Transient-state kinetics of the ADP-insensitive phosphoenzyme in sarcoplasmic reticulum: implications for transient-state calcium translocation.
The kinetics of formation of the ADP-sensitive (EP) and ADP-insensitive (E*P) phosphoenzyme intermediates of the CaATPase in sarcoplasmic reticulum (SR) were investigated by means of the quenched-flow technique. At 21 degrees C, addition of saturating ADP to SR vesicles phosphorylated for 116 ms with 10 microM ATP gave a triphasic pattern of dephosphorylation in which EP and E*P accounted for 33% and 60% of the total phosphoenzyme, respectively. Inorganic phosphate (Pi) release was less than stoichiometric with respect to E*P decay and was not increased by preincubation with Ca2+ ionophore. The fraction of E*P present after only 6 ms of phosphoenzyme formation was similar to that at 116 ms, indicating that isomerization of EP to E*P occurs very rapidly. Comparison of the time course of E*P formation with intravesicular Ca2+ accumulation measured by quenching with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid + ADP revealed that Ca2+ release on the inside of the vesicle was delayed with respect to E*P formation. Since Ca2+ should dissociate rapidly dissociation from the low-affinity transport sites, these results suggest that Ca2+ remains "occluded" after phosphoenzyme isomerization and that a subsequent slow transition controls the rate of Ca2+ release at the intravesicular membrane surface. Analysis of the forward and reverse rate constants for the EP to E*P transition gave an expected steady-state distribution of phosphoenzymes strongly favoring the ADP-insensitive form. In contrast, the observed ratio of EP to E*P was about 1:2. To account for this discrepancy, a mechanism is proposed in which stabilization of the ADP-sensitive phosphoenzyme is brought about by a conformational interaction between adjacent subunits in a dimer.
Froehlich
J P
JP
Heller
P F
PF
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D009132:Muscles / Q000201:enzymology*; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D015636:Magnesium Chloride; D000001:Calcimycin; D000244:Adenosine Diphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002122:Calcium Chloride; D002118:Calcium