Skeletal Muscle Pyruvate Kinase Research Articles

Purification and characterization of skeletal muscle pyruvate kinase from the hibernating ground squirrel, Urocitellus richardsonii: potential regulation by posttranslational modification during torpor.

Ground squirrel torpor during winter hibernation is characterized by numerous physiological and biochemical changes, including alterations to fuel metabolism. During torpor, many tissues switch from carbohydrate to lipid catabolism, often by regulating key enzymes within glycolytic and lipolytic pathways. This study investigates the potential regulation of pyruvate kinase (PK), a key member of the glycolytic pathway, within the skeletal muscle of hibernating ground squirrels. PK was purified from the skeletal muscle of control and torpid Richardson's ground squirrels, and PK kinetics, structural stability, and posttranslational modifications were subsequently assessed. Torpid PK displayed a nearly threefold increase in K m PEP as compared to control PK when assayed at 5°C. ProQ Diamond phosphoprotein staining as well as phospho-specific western blots indicated that torpid PK was significantly more phosphorylated than the euthermic control. PK from the torpid condition was also shown to possess nearly twofold acetyl content as compared to control PK. In conclusion, skeletal muscle PK from the Richardson's ground squirrel may be regulated posttranslationally between the euthermic and torpid states, and this may inhibit PK functioning during torpor in accordance with the decrease in glycolytic rate during dormancy.

Molecular cloning and expression of pyruvate kinase from globefish ( Fugu rubripes) skeletal muscle

A cDNA clone encoding pyruvate kinase (PK) was isolated from a skeletal muscle cDNA library of globefish ( Fugu rubripes), which is a kind of lower vertebrate. The full-length cDNA of globefish skeletal muscle pyruvate kinase (FM-PK) is approximately 2 kb and encodes a protein comprising 530 amino acids. The FM-PK gene is spanning approximately 4.8 kb and consists of 11 exons. FM-PK mRNA was detected in muscle and heart using Northern blots. The recombinant FM-PK (rFM-PK) was expressed in a baculovirus-insect cell system and purified using ion-exchange chromatography. The purified rFM-PK was shown to exist a 230 kDa homotetramer composed of 57 kDa subunits. Gel filtration showed 230 000 as the tetramer of the subunit. The apparent K m (or S 0.5) and the Hill coefficient for phosphoenolpyruvate (PEP) and ADP are 0.14 mM, 1.3 and 0.30 mM 0.98 at pH 7.4, respectively, when the enzyme is saturated with the second substrate. The rFM-PK is strongly activated by fructose-1,6-bisphosphate, the apparent K m for PEP changes to 0.059 mM and the Hill coefficient to 1.1. ATP, which is the product of the enzyme reaction, inhibits activity. This is the first report to show the full-length cDNA and amino acid sequence of PK for a species of fish.

Differential coupling of smooth and skeletal muscle pyruvate kinase to creatine kinase.

The interaction of pyruvate kinase from skeletal (SKPK) and smooth (SMPK) muscle with MM-creatine kinase (MMCK) and BB-creatine kinase (BBCK) was assessed using temporal absorbance changes, variations in absorbance at different wavelengths, concentration dependence, association in an electric field, and PK kinetic activity. SKPK exhibits a time course of absorbance increase in the presence of MMCK with a time constant of 29.5 min. This increase occurs at all wavelength from 240 to 1000 nm. At 195 nm, the combination of SKPK and MMCK produces a decrease in absorption with electric fields of both 0 and 204 V/cm. The change in SKPK-MMCK is saturable. SKPK activity is significantly increased by the presence of MMCK in solutions of 0-32% ethanol. These results indicate specific SKPK-MMCK interaction. SMPK and BBCK did not exhibit similar coupling when the BBCK concentration dependence of absorbance or SMPK activity in solutions of 0-32% ethanol was determined. Both MMCK and BBCK increased SKPK activity; neither MMCK nor BBCK increased SMPK activity. The ability to form diazymatic complexes with creatine kinase appears to reside in SKPK. This coupling may account for the increased flux through PK without significant substrate changes seen during skeletal muscle activation. This coupling will not occur in smooth muscle.

Reversible phosphorylation control of skeletal muscle pyruvate kinase and phosphofructokinase during estivation in the spadefoot toad, Scaphiopus couchii.

