Transmembrane gradient and ligand-induced mechanisms of adenosine 5'-triphosphate synthesis by sarcoplasmic reticulum adenosinetriphosphatase.
A series of experiments was performed in order to characterize ATP formation by sarcoplasmic reticulum adenosinetriphosphatase (ATPase). Comparative measurements were obtained by using native and leaky vesicles, in the presence and in the absence of a transmembrane Ca2+ gradient. ATP formation was started by addition of ADP to phosphoenzyme obtained by preincubation with acetyl phosphate and Ca2+ or by addition of ADP and Ca2+ to phosphoenzyme obtained by preincubation with inorganic phosphate (Pi) in the absence of Ca2+. Transient-state measurements were carried out to obtain a kinetic characterization of phosphoenzyme formation following addition of ATP to enzyme preincubated with Ca2+ (10(2) s-1) in the forward direction of the cycle and for ATP formation following addition of ADP to the phosphoenzyme-calcium complex (3 X 10(2) s-1) in the reverse direction of the cycle. The rate constants of ATP association (4.5 X 10(6) M-1) with and dissociation (50 s-1) from the catalytic site were also obtained. A slow (kapp = 20 s-1) step for ATP formation was observed when millimolar Ca2+ and ADP were added to phosphoenzyme obtained with Pi. This demonstrates a transition of this phosphoenzyme to a rapidly reactive state, before the occurrence of phosphoryl transfer to ADP. A match of the ATP hydrolysis and Ca2+ gradient potentials is consistent with ATP formation in the presence of a Ca2+ gradient but does not explain ATP formation in the absence of a gradient. A formulation is then introduced considering all the equilibrium constants for the partial reactions of the ATPase cycle.(ABSTRACT TRUNCATED AT 250 WORDS)
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000818:Animals; D007700:Kinetics; D008024:Ligands; D009132:Muscles / Q000201:enzymology; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D008024:Ligands; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases