Stimulation of pyruvate kinase phosphatase activity by insulin in isolated rat hepatocytes

Addition of insulin (10(-8)M) to hepatocytes, incubated either in the absence or in the presence of a suboptimal concentration of glucagon, caused the reactivation of pyruvate kinase and simultaneously provoked a transient stimulation of pyruvate kinase phosphatase activity (40-70% over control values). The stimulatory effect of insulin on pyruvate kinase phosphatase activity was dose-dependent (ED50 = 1 to 2 X 10(-11)M) and persisted after Sephadex G-25 filtration or ammonium sulfate precipitation of hepatocytes extracts. Our results demonstrate that insulin exerts a short-term regulation on hepatic pyruvate kinase phosphatase activity.     

Stimulation of pyruvate dehydrogenase phosphatase activity by polyamines

Pyruvate dehydrogenase phosphatase requires Mg2+ or Mn2+, and its activity in the presence of Mg2+ is markedly stimulated by Ca2+. At saturating Mg2+ and Ca2+ concentrations, the polyamines spermine, spermidine and putrescine stimulated the activity of pyruvate dehydrogenase phosphatase 1.5- to 3-fold. Spermine was the most active of the polyamines. At a physiological concentration of Mg2+ (1 mM) and saturating Ca2+ concentration, the stimulation by 0.5 mM spermine was 4- to 5-fold, and at 0.3 mM Mg2+, the stimulation was 20- to 30-fold. In the absence of Mg2+ or Ca2+, spermine had no effect. These results suggest that a polybasic factor may be involved in the regulation of pyruvate dehydrogenase phosphatase activity.     

Modulation of SHP‐1 phosphatase activity by monovalent and bivalent SH2 phosphopeptide ligands

A sequence derived from the epithelial receptor tyrosine kinase Ros (pY2267) represents a high‐affinity binding partner for protein tyrosine phosphatase SHP‐1 and was recently used as lead structure to analyze the recognition requirements for the enzyme's N‐SH2 domain. Here, we focused on a set of peptides comprising C‐terminally extended linear and conformationally constrained side chain‐bridged cyclic N‐SH2 ligands based on the consensus sequence LxpYhxh(h/b)(h/b) (x = any amino acid, h = hydrophobic, and b = basic residue). Furthermore, the bivalent peptides described were designed to modulate the activity of SHP‐1 through binding to both, the N‐SH2 domain as well as an independent binding site on the surface of the catalytic domain (PTP domain). Consistent with previous experimental findings, surface plasmon resonance experiments revealed dissociation constants of most compounds in the low micromolar range. One peptide, EGLNpYc[KVD]MFPAPEEE? NH₂, displayed favorable binding affinity, but reduced ability to stimulate SHP‐1. Docking experiments revealed that the binding of this ligand occurs in binding mode I, recently described to lead to an inhibited activation of SHP‐1. In summary, results presented in this study suggest that inhibitory N‐SH2 ligands of SHP‐1 may be obtained by designing bivalent compounds that associate with the N‐SH2 domain and simultaneously occupy a specific binding site on the PTP domain. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 102–112, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Association of phosphoenolpyruvate phosphatase activity with the cytosolic pyruvate kinase of germinating mung beans

