Ionic inhibition of catalytic phosphorylation of histone by bovine brain protein kinase.
The effects of various ions commonly found in protein kinase assays upon the rate of histone phosphorylation catalyzed by the highly purified bovine brain enzyme, protein kinase I, have been investigated. Sodium, potassium, and magnesium were found to inhibit histone phosphorylation by protein kinase I  in a similar manner. The degree of inhibition by any of these cations was demonstrated to be directly proportional to the square root of the ionic strength of the assay medium. The relationship between the ionic strength of the assay medium and the rate of histone phosphorylation catalyzed by protein kinase I was employed to correct the rate of histone phosphorylation at various magnesium acetate concentrations to a standard ionic strength. When this was done an analysis of the previously postulated rate law for histone phosphorylation c atalyzed by protein kinase I gave a binding constant for the magnesium-ATP complex which was in agreement with that expected for this complex on  the basis of various binding constants available in the literature. These results demonstrate that it is unnecessary to postulate a specific ion inhibition process for protein kinase I by the ions employed in this study. They also support the reasonable assumption that magnesium ion binds to ATP at or prior to the rate-determining step in histone phosphorylation catalyzed by protein kinase I. The expression developed in this paper for the effect of ionic strength upon protein kinase I activity can now be used to correct activity measurements made under various assay conditions to a standard assay state, allowing facile comparisons of kinetic data. It should be possible to develop similar expressions for other protein kinases and substrates to permit useful interpretation of kinetic data.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008433:Mathematics; D009994:Osmolar Concentration; D010085:Oxidative Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000494:pharmacology*; D006656:Protamine Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011485:Protein Binding; D047428:Protein Kinase Inhibitors; D012964:Sodium / Q000494:pharmacology*
D047428:Protein Kinase Inhibitors; D012964:Sodium; D006656:Protamine Kinase; D008274:Magnesium; D011188:Potassium