The present study analyzes the kinetic effect of phosphorylation of the muscle PFK from Austromegabalanus psittacus (a giant barnacle species), after in vitro exposure to the catalytic subunit of a cAMP-dependent protein kinase from bovine heart. The balanid PFK was partially purified by ion exchange chromatography on DEAE-cellulose by elution at 0.25 M NH4Cl (linear gradient of the salt). The activity of the endogenous protein kinase C of A. psittacus muscle was also measured, eluting at 0.08 M NH4Cl in the salt gradient. Bovine protein kinase A activated the cirripede PFK, increasing its affinity for fructose 6-P approximately 2.6 times (in the absence of allosteric effectors) and around 5 times in the presence of 0.2 mM AMP, and decreasing the enzyme’s cooperativity for Fru 6-P as a result of phosphorylation (in presence as well as the absence of 0.2 mM AMP). Allosteric activation of the barnacle muscle PFK by fructose 2,6-bisphosphate occurred at much lower concentrations of FBP after phosphorylation by bovine PKA, and the inhibitory effect of citrate on the balanid PFK was markedly reversed in the phosphorylated form of PFK. Endogenous PKC from A. psittacus muscular tissue also activated PFK in preliminary measurements, in a similar manner to that by bovine heart PKA. Using size exclusion chromatography (fast protein liquid chromatography, FPLC) analysis of the partially purified PFK in Superose 6B column, a native molecular weight of 602 kDa was obtained, which corresponds to an hexameric conformation of the enzyme.
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