Both pyruvate kinase (PK) and phosphofructokinase (PFK) occur in two different forms, separable by isoelectric focusing (IEF), in skeletal muscle of the spadefoot toad Scaphiopus couchii. During estivation (aerobic dormancy) the proportions of the two forms changed compared with controls; in both cases the amount of enzyme in Peak I (pI = 5.3-5.4) decreased whereas activity in Peak II (isoelectric point = 6.2-6.4) increased. In vitro incubation of crude muscle extracts with 32P-ATP under conditions that promoted the activity of cAMP-dependent protein kinase led to strong radiolabeling associated with Peak I, but not Peak II, and reverse phase HPLC confirmed that 32P was associated with the subunits of both PK and PFK found in Peak I. Specific radiolabeling of Peak I PK and PFK by protein kinase A was further confirmed using immunoprecipitation. In total, this information allowed identification of the Peaks I and II enzymes as the phosphorylated and dephosphorylated forms, respectively, and the effect of estivation was to increase the proportion of dephosphorylated PK and PFK in muscle. Analysis of the kinetic properties of partially purified PK and PFK revealed significant kinetic differences between the two forms of each enzyme. For PK, the Peak II (low phosphate) enzyme showed a 1.6-fold higher Km for phosphoenolpyruvate and a 2.4-fold higher Ka for fructose-1,6-bisphosphate than did the Peak I (high phosphate) form. These kinetic properties suggest that Peak II PK is the less active form, and coupled with the shift to predominantly the Peak II form during estivation (87% Peak II vs. 13% Peak I), are consistent with a suppression of PK activity in estivating muscle, as part of the overall metabolic rate depression of the estivating state. A similar shift to predominantly the Peak II, low phosphate, form of PFK (75% Peak II, 25% Peak I) in muscle of estivating animals is also consistent with metabolic suppression since phosphorylation of vertebrate skeletal muscle PFK is typically stimulated during exercise to enhance enzyme binding to myofibrils in active muscle. Peak II PFK also showed reduced sensitivity to inhibition by Mg:ATP (I50 50% higher) compared with the Peak I form suggesting that the enzyme in estivating muscle is less tightly regulated by cellular adenylate status than in awake toads. The data indicate that reversible phosphorylation control over the activity states of enzymes of intermediary metabolism is an important mechanism for regulating transitions between dormant and active states in estivating species.

Hydroxylamine cleavage of cat skeletal-muscle pyruvate kinase: Separation of the fragments and N-terminal sequence analysis

Hydroxylamine cleavage of cat skeletal-muscle pyruvate kinase: Separation of the fragments and <i>N</i>-terminal sequence analysis

Kinetic properties of human muscle pyruvate kinase.

The steady-state kinetics of human skeletal muscle pyruvate kinase (MA) and its RNA-complex (MB) has been examined and compared. Kinetic studies revealed significant differences in kinetic properties with respect to free and complex form of pyruvate kinase. The MA form follows a simple Michaelis-Menten kinetics in contrast with the MB form, which displays a negative cooperativity with respect to ADP. Vmax for the complex is 40-60% that for free enzyme. Heterologous RNA is a noncompetitive inhibitor of free enzyme but the kinetics of the complex (MB) is not affected. In presence of 1.0 mM ATP in an assay mixture the kinetic constants of the complex were unchanged except for Vmax, which increased by nearly 60%. Aged preparations of free enzyme (MA) were activated by 100% and more, but the native enzyme was inhibited by 22%. Inorganic phosphate is a potent activator of both forms of pyruvate kinase. In presence of 50 mM K-phosphate the apparent Michaelis constant and interaction coefficient are unchanged, but Vmax for free enzyme increases by 35% and for the complex by 70%, respectively. The specific activity of aged MA form can be restored to the original value after incubation of the enzyme in 50 mM K-phosphate, pH 7.6, or by addition of ATP (1.0 mM) to the assay mixture.

Single subunits of Sepharose-bound pyruvate kinase are inactive.

Bovine skeletal muscle pyruvate kinase was covalently coupled to Sepharose that had previously been activated by low concentrations of cyanogen bromide. Reaction conditions were chosen such that essentially all tetrameric enzyme molecules were covalently bound via a single subunit. Denaturation of the immobilized enzyme with guanidine hydrochloride followed by removal of noncovalently bound subunits amd denaturant resulted in essentially no enzymatic activity remaining bound to the resin. Thus, single immobilized subunits of bovine pyruvate kinase were inactive. Sepharose-bound enzymatic activity could be recovered by adding soluble renaturing enzyme subunits to the immobilized monomers. The former combine noncovalently with the latter, presumably resulting in re-formation of bound tetramers, and an average recovery of 61% of the original matrix-bound activity was observed. While interactions with other enzyme subunits appear to be necessary for catalytic activity of bovine muscle pyruvate kinase, these subunit interactions apparently can be provided by chemically modified subunits. Soluble, renaturing subunits from enzyme that had been inactivated by treatment with trinitrobenzenesulfonic acid were able to interact with matrix-bound single subunits, thereby restoring the enzymatic activity of the latter.

