Transient kinetics of formation of phosphorylated intermediate (EP) by fragmented sarcoplasmic reticulum from bullfrog skeletal muscle: ligand addition sequence-dependent rate of EP formation.
In order to obtain a better understanding of Ca2+-activated ATP hydrolysis by sarcoplasmic reticulum, the transient kinetics of phosphorylated intermediate (EP) formation was examined with different sequences of addition of Ca2+ and ATP to GEDTA-added (Ca2+-free) fragmented sarcoplasmic reticulum (FSR) from bullfrog skeletal muscle. With a short delay line (10-20 ms), the addition sequence of ATP followed by Ca2+ gives rise to a faster EP formation without any lag time. In the reverse sequence of ligand addition, a lag time of 2.5-3 ms was consistently observed irrespective of ATP concentration, and the rate of EP formation was lower. As the preincubation time with Ca2+ became longer, the rate constant for EP formation and the maximum level of EP attainable increased even in the presence of fixed concentrations of Ca2+ and ATP. The rate constant per the unit concentration of EP, however, remained constant, indicating second-order kinetics between Ca2+-activated FSR and ATP. With a preincubation time of 83.6 ms, no lag time was observed. In the addition sequence of ATP and 83.6 ms later Ca2+, a burst in EP formation was observed, which was followed by a usual exponential time-course of EP formation. Similar determinations in the presence of various concentrations of ATP led us to the following estimates of the rates of the reactions leading to EP formation at 15 degrees C. (formula: see text)
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D007700:Kinetics; D009132:Muscles / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011892:Rana catesbeiana; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D010750:Phosphoproteins; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium