Mitochondrial creatine kinase binding to liposomes and vesicle aggregation: effect of cleavage by proteinase K

Mitochondrial creatine kinase and its proteinase K nicked-derivative interaction with liposomes induced slight secondary structure changes evidenced by infrared spectra. In nondenaturing conditions, the N-terminal (K₁) and the C-terminal (K₂) fragments remained associated with each other and bound to liposomes. When the two fragments were separated by denaturation, K₂ was soluble, whereas most of K₁ was adsorbed onto liposomes. The three-dimensional structure of uncleaved mtCK suggests that the C-terminal moiety, which contains positively charged surface residues, interacted with membranes. After denaturation and renaturation of the nicked enzyme, both peptides did not refold properly and did not reassociate with each other. The misfolded K₁ fragment bound to the membrane through a stretch of positive residues, which were buried in the native enzyme. The lack of binding of the ill-folded K₂ peptide could be related to the disruption of the optimal disposition of its positive charges, responsible for the correct interaction of native mtCK with membrane.     

Creatine kinase activity in normal and hypertrophied rabbit urinary bladder tissue (following partial outlet obstruction)

The urinary bladder depends on intracellular ATP to support a number of essential intracellular processes including contraction. The concentration of ATP is maintained by mitochondrial oxidative phosphorylation, cytosolic glycolysis and the cytosolic activity of creatine kinase, the enzyme that catalysis the rapid transfer of a phosphate from creatine phosphate (CP) to ADP resulting in the formation of ATP.Prior studies in this lab and others have demonstrated that mitochondrial respiration is significantly lower in hypertrophied bladder tissue (induced by partial outlet obstruction of the white New Zealand Rabbit). In addition to decreased mitochondrial respiration, there are significant increases in glycolysis and lactic acid formation in the hypertrophied tissue.In view of the increased glycolysis and decreased mitochondrial function in the hypertrophied tissue, and the importance in creatine kinase in maintaining cytosolic levels of ATP, the current study was designed to determine if outlet obstruction induces any changes in the activity of creatine kinase.The following is a summary of the results: 1) The bladder mass increased from 2.2 ± 0.2 gm to 11.5 ±1.6 gm at 7 days following outlet obstruction. 2) The intracellular concentrations of both ATP and CP were significantly reduced in the bladder tissue following 7 days of obstruction. 3) The percent of protein (per tissue mass) was significantly lower in the obstructed bladders, although the percent of soluble protein was similar. 4) Creatine kinase activity of control bladders showed linear kinetics with a V_max = 1120 nmoles/mg protein/4 min and K_m = 147 µM CP. 2) The creatine kinase activity of obstructed bladders also displayed linear kinetics with a V_max = 1125 nmoles/mg protein/4 min tissue, and K_m = 276 µM CP.These studies demonstrate that whereas both control and obstructed bladders have virtually identical maximum creatine kinase activities, the K_m for the obstructed tissue is significantly higher than the K_m for the control tissue. This may indicate that under cellular conditions (at sub-maximum substrate concentrations), the creatine kinase activity of the obstructed bladders may be significantly lower than the activity of the control bladders. In addition, the reduced tissue concentrations of ATP and CP would certainly be consistent with the reduced functional response to bethanechol and field stimulation.     

Characterization of creatine kinase isoforms in herring (Clupea harengus) skeletal muscle

It is known that mitochondrial creatine kinase (MtCK) in mammals is always expressed in conjunction with one of the cytosolic forms of creatine kinase (CK), either muscle-type (MM-CK) or brain-type (BB-CK) in tissues of high, sudden energy demand. The two creatine kinase (CK) isoforms were detected in herring (Clupea harengus) skeletal muscle: cytosolic CK and mitochondrial CK (MtCK) that displayed the different electrophoretic mobility. These isoforms differ in molecular weight and some biochemical properties. Isolation and purification procedures allowed to obtain purified enzymes with specific activity of the 206 μmol/min/mg for cytosolic CK and 240 μmol/min/mg for MtCK. Native M_rs of the cytosolic CK and MtCK determined by gel permeation chromatography were 86.000 and 345.000, respectively. The results indicate that one of isoforms found in herring skeletal muscle is a cytosolic dimer and the other one, is a mitochondrial octamer. Octamerization of MtCK is not an advanced feature and also exists in fish. These values correspond well with published values for MtCKs and cytosolic CK isoforms from higher vertebrate classes and even from lower invertebrates.     

