Effect of Inorganic Phosphate on the Rate of ADP Release During Ramp Shortening in Activated Permeabilized Fibers from Rabbit Psoas Muscle

Abstract

A coumarin-labeled recombinant phosphorylated nucleoside diphosphate kinase (P∼NDPK-IDCC; West et al., 2009; Biophys.J. 96:3281-3294), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during steady shortening of single permeabilized rabbit psoas fibers at 12°C (pCa 4.5, pH 7.1, ATP 5.7mM). A fiber contracted from the relaxed state by immersion into a Ca2+ activation solution at 0°C. Temperature activation was then initiated by immersion of the fiber into silicone oil at 12°C. The activation solutions were prepared with either zero added Pi or with 10 mM added Pi at constant ionic strength (0.15 M). The decline in fluorescence intensity emission (470nm) associated with MgADP-dependent dephosphorylation of P∼NDPK-IDCC (60 μM) was monitored during activation and during a period of isovelocity shortening. Fluorescence emission (580 nm) of a rhodamine dye was measured simultaneously to correct the P∼NDPK-IDCC signal for the effects of fiber movements and volume changes. The rate of MgADP release in the absence of added Pi increased from 0.7 ± 0.07 mM·s−1 at 0.2 fiber-lengths·s−1 to approximately 3.4 ± 0.25 mM·s−1 for shortening velocities between 1 and 2 fiber-lengths·s−1. When 10 mM Pi was added, the rate of ADP release at 0.2 fiber-lengths·s−1 was 0.48 ± 0.05 mM·s−1 and 2.6 ± 0.4 mM·s−1 at 1-2 fiber-lengths·s−1. In the absence of added Pi, the rate of ATP hydrolysis calculated from the appearance of ADP is similar to that calculated previously from the appearance of Pi using MDCC-labeled phosphate binding protein, over the same range of fiber shortening speeds (He et al., 1999, J. Physiol. 517: 839-854). Thus Pi slows the ATPase rate by 25-30%, both in the isometric and isotonic state. The energetic consequences will be discussed.