Analysis of creatine kinase variation in some members of the family Acipenseridae

Creatine kinase (E.C. 2.7.3.2) was examined in stellate sturgeon Acipenser stellatus Pallas, Russian sturgeon A. gueldenstaedtii Brandt, European sterlet A. ruthenus L., Siberian sterlet A. ruthenus marsiglii Brandt, and great sturgeon (beluga) Huso huso L., using polyacrylamide gel electrophoresis. Two loci for creatine kinase were identified: CK-A* in white skeletal muscle and CK-C* in stomach wall muscle. Most species proved to be monomorphic at the CK-A* locus, showing the same phenotype represented by a single band. Heterogeneity and polymorphism in creatine kinase, determined by the CK-A* locus, were found only in Russian sturgeon. Based on the results of densitometric analysis of band staining intensity, we have advanced a hypothesis that synthesis of subunits of the CK-A* product in this species was controlled by eight genes. However, the genotype frequencies in the sample were significantly different from those theoretically expected upon free and independent gene recombination. The results of this study support the hypothesis on the absence of heterodimeric creatine kinase molecules in the skeletal muscle of Russian sturgeon. Locus CK-C* in sterlet was revealed as a single, intensely stained, rapidly migrating fraction, whereas in Russian sturgeon, the enzyme activity in this zone was very weak. No creatine kinase was found in liver, kidneys, spleen, heart, and intestine mucous tunic.     

Amino-acid Sequence Homology in the Muscle Aldolases from Sturgeons of Different Species

STUDIES on the primary structure of aldolases isolated from ox, pig and rabbit muscle show that the amino-acid sequence of fructose 1,6-diphosphate aldolase [EC 4.1.2.13] has been highly conserved throughout mammalian evolution¹. But comparison of the primary structure of the enzyme from these species with that from the muscle of a single North Sea sturgeon, presumably Acipenser sturio, indicated that although the proteins were homologous, a number of amino-acid replacements occurred between sturgeon aldolase and the aldolases of the phylogenetically distant mammalian species¹. As a knowledge of the nature and number of amino-acid replacements between homologous proteins caft provide information both about the functional role of individual residues and about evolution, further comparative studies of rabbit and sturgeon aldolases were undertaken and an account of the sequence homology around the active-site-lysine residue of aldolases from rabbit muscle, rabbit liver and the muscle of the river sturgeon of Eastern Canada, Acipenser fulvescens, has been given^2,3.     

Analysis of creatine kinase variation in some members of the family Acipenseridae

Creatine kinase (E.C. 2.7.3.2) was examined in stellate sturgeon Acipenser stellatus Pallas, Russian sturgeon A. gueldenstaedtii Brandt, European sterlet A. ruthenus L., Siberian sterlet A. ruthenus marsiglii Brandt, and great sturgeon (beluga) Huso huso L., using polyacrylamide gel electrophoresis. Two loci for creatine kinase were identified: CK-A* in white skeletal muscle and CK-C* in stomach wall muscle. Most species proved to be monomorphic at the CK-A* locus, showing the same phenotype represented by a single band. Heterogeneity and polymorphism in creatine kinase, determined by the CK-A* locus, were found only in Russian sturgeon. Based on the results of densitometric analysis of band staining intensity, we have advanced a hypothesis that synthesis of subunits of the CK-A* product in this species was controlled by eight genes. However, the genotype frequencies in the sample were significantly different from those theoretically expected upon free and independent gene recombination. The results of this study support the hypothesis on the absence of heterodimeric creatine kinase molecules in the skeletal muscle of Russian sturgeon. Locus CK-C* in stellate sturgeon was revealed as a single, intensely stained, rapidly migrating fraction, whereas in Russian sturgeon, the enzyme activity in this zone was very weak. No creatine kinase was found in liver, kidneys, spleen, heart, and intestine mucous tunic.     

Comparison of the subunit and primary structures of the pyruvate kinases from rabbit and sturgeon muscles.

