-CK knock-out mice occur early.     

Comparative subcellular fractionation of control and cold-adapted rat brown and white adipose tissue with special reference to peroxisomal and mitochondrial distributions

A new technique for single-step subcellular fractionation of adipose tissue homogenates by analytical sucrose density gradient centrifugation in a vertical pocket reorientating rotor is described. The density gradient distributions of mitochondrial and peroxisomal marker enzymes in brown and white adipose tissue of control and cold exposed rats are compared. The equilibrium density of brown fat mitochondria was found to be significantly increased compared with white fat mitochondria. GDP binding activity was localized solely to the mitochondria in both control and cold-adapted brown adipose tissue. Brown and white fat mitochondria fractions were isolated by differential centrifugation and the specific activities of various enzymes in the homogenate and mitochondrial preparations determined. The specific activity of creatine kinase in brown adipose tissue was found to be ten-fold higher than in white fat and subcellular fractionation studies showed the activity to have an exclusively cytosolic distribution in both tissues. GDP binding activity and some of the mitochondrial enzymes showed, in brown adipose, a striking increase in total activity in cold adapted rats compared to control animals. For some enzyme activities there was a small increase when expressed per mg tissue or per mg mitochondrial protein. When expressed per mg DNA i.e. per cell, there was a reduced specific activity of the mitochondrial and peroxisomal enzymes in both brown and white adipose tissue on cold adaptation.     

Compartmentation of ATP synthesis and utilization in smooth muscle: roles of aerobic glycolysis and creatine kinase

The phosphocreatine content of smooth muscle is of similar magnitude to ATP. Thus the function of the creatine kinase system in this tissue cannot simply be regarded as an energy buffer. Thus an understanding of its role in smooth muscle behavior can point to CK function in other systems. From our perspective CK function in smooth muscle is one example of a more general phenomenon, that of the co-localization of ATP synthesis and utilization. In an interesting and analogous fashion distinct glycolytic cascades are also localized in regions of the cell with specialized energy requirements. Similar to CK, glycolytic enzymes are known to be localized on thin filaments, sarcoplasmic reticulum and plasma membrane. In this chapter we will describe the relations between glycolysis and smooth muscle function and compare and contrast to that of the CK system. Our goal is to more fully understand the significance of the compartmentation of distinct pathways for ATP synthesis with specific functions in smooth muscle. This organization of metabolism and function seen most clearly in smooth muscle is likely representative of many other cell types.     

Once again about the functional coupling between mitochondrial creatine kinase and adenine nucleotide translocase

The synthesis of creatine phosphate (CP) by mitochondrial creatine kinase during oxidative phosphorylation was terminated when the mass action ratio of the creatine kinase reaction = [ADP]·[CP][ATP]·[Cr] became equal to the apparent equilibrium constant (K _eq ^app) of this reaction. Subsequent excess of over the K _eq ^app was due to an increase in the ADP concentration in the medium. A comparable increase in the ADP concentration also occurred in the absence of creatine (Cr) in the incubation medium. Increase in the ADP concentration was shown to be associated with a decrease in the rate of oxidative phosphorylation and with a relative increase in the ATPase activity of mitochondria during the incubation. A low concentration of ADP (<30 M) and relatively high concentrations (1-6 mM) of other components of the creatine kinase reaction prevented the detection of the reverse reaction within 10 min after exceeded the K _eq ^app, but the reverse reaction became evident on more prolonged incubation. The reverse reaction was accompanied by a further increase in . Low ADP concentration in the medium was also responsible for the lack of an immediate conversion of the excess creatine phosphate added although > K _eq ^app. The findings are concluded to be in contradiction with the concept of microcompartment formation between mitochondrial creatine kinase and adenine nucleotide translocase.     

Two fused proteins combining Stichopus japonicus arginine kinase and rabbit muscle creatine kinase

Two fused proteins of dimeric arginine kinase (AK) from sea cucumber and dimeric creatine kinase (CK) from rabbit muscle, named AK-CK and CK-AK, were obtained through the expression of fused AK and CK genes. Both AK-CK and CK-AK had about 50% AK activity and about 2-fold K _m values for arginine of native AK, as well as about 50% CK activity and about 2-fold K _m values for creatine of native CK. This indicated that both AK and CK moieties are fully active in the two fused proteins. The structures of AK, CK, AK-CK, and CK-AK were compared by collecting data of far-UV circular dichroism, intrinsic fluorescence, 1-anilinonaphthalene-8-sulfonate binding fluorescence, and size-exclusion chromatography. The results indicated that dimeric AK and CK differed in the maximum emission wavelength, the exposure extent of hydrophobic surfaces, and molecular size, though they have a close evolutionary relationship. The structure and thermodynamic stability of AK, CK, AK-CK, and CK-AK were compared by guanidine hydrochloride (GdnHCl) titration. Dimeric AK was more dependent on the cooperation of two subunits than CK according to the analysis of residual AK or CK activity with GdnHCl concentration increase. Additionally, AK and CK had different denaturation curves induced by GdnHCl, but almost the same thermodynamic stability. The two fused proteins, AK-CK and CK-AK, had similar secondary structure, tertiary structure, molecular size, structure, and thermodynamic stability, which indicated that the expression order of AK and CK genes might have little effect on the characteristics of the fused proteins and might further verify the close relationship of dimeric AK and CK. Published in Russion in Biokhimiya, 2006, Vol. 71, No. 9, pp. 1208–1214.