Studies on the cyclic 3': 5' AMP stimulated pig liver protein kinase reaction with pyruvate kinase as substrate

Lars Berglund, O. Ljungstrom, L. Engstrom

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

The phosphorylation of pig liver pyruvate kinase by cyclic adenosine 3':5' monophosphate dependent protein kinase has been studied. For comparison, mixed histone and a synthetic heptapeptide were also used as substrates. Protein kinase was purified by chromatography on DEAE cellulose, hydroxyapatite, and Sephadex G 200. The enzyme was stimulated by cyclic AMP with apparent K(a) values of 2.5 and 0.8 x 10-7M for pyruvate kinase and histone substrates, respectively. Divalent cations were essential for the activity of the protein kinase. Variation of the concentration of ATP resulted in approximately straight lines in Lineweaver Burk plots for the phosphorylation of both pyruvate kinase and mixed histone. The apparent K(m) values for ATP were 21 and 11 μm, respectively. The phosphorylation rate increased with the concentration of pyruvate kinase even at a concentration of 2 μm pyruvate kinase. At a high ionic strength, the phosphorylation rate of both pyruvate kinase and histone decreased. The phosphorylation rate varied markedly with pH in imidazole/HCl and Tris/HCl buffers. At slightly alkaline pH values, pyruvate kinase was phosphorylated at a much higher rate than at pH 7, but this was not the case for histone. At pH 8.5, the phosphorylation rate of pyruvate kinase was 3.5 times the rate at pH 7, while the corresponding increase for the histone phosphorylation was 50%. In potassium phosphate buffers, the phosphorylation rate of both substrates did not change significantly over the pH range studied. Arrhenius' plots of the protein kinase reaction resulted in a break at about 10° when pyruvate kinase was used as substrate, whereas a straight line was obtained when using histone. The negative allosteric effectors of pyruvate kinase, alanine, and phenylalanine, increased the phosphorylation rate of pyruvate kinase at pH 8 by 50 and 120%, respectively. The same effectors did not influence the phosphorylation rate of mixed histone or a synthetic heptapeptide. It is concluded that the conformations adopted by pyruvate kinase in the presence of allosteric inhibitors make it a better substrate for the protein kinase.

Original languageEnglish (US)
Pages (from-to)613-619
Number of pages7
JournalJournal of Biological Chemistry
Volume252
Issue number2
StatePublished - 1977
Externally publishedYes

Fingerprint

Pyruvate Kinase
Adenosine Monophosphate
Phosphorylation
Liver
Protein Kinases
Swine
Histones
Substrates
Buffers
Adenosine Triphosphate
DEAE-Dextran
DEAE-Cellulose
Arrhenius plots
Tromethamine
Divalent Cations
Durapatite
Chromatography
Ionic strength
Phenylalanine
Alanine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Studies on the cyclic 3' : 5' AMP stimulated pig liver protein kinase reaction with pyruvate kinase as substrate. / Berglund, Lars; Ljungstrom, O.; Engstrom, L.

In: Journal of Biological Chemistry, Vol. 252, No. 2, 1977, p. 613-619.

Research output: Contribution to journalArticle

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abstract = "The phosphorylation of pig liver pyruvate kinase by cyclic adenosine 3':5' monophosphate dependent protein kinase has been studied. For comparison, mixed histone and a synthetic heptapeptide were also used as substrates. Protein kinase was purified by chromatography on DEAE cellulose, hydroxyapatite, and Sephadex G 200. The enzyme was stimulated by cyclic AMP with apparent K(a) values of 2.5 and 0.8 x 10-7M for pyruvate kinase and histone substrates, respectively. Divalent cations were essential for the activity of the protein kinase. Variation of the concentration of ATP resulted in approximately straight lines in Lineweaver Burk plots for the phosphorylation of both pyruvate kinase and mixed histone. The apparent K(m) values for ATP were 21 and 11 μm, respectively. The phosphorylation rate increased with the concentration of pyruvate kinase even at a concentration of 2 μm pyruvate kinase. At a high ionic strength, the phosphorylation rate of both pyruvate kinase and histone decreased. The phosphorylation rate varied markedly with pH in imidazole/HCl and Tris/HCl buffers. At slightly alkaline pH values, pyruvate kinase was phosphorylated at a much higher rate than at pH 7, but this was not the case for histone. At pH 8.5, the phosphorylation rate of pyruvate kinase was 3.5 times the rate at pH 7, while the corresponding increase for the histone phosphorylation was 50{\%}. In potassium phosphate buffers, the phosphorylation rate of both substrates did not change significantly over the pH range studied. Arrhenius' plots of the protein kinase reaction resulted in a break at about 10° when pyruvate kinase was used as substrate, whereas a straight line was obtained when using histone. The negative allosteric effectors of pyruvate kinase, alanine, and phenylalanine, increased the phosphorylation rate of pyruvate kinase at pH 8 by 50 and 120{\%}, respectively. The same effectors did not influence the phosphorylation rate of mixed histone or a synthetic heptapeptide. It is concluded that the conformations adopted by pyruvate kinase in the presence of allosteric inhibitors make it a better substrate for the protein kinase.",
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