The structures of the pyruvate kinases isolated from rabbit and sturgeon muscles were compared. Both enzymes are composed of subunits of 56000 mol.wt. Amino acid compositions of the two enzymes are similar, but not identical. Examination of the peptides produced by CNBr cleavage demonstrated that there are at least some highly homologous regions in the two proteins. There are only two replacements between an 18-residue portion of the polypeptide chain of rabbit muscle pyruvate kinase and a portion of the polypeptide chain of the enzyme isolated from sturgeon muscle.     

Kinetic properties of cerebral pyruvate kinase

Partly purified guinea-pig brain pyruvate kinase is not activated by fructose 1,6-diphosphate and gives hyperbolic substrate-saturation curves with phosphoenolpyruvate. It is therefore different from the L-type pyruvate kinase of mammalian liver. Inhibition by MgATP(2-) was competitive for MgADP(-) but not for phosphoenolpyruvate, and the enzyme is therefore different from the M-type pyruvate kinase, which is said to be competitively inhibited by MgATP(2-) with respect to both substrates. The K(i)(MgATP(2-)) value of approx. 8mm for the brain enzyme is higher than the values (about 2mm) reported for the muscle enzyme. Stimulation of enzymic activity was observed at low (1-2mm) concentrations of MgATP(2-). Substrate kinetic constants were K(m) (MgADP(-))=0.47mm, K(m) (phosphoenolpyruvate)=0.08mm. Free Mg(2+) at very high concentrations (over 10mm) was inhibitory (K(i)=20-32mm). Neither ADP(3-) nor 5'-AMP(2-) inhibited the activity. The brain enzyme was concluded to be different from both the M-type and the L-type of other mammalian organs such as muscle and liver.     

Purification and characterisation of the insulin-stimulated protein kinase from rabbit skeletal muscle; close similarity to S6 kinase II.

The insulin-stimulated protein kinase (ISPK) was purified over 50,000-fold from extracts of rabbit skeletal muscle by a procedure involving chromatography on phosphocellulose, fractionation with ammonium sulphate, and further chromatography on DEAE-cellulose, phenyl-Superose, Mono S and Mono Q. About 10 micrograms enzyme was isolated from 800 g muscle (one rabbit) in four days with an overall recovery of 5%. The purified enzyme showed a single protein-staining band of apparent molecular mass 91 kDa when analysed by SDS/polyacrylamide gel electrophoresis. The ISPK comigrated during SDS/polyacrylamide gel electrophoresis with the enzyme S6 kinase II from Xenopus eggs, and was recognised in immunoblotting and immunoprecipitation experiments by antibodies raised against S6 kinase II. The substrate specificities of ISPK and S6 kinase II were also very similar and like S6 kinase II, ISPK that had been inactivated by protein phosphatase 2A could be reactivated by incubation with mitogen-activated protein kinase and MgATP. ISPK was distinct from an insulin-stimulated 70-kDa S6 kinase from rat liver in both substrate specificity and immunological cross reactivity. It is concluded that ISPK is closely related in structure to S6 kinase II and may be a mammalian equivalent of this enzyme. The possibility that ISPK is involved in mediating a number of the actions of insulin is discussed.     

Identification of a calmodulin-dependent glycogen synthase kinase in rabbit skeletal muscle, distinct from phosphorylase kinase

A glycogen synthase kinase that is completely dependent on Ca2+ and calmodulin has been identified in mammalian skeletal muscle, and purified approximately 3000-fold by chromatography on phosphocellulose and calmodulin--Sepharose. The presence of 50 mM NaCl in the homogenisation buffer was critical for extraction of the enzyme. The calmodulin-dependent glycogen synthase kinase (app. Mr 850 000) is distinct from myosin light-chain kinase and phosphorylase kinase, but phosphorylates the same serine residue on glycogen synthase as phosphorylase kinase. The physiological role of the enzyme is discussed.     

Properties of chicken skeletal muscle pyruvate kinase and a proposal for its evolutionary relationship to the other avian and mammalian isozymes.

