The genetic system of the L-type pyruvate kinase forms in man. Subunit structure, interrelation and kinetic characteristics of the pyruvate kinase enzymes from erythrocytes and liver

Pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from human liver and red cells has been purified to homogeneity; its subunit structure and some of its kinetic characteristics have been studied. The influence of a partial proteolysis by trypsin on the subunit structure, the isozymic pattern and the kinetic characteristics of red cell and liver enzyme have been investigated. From the results of this study we may conclude that: 1. Liver (L-type) pyruvate kinase is composed of 4 identical L subunits while the major form of erythrocyte enzyme (PK-R2) is a heterotetramer designated as L2L2', the molecular weight of L' being slightly higher than that of L subunits (63 000 and 58 000 respectively). Pyruvate kinase PK-R1, predominant in the erythroblasts and the young red cells, is composed of four identical L' subunits. 2. A mild tryptic attack is able to transform PK-R1 into PK-R2, then PK-R2 into pyruvate kinase L (PK-L). The same proteolytic treatment transforms the L' subunits into L ones. 3. Consequently L-type pyruvate kinase seems to be initially synthesized in the erythroid precursors as an L4' enzyme secondarily partially proteolysed into L2L2'. In liver a very active proteolytic system would be responsible for the total transformation into L4 pyruvate kinase. 4. L4' enzyme exhibits Michaelis-Menten kinetic behaviour with an apparent Michaelis constant of 3.8 mM whereas L4 enzyme shows both positive and negative homotropic interactions towards phosphoenolpyruvate and has [S] 0.5 of 1.2 mM. The characteristics of L2L2' are roughly intermediate between those of L4' and of L4. Fructose 1,6-biphosphate decreases [S]0.5 for these three pyruvate kinase forms without suppressing the differences in the apparent affinity for phosphoenolpyruvate of these enzymes. 5. L4 pyruvate kinase is more inhibited by Mg-ATP than L4', with L2L2' in the intermediate range. 6. Tryptic treatment of each enzyme form studied transforms its kinetic behaviour into that observed for L4.     

Molecular organization of human L' and L pyruvate kinases

Extremities, peptide maps and phosphorylatable site localization of human erythrocyte L' and liver L pyruvate kinases (EC 2.7.1.40) were investigated. L' and L subunits seemed to have similar, blocked NH2 termini and differ in their sensitivity to carboxypeptidase A, that is to say in their C-terminal ends. After digestion by Staphylococcus aureus V8 protease, the phosphorylated sites of both L' and L subunits were located on those peptides which were different in L' and L, that is to say on the C-terminal sides. A mild proteolytic attack of the native tetrameric enzymes by trypsin partially degraded the phosphorylatable peptides without removing the phosphoserine residue; in the same conditions, chymotrypsin split off this phosphorylated residue and subtilisin totally degraded the phosphorylated peptides. From these results it appears, therefore, that age-dependent proteolytic degradation of L' subunits in old red cells involves the C-terminal side of the molecules, ultimately resulting in cleavage of the phosphorylated site. Since erythrocyte L' and liver L subunits are encoded by different species of messenger RNAs, our results indicate, in addition, that these messenger RNA species should differ by their 3' coding sequences.     

Functional changes associated with the sequential transformation of L'4 into L4 pyruvate kinase.

The functional changes, associated with the sequential transformation of L'4 into L4 pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) were studied. L'4 enzyme from human erythrocytes shows strong hysteretic behaviour: the initial rate of the enzyme preincubated with an unsaturating concentration of phosphoenolpyruvate is much higher than of the enzyme preincubated with ADP, at the same phosphoenolpyruvate concentration, although the "final activity" (the activity of the linear part of the reaction progress curve) was the same in both cases. This phenomenon was observed both in the presence and absence of fructose 1,6-diphosphate. High concentrations of both Mg2+free and MgATP2- diminish the difference in initial rate, between the ADP and phosphoenolpyruvate preincubated enzymes: Mg2+free by stabilizing the phosphoenolpyruvate-induced form; ATPMg2- by stabilizing the ADP-induced form. The magnitude of the difference in initial rates of the ADP-or phosphoenolpyruvate-preincubated enzyme is a function of both substrates. L4 pyruvate kinase (either from human liver or trypsin treated L'4 enzyme) does not, or to a very slight extent, show such behaviour. L'2L2 pyruvate kinase shows behaviour intermediate between L'4 and L4 enzymes. A model is proposed to describe the kinetic behaviour of L'4 and L4 enzymes.     

