Lorentz Engström

The minimum substrate of cyclic AMP-stimulated protein kinase, as studied by synthetic peptides representing the phosphorylatable site of pyruvate kinase (type L) of rat liver

Synthetic peptides, representing part of the phosphorylatable site of rat liver pyruvate kinase, were phosphorylated by (32P)ATP and the catalytic subunit of cyclic AMP-stimulated protein kinase. The shortest peptide which could be significantly phosphorylated was Arg-Arg-Ala-Ser-Val, with an apparent Km of 0.08 mM. The apparent Km for Arg-Arg-Ala-Ser-Val-Ala was 0.02 mM and that for Leu-Arg-Arg-Ala-Ser-Val-Ala was less than 0.01 mM. Peptides in which threonine was substituted for serine, or leucine for the one or the other arginine of the pentapeptide were not detectably phosphorylated. Substitution of phenylalanine for valine increased, and substitution of lysine or glycine for valine considerably decreased the rate of phosphorylation.

Phosphorylation of purified rat liver pyruvate kinase by cyclic 3′,5′-AMP-stimulated protein kinase

Abstract 1. 1. Rat liver pyruvate kinase (type L) was purified by precipitation at pH5 and (NH4)2SO4 fractionation, followed by chromatography on DEAE-cellulose, hydroxylapatite and Sepharose 6B in the presence of glycerol in order to stabilize the enzyme. The enzyme preparation was nearly homogeneous as judged by Sepharose 6B chromatography and polyacrylamide gel electrophoresis in the presence of detergent. 2. 2. The enzyme was shown to be phosphorylated by [32P]ATP and cyclic 3′,5′-AMP-stimulated protein kinase. In preliminary experiments, the activity of the enzyme was decreased by phosphorylation, especially at low phosphoenolpyruvate concentrations. The results suggest that the L type of rat liver pyruvate kinase belongs to the enzymes whose activity is regulated by phosphorylation-dephosphorylation reactions.

Studies on calf-intestinal alkaline phosphatase I. Chromatographic purification, microheterogeneity and some other properties of the purified enzyme

Abstract Calf-intestinal alkaline phosphatase prepared according to Portmann has been chromatographed on triethylaminoethyl-cellulose, and has subsequently been shown to be microheterogeneous, since it could be separated into several active fractions by rechromatography and zone electrophoresis. It has not been possible to purify the main enzyme fraction further by chromatography on triethylaminoethyl- or Ecteola-cellulose, on hydroxyl apatite or by zone electrophoresis. Its activity has invariably reached approx. 400 000 Portmann units/mg N. On sedimentation analysis, the enzyme is found to show only one sharp boundary. A preliminary determination according to Archibald indicates a molecular weight of approx. 100 000 and a fairly high degree of homogeneity. The enzyme is found to contain neutral sugar and hexosamine in varying amounts, but no sialic acid. The enzyme has been shown by the dithizone method to contain approx. 0.2% Zn.

The Regulation of Liver Pyruvate Kinase by Phosphorylation—Dephosphorylation

Publisher Summary This chapter discusses the regulation of liver pyruvate kinase by phosphorylation–dephosphorylation. The rate and extent of the phosphorylation of L-type liver pyruvate kinase in vitro indicate that it is a specific reaction. This is supported by inhibition of the enzyme by the phosphorylation, which may be expected with regard to the known effect of cAMP on liver gluconeogenesis. The specificity of the phosphorylation reaction is also demonstrated by the fact that neither the A-type enzyme from pig kidney nor the M-type muscle enzyme from the rabbit or pig are substrates of cAMP-stimulated protein kinase. However, phosphorylation of the type-A enzyme from chicken liver has been preliminarily reported. The phosphorylation of liver pyruvate kinase in vitro is reversible, owing to the presence in rat liver cell sap of phosphoprotein phosphatase, which acts on phosphorylated pyruvate kinase. In vitro experiments strongly indicate that L-type liver pyruvate kinase is an enzyme whose activity is regulated by reversible protein phosphorylation in vivo .

Studies on calf-intestinal alkaline phosphatase II. Incorporation of inorganic phosphate into a highly purified enzyme preparation

Abstract Inorganic phosphate has been shown to be incorporated into calf-intestinal alkaline phosphatase, since radioactive phosphorylserine could be isolated from an acid hydrolysate of a highly purified enzyme preparation, incubated with radioactive inorganic phosphate and then inactivated by acid. Much smaller amounts of labelled phosphorylserine are isolated from acetone-precipitated or heat-inactivated enzyme. After alkali inactivation of the enzyme, no phosphorylserine can be demonstrated. With a phosphate concentration of 0.03 mM, the incorporation is found to be maximal at approx. pH 5, where it is very rapid. Addition of metal ions is not necessary for the reaction. It is found that 0.01 M EDTA, 0.025 M 1-10-phenanthroline and hydrogen sulphide strongly inhibit incorporation, indicating the participation of a metal. The possible role of phosphate incorporation in the enzyme mechanism is briefly discussed.

Regulation in vitro of rat liver pyruvate kinase by phosphorylation-dephosphorylation reactions, catalyzed by cyclic-AMP dependent protein kinases and a histone phosphatase

1. Cyclic-AMP dependent protein kinases, resolved by chromatography on DEAE-cellulose and hydroxylapatite, catalysed the phosphorylation of rat liver pyruvate kinase and calf thymus histones by [gamma32P]ATP. [32P]phosphopeptides, from acid hydrolysates of pyruvate kinase phosphorylated by the different protein kinase fractions, displayed identical electrophoretic patterns. Phosphorylation inhibited pyruvate kinase activity. 2. Full activity was restored when phosphorylated pyruvate kinase was dephosphorylated by a histone phosphatase from the soluble fraction of rat liver. These results are consistent with the hypothesis that pyruvate kinase is regulated by phosphorylation-dephosphorylation reactions.

Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3', 5'-AMP-stimulated protein kinase.

The kinetics of rat liver L-type pyruvate kinase (EC 2.7.1.40), phosphorylated with cyclic AMP-stimulated protein kinase from the same source, and the unphosphorylated enzyme have been compared. The effects of pH and various concentrations of substrates, Mg2+, K+ and modifiers were studied. In the absence of fructose 1, 6-diphosphate at pH 7.3, the phosphorylated pyruvate kinase appeared to have a lower affinity for phosphoenolpyruvate (K0.5=0.8 mM) than the unphosphorylated enzyme (K0.5=0.3 mM). The enzyme activity vs. phosphoenolpyruvate concentration curve was more sigmoidal for the phosphorylated enzyme with a Hill coefficient of 2.6 compared to 1.6 for the unphosphorylated enzyme. Fructose 1, 6-diphosphate increased the apparent affinity of both enzyme forms for phosphoenolpyruvate. At saturating concentrations of this activator, the kinetics of both enzyme forms were transformed to approximately the same hyperbolic curve, with a Hill coefficient of 1.0 and K0.5 of about 0.04 mM for phosphoenolpyruvate. The apparent affinity of the enzyme for fructose 1, 6-diphosphate was high at 0.2 mM phosphoenolpyruvate with a K0.5=0.06 muM for the unphosphorylated pyruvate kinase and 0.13 muM for the phosphorylated enzyme. However, in the presence of 0.5 mM alanine plus 1.5 mM ATP, a higher fructose 1, 6-diphosphate concentration was needed for activation, with K0.5 of 0.4 muM for the unphosphorylated enzyme and of 1.4 muM for the phosphorylated enzyme. The results obtained strongly indicate that phosphorylation of pyruvate kinase may also inhibit the enzyme in vivo. Such an inhibition should be important during gluconeogenesis.

Amino acid sequence of a (32P)phosphopeptide from pig liver pyruvate kinase phosphorylated by cyclic 3′,5′-AMP-stimulated protein kinase and γ-(32P)ATP

Abstract Pig liver pyruvate kinase (type L) was 32 P-labelled by incubation with ( 32 P)ATP and cyclic 3′,5′-AMP-stimulated protein kinase from the same source. One major ( 32 P)phosphopeptide was isolated from a peptic hydrolysate of the enzyme. Its amino acid sequence was Leu-Arg-Arg-Ala-( 32 P)SerP-Leu.

STUDIES ON BOVINE-LIVER ALKALINE PHOSPHATASE, PURIFICATION, PHOSPHATE INCORPORATION.

Abstract 1. 1. A highly purified preparation of bovine-liver alkaline phosphatase (ortho-phosphoric monoester phosphohydrolase, EC 3.I.3.I) has been obtained from a liver homogenate treated with butanol. Acetone and ethanol fractionation have been used, followed by two consecutive chromatographies on TEAE-cellulose and one on Sephadex G-200. 2. 2. Labelled phosphorylserine has been isolated from acid hydrolysates of the purified enzyme, which had been incubated with radioactive orthophosphate and then inactivated by acid. Most probably, the phosphate was bound to a particular serine molecule at the active site of the enzyme. This supports the view that an intermediate phosphorylenzyme is formed during the action of alkaline phosphatase.

Non-dependence on native structure of pig liver pyruvate kinase when used as a substrate for cyclic 3′,5′-AMP-stimulated protein kinase

Abstract Alkali-inactivated pig liver pyruvate kinase, type L, and a cyanogen bromide fragment from the same enzyme were shown to be phosphorylated by ( 32 P)ATP and cyclic 3′,5′-AMP-stimulated protein kinase. In both cases the rate of phosphorylation was higher than with the native enzyme. Pyruvate kinases types A and M were not phosphorylated under the same conditions. From the 32 P-labelled cyanogen bromide fragment ( 32 P)phosphorylserine was isolated. The electrophoretic pattern of ( 32 P)phosphopeptides obtained on partial acid hydrolysis of the fragment indicated that the phosphorylated site of the fragment was identical with that of the native pyruvate kinase.