The bioflavonoid, quercetin, inhibited the (Na+, K+)adenosine triphosphatase purified from the electric organ of electric eel (Electrophorus electricus) or from lamb kidney. An analysis of its mode of action revealed that the formation of phosphoenzyme from Pi but not from ATP was inhibited. Quercetin increased the amount of ADP-sensitive phosphoenzyme (E1--P), indicating an inhibition of the conversion of E1--P to the ADP-insensitive form (E2--P). The rate of dephosphorylation of the phosphoenzyme formed from ATP was slowed by quercetin. These results suggest that quercetin inhibits the formation of E2--P from either Pi or E1-P as well as the hydrolysis of the phosphoenzyme. Its mode of action is therefore different from that of ouabain and other inhibitors of the Na+, K+)adenosine triphosphatase.

The phosphorylation of (Na+, K+)ATPase from the electric organ of the electric eel is dependent on Mg2+. The amount of phosphoenzyme formed was increased by K+ and decreased by Na+. Kinetic analyses indicate that a ternary complex of ATPase, Pi and Mg2+ is formed prior to phosphorylation of the protein. Calorimetric studies revealed extraordinarily large enthalpy changes associated with the binding of Mg2+ (-49 kcal/mol) and of Pi (-42 kcal/mol), indicating a thermodynamically significant conformational change in the enzyme. The dissociation constant for the binding of Mg2+ and Pi derived from calorimetric measurements is in good agreement with the value obtained from the kinetic studies. These results indicate that ion binding induces a conformational change in the enzyme which is a prerequisite for phosphorylation by Pi.

The enthalpy change (delta H degrees ') associated with the binding of Mg2+ to the sarcoplasmic reticulum calcium adenosine 5'-triphosphatase [(Ca2+)ATPase] is -76 kcal/mol. The affinity constant for Mg2+ obtained from calorimetric measurements agrees with the Km value for Mg2+ in the phosphorylation of the enzyme by inorganic phosphate (Pi). The delta H degrees ' of binding of Pi to the enzyme is -23.5 kcal/mol, and the affinity constant for Pi obtained from the calorimetry also agrees with the Km value for Pi in the phosphorylation reaction. delta H degrees ' of Mg2+ binding is reduced to -35 kcal/mol in the presence of either 20 mM Pi or 1.2 mM Ca2+ without a significant change in the affinity of the enzyme for Mg2+. delta H degrees ' of Pi binding to the enzyme drops to -8.5 kcal/mol in the presence of 10 mM Mg2+ without a significant change in the affinity of the enzyme for Pi. On the other hand, the presence of Ca2+ does not affect the delta H degrees ' for the binding of the substrate analogue 5'-adenylyl beta,gamma-imidodiphosphate [App(NH)p], and the presence of this analogue does not affect the delta H degrees ' for Ca2+ binding. The results suggest a model in which a conformational change, largely controlled by Mg2+ binding to the enzyme, leads to the formation of the covalent phosphoprotein intermediate.

Synthesis of beta-lactamase, the product of the amp gene on pBR322, in Escherichia coli K-12 was reversibly repressed with a shift-up of the growth temperature from 30 to 42 degrees C. The temperature shift, however, did not affect the level of mRNA encoding beta-lactamase, which suggested the involvement of translational control.

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