The procedure of Malhotra and Kayastha ([1990] Plant Physiology 93: 194-200) for the purification to homogeneity of a phosphoenolpyruvate-specific alkaline phosphatase (PEP phosphatase) from germinating mung beans (Vigna radiata) was followed. Although a higher specific activity of 1.4 micromoles pyruvate produced per minute per milligram protein was obtained, the final preparation was less than 10% pure as judged by polyacrylamide gel electrophoresis. Attempts to further purify the enzyme resulted in loss of activity. The partially purified enzyme contained significant pyruvate kinase activity (0.13 micromole pyruvate produced per minute per milligram protein) when assayed at pH 7.2, but not at pH 8.5. The PEP phosphatase activity of the final preparation exhibited hysteresis; a lag time of 5 to 6 minutes was required before a steady-state reaction rate was attained. A western blot of the final preparation revealed an immunoreactive 57 kilodalton polypeptide when probed with monospecific rabbit polyclonal antibodies prepared against germinating castor bean cytosolic pyruvate kinase. No antigenic cross-reaction of the final preparation was observed with antibodies against castor bean leucoplast pyruvate kinase, or black mustard PEP-specific acid phosphatase. Nondenaturing polyacrylamide gel electrophoresis of the final preparation resulted in a single PEP phosphatase activity band; when this band was excised and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting, a 57 kilodalton silver-staining polypeptide was obtained that strongly cross-reacted with the anti-(cytosolic pyruvate kinase) immunoglobulin G. It is suggested that mung bean PEP-specific alkaline phosphatase activity is due to cytosolic pyruvate kinase, in which pyruvate and ortho-phosphate are formed in the absence of ADP.     

Synthetic peptide substrates for mammalian pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase

The specificities of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase were probed using synthetic peptides corresponding to the sequence around phosphorylation sites 1 and 2 on pyruvate dehydrogenase [Tyr-His-Gly-His-Ser(P1)-Met-Ser-Asp-Pro-Gly-Val-Ser(P2)-Tyr-Arg]. The dephosphotetradecapeptide containing aspartic acid at position 8 was a better substrate for the kinase than was the tetradecapeptide containing asparagine at position 8. The apparent Km and V values for the two peptides were 0.43 and 6.1 mM and 2.7 and 2.4 nmol of 32P incorporated/min/mg, respectively. Methylation of the aspartic acid residue also increased the apparent Km of the tetradecapeptide about 14-fold. These results indicate that an acidic residue on the carboxyl-terminal side of phosphorylation site 1 is an important specificity determinant for the kinase. Phosphate was incorporated only into site 1 of the synthetic peptide by the kinase. The phosphatase exhibited an apparent Km of 0.28 mM and a V of 2.3 mumol of 32P released/min/mg for the phosphorylated tetradecapeptide containing aspartic acid. Methylation of the aspartic acid residue had no significant effect on dephosphorylation. The octapeptide and phosphooctapeptide produced by cleavage of the aspartyl-prolyl bond by formic acid were poorer substrates for the kinase and phosphatase than were the tetradecapeptide and phosphotetradecapeptide, respectively. Modification of the amino terminal by acetylation or lysine addition had only a slight effect on the kinase and phosphatase activities.     

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 dual activity of pyruvate kinase type M2 from chromatin extracts of neoplastic cells

Pyruvate kinase type M(2) from Morris hepatoma 7777 tumour cell nuclei and cytosol, in contrast to types L and M(2) from nuclei and cytosol of normal rat liver, shows the histone H(1) kinase activity. Moreover, in the presence of L-cysteine and without ADP it converts 2-phosphoenolpyruvate (PEP) to pyruvate while in the presence of L-arginine or L-histidine does not. L-Cysteine markedly stimulates the activity of histone H(1) kinase transferring a phosphate group from PEP to, as results suggested, the epsilon -amino group of L-lysine of histone H(1). This, L-cysteine which is known to inhibit the activity of pyruvate kinase type M(2) from neoplastic cells transfering a phosphate from PEP to ADP, can act as a control factor champing the direction of enzymatic reaction in cancer cells.     

Rat liver pyruvate kinase: influence of ligands on activity and fructose 1,6-bisphosphate binding

The ability for various ligands to modulate the binding of fructose 1,6-bisphosphate (Fru-1,6-P2) with purified rat liver pyruvate kinase was examined. Binding of Fru-1,6-P2 with pyruvate kinase exhibits positive cooperativity, with maximum binding of 4 mol Fru-1,6-P2 per enzyme tetramer. The Hill coefficient (nH), and the concentration of Fru-1,6-P2 giving half-maximal binding [FBP]1/2, are influenced by several factors. In 150 mM Tris-HCl, 70 mM KCl, 11 mM MgSO4 at pH 7.4, [FBP]1/2 is 2.6 microM and nH is 2.7. Phosphoenolpyruvate and pyruvate enhance the binding of Fru-1,6-P2 by decreasing [FBP]1/2. ADP and ATP alone had little influence on Fru-1,6-P2 binding. However, the nucleotides antagonize the response elicited by pyruvate or phosphoenolpyruvate, suggesting that the competent enzyme substrate complex does not favor Fru-1,6-P2 binding. Phosphorylation of pyruvate kinase or the inclusion of alanine in the medium, two actions which inhibit the enzyme activity, result in diminished binding of low concentrations of Fru-1,6-P2 with the enzyme. These effectors do not alter the maximum binding capacity of the enzyme but rather they raise the concentrations of Fru-1,6-P2 needed for maximum binding. Phosphorylation also decreased the nH for Fru-1,6-P2 binding from 2.7 to 1.7. Pyruvate kinase activity is dependent on a divalent metal ion. Substituting Mn2+ for Mg2+ results in a 60% decrease in the maximum catalytic activity for the enzyme and decreases the concentration of phosphoenolpyruvate needed for half-maximal activity from 1 to 0.1 mM. As a consequence, Mn2+ stimulates activity at subsaturating concentrations of phosphoenolpyruvate, but inhibits at saturating concentrations of the substrate or in the presence of Fru-1,6-P2. Both Mg2+ and Mn2+ diminish binding of low concentrations of Fru-1,6-P2; however, the concentrations of the metal ions needed to influence Fru-1,6-P2 binding exceed those needed to support catalytic activity.     

Modulation of reconstituted cholesterol 7 alpha-hydroxylase by phosphatase and protein kinase

Cholesterol 7 alpha-hydroxylase activity was completely inhibited by incubation with alkaline phosphatase in a reconstituted enzyme system containing a cytochrome P-450, NADPH-cytochrome P-450 reductase and phospholipid. On the other hand, cAMP-dependent protein kinase stimulated cholesterol 7 alpha-hydroxylase activity by 2.5-fold. The modulation of cholesterol 7 alpha-hydroxylase activity was dependent on the amount of phosphatase or kinase added. The phosphatase inhibited enzyme activity was partially reversed by the treatment with protein kinase. These experiments indicate that the reconstituted cholesterol 7 alpha-hydroxylase activity is reversibly regulated by phosphorylation/dephosphorylation mechanism.     

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

Since the introduction of the concepts of allostery about four decades ago, much advancement has been made in elucidating the structure-function correlation in allostery. However, there are still a number of issues that remain unresolved. In this review we used mammalian pyruvate kinase (PK) as a model system to understand the role of protein dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM)(α/β)₈ barrel structural motif. PK is an ideal system to address basic questions regarding regulatory mechanisms about this common (α/β)₈ structural motif. The simplest model accounting for all of the solution thermodynamic and kinetic data on ligand-enzyme interactions involves two conformational states, inactive E^T and active E^R. These conformational states are represented by domain movements. Further studies provide the first evidence for a differential effect of ligand binding on the dynamics of the structural elements, not major secondary structural changes. These data are consistent with our model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the inactive E^T and active E^R represent the two extreme end states. Sequence differences and ligands can modulate the distribution of states leading to alterations of functions. The future work includes: defining the network of functionally connected residues; elucidating the chemical principles governing the sequence differences which affect functions; and probing the nature of mutations on the stability of the secondary structural elements, which in turn modulate allostery.     

