A new method for the time-resolved measurement of phosphate release in permeabilized muscle fibers.
A new method for the measurement of phosphate release in contracting and relaxed permeabilized muscle fibers is described. The assay is based on a genetically engineered phosphate-binding protein labeled with a coumarin fluorescent probe, which binds inorganic phosphate tightly and shows a fourfold increase in fluorescence upon binding. Measurements of Pi release on the millisecond time scale with sensitivity in the 10 microM range are obtained that provide new information about the relationship between ATP hydrolysis and force production.
Ferenczi
M A
MA
National Institute for Medical Research, Mill Hill, London, United Kingdom.
He
Z H
ZH
Chillingworth
R K
RK
Brune
M
M
Corrie
J E
JE
Trentham
D R
DR
Webb
M R
MR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002352:Carrier Proteins / Q000378:metabolism; D003374:Coumarins; D005456:Fluorescent Dyes; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D009119:Muscle Contraction / Q000502:physiology; D009126:Muscle Relaxation / Q000502:physiology; D009132:Muscles / Q000378:metabolism*; D010539:Permeability; D028044:Phosphate-Binding Proteins; D010710:Phosphates / Q000378:metabolism*; D011817:Rabbits
D002352:Carrier Proteins; D003374:Coumarins; D005456:Fluorescent Dyes; D028044:Phosphate-Binding Proteins; D010710:Phosphates; C088410:N-(2-(1-maleimidyl)ethyl)-7-(diethylamino)coumarin-3-carboxamide; D000255:Adenosine Triphosphate