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.     

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.     

Homogeneous chicken heart mitochondrial creatine kinase purified by dye-ligand and transition-state analog-affinity chromatography

A method for the preparation of homogeneous mitochondrial creatine kinase from chicken heart is presented. The two-column procedure, which can be completed in 2 days, uses Procion red dye and transition-state analog-affinity chromatography. The transition-state analog-affinity chromatographic system utilizes an ADP-hexane-agarose column in conjunction with the transition-state analog complex originally developed by E. J. Milner-White and D. C. Watts (1971, Biochem, J. 122, 727-740) composed of KNO3, MgCl2, creatine, and ADP. The enzyme is a dimer composed of 2 Mr 43,000 subunits. The sequence of the first N-terminal 20 amino acids shows that the enzyme is different from the cytosolic isozymes but similar to human mitochondrial creatine kinase. The enzyme has an extinction coefficient of epsilon 280 nm = 2.22 +/- 0.10 ml X mg-1 X cm-1 and a maximum velocity of 200 IU/ml at pH 7.0. The kinetic constants for the chicken heart mitochondrial isozyme are comparable to values for the canine and human heart isozyme.     

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.     

Sheep liver serine-threonine dehydratase. I. Purification and evidence for covalently linked alpha-ketobutyrate as its cofactor.

L-Serine-threonine dehydratase (EC 4.2.1.16) from sheep liver has been obtained as a highly purified preparation as shown by ultracentrifuge studies and analytical disc gel electrophoresis. The dehydratase has a molecular weight of 98,000 +/- 10,000 and is composed of two nonidentical subunits with molecular weights of 41,000 and 47,000. The 41,000 subunit is covalently linked to the carbonyl reagent-sensitive coenzyme which has been identified as alpha-ketobutyric acid.     

Trout (Salmo gairdneri R.) liver 5-aminolevulinate synthetase: subcellular distribution and properties

The subcellular distribution of trout liver 5-aminolevulinate synthetase has been studied. A cytosolic form of the enzyme has been found. Its activity was a 30% of the mitochondrial enzyme. The cytosolic form has a molecular weight of 110,000, larger than the mitochondrial enzyme (70,000). The two enzyme forms showed a pH optimum of 7.5. The kinetic characteristic of both forms suggest that the cytosolic form is a precursor of the mitochondrial ALA-synthetase.     

Studies on phosphagen synthesis by mitochondrial preparations

Mitochondrial preparations from muscles of a crab (Cancer pagurus), two fish (Trachurus trachurus and Scyliorhinus canicula) and a bird (Columba livia) are able to synthesise, through ATP, the phosphagen related to that species. This indicates the presence of a bound phosphagen kinase. Addition of creatine kinase and creatine to crab mitochondria results in the synthesis of phosphocreatine. Similarly, the addition of arginine kinase and arginine to mitochondrial preparations from the fish and bird results in the synthesis of phosphoarginine. In the crab, the mitochondrial form of arginine kinase released by sonication had the same kinetic affinity constants and electrophoretic mobility and could not be distinguished immunologically from the cytosolic form. The close similarity of bound and cytosolic forms of arginine kinase in this crustacean suggests that the two forms have not evolved separately as has creatine kinase in the mammal.     

Human thymidine kinase. Purification and properties of the cytosolic enzyme of placenta

Cytosolic thymidine kinase (EC 2.7.1.21) has been purified 5200-fold to apparent homogeneity from normal human placenta. The purification includes sequential affinity chromatography on blue-Sepharose and a thymidine column. The molecular weight of the enzyme determined by gel filtration and sucrose density ultracentrifugation is 92,000. The subunit molecular weight is 44,000, suggesting that the enzyme is a dimer in its native state. With isoelectric focusing, placental thymidine kinase demonstrated a single form with an isoelectric point of 9.1. The final purified enzyme preparation exhibits no immunological cross-reactivity with human mitochondrial thymidine kinase.     

Differences in plasma membrane organization of neuroblastoma cells grown in the differentiated and undifferentiated states.

Rabbit skeletal muscle creatine kinase is inactivated when stored at ?17 °C in the presence of either chloride or nitrate. Other anions are not effective. Associated with the inactivation is an altered electrophoretic mobility and the loss of four out of the eight titratable thiol groups in the dimeric catalytic protein of molecular weight 82,600. The altered inactive form is separated from the native active enzyme by electrofocusing, and its catalytic activity is restored by treatment with 2-mercaptoethanol. Gel electrophoresis in the presence and absence of 2-mercaptoethanol establishes that solutions of the inactive enzyme are heterogeneous, containing mostly a protomeric polypeptide of molecular weight 41,000, but also significant amounts of disulfide-linked dimers, trimers, and tetramers. Sedimentation equilibrium analysis confirms the existence of higher molecular weight aggregates along with the preponderant protein species of molecular weight 43,000.     

