Simultaneous internalization and binding of calcium during the initial phase of calcium uptake by the sarcoplasmic reticulum Ca pump.
The kinetics of Ca2+ transport mediated by the sarcoplasmic reticulum (SR) Ca-ATPase were investigated by rapid kinetic techniques that either measure the disappearance of Ca2+ from the medium [stopped-flow photometry of Ca2+ indicators or rapid filtration (method 1)] or directly detect the changes in the accessibility of Ca2+ to the exterior of the membrane, i.e., occlusion of Ca2+ within the Ca pump and Ca2+ transport into the lumen of SR vesicles [EGTA quench (method 2)]. SR vesicles were preincubated in micromolar Ca2+ to form the E.2Cacyt intermediate of the Ca-ATPase, and then Ca2+ transport was initiated by addition of ATP. It was found that Ca2+ uptake measured by method 1 began with no lag phase, in spite of the prediction of kinetic models of the Ca-ATPase. Instead, the time course of Ca2+ uptake was found to have two components: a fast and a slow phase, similar to that obtained using method 2, although the rate constant of the fast phase determined by method 1 was considerably lower than that measured by method 2. The fast phase of Ca2+ uptake measured by method 1 was not influenced by either Ca2+ ionophore or detergent treatment, whereas the slow phase was diminished.(ABSTRACT TRUNCATED AT 250 WORDS)
Mészáros
L G
LG
Department of Physiology and Endocrinology, Medical College of Georgia, Augusta 30912.
Bak
J
J
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D000252:Calcium-Transporting ATPases / Q000737:chemistry; D007700:Kinetics; D009132:Muscles / Q000737:chemistry; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000737:chemistry* / Q000187:drug effects / Q000201:enzymology; D013053:Spectrophotometry
D000252:Calcium-Transporting ATPases; D002118:Calcium