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PubMed1985

Isolation and characterization of acetylcholine receptor membrane-associated (nonreceptor v2-protein) and soluble electrocyte creatine kinases.

Creatine kinase has been identified as a most prominent component of Torpedo electric organ and a minority constituent of the acetylcholine receptor (AChR) membranes obtained therefrom. Purification by low temperature ethanol extraction, precipitation of the Mg2+-enzyme complex, and mercurial-agarose chromatography yield preparations of soluble kinase with specific activities greater than 550 units/mg protein. Retention times in ion-exchange high performance liquid chromatography, electrophoretic behavior, immunochemical properties, tryptic mapping, and amino acid composition enable the comparison of creatine kinase isoenzymes. The denatured subunits of the predominant species have pI values of 6.3-6.8 and Mr = 40,000-42,000 characteristic of the so-called v2 proteins and show cross-reactivity with antibodies against the BB ("brain" type) creatine kinase. The MM ("muscle" type) antigens could be detected in the total electrocyte, but not in the AChR membranes; they have a slightly lower molecular weight and higher pI. The in situ membrane association of the BB isoenzyme is confirmed by immunocytochemistry. The apparent Km values for the substrate creatine phosphate are 2.2 mM for the AChR membrane-associated enzyme and 2.5 mM for the muscle form. The apparent Km values for Mg2+-ADP are 0.54 and 0.22 mM, respectively. Thus, a 2-fold higher affinity in the binding of ADP to the binary enzyme-creatine-P complex results from membrane association.

Amino Acids↗

PubMed1985

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

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 Vmax. The values for Km (mM/L) derived from Lineweaver-Burke plots are shown: (Table: see text). 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.

Amino Acids↗

PubMed1995

Cyclocreatine in cancer chemotherapy.

Cyclocreatine, an analog of creatine, is an efficient substrate for creatine kinase, but its phosphorylated form is a poor phosphate donor in comparison with creatine phosphate. Cyclocreatine was not very cytotoxic upon 24 h of exposure of human SW2 small-cell lung cancer cells to concentrations of up to 5 mM. However, combinations of cyclocreatine (0.5 mM, 24 h) with each of four antitumor alkylating agents, cis-diamminedichloroplatinum(II), melphalan, 4-hydroperoxycyclophosphamide, and carmustine, resulted in additive to greater-than-additive cytotoxicity toward exponentially growing SW2 cells. The greatest levels of synergy were seen at higher concentrations of 4-hydroperoxycyclophosphamide and carmustine as determined by isobologram analysis. In vivo cyclocreatine (0.5 or 1 g/kg) was more effective if given i.v. rather than i.p. The longest tumor-growth delays, up to 10 days, were produced by extended regimens of cyclocreatine. Cyclocreatine was an effective addition to therapy with standard anticancer agents including cis-diamminedichloroplatinum(II), cyclophosphamide, Adriamycin, or 5-fluorouracil. No additional toxicity was observed when 10 days of cyclocreatine treatment was given with full standard-dose regimens of each drug. The resultant increases in tumor-growth delay were 1.7- to 2.4-fold as compared with those obtained for each of the drugs alone. These results indicate that cyclocreatine may be an effective single agent and an effective addition to combination chemotherapy regimens.

Adenocarcinoma↗

PubMed1983

Isolation of three creatine kinases MM from porcine skeletal muscle.

The purified creatine kinase MM of porcine skeletal muscle [Takasawa, T. & Shiokawa, H. (1981) J. Biochem. 90, 195-204] was separated into three distinct fractions by isoelectric focusing (IEF) in a sucrose gradient column, and the three active fractions were isolated by repeated IEF. There were one major fraction with isoelectric point (pI) 6.57 and two minor fractions with pI 6.74 and pI 6.34, respectively. No differences were observed in the IEF pattern of the enzyme in the presence and absence of dithiothreitol throughout the column. There was no interconversion from one form to another during IEF. The distribution of the three forms on IEF was not affected by adding protease inhibitor to the extraction medium. Of the three fractions, the major fraction had the highest specific activity. The three fractions differed from one another in their amino acid compositions. Not only porcine muscle but also rabbit muscle creatine kinase displayed this type of heterogeneity. Such microheterogeneities may occur widely in muscle creatine kinases.

Amino Acids↗

PubMed1977

Subcellular compartmentation of creatine kinase isoenzymes in guinea pig heart.

In an attempt to determine whether a subcellular compartmentation of creatine kinase exists and if so, whether there is a different distribution of the 3 isoenzymes of CK in the cell, studies were carried out with the guinea pig heart which had been subfractionated by either isopycnic density gradient centrifugation or differential pelleting. Isoenzyme analysis of CK in the isolated subcellular fractions by electrophoresis on agarose gels, revealed that the MM isoenzyme occurred in the cytosol, myofibrils, and the microsomes while the MB isoenzyme (which is cardio-specific) was only found in the cytosol. Trace amounts of the BB isoenzyme were detected in the cytosol. Considerable CK activity was associated with the mitochondria, this did not represent the MM, the MB, or the BB isoenzymes but was a distinct and additional mitochondrial-specific form of CK. PH optima and kinetic studies were carried out to characterise and distinguish the mitochondrial isoenzyme from other CK isoenzyme activity. The evidence for a differential compartmentation of MM, MB, BB, and mitochondrial CK is discussed in relation to their possible cellular roles.

Animals↗

PubMed1985

Redistribution of creatine kinase isoenzymes in chronically overloaded myocardium.

Chronic overload due to an experimental abdominal aortic stenosis in rats causes hypertrophy of ventricles and a parallel increase of the MB and BB isoforms of creatine kinase (CK) in cardiac tissue. The CK isoenzyme profile was determined using a new two-step method combining anion exchange chromatography and electrophoresis. In overloaded ventricles a shift was observed from the MM isoform which decreased (from 407 +/- 10 mumol X min-1 X g-1 ww in sham-operated to 370 +/- 0.13, in the overloaded group, p less than 0.05) towards the MB and BB forms whose activity was enhanced (from 56 +/- 4 and 6.5 +/- 0.7 to 77 +/- 6 and 10.1 +/- 1.2, respectively, p less than 0.02). These modifications were more pronounced (318 +/- 15, 83 +/- 15 and 15.1 +/- 2.5, for the MM, MB and BB forms respectively, p less than 0.01) in rats having a very marked hypertrophy and whose ventricular/body weight ratio (expressed in mg of ventricles per g) was above 3. Moreover this ratio correlates both with the amount of the MB form (r = 0.32, p less than 0.05) and with the percentage of the B monomer (r = 0.51, p less than 0.01). This shift, like that previously described for lactate dehydrogenase and myosin, favoured the fetal form and this supports the hypothesis that overloaded myocytes improve their efficiency by expressing some of the isoforms normally present in the fetus.

Animals↗

PubMed1983

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.

Animals↗

PubMed1985

Reaction rates of creatine kinase and ATP synthesis in the isolated rat heart. A 31P NMR magnetization transfer study.

The NMR technique of magnetization transfer can be used to define intracellular reaction kinetics. In order to determine the relationship between ATP synthesis and flux through the creatine kinase reaction in the intact heart, we used this technique to measure flux through the creatine kinase reaction in the isolated, isovolumic rat heart at five levels of cardiac performance and oxygen consumption. The unidirectional reaction rate constants (s-1) calculated from a two-site exchange model for both the forward and reverse creatine kinase reactions increased with cardiac performance and oxygen consumption. As the rate-pressure product varied from 0 to 44.7 X 10(3) mm Hg/min and oxygen consumption rose from 5.9 to 45.8 mumol of O2/g dry weight/min, kforward increased from 0.27 to 1.30 and kreverse increased from 0.31 to 1.14. The relationship between creatine kinase flux and oxygen consumption, and thus ATP synthesis, took the form of the Michaelis-Menten equation. Rates of ATP synthesis estimated from magnetization transfer were similar to values calculated from oxygen consumption. The longitudinal relaxation time of creatine phosphate (2.06 s), the gamma-phosphorus atom of ATP (0.75 s), and inorganic phosphate (0.81 s) did not change with cardiac performance. These results show that myocardial energy transfer via the creatine kinase reaction is closely coupled to energy production.

Adenosine Triphosphate↗

PubMed1993

Altering creatine kinase isoenzymes in transgenic mouse muscle by overexpression of the B subunit.

To change the levels of expression and isoenzyme distribution of creatine kinase (CK) in muscle, transgenic technology was used to express the B subunit of CK in mouse muscle. Normally, mammalian skeletal muscle contains the MM dimer of CK. The BB dimer and MB heterodimer of CK can be found in brain and heart, respectively. Heterologous genes consisting of skeletal and cardiac muscle-specific actin promoters fused to the genomic coding region of the B form of CK were used to create transgenic mice. Lines were established from the three highest expressing founders. Analysis of skeletal muscle extracts revealed that all three lines had an increase in total CK activity measured under maximal velocity conditions. The highest expressing line, 7001, had a CK activity 150% that of control muscle. Nuclear magnetic resonance saturation transfer was used to measure the in vivo rate of the CK reaction. In 7001 hindlimb muscles, the CK catalyzed reaction was 200% that of control muscle. The elevation in CK activity in transgenic muscle was accompanied by significant changes in the composition of the cytosolic isoenzyme ratio of CK. In control, 100% of CK was MM, whereas 7001 had 60 +/- 18% MM, 32 +/- 10% MB, and 8 +/- 2% BB. There were no changes in ATP, phosphocreatine, Pi, or creatine levels in transgenic muscle compared with control. Immunofluorescence of myofibrils isolated from control and transgenic muscle revealed specific association of CK to the M line. Small amounts of MB CK were detected on myofibrils from transgenic mice. Transgenic mice expressing the B subunit of CK in muscle represent a first step toward altering CK isoforms so as to elucidate the specific roles of these isoforms in energy metabolism.

Animals↗

PubMed1983

A "column-batch" method for separating MB and LD1 in a single fraction.

In this "column-batch" method for separating the MB and BB isoenzymes of creatine kinase and the LD1 isoenzyme of lactate dehydrogenase, one can, alternatively, separate MB from BB or obtain a combined fraction containing MB, BB, and LD1. The principal advantage is that the resulting fractions are twofold as concentrated as was the applied sample. Thus, activity can be measured by conventional automated methods, with no need for the modifications to compensate for diluted fractions that are required by other ion-exchange methods. Another advantage is the total absence of interference by the MM isoenzyme. A strong anion exchanger (AG-MP1, Bio-Rad) is used in the acetate form at pH 6.3. There is no retention of MM; retained MB, BB, and LD1 are eluted with a solution of magnesium acetate. Results are compared with those obtained for subunit B and LD1 by immunoinhibition. Results with patients are considered consistent with myocardial infarction if MB exceeds 20 U/L and 3% of the total CK and LD1 exceeds 130 U/L or 28% of the total LD activity.

Chromatography, Ion Exchange↗

PubMed1980

Macro creatine kinase: a study on CK-linked immunoglobulin.

We describe three cases in which creatine kinase (CK, EC 2.7.3.2) was linked to immunoglobulin in serum. In this study, its prevalence was 0.8%. Enzyme-immunofixation electrophoresis revealed that the heavy chain of CK-linked immunoglobulins was of class alpha in all cases. The light-chain type was identified as lambda in two cases and as both lambda and kappa in one case. The complexes were dissociated at pH 3.4 and re-formed with CK isoenzymes MM and MB at pH 7.4. The complex fraction obtained by gel filtration was not inhibited by anti-CK-M antibodies. Treatment of the serum with urea after heating shows residual CK-MM activity; in contrast, normal CK activity disappeared entirely after this treatment. The present study suggests that CK-linked immunoglobulins may be one of the circulating immune complexes and must be distinguished from other macro-CK in the strict sense. The results obtained show that the presence of the complexes results in false-positive CK-B activity in the immuno-inhibition test, and they may provide interesting insights into the mode of binding of the CK-linked immunoglobulins.

Adult↗

PubMedDate not supplied

[Kinetic characteristics of creatine kinase and hexokinase from rat skeletal muscles during dietary deficit of vitamin K and administration of pelentane].

Kinetic parameters of rat creatine kinase isozymes at different vitamin K supply and treatment with antivitamin K--pelentan have been determined. MM-isozyme (skeletal muscle) has selective sensitivity to the vitamin K deficit, while BB and MB-isozymes (brain, kidney and heart) have not. The value KM for ATP of MM-isozymes increases, while maximal activity decreases. Pelentan treatment does not lead to the change of MM-creatine kinase affinity to ATP. Soluble hexokinase of skeletal muscle in rats with vitamin K deficiency and treated with pelentan has higher affinity to glucose as compared to normal rat enzyme. It has been supposed that skeletal muscle hexokinase exists in a particular molecular form under vitamin K deficiency.