Isolation and characterization of mitochondrial alanine aminotransferase from porcine tissue.

Mitochondrial alanine aminotransferase L-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2) has been isolated in homogeneous form from both porcine liver and kidney cortex, but in low yield. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate or 8 M urea gave a single band. An isoelectric point of 8.5 +/- 0.5 and a molecular weight of 75--80 000 were obtained. The enzyme is specific for L-alanine and is inhibited by D-alanine, aminooxyacetate and cyclosterine. The Km for pyruvate and glutamate is 0.4 mM and 32 mM, respectively. These values are similar to those determined for the cytoplasmic enzyme; however, at high concentrations, both compounds strongly inhibit the mitochondrial enzyme, an inhibition not observed with cytosolic alanine aminotransferase. These characteristics and the fact that the mitochondrial alanine aminotransferase was inactivated by procedures effective in the preparation of the cytosolic enzyme, clearly differentiate the two proteins and further support different roles for the two alanine aminotransferases in vivo.     

Creatine kinase isoenzymes in Torpedo californica: absence of the major brain isoenzyme from nicotinic acetylcholine receptor membranes

Creatine kinase, actin, and nu 1 are three proteins of Mr 43 000 associated with membranes from electric organ highly enriched in nicotinic acetylcholine receptor. High levels of creatine kinase are required to maintain adequate ATP levels, while actin may play a role in maintaining the synaptic cytoskeleton. Previous investigations have prompted the conclusion that postsynaptic specializations at the receptor-enriched membrane domains in electroplax contain the brain form of creatine kinase rather than the form of creatine kinase predominantly found in muscle. We have examined this conclusion by purifying Torpedo brain creatine kinase to virtual homogeneity in order to examine its immunochemical, molecular, and electrophoretic properties. On the basis of immunological cross-reactivity and isozyme analysis, the receptor-associated creatine kinase is identified to be of the muscle type. When the molecular characteristics of Torpedo brain and muscle creatine kinase are compared, the brain enzyme is positioned at a more basic pH during chromatofocusing and on two-dimensional gel electrophoresis (pI = 7.5-7.9). Furthermore, electrophoretic mobilities of the brain and muscle forms of creatine kinase differ in sodium dodecyl sulfate electrophoresis: the brain isozyme of creatine kinase has lower apparent molecular weight (Mr 41 000) when compared with the muscle enzyme (Mr 43 000). On the basis of the results of our current investigations, the hypothesis that the brain isozyme of creatine kinase is a component of the postsynaptic specializations of the Torpedo californica electroplax must be abandoned. Recent sequence data have established close homology between Torpedo and mammalian muscle creatine kinases. On the basis of electrophoretic criteria, our results indicate that a lower degree of homology exists between the brain isozymes.     

Characterization of a brain particulate bound form of creatine kinase

A bound form of creatine kinase associated with brain particulate was characterized by isoelectric focusing, antigenicity and chromatography and compared to muscle (MM), brain (BB), and heart mitochondrial isoenzymes. On partial purification and isoelectric focusing, the solubilized enzyme has a pl of 7.3, similar to the pl of muscle creatine kinase MM, pl 6.8, but different from brain creatine kinase BB, which precipitates on isoelectric focusing in sucrose or glycerol stabilized media at its calculated pl of 5.6. Gel filtration chromatography of deoxycholate solubilized particulate creatine kinase on Sephadex Gl50 reveals an estimated molecular weight of approximately 80,000 daltons. The brain particulate enzyme is antigenically distinct from both muscle and rat heart mitochondrial creatine kinase isoenzymes but has antigenic similarity with soluble cytoplasmic brain BB. The situation may be analogous to that found with rat heart mitochondria and rat heart cytoplasmic isoenzymes which we have shown to exhibit antigenic similarity even though differences in electrophoretic and amino acid composition have been demonstrated; however, the confident determination that the particulate enzyme is a separate isoenzyme will have to await amino acid analysis.     

Determination of isoelectric points of oligomeric forms of mitochondrial creatine kinase

Using isoelectrofocusing in three pH gradients differing in the initial pH value of the ampholyte gel mixture and in gradient pH range, the isoelectric points for the dimeric and octameric forms of mitochondrial creatine kinase from bovine heart and pigeon breast muscle were determined. The isoelectric points for the dimer and octamer are equal to 9.67 +/- 0.01 and 8.93 +/- 0.05 for the heart enzyme and to 9.56 +/- 0.08 and 8.91 +/- 0.23 for the skeletal muscle enzyme. The correctness of identification of the oligomeric forms of mitochondrial creatine kinase was confirmed by ultracentrifugation in a sucrose density linear gradient. Since creatine kinase is known to bind to mitochondrial membrane cardiolipin by electrostatic forces, it can be assumed that both oligomeric forms of the enzymes can bind to the membranes. However, the properties of the creatine kinase dimer suggest its greater ability to bind to mitochondrial membranes.     

Purification and properties of the bifunctional proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase from Pseudomonas aeruginosa

Proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase (Pro/P5C dehydrogenase), a bifunctional enzyme catalyzing the two consecutive reactions of the oxidation of proline to glutamic acid, was purified from Pseudomonas aeruginosa strain PAO1. Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L-glutamic acid. The purification procedure involved DEAE-cellulose chromatography, affinity chromatography on Matrex gel red A and gel filtration on Sephadex G-200. It resulted, after 40-fold purification with 11% yield, in a homogeneous preparation (greater than 98% pure). The molecular weight of the single subunit was determined as 119,000. Gel filtration of purified Pro/P5C dehydrogenase yielded a molecular weight of 242,000 while polyacrylamide gel electrophoresis under native conditions led to the appearance of two catalytically active forms of the enzyme with molecular weights of 241,000 and 470,000. Manual Edman degradation revealed proline, alanine and aspartic acid as the N-terminal amino acid sequence. Pro/P5C dehydrogenase was highly specific for the L-forms of proline and delta 1-pyrroline-5-carboxylic acid. Its apparent Km values were 45 mM for L-proline, 0.03 mM for NAD and 0.17 mM for NADP. The saturation function for delta 1-pyrroline-5-carboxylic acid was non-hyperbolic.     

Multiple molecular forms of murine thymocyte-stimulating factor

Murine thymocyte stimulating factor (TSF) was found to sediment in sucrose density gradients on a broad band with peaks at about 2.60 S and 2.0 S. Two main peaks of TSF activity (with buoyant densities of 1.34 and 1.28 g/ml) were found in CsCl density gradients. Gel chromatography on Sephadex G-100 columns of the material sedimented in sucrose or CsCl density gradients originated multiple peaks of TSF activity with various molecular weights. Heterogeneity of molecular forms of TSF was also found upon dilution of the factor. The lowest molecular weights found were 4000 and 4700 daltons. When Sephadex fractions containing the low molecular weight material were pooled and rerun on Sephadex columns, molecular species with a wide range of molecular weights were found. Temperature also affects the appearance of the low molecular weight forms of TSF. Most of the experiments presented in this work were carried out with Sephadex-purified TSF. Multiple molecular forms, however, and, in particular, the forms with molecular weights of 4000 and 4700 daltons were found also with TSF-Fraction IIIa, a highly purified preparation of this factor.     

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.     

Specific inhibition of ATP-ADP translocase in cardiac mitoplasts by antibodies against mitochondrial creatine kinase

Mitochondrial creatine kinase was purified from rat hearts and used to produce antibodies in chicken and rabbits. Antibodies were purified to a high degree of homogeneity by an affinity chromatography method. Chicken antibodies against mitochondrial creatine kinase inhibited this enzyme in rat-heart mitochondrial inner membrane and matrix preparation, and simultaneously blocked oxidative phosphorylation. Under these conditions respiratory chain activities remained unchanged, but adenine nucleotide translocase was inhibited. Removal of mitochondrial creatine kinase from the membrane by pretreatment with 0.15 M KCl and 20 mM ADP completely abolished the effect of antibodies against mitochondrial creatine kinase on oxidative phosphorylation. Noninhibitory antibodies from rabbit with high affinity to rat mitochondrial creatine kinase inhibited neither creatine kinase activity nor oxidative phosphorylation. These data show close and specific spatial arrangement of mitochondrial creatine kinase and adenine nucleotide translocase in mitochondria. It is supposed that there is a fixed orientation of these proteins in the cardiolipin domain in the membrane and that their interaction may occur by a frequent collision due to their lateral movement.     

