Hormone‐Induced Changes in Pyruvate Kinase

Berkel, Theo J.C. Van; Kruijt, J. Kar; Koster, J.F.

doi:10.1111/j.1432-1033.1977.tb11967.x

Cited by 40 publications

(15 citation statements)

References 36 publications

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“…This raises the problem of the physiological significance of such a phosphorylation mechanism in red cells. The role of this system is well understood in liver where pyruvate kinase (and glycolysis) must be inhibited under gluconeogenic conditions [4,5,7,9]. In red cells, by contrast, the gluconeogenic pathway does not exist, so the role of phosphorylation of pyruvate kinase would be more difficult to understand.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of Human Erythrocyte Pyruvate Kinase by Soluble Cyclic‐AMP‐Dependend Protein Kinases

Marie

Buc

Simon

et al. 1980

European Journal of Biochemistry

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Human red cell contain soluble adenosine-3', 5'-phosphate-dependent protein kinases, which are able to phosphorylate the L subunits of erythrocyte pyruvate kinase. Efficiency and maximum level of phosphorylation are very comparable in human liver and red cells. Phosphorylation of red cell pyruvate kinase results in the same kinetic modifications as for liver enzyme, namely a shift towards a 'T' allosteric state characterized by a decreased affinity for phosphoenolpyruvate and increased inhibition by the allosteric inhibitors ATP and alanine. In the course of red cell aging a small amount of partially proteolysed pyruvate kinase, devoid of the phosphorylatable site, appears; it resembles the subtilisin-treated L b enzyme and accounts for less than 20% of total pyruvate kinase subunits.Endogenous phosphorylation of pyruvate kinase from erythrocytes incubated in the presence of cyclic nucleotides produces the same kinetic modifications as phosphorylation in partially purified extract; this, however, does not change glucose consumption, lactate production and glycolytic intermediate concentrations of the incubated cells.Regulation of liver L-type pyruvate kinase by a phosphorylation mechanism has been intensively studied for several years. Ljungstrom et al. were the first to report that rat liver L-type pyruvate kinase can be phosphorylated by an adenosine-3',5'-phosphatedependent (cyclic-AMP-dependent) protein kinase [l]. Phosphorylation of pig-liver L-type enzyme has also been reported [2,3]. Glucagon induces phosphorylation of liver L-type pyruvate kinase in vivo through a cyclic-AMP-dependent mechanism [4-61. This phosphorylation of L-type pyruvate kinase causes important changes in the kinetics of the enzyme: decreased affinity for phosphoenolpyruvate, and increased inhibition by the allosteric effectors ATP and alanine [2,7-111. These kinetic modifications are expected to play an important physiological role in the dynamic balance between glycolysis and gluconeogenesis by blocking the pyruvate kinase reaction when gluconeogenesis is the predominant pathway, thus impairing a futile cycle between phosphoenolpyruvate and pyruvate.Ahhreviutzon. Cyclic AMP, adenosine 3',5'-phosphate.We have previously shown that human L-type pyruvate kinase seems to undergo, after its synthesis, a progressive proteolytic processing, which is different in liver and red cells. In these latter cells the Lb precursor (composed of four identical subunits of M , 63000) is partially proteolysed with red cell maturation and aging, and this gives rise to heterotetrameric forms composed of L and partially proteolysed subunits [12,13]. In liver the L-type pyruvate kinase consists almost entirely of a form composed of four identical subunits of M , about 60000 closely resembling trypsin-treated L-type subunits [12-141. We thus hypothesized that in liver the L subunits are immediately proteolysed by a specific proteolytic system after they have been synthesized. We [13] and other authors [15] proved that proteolytic processing of L-type pyruva...

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Section: Discussionmentioning

confidence: 99%

Phosphorylation of Human Erythrocyte Pyruvate Kinase by Soluble Cyclic‐AMP‐Dependend Protein Kinases

Marie

Buc

Simon

et al. 1980

European Journal of Biochemistry

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“…This was demonstrated for the formation of cyclic AMP (224) and for the inactivation of pyruvate kinase (90, 91) and of phos phofructokinase (166); the latter inactivation is now known to he mediated by the disappearance offructose 2,6-bisphosphate. Fasting and diabetes are two conditions in which the concentration of cyclic AMP in the liver is increased (4,224,225); this offers an explanation fo r the presence of pyruvate kinase in its inactive form (86,97,226) and a low content in fr uctose 2,6-bisphosphate (see next section).…”

Section: The Antagonism By Insulinmentioning

confidence: 95%

“…Furthermore, the proportion of the enzyme in the inactive form is greater during starvation and diabetes than under control conditions (86,97). No effect of glucagon on pyruvate kinase activity has however been observed in chicken hepatocytes, although the hormone stimulates gluconeogenesis (98).…”

Section: Hormonal Effectsmentioning

confidence: 95%

Gluconeogenesis and Related Aspects of Glycolysis

Hers¹,

Hue²

1983

Annu. Rev. Biochem.

516

259

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“…After the incubation with 5 mM MgCl2, erythrocyte PK activity was elevated 1.20-fold, and L-type PK was elevated 1.22-fold. Erythrocyte PK L-Type PK PK in vitra (in the cell-free system of hepatocytes (7,11), isolated hepatocytes (2,12), and liver slices (6)) and in viva (10). From these observations, phosphorylation is thought to play an important role in in viva regulation of Ltype PK activity.…”

Section: Resultsmentioning

confidence: 99%

EVIDENCE OF IN VIVO PHOSPHORYLATION OF ERYTHROCYTE AND LIVER PYRUVATE KINASES

Fujii¹,

Nakashima²,

Kaneko³

1981

Biomed. Res.

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Rat red cells and liver were obtained 4 hr after intravenous injection of ortho[32P]-phosphate. Erythrocyte pyruvate kinase (PK; EC 2.7.1.40) and L-type PK were recovered by the double antibody technique and applied to SDS-polyacrylamide gel electrophoresis. Incorporation of "P into erythrocyte PK and L-type PK was detected on autoradiograms of the electrophoresis corresponding to the L' and L subunits, respectively. The results demonstrated directly in viva phosphorylation of rat erythrocyte and L-type PKs.Rat and human erythrocyte PKs of the partially purified samples were phosphorylated using [T-32P]ATP. These phosphorylated PKs labelled with "P were incubated with MgCl2, resulting in the dephosphorylation and reactivation ofthe PKs, which indicate in viva dephosphorylation by the endogenous phosphoprotein phosphatase. Under the identical experimental conditions, samples freshly prepared from human red cells and human liver incubated with MgCl, resulted in the elevation of PK activity, indicating that human erythrocyte and L-type PKs are dephosphorylated by endogenous phosphatase in the presence of MgCl2. These results present indirect evidence that human erythrocyte a.nd L-type PKs are regulated in viva by the phosphorylation-dephosphorylation process.KEY WORDS phosphorylation / erythrocyte pyruvate kinase / L-type pyruvate kinase Erythrocyte pyruvate kinase (PK; EC 2.7.1.40) and L-type PK are phosphorylated by cyclic AMP-dependent protein kinase, and phosphorylated PKs exhibit a lower affinity for phosphoenolpyruvate (PEP) than unphosphorylated PKs in virra (2-9, ll, l2). The reactivation of both erythrocyte and L-type PKs by dephosphorylation has also been reported in virra (5, ll). These observations suggest that PK activity may be regulated in viva by a phosphorylation-dephosphorylation process. L-type PK was shown to be phosphorylated in viva in the rat (10), but there has been no evidence that the phosphorylation of erythrocyte PK really takes place in viva.This paper presents direct evidence of in viva phosphorylation of rat erythrocyte and L-type PKs and indirect evidence of in viva phosphorylation of human erythrocyte and L-type PKs. In addition, the dephosphorylation of erythrocyte PK by endogenous phosphatase was demonstrated, though dephosphorylation of erythrocyte PK was reported using exogenous phosphatase (5). MATERIALS AND METHODSAssay and SDS-Pa/yacrylamide Gel Electrophoresis afPK PK was assayed as reported previously (4) without the addition of ATP in the assay medium. SDS-polyacrylamide slab gel electrophoresis was performed according to Fairbanks er al. (1).

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Hormone‐Induced Changes in Pyruvate Kinase

Cited by 40 publications

References 36 publications

Phosphorylation of Human Erythrocyte Pyruvate Kinase by Soluble Cyclic‐AMP‐Dependend Protein Kinases

Phosphorylation of Human Erythrocyte Pyruvate Kinase by Soluble Cyclic‐AMP‐Dependend Protein Kinases

Gluconeogenesis and Related Aspects of Glycolysis

<b>EVIDENCE OF <i>IN VIVO</i> PHOSPHORYLATION OF ERYTHROCYTE AND LIVER PYRUVATE </b><b>KINASES </b>

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