Characterization of human red blood cell tyrosine kinase

A new tyrosine kinase in human red blood cells has been characterized and partially purified. The major substrate was a protein of molecular weight 93 K which could be phosphorylated both in whole red blood cells incubated with inorganic [32P] orthophosphate and in ghost preparations incubated with [gamma 32P] ATP. This tyrosine kinase displayed an alkaline isoelectric pH (around 8.5), a molecular weight of 32-33 K and does not seem to be autophosphorylable. Some kinetics of the enzyme are reported. This red blood cell tyrosine kinase is unrelated to EGF and insulin or insulin-like receptor subunits. This enzyme may represent a novel class of tyrosine kinases.     

Proteolytic processing of human L-type pyruvate kinase increases its ability to be phosphorylated

Red cell soluble cyclic 3′-5′ AMP-dependent protein kinase phosphorylates more efficient L₄ liver pyruvate kinase or the L_b partially proteolysed form of erythrocyte enzyme than the L′₄ precursor. Affinity of protein kinase for liver L₄ and L′₄ as substrates is similar (10 μM at 0.1 M ATP and 1 μM cyclic AMP), but maximal velocity of the phosphorylation reaction is twice higher with L₄ than L′₄. Thus it appears that proteolytic processing of pyruvate kinase increases its ability to be phosphorylated, in the same way that it increases its allosteric properties.     

Endogenous phosphorylation of soluble enzymes in human red cells. Cyclic 3',5'-AMP-dependent phosphorylation of phosphofructokinase without detectable regulatory effect

ATP-depleted human red cells have been incubated in a glucose-containing medium with [32P]orthophosphate in the presence and in the absence of cyclic 3',5'-AMP and dibutyril cyclic 3',5'-AMP. Spectrin, pyruvate kinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and hemoglobin A1 have been purified and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein-bound radioactivity has been measured from the sodium dodecyl sulfate polyacrylamide gels and the trichloroacetic acid-precipitated proteins. In the cytosol, the most intense phosphorylation was found for pyruvate kinase whose, in the presence of cyclic AMP, specific radioactivity was comparable to that of the membrane protein and spectrin. In the absence of cyclic nucleotides it was five times less phosphorylated. Phosphofructokinase was only phosphorylated when the red cells were incubated with cyclic nucleotides; the extent of phosphorylation was four times less than for pyruvate kinase. Hemoglobin, glucose-6-phosphate dehydrogenase and a contaminant protein copurified with phosphofructokinase were not phosphorylated: the 'background' of the radioactivity found for these proteins was 100 times less than for pyruvate kinase and spectrin, and 20 times less than for phosphofructokinase (+cyclic AMP).     

Control of pyruvate kinase activity by glucagon in isolated hepatocytes

Incubation of hepatocytes with 10 nM glucagon led to an increase in the K_0.5 for phosphoenolpyruvate for pyruvate kinase measured in homogenates of the cells. Incubation of partially purified rat liver pyruvate kinase with protein kinase and ATP led to a similar result. In addition, both the phosphorylated enzyme and homogenates prepared from cells incubated with glucagon exhibited an apparently decreased sensitivity to stimulation by fructose diphosphate when activity was measured in the presence of physiological concentrations of ATP and alanine. These similarities suggest that the effect of glucagon to inhibit hepatocyte pyruvate kinase may be mediated at least in part by a phosphorylation-dephosphorylation mechanism.     

The effects of triethyltin bromide on red cell and brain cyclic AMP-dependent protein kinases

Triethyltin bromide activates the cyclic AMP-dependent protein kinases of human red cell membranes and of bovine brain. Additions of 25-500 microM triethyltin to red cell ghosts resulted in enhanced phosphorylation of ghost proteins. When added to partially purified cyclic AMP-dependent protein kinases from red cell ghosts or bovine brain, stimulation of the phosphorylation of calf thymus histone was observed. The enhancement of kinase activity was due to release of catalytic subunits from the intact protein kinase. Brief exposure of the partially purified enzymes to triethyltin, followed by DE52 chromatography, resulted in elution profiles for regulatory and catalytic subunits that were similar to the profile resulting after cyclic AMP activation. Triethyltin interacts with both regulatory and catalytic subunits. When it was added to the partially purified cyclic AMP-dependent protein kinases from human red cell ghosts or bovine brain, noncompetitive inhibition of cyclic AMP binding to the regulatory subunit of the enzyme was observed. It interacted with the catalytic subunit to produce slow inhibition of catalytic activity. The inhibition was non-competitive with respect to both histone and ATP. When intact red cells were subjected to brief exposure with triethyltin, enhanced phosphorylation of certain membrane proteins occurred, suggesting that the activation of the cyclic AMP protein kinases by triethyltin may be physiologically significant.     