Cytosolic L-alanine:4,5-dioxovalerate transaminase differs from the mitochondrial form

L-Alanine:4,5-dioxovalerate transaminase was detected in the kidney cytosolic fraction with a lower specific activity than the mitochondrial enzyme. The enzyme was purified from the cytosol to homogeneity with a yield of 32%, and comparative analysis with the mitochondrial form was performed. Both forms of the enzyme have identical pH and temperature optima and also share common antigenic determinants. However, differences in their molecular properties exist. The molecular mass of the native cytoplasmic enzyme is 260 kDa, whereas that of the mitochondrial enzyme is 210 kDa. In addition, the cytoplasmic L-alanine: 4,5-dioxovalerate transaminase had a homopolymeric subunit molecular mass of 67 kDa compared to a subunit molecular mass of 50 kDa for the mitochondrial L-alanine:4,5-dioxovalerate transaminase. This is the first report of two forms of L-alanine:4,5-dioxovalerate transaminase. The different responses of cytosolic and mitochondrial L-alanine:4,5-dioxovalerate transaminases to hemin supplementation both in vitro and in vivo was demonstrated. Maximum inhibition of mitochondrial L-alanine:4,5-dioxovalerate transaminase activity was demonstrated with hemin injected at a dose of 1.2 mg/kg body mass, whereas the same dose of hemin stimulated the cytosolic enzyme to 150% of the control. A one-dimensional peptide map of partially digested cytosolic and mitochondrial L-alanine:4,5-dioxovalerate transaminase shows that the two forms of the enzymes are structurally related. Partial digestion of the cytosolic form of the enzyme with papain generated a fragment of 50 kDa which was identical to that of the undigested mitochondrial form (50 kDa). Moreover, papain digestion resulted in a threefold increase in cytosolic enzyme activity over the native enzyme, and such enhancement was comparable to the activity of the mitochondrial form of the enzyme. Therefore, we conclude that the cytosolic form of L-alanine: 4,5-dioxovalerate transaminase is different from the mitochondrial enzyme. Furthermore, immunoblot analysis indicated that the mitochondrial enzyme has antigenic similarity to the cytosolic enzyme as well as to the papain-digested cytosolic enzyme 50-kDa fragment.     

Neuroprotective effects of creatine in a transgenic animal model of amyotrophic lateral sclerosis

Mitochondria are particularly vulnerable to oxidative stress, and mitochondrial swelling and vacuolization are among the earliest pathologic features found in two strains of transgenic amyotrophic lateral sclerosis (ALS) mice with SOD1 mutations. Mice with the G93A human SOD1 mutation have altered electron transport enzymes, and expression of the mutant enzyme in vitro results in a loss of mitochondrial membrane potential and elevated cytosolic calcium concentration. Mitochondrial dysfunction may lead to ATP depletion, which may contribute to cell death. If this is true, then buffering intracellular energy levels could exert neuroprotective effects. Creatine kinase and its substrates creatine and phosphocreatine constitute an intricate cellular energy buffering and transport system connecting sites of energy production (mitochondria) with sites of energy consumption, and creatine administration stabilizes the mitochondrial creatine kinase and inhibits opening of the mitochondrial transition pore. We found that oral administration of creatine produced a dose-dependent improvement in motor performance and extended survival in G93A transgenic mice, and it protected mice from loss of both motor neurons and substantia nigra neurons at 120 days of age. Creatine administration protected G93A transgenic mice from increases in biochemical indices of oxidative damage. Therefore, creatine administration may be a new therapeutic strategy for ALS.     

Progressive decrease of phosphocreatine, creatine and creatine kinase in skeletal muscle upon transformation to sarcoma

In vertebrates, phosphocreatine and ATP are continuously interconverted by the reversible reaction of creatine kinase in accordance with cellular energy needs. Sarcoma tissue and its normal counterpart, creatine-rich skeletal muscle, are good source materials to study the status of creatine and creatine kinase with the progression of malignancy. We experimentally induced sarcoma in mouse leg muscle by injecting either 3-methylcholanthrene or live sarcoma 180 cells into one hind leg. Creatine, phosphocreatine and creatine kinase isoform levels decreased as malignancy progressed and reached very low levels in the final stage of sarcoma development; all these parameters remained unaltered in the unaffected contralateral leg muscle of the same animal. Creatine and creatine kinase levels were also reduced significantly in frank malignant portions of human sarcoma and gastric and colonic adenocarcinoma compared with the distal nonmalignant portions of the same samples. In mice, immunoblotting with antibodies against cytosolic muscle-type creatine kinase and sarcomeric mitochondrial creatine kinase showed that both of these isoforms decreased as malignancy progressed. Expressions of mRNA of muscle-type creatine kinase and sarcomeric mitochondrial creatine kinase were also severely downregulated. In human sarcoma these two isoforms were undetectable also. In human gastric and colonic adenocarcinoma, brain-type creatine kinase was found to be downregulated, whereas ubiquitous mitochondrial creatine kinase was upregulated. These significantly decreased levels of creatine and creatine kinase isoforms in sarcoma suggest that: (a) the genuine muscle phenotype is lost during sarcoma progression, and (b) these parameters may be used as diagnostic marker and prognostic indicator of malignancy in this tissue.