Modulation of creatine kinase activity by ruthenium complexes

Creatine kinase is a crucial enzyme for brain, heart and skeletal muscle energy homeostasis, and a decrease of its activity has been associated with cell death. Many biological properties have been attributed to ruthenium complexes. In this context, this work was performed in order to evaluate creatine kinase activity from rat brain, heart and skeletal muscle (quadriceps) after administration of ruthenium complexes, trans-[RuCl(2)(nic)(4)] (nic=3-pyridinecarboxylic acid) 180.7 micromol/kg (complex I), trans-[RuCl(2)(i-nic)(4)] (i-nic=4-pyridinecarboxylic acid) 13.6 micromol/kg (complex II), trans-[RuCl(2)(dinic)(4)] (dinic=3,5-pyridinedicarboxylic acid) 180.7 micromol/kg (complex III) and trans-[RuCl(2)(i-dinic)(4)] (i-dinic=3,4-pyridinedicarboxylic acid) 180.7 micromol/kg (complex IV). Our results showed that complex I caused inhibition of creatine kinase activity in hippocampus, striatum, cerebral cortex, heart and skeletal muscle. Besides, complex II did not affect the enzyme activity. complexes III and IV increased creatine kinase activity in hippocampus, striatum, cerebral cortex and heart, but not in skeletal muscle. Besides, none of the complexes in vitro altered creatine kinase activity, suggesting that enzymatic activity is indirectly affected by complexes I, III and IV. It is believed that diminution of creatine kinase in brain of rats caused by complex I may be related to results from other study reporting memory impairment caused by the same complex. Further research is necessary in order to elucidate the effects of ruthenium complexes in other important metabolic enzymes.     

Modification of pyruvate kinase activity by proteins from chicken liver

The partial purification of a protein fraction inhibiting pyruvate kinase isoenzymes is described. The fraction was isolated from the (NH4)2SO4 step of the purification procedure for pyruvate kinase isoenzymes from chicken liver (Eigenbrodt, E. & Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1033-1046) by extraction with 1N NaOH, acidification to pH 3, ethanol precipitation and chromatography of the supernatant on DEAE-cellulose. The inhibitor fraction was further purified by disc gel electrophoresis using a gel gradient from 10 to 25%; this procedure separated activating proteins from the inhibitor fraction. The inhibitor fraction inhibited the pyruvate kinase isoenzymes from chicken in the sequence of decreasing effect: M2 greater than L greater than M1. The inhibition was due to a decrease in the affinity for phosphoenolpyruvate. The inhibitor is stable against heating for 5 min in 1% sodium dodecyl sulfate at 100 degrees C; it is destroyed by pepsin digestion. The inhibitor fraction could be purified further only by dodecyl sulfate gel electrophoresis. This resulted in the separation of 2 inhibitors (Mr = 33,500 +/- 8500 and ca. 5000), an activator (Mr = 15,100 +/- 5200), and an unidentified protein (Mr = 27,000).     

Modulation by fumarate of a P i -insensitive pyruvate kinase from Rhodopseudomonas capsulata

Cold lability was found to be responsible for the initial failure to detect pyruvate kinase activity in extracts of the facultative phototroph, Rhodopseudomonas capsulata. Taking advantage of the reversal of cold inactivation by high concentrations of monovalent cations, the enzyme could be partially purified by (NH₄)₂SO₄-precipitation and gelfiltration. In contrast to the enzyme from Rhodospirillum rubrum, the pyruvate kinase from R. capsulata is nearly insensitive to inorganic phosphate. Instead, it is susceptible to allosteric inhibition by fumarate. Adenosinemonophosphate and sugar phosphates as activators prevent the inhibitory action of fumarate.     

Peptides derived from pyruvate dehydrogenase as substrates for pyruvate dehydrogenase kinase and phosphatase.

Evidence is presented that phosphopeptides produced by tryptic digestion of phosphorylated pyruvate dehydrogenase are effective substrates for pyruvate dehydrogenase phosphatase and that the dephosphopeptides can serve as substrates for pyruvate dehydrogenase kinase. These findings indicate that the phosphatase and the kinase do not require an intact tertiary structure in pyruvate dehydrogenase, but apparently recognize components of the local primary sequence around the phosphorylation sites.