Purification and crystallization of creatine kinase from rabbit skeletal muscle

Crystallization is the primary rate-limiting step in protein structure determination. It has been our experience over approximately 10 years that crystals are obtained in about 20% of the proteins attempted and that only about 10% of these crystals are sufficiently well ordered to permit atomic resolution structure analysis. In attempts to overcome this limitation, we have investigated the effect on crystallization of microheterogeneity in a protein regarded as pure by conventional criteria. Creatine kinase was purified from rabbit skeletal muscle and crystallized from methylpentanediol. The protein appeared to be nearly pure judging by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high specific activity. The crystals that were obtained were of poor quality, and an extensive survey of precipitants, crystallization conditions, and additives failed to discover conditions from which usable crystals could be obtained. The enzyme was then subjected to a series of further purification steps. After each purification step, the quality of the crystals obtained under almost identical conditions improved. The final purification step was flat-bed isoelectric focusing. Crystals grown from focused creatine kinase are well ordered and diffract to approximately 3-A resolution.     

Consequences of a six residual deletion from the N-terminal of rabbit muscle creatine kinase

A mutant of dimeric rabbit muscle creatine kinase (CK), in which six residues (residues 2-7) at the N-terminal were removed by the PCR method, was studied to assess the role of these residues in dimer cohesion and to determine the structural stability of the protein. The specific activity of the mutant was 70.39% of that of the wild-type CK, and the affinity for Mg-ATP and CK substrates was slightly reduced compared with the wild-type protein. The structural stability of the mutant was investigated by a comparative equilibrium urea denaturation study and a thermal denaturation study. The data acquired by intrinsic fluorescence and far-UV circular dichroism (CD) during urea unfolding indicated that, the secondary and tertiary structures of the mutant were more stable than those of wild-type CK. Furthermore, results of 8-anilino-1-naphthalene-sulfonic acid (ANS) fluorescence demonstrated that the hydrophobic surface of the mutant CKND(6) was more stable during urea titration. Data from size exclusion chromatography (SEC) experiments indicated that deletion of the six N-terminal residues resulted in a relatively loose molecular structure, but the dissociation of the mutant CKND(6) occurred later during the unfolding process than for wild-type CK. Consistent with this result, the differential scanning calorimetry (DSC) profiles demonstrated that the thermal stability of the enzyme was increased by removal of the six N-terminal residues. We conclude that a more stable quaternary structure was obtained by deletion of the six residues from the N-terminal of wild-type CK.     

Properties of creatine kinase from skeletal muscle mitochondria

Creatine kinase from pigeon breast muscle was obtained in a homogeneous (as evidenced from polyacrylamide gel SDS electrophoresis) state. The molecular mass of the enzyme monomer is 43,000. Ultracentrifugation in a sucrose density gradient and gel filtration revealed that the enzyme is present in solution as a mixture of two major forms, i.e., octamer and dimer, which differ in their activity. The decrease of ionic strength from 0.25 to 0.02 results in reversible dissociation of the octameric form. A temperature rise from 5 degrees to 20 degrees C or the nature of monovalent anions (e.g., Cl-, CH3COO-, NO3-) and cations (K+, Na+) present in the medium do not influence the distribution of oligomeric forms. At pH 6.0 the major form is represented by the octamer; its dissociation is caused by an increase of pH. The octamer dissociation occurs in a mixture of substrates of the creatine kinase reaction in the presence of Mg2+; no such dissociation is observed in the absence of Mg2+ and in the presence of each of the reaction substrates. The non-interacting pair of substrates--ADP and creatine--causes the dissociation of the octamer in the presence of nitrate ions but not acetate. It is concluded that the dissociating effect of substrates is due to the conformational changes of subunits during catalysis. At physiological concentrations of nucleotide substrates the degree of octamer dissociation depends on the ratio of creatine phosphate and creatine concentrations, as well as on the presence of chlorine and phosphate ions. A qualitative estimation of the rate of pH- and substrate-dependent dissociation of creatine kinase octamer revealed that under the given experimental conditions the pH-dependent dissociation is completed within hours, whereas the substrate-dependent one--within seconds or minutes. According to its properties, mitochondrial creatine kinase from pigeon breast muscle is close to its bovine heart counterpart; the observed differences were found to be quantitative.     

Anion activation of monkey muscle creatine kinase

Creatine kinase from rhesus monkey skeletal muscle is activated by acetate and other short chain fatty acids. Activation is associated with lower Km and higher Vmax values at less than saturating substrate concentrations but does not occur when both substrates are saturating. No co-operativity between subunits is evident in the activation process. It appears that acetate promotes the mutual enhancement by substrates in their binding by inducing the optimum enzyme conformation normally associated with substrate saturation. Conservation of this activation effect through the evolution of the phosphagen kinases implies that it may well be of physiological significance.     