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.     

Expressing creatine kinase in transgenic tobacco – a first step towards introducing an energy buffering system in plants

Creatine kinase a key enzyme in cellular energy homeostasis of vertebrates offers the promise of engineering plants with enhanced stress tolerance. In order to provide plants with such an energy buffering system, tobacco was transformed with a cDNA, encoding the cytosolic brain-type isoform of chicken creatine kinase (BB-CK), the expression of which was under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter. Transgenic tobacco plants were selected and suspension cultures generated. Both transgenic plants and suspension cultures were shown to stably express enzymatically active BB-CK in vitro and in vivo, and in most cases for three successive generations (T₀–T₂). Exogenously supplied creatine was shown to enter the plant cells and resulted in only a slight reduction in root growth at concentrations up to 10mM. Furthermore, the BB-CK expressing tobacco plants and cell suspension cultures were able to convert creatine into phosphocreatine.     

The Antioxidant Vitamin E Modulates Amyloid β-Peptide-Induced Creatine Kinase Activity Inhibition and Increased Protein Oxidation: Implications for the Free Radical Hypothesis of Alzheimer's Disease

Amyloid -peptide (A), the main constituent of senile plaques in Alzheimer's disease (AD) brain, is hypothesized to be a key factor in the neurodegeneration seen in AD. Recently it has been shown by us and others that the neurotoxicity of A occurs in conjunction with free radical oxidative stress associated with the peptide. A(1–40) and several other fragments of the A sequence are associated with free radicals in solution that are detectable using electron paramagnetic resonance spectroscopy. These free radicals were shown to attack brain cell membranes, initiate lipid peroxidation, increase Ca²⁺ influx and damage membrane and cytosolic proteins. In AD brain obtained under rapid autopsy protocol, the activity of the oxidatively-sensitive enzyme creatine kinase was shown to be significantly reduced. We reasoned that A-associated free radical-induced modification of creatine kinase activity and other markers of cellular damage might be modulated by free radical scavengers. Accordingly, this study demonstrates that vitamin E can modulate A(25–35)-induced oxidative damage to creatine kinase and cellular proteins in cultured embryonic hippocampal neurons. These results, consistent with the hypothesis of free radical-mediated A toxicity in AD, are discussed with deference to potential free radical scavengers as therapeutic agents for slowing the progression of AD.     

A theoretical model of some spatial and temporal aspects of the mitochondrion creatine kinase myofibril system in muscle

We describe a model of mitochondrial regulation in vivo which takes account of spatial diffusion of high-energy (ATP and phosphocreatine) and low-energy metabolites (ADP and creatine), their interconversion by creatine kinase (which is not assumed to be at equilibrium), and possible functional 'coupling' between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on relationships between oxidative ATP synthesis rate and spatially-averaged metabolite concentrations. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, to a degree which depends on the degree of coupling. At high creatine kinase activity, the fraction of flow carried by ATP is small. Lowering the creatine kinase activity raises this fraction, especially when there is little functional coupling. All metabolites show small spatial gradients, more so at low cytosolic creatine kinase activity, and unless there is near-complete coupling, so does net creatine kinase flux. During workjump transitions, spatial-average responses exhibit near-exponential kinetics as expected, while concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. (Mol Cell Biochem 174: 29–32, 1997)     

Change in kinetic regime of protein aggregation with temperature increase. Thermal aggregation of rabbit muscle creatine kinase

