A flash photolysis fluorescence/light scattering apparatus for use with sub microgram quantities of muscle proteins.
Transient kinetic methods such as stopped flow and quenched flow have been used to elucidate many of the fundamental features of the molecular interactions which underlie muscle contraction. However, these methods traditionally require relatively large amounts of protein (10(-3) g) and so have been used most effectively for the proteins purified from bulk muscle tissue of large animals or where the proteins can be expressed in large amounts (e.g.. Dictyostelium). We have investigated the use of flash photolysis of an inert precursor of ATP (cATP) to initiate the dissociation of acto.S1 and acto.myosin and the subsequent ATP turnover reaction. Using a sample volume of 10 microl we show that a significant amount of information on the transient and steady-state kinetics of the system can be obtained from a sample containing just 50 nM of acto.myosin or acto.S1 complex in solution. Therefore in presence of excess of one protein component the measurements require only 250 ng myosin, 62 ng S1 or 25 ng actin. This is therefore the method of choice for kinetic analysis of acto.myosins which are only available in microgram quantities. We report for the first time the determination of the second order rate constant of ATP-induced dissociation of actin from the myosin extracted from a single fibre from a rabbit psoas muscle.
Weiss
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S
Research School of Biosciences, University of Kent, Canterbury, Kent, UK.
Chizhov
I
I
Geeves
M A
MA
D000199:Actins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000205:Actomyosin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D007700:Kinetics; D007834:Lasers; D009119:Muscle Contraction / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000378:metabolism; D009124:Muscle Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015879:Myosin Subfragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010775:Photic Stimulation / Q000295:instrumentation / Q000379:methods*; D010782:Photolysis; D011817:Rabbits
D000199:Actins; D009124:Muscle Proteins; D015879:Myosin Subfragments; C029273:P(3)-1-(2-nitro)phenylethyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin