Studies on the phosphorylation and dephosphorylation of L-type pyruvate kinase

Hsu, Ying Chang (1988) Studies on the phosphorylation and dephosphorylation of L-type pyruvate kinase. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The hormonal control of L-type pyruvate kinase phosphorylation was studied using hepatocytes isolated by the collagenase perfusion method. The hepatocytes obtained were stimulated with various hormones and pharmacological effectors in the presence of H₃³²PO₄. The L-type pyruvate kinase was isolated by either immunoprecipitation or by affinity chromatography techniques. The effect of the various agents on the state of phosphorylation of the enzyme was examined by autoradiography after separation by SDS-PAGE. The incorporation of ³²P into L-type puruvate kinase was stimulated in the presence of 1 μM glucagon by 3.5-4.0 fold over that of the control. The effect of 10 μM phenylephrine on phosphorylation of pyruvate kinase is approximately half of that seen with glucagon. Maximal incorporation of ³²P in cells exposed to either 10 μM phenylephrine or 1 μM glucagon was observed after 6-8, or 2-4 minutes, respectively. Phorbol 12-myristate 13-acetate was not found to have any significant influence on the phosphorylation of L-type pyruvate kinase. However, the Ca²⁺ ionophore A23187 and vasopressin did increase the phosphorylation of the enzyme by 1.8 and 1.6 fold, respectively, over that of control. The site of phosphorylation of L-type pyruvate kinase isolated from hepatocytes stimulated by glucagon was found to be located in a Mr= 17,000 peptide after specific cleavage of the protein between neighbouring aspartate and proline residues. The peptide was also phosphorylated by treatment of L-type pyruvate kinase in vitro with cAMP-dependent kinase followed by specific aspartyl-prolyl cleavage. Stimulation of hepatocytes with phenylephrine also produced the phosphorylation of the same Mr= 17,000 fragment. A minor fragment of Mr= 14,000 was also observed but contained no more than 10% of the radioactivity. Phosphoamino acid analysis of [³²P] phosphoryl L-type pyruvate kinase showed that all of the ³²P was associated with phosphoserine (not phosphothreonine nor phosphotyrosine) following treatment with glucagon or phenylephrine. The in vivo half-life for the dephosphorylation of the [³²P]-phosphoryl pyruvate kinase was measured as being between 22-24 minutes. This apparent half-life will be longer than the true half-life due to the inevitable re-incorporation of ³²P into pyruvate kinase via ATP. Insulin increased, whilst NaF decreased, the rate of dephosphorylation of [³²P]-phosphoryl pyruvate kinase. However, glucagon did not have any observable effect on the rate of dephosphorylation measured in these conditions.