Creatine kinase thermal aggregation kinetics has been studied in 30 mM Hepes-NaOH buffer, pH 8.0, at two temperatures: 50.6 and 60°C. Aggregation kinetics was analyzed by measuring the growth of apparent absorption (A) at 400 nm. It was found that the limiting value of apparent absorption (A _lim) is proportional to protein concentration at both temperatures. The first order rate constant (k _I) does not depend on protein concentration in the range 0.05–0.2 mg/ml at temperature 50.6°C, but at temperature 60°C it increases with the growth of protein concentration in the range 0.1–0.4 mg/ml. Kinetic curves, shown in coordinates {A/A _lim; t}, in experiments at 50.6°C fuse to a common curve, which coincides with the theoretical curve of creatine kinase denaturation calculated using the denaturation rate constant determined from differential scanning calorimetry. At temperature 60°C, half-transformation time t _1/2 = ln2/k _I decreases when protein concentration grows. We conclude that when temperature increased from 50.6 to 60°C, change in the kinetic regime of thermal creatine kinase aggregation took place: at 50.6°C aggregation rate is limited by the stage of protein molecule denaturation, but at 60°C it is limited by the stage of protein aggregate growth, which proceeds as a reaction of pseudo-first order. Small heat shock protein Hsp 16.3 Mycobacterium tuberculosis suppresses the creatine kinase aggregation. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 3, pp. 408–416.     

Shared structural determinants for the calcium-independent liposome membrane permeabilization and sarcolemma depolarization in Bothropstoxin-I,a LYS49-PLA2 from the venom of Bothrops jararacussu

The structural determinants of myotoxicity of bothropstoxin-I (BthTX-I), a Lys49 phospholipase A₂ from Bothrops jararacussu venom, were studied by measuring the resting membrane potential in the mouse phrenic nerve-diaphragm preparation. This method proved to be around 100-fold more sensitive than the creatine kinase release assay, and was used to evaluate a total of 31 site-directed BthTX-I alanine scanning mutants. Mutants that reduced the resting membrane potential were located in a surface patch defined by residues in the C-terminal loop (residues 115–129), positions 37–39 in the membrane interfacial recognition surface (Y46 and K54), and residue K93. These results expand the known structural determinants of the biological activity as evaluated by previous creatine kinase release experiments. Furthermore, a strong correlation is observed between the structural determinants of sarcolemma depolarization and calcium-independent disruption of liposome membranes, suggesting that a common mechanism of action underlies the permeabilization of the biological and model membranes.     

Spin biochemistry

The rates of adenosine triphosphate (ATP) production by isolated mitochondria and mitochondrial creatime kinase incubated in isotopically pure media containing, separately, ²⁴Mg²⁺, ²⁵Mg²⁺, and ²⁶Mg²⁺ ions were shown to be strongly dependent on the magnesium nuclear spin and magnetic moment. The rate of adenosine 5′-diphosphate phosphorylation in mitochondria with magnetic nuclei²⁵Mg is about twice higher than that with the spinless, nonmagnetic nuclei^24.26Mg. When mitochondrial oxidative phosphorylation was selectively blocked by treatment with 1-methylnicotine amide, ²⁵Mg²⁺ ions were shown to be nearly four times more active in mitochondrial ATP synthesis than ^24,26Mg²⁺ ions. The rate of ATP production associated with creatine kinase is twice higher for ²⁵Mg²⁺ than for ^24.26Mg and does not depend on the blockade of oxidative phosphorylation. There is no difference between ²⁴Mg²⁺ and ²⁶Mg²⁺ effects in both oxidative and substrate phophorylation. These observations demonstrate that the enzymatic phosphorylation is a nuclear spin selective process controlled by magnetic isotope effect. The reaction mechanism proposed includes a participation of intermediate ion-radical pairs with Mg⁺ cation as a radical partner. Therefore, the key mitochondrial phosphotransferases work as a magnesium nuclear spin mediated molecular machines.     

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.     

Purification and localization of brain-type creatine kinase in sodium chloride transporting epithelia of the spiny dogfish, Squalus acanthias.