Pyruvate kinase (EC 2.7.1.40) was isolated and purified from chicken and turkey breast muscle with a purification procedure very similar to that used for the bovine skeletal muscle isozyme (Cardenas, J., Dyson, R., and strandholm, J. (1973), J. Biol. Chem. 248,6931). A study of the chemical and physical properties of the chicken enzyme revealed that it is a tetramer of four apparently identical subunits, closely resembling in this and most other respects the mamalian type 7 isozyme. The properties of these two enzymes are similar enough to permit subunits of chicken type M pyruvate kinase to combine with subunits of mammalian type L (one of the three mammalian isozymes) to form interspecies tetrameric hybrid isozymes in relative quantities that do not differ makedly from those formed when both the M and L isozymes are of mammalian origin. The similarity between the mammalian and avian type M pyruvates kinases suggests a close evolutionary relationship. Further comparisons among the three mammalian and two avian isozymes of pyruvate kinase are consistent with a common evolutionary origin, perhaps from an ancestral form of the type K isozyme, which is the only pyruvate kinase identified in mammalian and avian embryos.     

Metabolic regulation of glycolysis in sea bass (Dicentrarchus labrax L.) muscle. I. Kinetic study and characteristic modulators of pyruvate kinase

White muscle pyruvate kinase from sea bass presents positive cooperativity with respect to PEP substrate. The enzyme is regulated by F-1.6-P2 and L-Phenylalanine. The activator effect of F-1.6-P2 in experiments carried out for the substrate PEP with crude extract seems to indicate that the enzyme is activated in vivo by this compound. The enzyme was not inhibited by either alanine or ATP but was inhibited by L-phenylalanine. Therefore this enzyme presents kinetic and regulatory properties similar to those of the mammalian isozyme M2.     

Adenylate kinase of Mycobacterium marinum. Amino acid composition, secondary structure and other aspects

1. Adenylate kinase purified from Mycobacterium marinum shares homology with the enzyme from mammalian muscle and from other bacteria with respect to amino acid composition, total residues and Mr. 2. However, it differs from others in not containing a free sulfhydryl or disulfide group. 3. It is much more sensitive to inhibition by adenosine (5')pentaphospho(5')adenosine than is the enzyme from other sources. 4. The enzyme contains 24% alpha-helix, 30% beta-structure and 46% random coil.     

The purification and kinetic characterization of eel white muscle pyruvate kinase

A stable, homogeneous preparation of pyruvate kinase from white muscle of the American eel, Anguilla rostrata with a specific activity of 350 units/mg has been obtained. The enzyme has a pH optimum in the range 6.3-6.5 and requires Mg2+ and K+ for maximum activity. Eel muscle pyruvate kinase exhibits slight co-operativity in the binding of the substrate phosphoenol-pyruvate. It is activated by fructose-1,6-bisphosphate in a pH dependent manner and is inhibited by both alanine and phenylalanine. These properties are very similar to the properties of the mammalian M2 isozyme.     

Monoclonal antibody assessment of tissue- and species-specific myosin light chain kinase isozymes

Monoclonal antibodies raised against chicken gizzard smooth muscle myosin light chain kinase were used for immunological and structural studies of this enzyme. Epitope mapping of trypsin-digested chicken gizzard enzyme showed that MM-1, 2, 3, 4, 5, 6, and 7 bind to 65 kDa (trypsin-digested) and 60 kDa (chymotrypsin-digested) fragments which contain the catalytic domain of the kinase. Kinetic analysis demonstrated that MM-7 inhibited kinase activity competitively with respect to ATP and noncompetitively with respect to myosin light chain, thereby indicating that MM-7 binds at or near the ATP binding site of the enzyme. Immunoblot analysis revealed that all these antibodies (MM-1 to 12) reacted with the enzyme (130 kDa) from intestinal and vascular smooth muscles, whereas 5 (MM-1, 3, 4, 6, and 9) or 3 (MM-1, 3, and 4) of 12 antibodies did not cross-react with chicken cardiac muscle or with blood platelet myosin light chain kinase (130 kDa), respectively. None of these antibodies showed cross-reactivity against skeletal muscle myosin light chain kinase. As for mammalian species, MM-11 and 12 reacted with myosin light chain kinase of vascular smooth muscle (140 kDa) and MM-11 cross-reacted with the enzyme (140 kDa) from cardiac muscle of rat and rabbit. These data suggest the existence of at least 4 subspecies of myosin light chain kinase in chicken tissues and the heterogeneity of tissue- and species-specific isozyme forms.     

