The Use of Fast Kinetic Systems for the Study of ng-μg Quantities of Purified 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 (several mg) 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 been developing methods which would allow fast transients to be studied on the much smaller quantities of protein available from individual drosophila muscles, single mammalian muscle fibres, human biopsies and non-muscle myosins.
The use of fluorescent labels on actin, myosin or ATP can be used in modern stopped-flow equipment at concentrations as low as 10 nM. These labels can report on the interactions between the three molecules using a few μ;g of protein. However the stopped flow systems requires large volumes of sample to give sufficient acceleration of the sample to achieve good mixing. We have therefore been using a similar optical set up but initiating the reaction using caged ATP. In this device the same measurements can be made using 10–20 fold less material. This is the quantity of myosin which can be purified from a few mm of a single mammalian muscle fibre. Using these methods the following measurements can be made: rate of ATP induced dissociation of actomyosin, the affinity of ADP for actomyosin, the rate of ADP release from actomyosin and the affinity of actin for myosin and myosin ADP.
KeywordsIndirect Flight Muscle Actin Concentration Light Scattering Signal Fast Transient Stop Flow System
1-(2-nitrophenyl)ethyl phosphate ester of ATP
actin labelled at csy374 with pyrene iodoacetamide
the first 761 amino acids of cytoplasmic myosin II from Dictyostelium discoideum
myosin subfagment 1
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- 8.Cremo C. R. and Geeves, M. A. Biochem. in press.Google Scholar