The targeting of creatine kinase isoenzymes to specific sites within muscle cells provides a system for the regeneration of ATP in situ from ADP and creatine phosphate. We have recently reported the colocalization of brain-type (B) creatine kinase and the nonsarcomeric mitochondrial creatine kinase isoenzymes in the thick ascending limb of the loop of Henle in the rat kidney, suggesting that creatine kinase may regenerate ATP for sodium transport (Friedman, D.L., and Perryman, M.B. (1991) J. Biol. Chem. 266, 22404-22410). In order to test the hypothesis regarding the association of B creatine kinase with sodium transport, we examined the creatine kinase enzymes in the rectal (salt-secreting) gland of the dogfish shark which contains high levels of the Na+/K(+)-ATPase. The creatine kinase isoform composition was determined by non-denaturing electrophoresis, immunoblotting, protein purification, and amino acid sequence analysis. The results demonstrate both B creatine kinase and mitochondrial creatine kinase proteins are present in the rectal gland, an isoform composition which is the same as in the mammalian kidney. By using a combination of chromatographic techniques, shark B creatine kinase was purified to homogeneity and partial sequence data was obtained from two cyanogen bromide peptide fragments. One of these fragments contains the active site and is identical at all sequenced residues with the corresponding region from the echinoderm sperm flagellar creatine kinase, and is 96% homologous with both chicken and rat B creatine kinase subunits. The other fragment corresponds to a region near the N-terminal of mammalian creatine kinases and is 89% homologous with B creatine kinase from chicken. The localization of these isoforms was examined by immunocytochemistry using subunit specific antisera. Mitochondrial creatine kinase and B creatine kinase immunoreactivity are detected in all tubules, and is restricted to the basal region of the cells, which is the site of the Na+/K(+)-ATPase. The conservation of creatine kinase isoform expression in excretory tissue, and the localization of creatine kinase immunoreactivity in the basal region of the tubule cells, demonstrate that subcellular compartmentation of B creatine kinase may underly the functional coupling of creatine kinase activity with sodium transport.     

Differentiation markers of mouse C2C12 and rat L6 myogenic cell lines and the effect of the differentiation medium

Summary The differentiation grade of cells in culture is dependent on the composition of the culture medium. Two commonly used myogenic cell lines, mouse C₂C₁₂ and rat L₆, usually differentiate at a low concentration of horse serum. In this study we compared the effect of horse serum with a medium containing a low percentage of Ultroser G and rat brain extract. The maturation grade was evaluated on the basis of various biochemical, (immuno)histochemical and cell-physiological parameters. Substitution of horse serum by Ultroser G and rat brain extract during the differentiation phase resulted in a higher maturation grade of the myotubes of both cell lines, on the basis of creatine kinase activity and the diameter of the myotubes. In addition, the C₂C₁₂ myotubes display cross-striation, contain a higher percentage of creatine kinase muscle-specific isoenzyme MM, show a ninefold increase in acetylcholine receptor (AChR) clusters, form a continuous basement membrane, and have a lower resting cytosolic Ca²⁺ concentration. L₆ myotubes show a fivefold increase in AChR clusters and a twofold increase in the expression of the mRNA of the ɛ-subunit of AChR. C₂C₁₂ cells show spontaneous contraction and response of cytosolic Ca²⁺ to various stimulants in contrast to L₆ cells which do not. These studies established that the Ultroser G/brain extract medium leads to a higher differentiation grade of both cell lines, but parameters appropriate for use as differentiation markers appear to differ between both cell lines.     

Role of the carboxyl terminus on the catalytic activity of protein kinase CK2alpha subunit

Protein kinase CK2 (also known as casein kinase 2) has catalytic (alpha, alpha') and regulatory (beta) subunits. The role of carboxyl amino acids in positions from 324 to 328 was studied for Xenopus laevis CK2alpha. Deletions and mutations of these residues were produced in recombinant CK2alpha, which was assayed for kinase activity. Activity dropped 7000-fold upon deletion of amino acids 324-328. The key residues are isoleucine 327 and phenylalanine 324. A three dimensional model of CK2alpha indicates that these hydrophobic residues of helix alphaN may interact with hydrophobic residues in helix alphaE which is linked to the catalytic center.     

Structure-function relationship of myotoxin a using peptide fragments.

Myotoxin a, a small basic polypeptide isolated from the venom of prairie rattlesnake (Crotalus viridis viridis), has been shown to bind to sarcoplasmic reticulum (SR) Ca(2+)-ATPase. The attachment of myotoxin a to Ca(2+)-ATPase is believed to cause uncoupling of the calcium pump. In order to further elucidate which portion of myotoxin a is important for the uncoupling action, five peptides were synthesized and two peptide fragments were obtained by chemical cleavage. These peptides correspond to discrete portions of the primary sequence of myotoxin a. The peptides are equivalent to the primary sequence of myotoxin a from 1 to 16 residues, 7 to 22 residues, 13 to 28 residues, 19 to 34 residues, and 25 to 42 residues. Chemically produced fragments are equivalent to 1 to 28 residues and 29 to 42 residues of myotoxin a. Peptides of the sequences "YKQCHKKGGHCFPKEK" and "LGKMDCRWKWKCCKKGSG" of myotoxin a inhibited 45Ca uptake into isolated SR and bound to Ca(2+)-ATPase. The same peptides caused weak skeletal muscle vacuolization similar to that caused by native myotoxin a and increased serum creatine kinase activity. The active peptides correspond to the N-terminal and C-terminal portions of myotoxin a. The inactive or less active peptides have sequences which correspond to the middle sequence of myotoxin a. From this study, both the N-terminal and the C-terminal regions of primary sequence of myotoxin a are required to express myotoxin a's biological activity.     

Phosrestide-1, a peptide derived from the Drosophila photoreceptor protein phosrestin I, is a potent substrate for Ca/calmodulin-dependent protein kinase II from rat brain

Multifunctional Ca²⁺/calmodulin-dependent protein kinase type II (CaMK II) plays a crucial role in mediation of cellular responses to rising cytosolic Ca²⁺ levels. We find that the novel peptide substrate PGTIEKKRSNAMKKMKSIEQHR serves as a highly potent substrate for CaMK II enzymes purified from both Drosophila and rat. The peptide is derived from a photoreceptor-specific protein, phosrestin I, of the Drosophila compound eye and is designated as phosrestide-1. Using saturating substrate concentrations, the enzymes from both species transfer the γ-phosphoryl group of ATP to phosrestide-1 at a level three to ten times greater than to the commercially available mammalian-derived CaMK II substrates, autocamtide-3 and syntide-2. This indicates a conservation of substrate preferences for CaMK II derived from distantly related species, a dipteran fly and a mammal. Although phosrestide-1 contains two potential serine residues for CaMK II phosphorylation, we find that only the C-terminal serine is phosphorylated by rat CaMK II. However, removal of the upstream sequence containing the N-terminal serine substantially reduced the potency of phosrestide-1 as a CaMK II substrate to a level comparable to that of syntide-2 or autocamtide-3. We also find that a peptide representing the N-terminal segment of phosrestide-1 does not inhibit either CaMK II. Therefore, the enhanced potency of phosrestide-1 as a CaMK II substrate is likely to be due to a preferred conformation of the peptide induced by the N-terminal segment rather than to a specific binding of the enzymes to the N-terminus of the peptide. To the best of our knowledge, phosrestide-1 is the first CaMK II substrate which is designed based on an invertebrate sequence. The high phosphorylation level of phosrestide-1 by CaMK II of mammalian origin may reflect highly conserved CaMK II signaling cascades between vertebrates and invertebrates.     

A P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

Monoclonal antibody studies of creatine kinase. The ART epitope: evidence for an intermediate in protein folding.

