Time course and strain dependence of ADP release during contraction of permeabilized skeletal muscle fibers.
A phosphorylated, single cysteine mutant of nucleoside diphosphate kinase, labeled with N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide (P approximately NDPK-IDCC), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during contraction of single permeabilized rabbit psoas fibers. The dephosphorylation of the phosphorylated protein by MgADP occurs within the lattice environment of permeabilized fibers with a second-order rate constant at 12 degrees C of 10(5) M(-1) s(-1). This dephosphorylation is accompanied by a change in coumarin fluorescence. We report the time course of P approximately NDPK-IDCC dephosphorylation during the period of active isometric force redevelopment after quick release of fiber strain at pCa(2+) of 4.5. After a rapid length decrease of 0.5% was applied to the fiber, the extra NDPK-IDCC produced during force recovery, above the value during the approximately steady state of isometric contraction, was 2.7 +/- 0.6 microM and 4.7 +/- 1.5 microM at 12 and 20 degrees C, respectively. The rates of P approximately NDPK-IDCC dephosphorylation during force recovery were 28 and 50 s(-1) at 12 and 20 degrees C, respectively. The time courses of isometric force and P approximately NDPK-IDCC dephosphorylation were simulated using a seven-state reaction scheme. Relative isometric force was modeled by changes in the occupancy of strongly bound A.M.ADP.P(i) and A.M.ADP states. A strain-sensitive A.M.ADP isomerization step was rate-limiting (3-6 s(-1)) in the cross-bridge turnover during isometric contraction. At 12 degrees C, the A.M.ADP.P(i) and the pre- and postisomerization A.M.ADP states comprised 56%, 38%, and 7% of the isometric force-bearing AM states, respectively. At 20 degrees C, the force-bearing A.M.ADP.P(i) state was a lower proportion of the total force-bearing states (37%), whereas the proportion of postisomerization A.M.ADP states was higher (19%). The simulations suggested that release of cross-bridge strain caused rapid depopulation of the preisomerization A.M.ADP state and transient accumulation of MgADP in the postisomerization A.M.ADP state. Hence, the strain-sensitive isomerization of A.M.ADP seems to explain the rate of change of P approximately NDPK-IDCC dephosphorylation during force recovery. The temperature-dependent isometric distribution of myosin states is consistent with the previous observation of a small decrease in amplitude of the P(i) transient during force recovery at 20 degrees C and the current observation of an increase in amplitude of the ADP-sensitive NDPK-IDCC transient.
West
Timothy G
TG
Imperial College London, Molecular Medicine Section, National Heart and Lung Institute, London, United Kingdom.
Hild
Gabor
G
Siththanandan
Verl B
VB
Webb
Martin R
MR
Corrie
John E T
JE
Ferenczi
Michael A
MA
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003198:Computer Simulation; D003374:Coumarins; D005260:Female; D005453:Fluorescence; D007700:Kinetics; D016014:Linear Models; D009119:Muscle Contraction / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D053580:Muscle Strength; D020125:Mutation, Missense; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D016658:Psoas Muscles / Q000378:metabolism; D011817:Rabbits
D003374:Coumarins; D020033:Protein Isoforms; D000244:Adenosine Diphosphate; C030123:coumarin; D009701:Nucleoside-Diphosphate Kinase; D002118:Calcium