Glucagon-induced phosphorylation of pyruvate kinase (type L) in rat liver slices.

The effect of glucagon on the phosphorylation of pyruvate kinase in ³²P-labelled slices from rat liver was investigated. Pyruvate kinase was isolated by immunoadsorbent chromatography. The enzyme was partially phosphorylated in the absence of added hormone (0.2 mol of phosphate/mol of enzyme subunit). Upon incubation with 10^?7 M glucagon, the incorporation of [³²P]phosphate was 0.6–0.7 mol/mol of enzyme subunit. Concomitantly, the concentration of intracellular cyclic 3′,5′-AMP increased from 0.3 to 3.2 μM. The phosphorylation inhibited the enzyme activity at low concentrations of phosphoenolpyruvate (60% at 0.5 mM). Almost maximal phosphorylation of the enzyme was reached within 2 min after the addition of glucagon. The concentration of hormone giving half maximal effect on the pyruvate kinase phosphorylation was about 7×10^?9M. The inactivation of the enzyme paralleled the increase in phosphorylation. It is concluded that pyruvate kinase is phosphorylated in the intact liver cell.     

Cloning of uvrA, lexC and ssb genes of Escherichia coli.

The L' subunits, phosphorylatable precursors of L-type pyruvate kinase, can be proteolyzed in vivo and in vitro, and transformed into several molecular species.Trypsin induces the appearance of a phosphorylatable form similar to liver L-type enzyme.Subtilisin splits the phosphorylatable site and induces the appearance of a form similar to the minor form occuring with red cell aging.Sensitivity to proteolysis of some pyruvate kinase variants is modified.     

Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase

Rat liver L-type pyruvate kinase was phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver. The calmodulin (CaM)-dependent kinase catalyzed incorporation of up to 1.7 mol of 32P/mol of pyruvate kinase subunit; maximum phosphorylation was associated with a 3.0-fold increase in the K0.5 for P-enolpyruvate. This compares to incorporation of 0.7 to 1.0 mol of 32P/mol catalyzed by the cAMP-dependent protein kinase with a 2-fold increase in K0.5 for P-enolpyruvate. When [32P]pyruvate kinase, phosphorylated by the CaM-dependent protein kinase, was subsequently incubated with 5 mM ADP and cAMP-dependent protein kinase (kinase reversal conditions), 50-60% of the 32PO4 was removed from pyruvate kinase, but the K0.5 for P-enolpyruvate decreased only 20-30%. Identification of 32P-amino acids after partial acid hydrolysis showed that the CaM-dependent protein kinase phosphorylated both threonyl and seryl residues (ratio of 1:2, respectively) whereas the cAMP-dependent protein kinase phosphorylated only seryl groups. The two phosphorylation sites were present in the same 3-4-kDa CNBr fragment located near the amino terminus of the enzyme subunit. These results indicate that the CaM-dependent protein kinase catalyzed phosphorylation of L-type pyruvate kinase at two discrete sites. One site is apparently the same serine which is phosphorylated by the cAMP-dependent protein kinase. The second site is a unique threonine residue whose phosphorylation also inactivates pyruvate kinase by elevating the K0.5 for P-enolpyruvate. These results may account for the Ca2+-dependent phosphorylation of pyruvate kinase observed in isolated hepatocytes.     

Epinephrine-stimulated phosphorylation of pyruvate kinase in hepatocytes

Incubation of rat liver parenchymal cells with 10^?5m epinephrine or norepinephrine resulted in a rapid incorporation of ³²P into pyruvate kinase. Inclusion of α-adrenergic blocking agents (phenoxybenzamine or phentolamine) in the hepatocyte incubation medium prior to addition of epinephrine suppressed the subsequent phosphorylation of pyruvate kinase. On the other hand, inclusion of the β-adrenergic antagonist, propranolol, in the hepatocyte incubation medium prior to addition of epinephrine did not suppress the epinephrine-elicited phosphorylation of pyruvate kinase. Exogenous addition of either cyclic AMP or cyclic GMP to the hepatocyte incubation medium also resulted in increased phosphorylation of pyruvate kinase. To investigate whether the same amino acid residue(s) of liver pyruvate kinase was being phosphorylated in each instance, ³²P-labeled pyruvate kinase was isolated from hepatocytes after incubation in the presence or absence of either glucagon or epinephrine. In addition, purified liver pyruvate kinase was phosphorylated in vitro with a rat liver cyclic AMP-dependent protein kinase. Each ³²P-labeled pyruvate kinase was then subjected to tryptic digestion, two-dimensional thin-layer peptide mapping, and autoradiography. Each ³²P-labeled pyruvate kinase sample yielded 44 to 48 tryptic peptides upon staining with ninhydrin and 4 peptides that contain ³²P as detected by autoradiography. Furthermore, the same 4 peptides of pyruvate kinase were radiolabeled in each instance. Thus phosphorylation of pyruvate kinase in vitro with [γ-³²P]ATP or upon addition of either glucagon or epinephrine to hepatocytes incubated with ³²P_i resulted in phosphorylation of the same amino acid residues.     

Calcium-calmodulin dependent phosphorylation of erythrocyte pyruvate kinase

In the red cell incubated with ortho-[32P] phosphate, CaCl₂ and calcium ionophore A 23187, phosphorylation of erythrocyte pyruvate kinase was demonstrated using the double antibody technique and autoradiography. Phosphorylation was inhibited by calmodulin inhibitors, trifluoperazine or ZnCl₂. In the presence of purified erythrocyte calmodulin, CaCl₂ and [γ-³²P] ATP, the partially purified erythrocyte pyruvate kinase containing cytozol protein kinases was phosphorylated. This was also inhibited by trifluoperazine or ZnCl₂. From these results, it was concluded that erythrocyte pyruvate kinase is phosphorylated by a calcium-calmodulin dependent process.     

Partial proteolysis of human L-type phosphofructokinase

L (liver) type phosphofructokinase subunits purified from human leukocytes are slightly lighter than L subunits from liver and red blood cells. A mild treatment of red blood cell L4 enzyme with subtilisin converts its subunits into forms of similar molecular weight to leukocyte enzyme. From a kinetical point of view, subtilisin-treated L4 phosphofructokinase and leukocyte enzymes are characterized by a decrease of the allosteric properties as compared to non-treated red cell L4 phosphofructokinase.     

Pyruvate kinase type M2 is phosphorylated in the intact chicken liver cell

Isolated hepatocytes from 24 h starved chicks were depleted of phosphate and incubated in the presence of 32P-orthophosphate. Pyruvate kinase type M2 from crude cell extracts was partially purified by chromatography on DEAE-Sephacel and hydroxylapatite. SDS slab gel electrophoresis of the fractions containing the enzyme and immunoprecipitation with antisera showed the phosphorylation of pyruvate kinase type M2. Phosphoamino acid analysis identified serine as phosphate acceptor.     

Antagonizing effects of phorbol 12-myristate 13-acetate on hormonally stimulated gluconeogenesis in isolated rat hepatocytes involve activity changes of pyruvate kinase

The tumor-promoting phorbol ester phorbol 12-myristate 13-acetate partially neutralized the stimulatory effects of epinephrine (alpha 1-adrenergic actions), glucagon, and dibutyryl-cAMP on gluconeogenesis in isolated hepatocytes of fasted rats, when lactate or dihydroxyacetone was used as the substrate. By constructing metabolic crossover plots and by comparing rates of lactate production from dihydroxyacetone with K0.5 values of extracted pyruvate kinase for phosphoenolpyruvate, we obtained evidence that phorbol ester actions on hormonally stimulated gluconeogenesis were accompanied by proportionate increases in activity of pyruvate kinase. Although purified pyruvate kinase from rat liver was a substrate for protein kinase C in vitro, phosphorylation was not accompanied by modulation of kinetic parameters. Furthermore, incubation of pyruvate kinase extracted from hormone-treated hepatocytes with protein kinase C revealed no activation of the prephosphorylated enzyme. This and the absence of effects of the phorbol ester on basal rates of gluconeogenesis and lactate production suggest that effects of protein kinase C on pyruvate kinase activity in hepatocytes may result from impairment of steps at the level of hormone-induced signal transduction.     

Hepatic pyruvate kinase. Regulation by glucagon, cyclic adenosine 3'-5'-monophosphate, and insulin in the perfused rat liver.