Animals↗

PubMed1986

Creatine kinase isoenzyme MM variants in skeletal muscle and plasma from marathon runners.

We investigated the patterns of variants of creatine kinase isoenzyme MM (CK-MM) in gastrocnemius muscle and plasma sampled from male and female long-distance runners before and after a marathon race. The proportions of CK-MM variants MM1 (pI 6.90) and MM2 (pI 6.62), identified in the skeletal muscle from both sexes, did not differ significantly from those in skeletal muscle from nonrunning controls or from heart muscle. CK-MM1 was the major (84-85% of total CK-MM) variant form. Patterns of CK-MM in plasma collected from male runners 24, 48, 72, and 96 h after the race were similar to those for female runners, but we detected two new additional variants, which we designate MM1B (pI 6.76) and MM2B (pI 6.49). For both sexes the total CK-MM activities in plasma were significantly (p less than 0.05) greater after the race, but the women's total CK-MM activities were significantly (p less than 0.05) less than the men's. The rates of disappearance of MM1, MM2, and MM3 from plasma after the race differed significantly (p less than 0.05) between men and women, MM1 clearing the fastest. Determination of the CK-MM variants in plasma after strenuous exercise may be of help in assessing CK release from injured skeletal muscle.

Adult↗

PubMed1990

Creatine kinase and lactate dehydrogenase isoenzyme patterns in cultured normal and pathological gingival fibroblasts.

The isoenzyme patterns of creatine kinase (CK) and lactate dehydrogenase (LDH) were examined in cultured fibroblasts derived from the gingival tissue of a healthy person and patients with periodontitis and gingival fibromatosis. Human foreskin fibroblasts were also included for comparison. Cytosol fractions were prepared by sonication and centrifugation. Gingival fibroblasts from normal subjects (A), and from patients with periodontitis (B) or fibromatosis (C), exhibited CK-MM (muscle form) as a predominant isoenzyme and CK-BB (brain form) as a minor fraction. There was no significant difference in specific activities of CK-BB fraction in all 3 types of fibroblasts. However, B fibroblasts contained a higher proportion of MM type isoenzyme than A fibroblasts. C fibroblasts showed the lowest amount of CK-MM isoenzyme as compared to either A or B fibroblasts. In contrast, fibroblasts derived from human foreskin (D) contained only a small amount of CK-BB isoenzyme, but CK-MM was not present. In the studies of LDH isoenzymes, A, B, and C fibroblasts possessed only 3 isoenzymes, namely, LDH-3, LDH-4 and LDH-5. In terms of the relative abundance of M vs H subunits, A, B, and C fibroblasts were shown to have a preponderance of M subunits (85-87% M vs 13-15% H subunits). In comparison, D fibroblasts contained 4 isoenzymes (LDH-2, LDH-3, LDH4, LDH-5) with 73% M and 27% H subunits. The ratio of LDH-5 to LDH-4 was calculated for all fibroblasts tested, and the values were 1.11 in A, 1.39 in B, 1.16 in C and 0.64 in D.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1982

Macro creatine kinases: results of isoenzyme electrophoresis and differentiation of the immunoglobulin-bound type by radioassay.

In 2.9% of sera from 1253 unselected patients we detected two different types of macromolecular creatine kinases (CK; EC 2.7.3.2). One macro type was represented by immunoglobulin-linked CK: in sera containing macro CK-BB isoenzyme, 125I-labeled CK-BB was bound with high affinity to the immunoglobulin fraction. Furthermore, during electrophoresis, macro CK-BB mostly migrated between CK-MB and CK-MM, and was fixed to Protein A from Staphylococcus aureus. We therefore propose radioelectrophoresis as a specific, highly sensitive, and simple method for detecting this type of macro CK. This form occurs predominantly in elderly women, is not correlated to any specific disease, and persists in blood over a long period of time. In contrast, a second type (macro-CK type 2) never bound radiolabeled CK isoenzymes, and was not adsorbed to Protein A. Electrophoretic migration of this macro-CK type 2 was generally cathodic to CK-MM. We observed this type in severely ill patients, frequently those suffering from malignant tumors. Clinical observations and biochemical data suggest that macro-CK type 2 is of mitochondrial origin.

Age Factors↗

PubMed1997

High salt concentrations induce dissociation of dimeric rabbit muscle creatine kinase. Physico-chemical characterization of the monomeric species.

Incubation of dimeric MM-creatine kinase (MM-CK) with high NaCl or LiCl concentrations results in dissociation of the subunits and complete enzyme inactivation. In NaCl, this process, which depends on protein concentration, may be described according to a two-state model where the dimer can be reversibly converted into compact folded monomers (D <--> 2M). At LiCl concentrations higher than 2-2.5 M, MM-CK is recovered in two monomeric states: an inactive compact species (M) and a more expanded form (EF), which represents 15-20% of the population. Thus, in LiCl, a three-state model (D <--> 2M --> 2EF) more adequately accounts for our experimental results. The monomeric species (M) obtained in NaCl and LiCl exhibits some properties of the molten globule state described in guanidine hydrochloride. Indeed, this form is compact and devoid of any enzymatic activity; it maintains a high degree of secondary structure and binds 8-anilino-1-naphthalenesulfonate. The formation of this intermediate induces the exposure of a second tryptophan (among the four present) which is located at the monomer-monomer interface in the native structure. In LiCl, the monomeric species (M) is irreversibly converted into a less compact form (EF) which seems to have lost a large part of its secondary structure.

Animals↗

PubMed2001

Functional aspects of creatine kinase isoenzymes in endothelial cells.

To characterize the isoenzyme distribution of creatine kinase (CK) in endothelial cells (ECs) and its functional role during substrate depletion, ECs from aorta (AECs) and microvasculature (MVECs) of pig and rat were studied. In addition, high- energy phosphates were continuously monitored by (31)P NMR spectroscopy in pig AECs attached to microcarrier beads. CK activity per milligram of protein in rat AECs and MVECs (0.08 +/- 0.01 and 0.15 +/- 0.08 U/mg, respectively) was <3% of that of cardiomyocytes (6.46 +/- 1.02 U/mg). Rat and pig AECs and MVECs displayed cytosolic BB-CK, but no MM-CK. Gel electrophoresis of mitochondrial fractions of rat and pig ECs indicated the presence of mitochondrial Mi-CK, mostly in dimeric form. The presence of Mi(a)-CK was demonstrated by indirect immunofluorescence staining using Mi(a)-CK antibodies. When perifused with creatine-supplemented medium, phosphocreatine (PCr) continuously increased with time (1.2 +/- 0.6 nmol x h(-1) x mg x protein(-1)), indicating creatine uptake and CK activity. Glucose withdrawal from the medium induced a rapid decrease in PCr, which was fully reversible on glucose addition, demonstrating temporal buffering of an energy deficit. Because both cytosolic and mitochondrial CK isoforms are present in ECs, the CK system may also contribute to energy transduction ("shuttle hypothesis").

Animals↗

PubMed1985

MM subisoenzymes of creatine kinase as an index of disease activity in polymyositis.

Creatine kinase (CK; EC 2.7.3.2), although the most commonly measured enzyme for assessing disease activity in polymyositis, is not always a reliable indicator of the extent and severity of myositis. Recently, the CK-MM isoenzyme has been found to undergo post-synthetic modifications upon release into the serum, such that electrophoretically identifiable sub-bands or subisoenzymes--MM1, MM2, and MM3--are produced. To determine the diagnostic and discriminative value of these subisoenzymes in polymyositis, we analyzed CK and its MM subisoenzyme forms in serum samples from 22 patients with myositis and from 23 controls. In the presence of inflammatory myositis and increased total CK activity, MM-patterns correlated with the clinical trend, often more accurately than did measurements of total CK. MM1 proportions greater than 30% of total CK-MM or ratios of MM3 to MM1 less than 1 were associated with an improving or stable condition, whereas MM1 activity less than 30% or MM3/MM1 greater than 1 reflected a deteriorating course of disease. Patients whose disease was assessed to be clinically deteriorating were clearly distinguished from patients with improving disease by their subisoenzyme patterns (p less than 0.01). Thus these patterns add significantly to the information obtainable by routine blood analysis.

Creatine Kinase↗