Properties of matrix-bound dimer and monomer derivatives of immobilized creatine kinase from rabbit skeletal muscle.

Dimeric creatine kinase (EC 2.7.3.2) from rabbit skeletal muscle can be immobilized via a single subunit to CNBr-activated Sepharose 4B and subsequently treated with guanidine hydrochloride followed by renaturation to yield a catalytically active matrix-bound subunit derivative. The importance of the intact dimeric structure in the activation of the enzyme by acetate was demonstrated. Immobilization did not appear to alter the pH optimum of the enzyme, and the kinetic parameters fot the matrix-bound derivatives were generally similar to those for the soluble enzyme, but the matrix-bound derivatives showed higher thermal stability and greater resistance to denaturation than did the soluble enzyme. The rates of reaction of thiol groups of the matrix-bound derivatives with iodoacetamide in the absence and in the presence of combinations of substrates were similar to those of the soluble enzyme. Studies with 5,5'-dithiobis-(2-nitrobenzoic acid) and with iodoacetamide revealed the presence of an additional reactive thiol group in the matrix-bound subunit derivative, which is presumably masked in the dimeric derivatives.     

Effects of acrylamide on creatine kinase from rabbit muscle

The mechanism of inhibition of creatine kinase (CK) by acrylamide (Acr) has been examined (in vitro). Within the concentration range of 0 to 1 M, Acr markedly inhibited CK and depleted the protein thiols. Both inactivation and thiol depletion were time- and Acr concentration-dependent. Addition of dithiothreitol (DTT) did not reactivate CK inactivated by Acr. However, CK with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) pre-blocked thiols can be reactivated by DTT after incubation with Acr. The transition-state analogue also had a significant protective effect on CK against Acr inhibition. We conclude that thiol alkylation is a critical event in inactivation of CK by Acr. Furthermore, Acr binding to CK changed its surface charge, which may be the same effect for the toxicity of Acr towards other proteins.     

Isolation and sequence analysis of cDNA clones coding for rat skeletal muscle creatine kinase

A series of cDNA clones corresponding to 1494 bases of rat muscle creatine kinase mRNA has been isolated and characterized. The identity of these clones has been confirmed by DNA sequence analysis and by comparison of the predicted amino acid sequence with that determined for the purified protein. The cDNA sequence accounts for the entire coding sequence of the creatine kinase protein in addition to the complete 3' untranslated region and 68 bases of 5' noncoding region. Sequences corresponding to the active site region of the protein, the initiation codon, the termination codon, and poly(A) addition signal have been identified.     

Amino acid sequence of rabbit skeletal muscle myosin light chain kinase

The amino acid sequence of the amino-terminal, 235-residue segment of rabbit skeletal muscle myosin light chain kinase has been determined. Together with the carboxyl-terminal segment previously described [Takio, K., Blumenthal, D. K., Edelman, A. M., Walsh, K. A., Krebs, E. G., & Titani, K. (1985) Biochemistry 24, 6028], the present work completes the 603-residue sequence of this protein. The amino-terminal segment that has been analyzed herein corresponds to a domain reported to be of highly asymmetrical shape and as yet unknown function. Secondary structure calculations failed to provide any evidence of alpha-helix or beta-structures, but polyproline II like helical structure is possible. Sequence analysis indicates the presence of approximately equal quantities of two isoforms differing in a single amino acid replacement. Unexpected difficulties were encountered in the present sequence analysis due to the presence of acid-labile Asp-Pro bonds and to five separable variants of a blocked 21-residue amino-terminal peptide, arising from rearrangement at an Asn-Gly bond.     

Phosphotransfer dynamics in skeletal muscle from creatine kinase gene-deleted mice

To assess the significance of energy supply routes in cellular energetic homeostasis, net phosphoryl fluxes catalyzed by creatine kinase (CK), adenylate kinase (AK) and glycolytic enzymes were quantified using 18O-phosphoryl labeling. Diaphragm muscle from double M-CK/ScCKmit knockout mice exhibited virtually no CK-catalyzed phosphotransfer. Deletion of the cytosolic M-CK reduced CK-catalyzed phosphotransfer by 20%, while the absence of the mitochondrial ScCKmit isoform did not affect creatine phosphate metabolic flux. Contribution of the AK-catalyzed phosphotransfer to total cellular ATP turnover was 15.0, 17.2, 20.2 and 28.0% in wild type, ScCKmit, M-CK and M-CK/ScCKmit deficient muscles, respectively. Glycolytic phosphotransfer, assessed by G-6-P 18O-phosphoryl labeling, was elevated by 32 and 65% in M-CK and M-CK/ScCKmit deficient muscles, respectively. Inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)/phosphoglycerate kinase (PGK) in CK deficient muscles abolished inorganic phosphate compartmentation and redirected high-energy phosphoryl flux through the AK network. Under such conditions, AK phosphotransfer rate was equal to 86% of the total cellular ATP turnover concomitant with almost normal muscle performance. This indicates that near-equilibrium glycolytic phosphotransfer reactions catalyzed by the GAPDH/PGK support a significant portion of the high-energy phosphoryl transfer in CK deficient muscles. However, CK deficient muscles displayed aberrant ATPase-ATPsynthase communication along with lower energetic efficiency (P/O ratio), and were more sensitive to metabolic stress induced by chemical hypoxia. Thus, redistribution of phosphotransfer through glycolytic and AK networks contributes to energetic homeostasis in muscles under genetic and metabolic stress complementing loss of CK function.     

Amino acid sequence of acylphosphatase from rabbit skeletal muscle

The complete amino acid sequence of acylphosphatase from rabbit skeletal muscle has been elucidated by automatic Edman degradation of peptides obtained from staphylococcal protease and trypsin digestions. The enzyme consisted of a single polypeptide chain of 98 amino acid residues, lacking only histidine. Its amino (N)-terminus was blocked by an acetyl group. The presented sequence of rabbit muscle enzyme was compared with those of equine and porcine muscle enzymes. There were four unique replacements, i.e., Arg-4, Asp-28, Arg-31, and Glu-56 in the sequences of both equine and porcine muscle enzymes were replaced by Gly, Gly, Lys, and Asp, respectively, in that of rabbit muscle enzyme. Extensive structural homology was observed among the three enzymes.     

Cooperative self-association of phosphorylase kinase from rabbit skeletal muscle

Ca(2+)- and Mg(2+)-induced association of phosphorylase kinase (PhK) from rabbit skeletal muscle has been studied at the magnitudes of the ionic strength close to the physiological values (40 mM Hepes, pH 6.8, containing 0.1 M NaCl, 0.1 mM Ca(2+), 10 mM Mg(2+); 25 degrees C) and under the molecular crowding conditions produced by high concentrations (1 M) of the natural osmolyte, trimethylamine N-oxide (TMAO). In the presence of 0.1 M NaCl two forms of PhK were registered, namely the "basic form" and "highly associated form", suggesting that PhK association may be treated as an example of cooperative association. According to the data on dynamic light scattering the average hydrodynamic radii of these forms were 16 and 144 nm. The addition of 1 M TMAO produces the time dependent increase in the light scattering intensity caused by the conversion of the basic form into the highly associated form. According to the data of the sedimentation analysis the basic form of PhK comprises a hexadecamer (M(r)=1320 kDa) and its small associates. The removal of Ca(2+) by addition of EGTA results in the reverse conversion of the highly associated form into the basic form suggesting reversibility of self-association of PhK. FAD, the ligand that is specifically bound to PhK, blocks the conversion of the basic form of PhK into the highly associated form.     

Unraveling multistate unfolding of rabbit muscle creatine kinase

GdmCl-induced unfolding of rabbit muscle creatine kinase, CK, has been studied by a variety of physico-chemical methods including near and far UV CD, SEC, intrinsic fluorescence (intensity, anisotropy and lifetime) as well as intensity and lifetime of bound ANS fluorescence. The formation of several stable unfolding intermediates, some of which were not observed previously, has been established. This was further confirmed by representation of fluorescence data in terms of "phase diagram", i.e. I(lambda1) versus I(lambda2) dependence, where I(lambda1) and I(lambda2) are fluorescence intensity values measured on wavelengths lambda(1) and lambda(2) under the different experimental conditions for a protein undergoing structural transformations. The unfolding behavior of CK was shown to be strongly affected by association of partially folded intermediates. A model of CK unfolding, which takes into account both structural perturbations and association of partially folded intermediates has been elaborated.     

Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice.