A reversible interconversion of two kinetically distinct forms of hepatic pyruvate kinase regulated by glucagon and insulin is demonstrated in the perfused rat liver. The regulation does not involve the total enzyme content of the liver, but rather results in a modulation of the substrate dependence. The forms of pyruvate kinase in liver homogenates are distinguished by measurements of the ratio of the enzyme activity at a subsaturating concentration of P-enolpyruvate (1.3 mM) to the activity at a saturating concentration of this substrate (6.6 mM). A low ratio form of pyruvate kinase (ratio between 0.1 and 0.2) is obtained from livers perfused with 10(-7) M glucagon or 0.1 mM adenosine 3':5'-monophosphate (cyclic AMP). A high ratio form of the enzyme is obtained from livers perfused with no hormone (ratio = 0.35 to 0.45). The regulation of pyruvate kinase by glucagon and cyclic AMP occurs within 2 min following the hormone addition to the liver. Insulin (22 milliunits/ml) counteracts the inhibition of pyruvate kinase caused by 5 X 10(-11) M glucagon, but has only a slight influence on the enzyme properties in the absence of the hyperglycemic hormone. The low ratio form of pyruvate kinase obtained from livers perfused with glucagon or cyclic AMP is unstable in liver extracts and will revert to a high ratio form within 10 min at 37 degrees or within a few hours at 0 degrees. Pyruvate kinase is quantitatively precipitated from liver supernatants with 2.5 M ammonium sulfate. This precipitation stabilizes the enzyme and preserves the kinetically distinguishable forms. The kinetic properties of the two forms of rat hepatic pyruvate kinase are examined using ammonium sulfate precipitates from the perfused rat liver. At pH 7.5 the high ratio form of the enzyme has [S]0.5 = 1.6 +/- 0.2 mM P-enolpyruvate (n = 8). The low ratio form of enzyme from livers perfused with glucagon or cyclic AMP has [S]0.5 = 2.5 +/- 0.4 mM P-enolpyruvate (n = 8). The modification of pyruvate kinase induced by glucagon does not alter the dependence of the enzyme activity on ADP (Km is approximately 0.5 mM ADP for both forms of the enzyme). Both forms are allosterically modulated by fructose 1,6-bisphosphate, L-alanine, and ATP. The changes in the kinetic properties of hepatic pyruvate kinase which follow treating the perfused rat liver with glucagon or cyclic AMP are consistent with the changes observed in the enzyme properties upon phosphorylation in vitro by a clyclic AMP-stimulated protein kinase (Ljungstr?m, O., Hjelmquist, G. and Engstr?m, L. (1974) Biochim. Biophys. Acta 358, 289--298). However, other factors also influence the enzyme activity in a similar manner and it remains to be demonstrated that the regulation of hepatic pyruvate kinase by glucagon and cyclic AMP in vivo involes a phosphorylation.     

Pyruvate kinase isozymes in man

Summary By focusing in sucrose, gradient L-type pyruvate kinase from human liver could be separated into 2 major forms (pI 6.28±0.03 and 5.85±0.09) and a minor more acid form (pI5). These different forms could also be detected by focusing in acrylamide-ampholine slab gel. The major forms were interconvertible, the equilibrium being shifted toward the acid form by fructose 1,6-diphosphate and SH reagents, and toward the alkaline form by proteinic factors extracted by ammonium sulphate fractionation from liver extracts and from hemolysates. These factors seemed to be responsible for the stabilization of the liver crude extract enzyme in its alkaline conformation.By acrylamide slab gel electrofocusing, erythrocyte pyruvate kinase from whole hemolysates exhibited a complex pattern composed of at least 3 interconvertible forms. The in vitro aging of the red blood cells and the storage of the hemolysates resulted in a progressive disappearance of the acid forms and in a strengthening of the alkaline form. Partially purified erythrocyte enzyme focused in 2 major bands, interconvertible under the influence of the same factors as those described for L-type pyruvate kinase. Although closely related, the focusing patterns of L-type and erythrocyte-type were never exactly identical.Double immunodiffusion against antihuman L-type serum showed a complete identity reaction between erythrocyte-and L-type pyruvate kinases. Moreover, antihuman M₂-type serum was unable to neutralize erythrocyte pyruvate kinase as well as to change its electrophoretic mobility.Consequently, we conclude that both human erythrocyte-and liver L-type pyruvate kinases existed under several conformers interconvertible under the influence of the same ligands or proteinic factors; erythrocyte-type enzyme seems to include L-type subunit and not M₁- or M₂-type subunits. The erythrocyte- and L-type enzymes, however, are not identical and the nature of the differences between them is discussed.Chargé de recherche INSERM.     

Post-translational modification of human erythrocyte pyruvate kinase

Upon storage, partially purified human erythrocyte pyruvate kinase (ATP: pyruvate-phosphotransferase, E.C. 2.7.1.40) from normal individuals was found to undergo a spontaneous oxidation to a form which displayed markedly reduced activity. This modified form of the enzyme exhibited kinetic patterns similar to those frequently reported for the enzyme in cases of nonspherocytic hemolytic anemia. The data are discussed in relation to the recently proposed theory that post-translational modification of pyruvate kinase is responsible for the abnormal kinetic patterns frequently encountered for this enzyme in the disease state. [Van Berkel, T. J. C., Koster, J. F., Kruyt, J. K. and Staal, G. E. J. 1973 $\text{Biochim. Biophys. Acta 321}$, 496–502].