Concentration of skeletal and cardiac muscle pyruvate kinase determined by specific radioimmunoassay

A specific radioimmunoassay has been developed for rabbit skeletal muscle pyruvate kinase (ATP:pyruvate 2- O-phosphotransferase, EC 2.7.1.40) and applied to extracts of skeletal and cardiac muscle from rabbits subjected to differing dietary states and alloxan diabetes. In animals fed Purina Checkers ® rabbit chow the concentration was 11.1 ± 3.3 μM is skeletal and 3.1 ± 0.6 μM (μmol/1000 g wet weight) in heart muscle. Compared to the chow-fed animals the enzyme concentration increased approximately 2-fold in both tissues after a 4 day fast or after fat feeding, without any change in enzyme activity. Using slab gel electrophoresis, we demonstrated that fat feeding and fasting evidently cause the muscle cell to accumulate inactive or less active, tetrameric pyruvate kinase. Alloxan diabetes had no influence on the concentration of the enzyme in chow-fed animals. Pyruvate kinase activity was measurement in the same extracts by a conventional assay. From the data specific activities were calculated: for example, 40 units/nmol skeletal muscle enzyme and 19 units/nmol heart muscle enzyme in chow-fed animals. It is only by the combined application of activity and chemical concentration assays to tissue extracts could such data be obtained. Thus, in addition to measuring enzyme concentration an approach is provided to detect differences in the catalytic state of the enzyme. Our data show that assayable activity of this enzyme does not always reflect enzyme concentration and that regulation of enzyme concentration and enzyme activity can occur independently.

Use of methanethiolation to investigate the catalytic role of sulphydryl groups in rabbit skeletal muscle pyruvate kinase

Incubation of rabbit skeletal muscle pyruvate kinase (ATP:pyruvate 2-O- phosphotransferase , EC 2.7.1.40) with methyl methanethiosulphonate resulted in the time- and inhibitor concentration-dependent loss of enzyme activity. Substrates or products of the catalytic reaction prevented the loss of activity caused by methanethiolation. Their effectiveness as protecting agents was placed in the order ADP > ATP > Mg 2+ > phospho enol pyruvate > pyruvate . The essential catalytic cation, K +, had no effect on the methanethiolation reaction. [Me- 3 H]Methanethiosulphonate modified all the available cysteine thiol groups which correlated to the incorporation of four SC 3H 3 groups per protomer. Four radioactive peptides were obtained on tryptic peptide mapping. When methanethiolation was carried out in the presence of Mg 2+ alone or with Mg 2+ and ATP together, then only three SC 3H 3 groups were incorporated into each subunit. If MgATP protected methanethiolated pyruvate kinase was reacted with iodo[2- 3H]acetic acid then 1.37±0.2 groups per protomer were carboxymethylated. 70% of the radioactivity was located in a single peptide on tryptic peptide mapping. This peptide was isolated and contained the segment carboxymethyl cysteine (Glx, Asx, Ser) Arg. Collectively these data indicate that although all thiol groups are equally accessible to methyl methanethiosulphonate, only a single thiol group participates in the catalytic event. An additional role in the maintenance of structure for this thiol group was also shown in studied of reduction and thermal denaturation of the enzyme.

Some kinetic properties of skeletal muscle pyruvate kinase from air-breathing and water-breathing fish of the Amazon

The kinetic properties of pyruvate kinase from skeletal muscle were studied in two species of air-breathing fish, Hoplerythrinus unitaeniatus and Arapaima gigas, and two species of water-breathing fish, Hoplias malabaricus and Osteoglossum bicirrhosum. It was found that the enzymes from Hoplias and Hoplerythrinus showed hyperbolic saturation kinetics for all substrates, were activated slightly by fructose 1,6-diphosphate, and were inhibited by phosphocreatine and citrate. The enzyme from Hoplias was inhibited by alanine, whereas the enzyme from Hoplerythrinus was not. The enzymes from Arapaima and Osteoglossum showed hyperbolic saturation kinetics for adenosine diphosphate, but the saturation kinetics for phusphoenol-pyruvate were sigmoidal. These enzymes were strongly activated by fructose 1,6-diphosphate and strongly inhibited by alanine, the former completely reversing the inhibition by the latter. Phosphocreatine and citrate were also found to be inhibitors of these enzymes, but the inhibition by phosphocreatine was not reversed by additions of fructose 1,6-diphosphate. The enzymes from the water-breathing fish were more sensitive to inhibition by alanine than were those from the air-breathing fish, but in other respects the enzymes were very similar.