Chemical cleavage at cysteine residues with nitrothiocyanobenzoic acid shows that the last 98 amino acids of the 380-amino-acid sequence of chick muscle creatine kinase are sufficient for binding of the monoclonal antibody CK-ART. Removal of the last 30 amino acids by cleavage at methionine residues with CNBr results in loss of CK-ART binding. CK-ART binding is also lost when these C-terminal methionine residues are oxidized to sulphoxide, but binding is regained on reduction. Proteinase K 'nicks' native CK at a single site near the C-terminus and two fragments of 327 amino acides and 53 amino acids can be separated by subsequent SDS or urea treatment. CK-ART still binds normally to 'nicked' CK, which is enzymically inactive. After treatment with either urea (in a competition enzyme-linked immunosorbent assay) or SDS (on Western blots), however, CK-ART binds to neither of the two fragments, although these treatments do not affect binding to intact CK. This suggests that parts of both CK fragments contribute to the CK-ART epitope. CK-ART is both species- and isoenzyme-specific, binding only to chick M-CK. The only C-terminal regions containing chick-specific sequences are residues 300-312 and residues 368-371, the latter group being close to the essential methionine residues. We suggest that one, or possibly both, of these regions is involved in forming the conformational epitope on the surface of the CK molecule which CK-ART recognizes. Native CK is resistant to trypsin digestion. The C-terminal half of urea-treated and partly-refolded CK is also resistant to trypsin digestion, whereas the N-terminal half is readily digested. The results suggest a C-terminal region which can refold more rapidly than the rest of the CK molecule and provide evidence for an intermediate in CK refolding.     

Role of C-terminal sequences in the folding of muscle creatine kinase

Proteinase K selectively nicks the native homodimeric muscle creatine kinase (MM-CK) into two 37.1 kDa N-terminal (K1) and two 5.8 kDa C-terminal (K2) fragments that remain firmly associated in a native-like, although inactive, heterotetrameric structure. This truncated protein has been named (K1K2)(2). To analyze the role of the C-terminal peptide in the protein structure acquisition, we studied in vitro refolding of the guanidinium chloride-denatured (K1K2)(2). Although they never reassociate with K2, in selected conditions the K1 fragments refold slowly to a dimeric state as shown by size exclusion chromatography data. This K1 dimer exhibits a fluorescence emission lambda max of 335 nm, a high degree of tyrosine exposure, strongly binds ANS but not MgADP, a CK substrate, and according to these structural characteristics, could be a dimeric molten globule species. We propose a folding model that takes into account the existence of a new transient intermediate state in the MM-CK refolding process. Besides two monomeric premolten and molten globule kinetic intermediates and the active final dimeric form, an inactive dimer, with partly compacted monomers, must ephemerally exist. Our results strongly suggest that the C-terminal end of the protein accelerates folding and plays a critical role for monomer final packing into a native-like conformation. The data also indicate that MM-CK catalytic efficiency is only acquired after dimerization.     

Crystal structure of brain-type creatine kinase at 1.41 A resolution

Excitable cells and tissues like muscle or brain show a highly fluctuating consumption of ATP, which is efficiently regenerated from a large pool of phosphocreatine by the enzyme creatine kinase (CK). The enzyme exists in tissue--as well as compartment-specific isoforms. Numerous pathologies are related to the CK system: CK is found to be overexpressed in a wide range of solid tumors, whereas functional impairment of CK leads to a deterioration in energy metabolism, which is phenotypic for many neurodegenerative and age-related diseases. The crystal structure of chicken cytosolic brain-type creatine kinase (BB-CK) has been solved to 1.41 A resolution by molecular replacement. It represents the most accurately determined structure in the family of guanidino kinases. Except for the N-terminal region (2-12), the structures of both monomers in the biological dimer are very similar and closely resemble those of the other known structures in the family. Specific Ca2+-mediated interactions, found between two dimers in the asymmetric unit, result in structurally independent heterodimers differing in their N-terminal conformation and secondary structure. The high-resolution structure of BB-CK presented in this work will assist in designing new experiments to reveal the molecular basis of the multiple isoform-specific properties of CK, especially regarding different subcellular locations and functional interactions with other proteins. The rather similar fold shared by all known guanidino kinase structures suggests a model for the transition state complex of BB-CK analogous to the one of arginine kinase (AK). Accordingly, we have modeled a putative conformation of CK in the transition state that requires a rigid body movement of the entire N-terminal domain by rms 4 A from the structure without substrates.