The binding of nicotinamide-adenine dimucleotide to glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus.

The binding of NAD+ to glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Bacillus stearothermophilus has been studied by measurement of protein fluorescence quenching. Slight negative co-operativity was observed in the binding of the third and fourth coenzyme molecules to the tetrameric enzyme. The first two coenzyme molecules were tightly bound. In this respect the enzyme resembles that from sturgeon muscle rather than that from yeast.     

Purification and general properties of guanosine 3':5'-monophosphate-dependent protein kinase from guinea pig fetal lung.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase was purified from the guinea pig fetal lung, a tissue shown to be the richest in this enzyme in all mammalian sources examined, and its general properties studied. The enzyme was purified 150-fold from crude extract by steps of pH 5.4 isoelectric precipitation, Sephadex G-200 filtration, hydroxylapatite treatment and DEAE-cellulose chromatography. The purified enzyme, free from contamination with adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase, had a specific activity at least equivalent to 600-fold purification of the enzyme from the adult lung. The pulmonary enzyme exhibited an absolute requirement of protein kinase modulator (prepared from various mammalian tissues with an exception of skeletal muscle) for its activity. Inhibitor protein of cyclic AMP-dependent protein kinase purified from rabbit skeletal muscle could not stimulate nor inhibit the cyclic GMP target enzyme, indicating the factors from mammalian sources regulating the two classes of protein kinases may not be the same. The enzyme had Ka values of 1.3 times 10(-8) and 3.3 times 10(-8) M for 8-bromo cyclic GMP and cyclic GMP, respectively, compared to 3.0 times 10(-6) M for cyclic AMP. Cyclic GMP lowered the Km of the enzyme for ATP from 6.3 times 10(-5) M in its absence to 2.1 times 10(-5) M in its presence, accompanied by an approximate doubling of the Vmax. The molecular weight of the enzyme (assayed by its catalytic and cyclic GMP-binding abilities) was estimated to be 123,000, corresponding to a sedimendation coefficient of 7.06 S, by means of sucrose density gradient ultracentrifugation. The cyclic GMP-dependent enzyme required Mg2+ and Co2+ for its activity with optimal concentrations of about 30 and 0.7 mM, respectively. The maximal activity seen in the presence of Mg2+, however, was nearly twice as high as that seen in the presence of Co2+. Histones were generally effective substrates for the enzyme, whereas protamine, casein, phosvitin, phosphorylase kinase, and activator protein of phosphodiesterase were not. The cyclic GMP-dependent enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent enzyme in the presence of Mg2+.     

Study of the fast-reacting cysteines in phosphoglycerate kinase using chemical modification and site-directed mutagenesis

Horse muscle phosphoglycerate kinase, like other mammalian phosphoglycerate kinases, contains seven cysteine residues of which two react rapidly with 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) following second-order kinetics (k = 640 M-1.s-1). Selective cyanylation of the fast-reacting cysteines, followed by chemical cleavage and subsequent sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis of the resulting polypeptides, suggested that these cysteines are at positions 378 and 379. Cysteine residues were introduced into yeast phosphoglycerate kinase by site-directed mutagenesis. Mutant enzymes, each containing only one cysteine residue at position 364, 376, or 377, were constructed from a mutant devoid of cysteine (Cys97----Ala). In the last two mutants, the cysteines were at positions corresponding to Cys378 and Cys379, respectively, in the horse muscle enzyme. The chemical reactivity of the cysteine groups in these latter two yeast mutant enzymes was similar to that of the fast-reacting cysteines in the horse muscle enzyme. Furthermore, they were similarly modified upon substrate binding. All these data demonstrate unambiguously that the fast-reacting cysteines in the horse muscle enzyme are Cys378 and Cys379.     