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.

The reversibility of skeletal muscle pyruvate kinase and an assessment of its capacity to support glyconeogenesis.

The kinetics of pyruvate phosphorylation by rabbit skeletal muscle pyruvate kinase (EC 2.7.1.40) has been studied with a coupled assay using P-enolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37). The reaction sequence is (See journal for formula). Although the equilibrium of the pyruvate kinase reaction by itself strongly favors pyruvate production, the over-all equilibrium of this coupled system favors the depletion of pyruvate, thus greatly reducing the problem of back reaction during the assay. In addition, the oxidation of NADH by malate dehydrogenase makes it possible to monitor the system with a spectrophotometer. The Michaelis constant of pyruvate kinase was found to be 0.9 mM for ATP and 7 mM for pyruvate, values that agree reasonably well with earlier studies using direct assays. However, the maximum velocity is about 6 mumol of pyruvate phosphorylated/min/mg of enzyme, which is very much faster than that indicated by earlier studies. These results suggest that the metabolic significance of the reverse reaction of muscle pyruvate kinase may have been underestimated. In particular, the data given here suggest that its rate in vivo is probably comparable to the observed rate of glycogen synthesis from lactate, making possible glyconeogenesis in muscle by pyruvate kinase reversal without the need for an enzymatic bypass of the kind employed by liver and kidney.

Bovine Pyruvate Kinases: III. HYBRIDS OF THE LIVER AND SKELETAL MUSCLE ISOZYMES

Abstract Bovine pyruvate kinases from skeletal muscle and from liver have been hybridized in vitro, producing an equally spaced, five-membered set of electrophoretically distinct species. This set consists of the tetrameric parental forms, designated L4 and M4 for the liver and muscle isozymes, respectively, plus three hybrids designated L3M, L2M2, and LM3 according to their content of type L and type M subunits. The hybrids were separated and isolated by isoelectric focusing. Neither the native enzymes nor the isolated hybrids showed any tendency to exist in more than one electrophoretic form. However, denaturation and renaturation of each isolated hybrid produced a reassortment of its subunits with the subsequent appearance of other members of the electrophoretic set. The subunit designations assigned to the hybrids, and the fact that they are tetramers, is inferred from their equal spacing between the parental enzymes after electrophoresis. In addition, the tetrameric nature of L2M2 was demonstrated directly by showing that it sediments in a sucrose density gradient at a velocity midway between native L4 and M4. Skeletal muscle pyruvate kinase is known to exhibit hyperbolic kinetics (Hill coefficient, nh = 1) and to be unaffected by fructose diphosphate. On the other hand, liver pyruvate kinase exhibits sigmoidal kinetics with varying phosphoenolpyruvate concentrations (nh = 2.4) and is activated by fructose diphosphate. Because the kinetic properties of these two native forms are so very different, we examined the catalytic behavior of the isolated hybrids with the hope of establishing the contribution to total hybrid activity made by the type L and type M subunits. The LM3 hybrid has hyperbolic kinetics with P-enolpyruvate and is unaffected by fructose diphosphate. L2M2, on the other hand, has hyperbolic kinetics with P-enolpyruvate, but has increased activity in the presence of fructose diphosphate. In contrast to these two species, L3M has sigmoidal kinetics with P-enolpyruvate (nh = 1.5) and hyperbolic kinetics only when fructose diphosphate is added. The kinetic properties of the hybrids were compared to mixtures of L4 and M4 having an equivalent ratio of type L to type M subunits. These comparisons showed that the hybrids do not have the same kinetic characteristics as equivalent mixtures of L4 and M4, indicating that the catalytic properties of a subunit are affected by the presence of nonhomologous subunits in the same molecule. It appears that the presence of three type M subunits in the LM3 hybrid, and two in the L2M2 hybrid, induce the L subunits in those species to exhibit hyperbolic kinetics. Conversely, the presence of three type L subunits in the L3M hybrid induces the M subunit in that species to exhibit sigmoidal kinetics. The data obtained in this study suggest that the properties of pyruvate kinase hybrids result from changes in an equilibrium between active and less active forms in agreement with the concerted model of Monod et al. ((1965) J. Mol. Biol. 12, 88–118), although other models may, of course, be equally applicable. It appears that a high proportion of type M subunits favors the active state and produces hyperbolic kinetics, while a high proportion of type L subunits in a hybrid favors the less active state and produces sigmoidal kinetics.

