Volume changes in high-affinity calcium binding of the sarcoplasmic reticulum calcium-transport enzyme.
The effect which hydrostatic pressure exerts on the hydrolysis of dinitrophenyl phosphate and nitrophenyl phosphate by the sarcoplasmic reticulum calcium-transport enzyme was determined. Activation volumes for substrate hydrolysis at saturating and non-saturating concentrations of calcium were determined and used to evaluate volume increments for initial calcium binding. A reaction scheme in which two unidirectional substrate-driven reactions transfer high-affinity into low-affinity calcium-binding sites was applied to determine binding-volume increments. It has been inferred from the pressure dependence of the volume-generating function, defined as the difference between the reciprocal reaction rates of the saturated and the unsaturated enzyme, that calcium binding proceeds in two steps. The two associated binding constants are endowed with large binding-volume increments of opposite signs (+84 to +207 ml/mol and -3 to -136 ml/mol). Under different experimental conditions, with respect to the temperature, degree of calcium saturation and absence or presence of Me2SO, they add up to the same integral volume increment of 73 +/- 3.5 ml/mol for the entry of two calcium ions into the reaction cycle. In aqueous media, the two binding constants contribute about equally to binding and to the observed binding-volume increment. The presence of Me2SO strongly favours the first binding step. The size of the integral volume increment is in line with that determined for the interaction of calcium with calmodulin [Kupke, D.W. & Dorrier, T.E. (1986) Biochem. Biophys. Res. Commun. 38, 199-204].
Stephan
S
S
Max-Planck-Institut für Medizinische Forschung, Abteilung Physiologie, Heidelberg, Federal Republic of Germany.
Hasselbach
W
W
D019297:2,4-Dinitrophenol / Q000031:analogs & derivatives*; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D004140:Dinitrophenols / Q000378:metabolism; D006868:Hydrolysis; D009596:Nitrophenols / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D011312:Pressure; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D004140:Dinitrophenols; D009596:Nitrophenols; D010755:Organophosphates; D009943:Organophosphorus Compounds; C047468:2,4-dinitrophenylphosphate; C008644:nitrophenylphosphate; D019297:2,4-Dinitrophenol; D002118:Calcium