Muscle creatine kinase (MCK) is expressed at high levels only in skeletal and cardiac muscle tissues. Previous in vitro transfection studies of skeletal muscle myoblasts and fibroblasts had identified two MCK enhancer elements and one proximal promoter element, each of which exhibited expression only in differentiated skeletal muscle. In this study, we have identified several regions of the mouse MCK gene that are responsible for tissue-specific expression in transgenic mice. A fusion gene containing 3,300 nucleotides of MCK 5' sequence exhibited chloramphenicol acetyltransferase activity levels that were more than 10(4)-fold higher in skeletal muscle than in other, nonmuscle tissues such as kidney, liver, and spleen. Expression in cardiac muscle was also greater than in these nonmuscle tissues by 2 to 3 orders of magnitude. Progressive 5' deletions from nucleotide -3300 resulted in reduced expression of the transgene, and one of these resulted in a preferential decrease in expression in cardiac tissue relative to that in skeletal muscle. Of the two enhancer sequences analyzed, only one directed high-level expression in both skeletal and cardiac muscle. The other enhancer activated expression only in skeletal muscle. These data reveal a complex set of cis-acting sequences that have differential effects on MCK expression in skeletal and cardiac muscle.     

Purification and properties of troponin T kinase from rabbit skeletal muscle

A protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) which catalyzes the phosphorylation of troponin T, phosvitin and casein has been purified over 2000 fold from rabbit skeletal muscle. The partial purification of this new enzyme, designated troponin T kinase, involves precipitation of contaminating proteins at pH 6.1, fractionation of the supernatant with (NH₄)₂SO₄ and succesive column chromatographies on DEAE-cellulose, hydroxyapatite and Sepharose 6B. The chromatographic patterns on DEAE-cellulose and hydroxyapatite columns show two peaks of troponin T kinase activity. Gel filtration experiments indicate the existence of multiple, possibly aggregated, forms of the enzyme. The purified enzyme does not catalyze the phosphorylation of phosphorylase b, troponin I, troponin C, tropomyosin, protamine, or myosin light chain 2 nor does it catalyze the interconversion of glycogen synthase I into the D form. Troponin T kinase is not affected by the addition of cyclic nucleotides or AMP to the reaction mixture. Divalent cations (other than Mg²⁺, required for the reaction) do not stimulate the enzyme, and several are inhibitory. Other characteristics of the reaction catalyzed by troponin T kinase, such as K_m values for ATP and substrate proteins, pH optima, effect of the concentration of Mg²⁺, substitution of ATP for GTP have also been studied.     

A preliminary crystallographic investigation of rabbit muscle creatine kinase

Three crystal forms of rabbit muscle creatine kinase have been grown, one of which seems suited to a high resolution X-ray diffraction study. The first form is of monoclinic space group P2₁ with $\text{a = 54}\text{A}\text{?}\text{, b = 114}\text{A}\text{?}\text{, c = 145}\text{A}\text{?}\text{, β = 91 °}$ and has as the asymmetric unit two molecules of total molecular weight 160, 000. The second form, grown in the presence of mercurials, is of space group A2 with $\text{a = 52}\text{A}\text{?}\text{, b = 165}\text{A}\text{?}\text{, c = 237}\text{A}\text{?}\text{, β = 91 °}$ and also has two molecules in the asymmetric unit. The third crystal form, grown in the presence of a high concentration of cysteine, is of apparent space group P2₁2₁2₁, but evidence indicates that the true space group may be P2₁22₁. The dimensions of the orthorhombic unit cell are $\text{a = 47}\text{A}\text{?}\text{, b = 86}\text{A}\text{?}\text{, c = 125}\text{A}\text{?}$, and the asymmetric unit contains a single protein subunit. Assuming the latter space group, then the creatine kinase molecule possesses a twofold axis relating two identical subunits.     

Effects of lactic acid and NaCl on creatine kinase from rabbit muscle.

The lactic acid induced unfolding and the salt-induced folding of creatine kinase (CK) were studied by enzyme activity, fluorescence emission spectra, circular dichroism spectra, and native polyacrylamide gel electrophoresis. The results showed that the kinetics of CK inactivation was a monophase process. Lactic acid caused inactivation and unfolding of CK with no aggregation during CK denaturation. The unfolding of the whole molecule and the inactivation of CK in solutions of different concentration of lactic acid were compared. Much lower lactic acid concentration values were required to bring about inactivation than were required to produce significant conformational changes of the enzyme molecule. At higher concentrations of lactic acid (more than 0.2 mM) the CK dimers were partially dissociated, as proved by native polyacrylamide gel electrophoresis. NaCl induced the molten globule state with a compact structure after CK was denatured with 0.8 mM lactic acid, and the increasing of anions led to a tight side-chain. The above results suggest that the effect of lactic acid differed from that of other denaturants such as guanidine hydrochloride, HCI, or urea during CK folding, and the molten globule state indicates that intermediates exist during CK folding.