Inhibition of Muscle Pyruvate Kinase by Creatine Phosphate

Abstract At neutral pH, concentrations of creatine phosphate comparable to those found in skeletal muscle and heart are highly inhibitory toward rabbit skeletal muscle pyruvate kinase. Creatine phosphate appears to be competitive with phosphoenolpyruvate with a Ki of 2.0 mm. That it is not a simple competition, however, is indicated by the following evidence. The inhibition is increased in the presence of high concentrations of ADP. ATP, a known inhibitor of pyruvate kinase, acts synergistically with creatine phosphate; that is, in the presence of both ATP and creatine phosphate the inhibition is much greater than the sum of the actions of the inhibitors separately. No synergism is seen between ATP and the inhibitory substrate analogue, 2-phosphoglyceric acid. The characteristics of the creatine phosphate effect are quite distinct from the inhibition noted previously with phenylalanine. Phenylalanine inhibition is maximal above pH 8.0 whereas creatine phosphate is more potent at neutral pH. Alanine, which reverses phenylalanine inhibition, has no effect on creatine phosphate inhibition. Phenylalanine does not inhibit the Mn2+-activated enzyme whereas creatine phosphate inhibition is more potent in the presence of manganous ion. Rabbit erythrocyte pyruvate kinase is only slightly inhibited by creatine phosphate. In the presence of the activator, fructose 1,6-diphosphate, the inhibition is much more pronounced. The results are discussed in relation to the regulation of muscle glycolysis and it is suggested that falling creatine phosphate levels may be one of the primary factors contributing to the increase in glycolytic flux that accompanies muscle contraction.

Allosteric Properties of Skeletal Muscle Pyruvate Kinase

Abstract The phenylalanine inhibition of skeletal muscle pyruvate kinase has been further studied. Even at high levels of the inhibitor the plots of reaction rates as a function of phosphoenolpyruvate concentration give hyperbolic curves; the inhibition by phenylalanine is of the mixed type. The enzyme exhibits a homotropic cooperative effect with respect to this inhibitor. This property is strongly dependent on pH. Serine, cysteine, and alanine reversed this inhibition. The reactivating amino acids greatly increase the I0.5 but do not alter the cooperativity of the inhibitor. These effects were not found with 2-phosphoglyceric acid, an isosteric inhibitor. In fact, the inhibition is independent of pH and it is not reversed by the amino acids. Furthermore the enzyme follows Michaelis-Menten kinetics with respect to this inhibitor. The reassociation of the enzyme from its subunits markedly decreases the homotropic cooperative effect of phenylalanine. It is concluded that this amino acid is an allosteric inhibitor.

Pyruvate kinase isozymes in adult tissue and eggs of Rana pipiens. II. Physical and kinetic studies of purified skeletal and heart muscle pyruvate kinases

1. 1. Pyruvate kinase (ATP:phosphotransferase, EC 2.7.1.40) from skeletal muscle of Rana pipiens has been purified to homogeneity as judged by sedimentation velocity studies, immunoelectrophoresis, double diffusion experiments and acrylamide-gel electrophoresis. The enzyme has a molecular weight of approx. 220 000 as estimated by gel-filtration studies and a subunit molecular weight of approx. 55 000 as measured by acrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. 2. 2. The properties of the frog skeletal and partially purified cardiac pyruvate kinase have been compared. Both enzymes have the same molecular weight as estimated by gel-filtration experiments and the cardiac enzyme cross-reacts with the antibody to the skeletal muscle pyruvate kinase. However, the cardiac enzyme exhibits different chromatographic and kinetic properties. Unlike the muscle enzyme, heart pyruvate kinase is retained on DEAE-Sephadex, displays temperature sensitivity, is inhibited by l-alanine and dl-phenylalanine and is activated by fructose 1,6-diphosphate. With respect to the extent of inhibition or activation by the latter compounds, present data suggest the possibility that there are two forms of the cardiac enzyme, one of which is highly sensitive, and a second, which is markedly less sensitive, to the effects of these modifiers.

Temperature effects on enzymes from homeothermic and heterothermic tissues of the harbor seal ( Phoca vitulina)

1. 1. Lactate dehydrogenase and pyruvate kinase of arterial smooth muscle of the harbor seal ( Phoca vitulina) exhibit thermal-kinetic properties similar to those observed for enzymes of poikilotherms. Enzyme-substrate affinity is inversely proportional to temperature over the range of temperatures the heterothermic flipper tissues experience. Consequently, the temperature coefficients of the enzymatic reactions are greatly reduced at physiological substrate concentrations. 2. 2. Kinetic comparisons of enzymes from homeothermic and heterothermic arteries revealed no differences in isoenzyme composition. 3. 3. Major differences in thermal-kinetic effects were found between seal arterial pyruvate kinase and rat skeletal muscle pyruvate kinase.