STOPPED-FLOW KINETIC INVESTIGATIONS OF CONFORMATIONAL-CHANGES OF PIG-KIDNEY NA-ATPASE(,K+)

The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase w ere investigated via the stopped-flow technique using the fluorescent labels RH421 and BLPM. After the enzyme is mixed with MgATP, both labe ls give almost identical kinetic responses. Under the chosen experimen tal conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau(1) approximate to 180 s(-1) (sat urating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to ph osphorylation of the enzyme and a subsequent conformational change (E( 1)ATP(Na+)(3) --> E2P(Na+)(3) + ADP). The rate of the phosphorylation reaction measured by the acid quenched-flow technique was 190 s(-1) at 100 mu M ATP, suggesting that phosphorylation controls the kinetics o f the RH421 signal and that the conformational change is very fast (gr eater than or equal to 600 s(-1)). The rate of the RH421 signal was op timal at pH 7.5. The Na+ concentration dependence of 1/tau(1) showed h alf-saturation at a Na+ concentration of 8-10 mM with positive coopera tivity involved in the occupation of the Na+ binding sites. The appare nt dissociation constant of the high affinity ATP binding site determi ned from the ATP concentration dependence of 1/tau(1) was 7.0 (+/-0.6) mu M, while the apparent K-d for the low affinity site and the rate c onstant for the E-2 to E-1 conformational change evaluated in the abse nce of Mg2+ were 143 (+/-17) mu M and less than or equal to 28 s(-1) A t RH421 concentrations in the micromolar range, a decrease in the valu e of 1/tau(1) is observed. On the basis of rapid quenched-flow measure ments, this inhibition can be attributed to a reaction step subsequent to phosphorylation. This accounts for previously observed kinetic dis crepancies between RH421 and BIPM.