Kinetic and thermodynamic control of ATP synthesis by sarcoplasmic reticulum adenosinetriphosphatase
Several experimental parameters, critical to the analysis of ATP synthesis by sarcoplasmic reticulum ATPase, were determined experimentally. 1) The phosphorylated enzyme intermediate obtained with acetylphosphate in the presence of a Ca/sup 2 +/ gradient was shown to be entirely ADP sensitive but quite stable in the absence of added ADP. On the contrary, the phosphoenzyme obtained with ATP is unstable due to the ADP formed during the phosphoryl transfer reaction. For this reason, addition of ADP to (/sup 32/P)phosphoenzyme obtained with (/sup 32/P)acetylphosphate provides the simplest conditions for kinetic studies on (gamma-/sup 32/P)ATP synthesis. 2) The dissociation rate constant of newly synthesized ATP (in the reverse direction of the ATPase cycle) was measured experimentally and found to be 16 s-1. This value agrees well with the dissociation rate constant determined for adenyl-5'-yl imidodiphosphate bound to this enzyme. 3) ATP synthesis observed in the absence of a Ca/sup 2 +/ gradient was shown to be a kinetic overshoot due to ligand-induced perturbation of a limited number of partial reactions and occurring before equilibration of the entire system. Most of the ATP formed under these conditions was subsequently hydrolyzed as the overall equilibrium was reached. 4) Based on these and other (previously characterized) parameters, satisfactory simulations of single and multiple cycle ATP synthesis, in the presence and in the absence of a Ca/sup 2 +/ gradient, were obtained.
- Research Organization:
- Univ. of Maryland School of Medicine, Baltimore
- OSTI ID:
- 5630311
- Journal Information:
- J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 262:27; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ADP
ALKALINE EARTH METAL COMPOUNDS
ANIMALS
ATP
ATP-ASE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOSYNTHESIS
CALCIUM COMPOUNDS
CELL CONSTITUENTS
DAYS LIVING RADIOISOTOPES
ENZYME ACTIVITY
ENZYMES
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
MUSCLES
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
OXYGEN COMPOUNDS
PHOSPHATES
PHOSPHOHYDROLASES
PHOSPHORUS 32
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
RABBITS
RADIOISOTOPES
REACTION KINETICS
SARCOPLASMIC RETICULUM
SYNTHESIS
THERMODYNAMICS
TRACER TECHNIQUES
VERTEBRATES