In vivo and in vitro phosphorylation of yeas pyruvate kinase isoforms by protein kinase A

P.PORTELA, S.HOWELL, S.MORENO AND S.ROSSI

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2002 vol. 277 p. 30477 - 30487

0021-9258

Saccharomyces cerevisiae pyruvate kinase 1 (Pyk1) was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HATpk1 was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HATpk1 was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HATpk1 pyruvate kinase 1 (Pyk1) was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HATpk1 Bcy1) and to be phosphorylated in a cAMP-dependent process. Both glutathione S-transferase (GST)-Pyk1 and GST-Pyk2 were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated (GST)-Pyk1 and GST-Pyk2 were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated (GST)-Pyk1 and GST-Pyk2 were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated S-transferase (GST)-Pyk1 and GST-Pyk2 were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated in vivo, in intact cells overexpressing the protein, or in vitro using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK vitro using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK vitro using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK , in intact cells overexpressing the protein, or in vitro using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPKTPK gene with an attenuated mutation (tpk1w1). The effect of phosphorylation on Pyk activity was assayed in partially purified preparations from three strains, containing different endogenous protein kinase A activity levels. Pyk1 activity was measured at different phosphoenolpyruvate concentrations in the absence or in the presence of the activator fructose 1,6-bisphosphate at 1.5 mM. Preliminary kinetic results derived from the comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. of phosphorylation on Pyk activity was assayed in partially purified preparations from three strains, containing different endogenous protein kinase A activity levels. Pyk1 activity was measured at different phosphoenolpyruvate concentrations in the absence or in the presence of the activator fructose 1,6-bisphosphate at 1.5 mM. Preliminary kinetic results derived from the comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. of phosphorylation on Pyk activity was assayed in partially purified preparations from three strains, containing different endogenous protein kinase A activity levels. Pyk1 activity was measured at different phosphoenolpyruvate concentrations in the absence or in the presence of the activator fructose 1,6-bisphosphate at 1.5 mM. Preliminary kinetic results derived from the comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost null protein kinase A activity. with almost null protein kinase A activity. activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6- bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity. with almost n