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.     

Properties of a monoclonal antibody directed to the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase

A synthetic peptide representing the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase (K-R-R-W-K-K-N-F-I-A-V-S-A-A-N-R-F-K-K-I-S-S-S-G-A-L) was used as an antigen to produce a monoclonal antibody. The antibody (designated MAb RSkCBP1, of the IgM class) reacted with similar affinity (KD approximately 20 nM) by competitive enzyme-linked immunoassay (ELISA) with the antigen peptide and intact rabbit skeletal muscle myosin light chain kinase. MAb RSkCBP1 inhibited rabbit skeletal muscle myosin light chain kinase activity competitively with respect to calmodulin (Ki = 20 nM). The antibody also inhibited myosin light chain kinase activity in extracts of skeletal muscle from several mammalian species (rabbit, sheep, and bovine) and an avian species (chicken). The concentration of MAb RSKCBP1 required for 50% inhibition of enzyme activity was similar for the mammalian species (80 nM) but was significantly higher for the avian species (1.2 microM). A competitive ELISA protocol was used to analyze weak cross-reactivity to other calmodulin-binding peptides and proteins. This assay demonstrated no cross-reactivity with the venom peptides melittin or mastoparan; smooth muscle myosin light chain kinases from hog carotid, bovine trachea, or chicken gizzard; bovine brain calmodulin-dependent calcineurin; or rabbit skeletal muscle troponin I. These data support the contention that the synthetic peptide used as the antigen represents the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase and that the calmodulin-binding domains of different calmodulin-regulated proteins may have distinct primary and/or higher order structures.     

Fructose 1,6-bisphosphate aldolase of Drosophila melanogaster: comparative sequence analyses around the substrate-binding lysyl residue

The amino acid sequence of a 103 residue segment encompassing the substrate-binding active site lysyl residue of fructose 1,6-bisphosphate aldolase from Drosophila melanogaster is determined. The sequence is identical to more than 70% with the structure of rabbit muscle aldolase and with the known partial sequences of the sturgeon muscle, trout muscle, and ox liver enzymes. The homology of the insect enzyme with the vertebrate aldolases strongly implies a similar tertiary structure folding.     

Protein phosphatase activity is controlled by an inhibitor phosphoprotein in tick salivary glands

Protein phosphatase activity in tick salivary glands was inhibited by heat-stable protein(s) from tick salivary glands as well as by an inhibitor protein from rabbit skeletal muscle. Inhibitor activity was increased after phosphorylation of inhibitor proteins with the catalytic subunit (C) of cyclic AMP-dependent protein kinase and ATP. C inhibited protein phosphatase activity of the partially purified enzyme, while purified cyclic AMP-dependent protein kinase inhibitor protein prevented inhibition of tick salivary gland protein phosphatase by C suggesting that the inhibitor phosphoprotein coelutes with the partially purified enzyme. A soluble heat-stable protein with a molecular weight of approx. 26 kDa was phosphorylated by C, suggesting that a protein phosphatase inhibitor protein similar to inhibitor-1 in mammalian tissue, is present in tick salivary glands.     

Activities of Lactate and Malate Dehydrogenase of the Sterlet and Russian Sturgeon under Conditions of Massive Spread of Muscle Pathology

As a result of the performed study, the basic data were obtained on the level of lactate dehydrogenate (LDH) and malate dehydrogenase (MDH) activity in two species of sturgeons caught in various regions of the Volga basin under conditions of spreading in the population of a pathology, one of its manifestations being stratification of muscle tissue. In the sterlet, a statistically significant increase of muscle LDH was revealed in fish with a pronounced stratification, while in the Russian sturgeon a tendency for such increase was noticed. In the group of sick fish a marked increase of dispersion of individual values of the enzyme activities in tissues was observed, which appears to indicate a disbalance of biochemical processes in the organism. The data obtained indicate that the stratification is not accompanied by a damage of myofibril sheaths and of liver cells. No obvious interspecies differences in the levels of LDH and MDH were observed between the sterlet and Russian sturgeon.