Observation of ATP turnover during in vitro motility assays

Abstract

Recent advances in the development of in virro motility assays have allowed the force and displacement of single actomyosin interactions to be measured [l]. In order to calculate the step-size of the crossbridge cycle, and hence determine the nature of the mechanochemical coupling, it is also necessary to investigate the ATPase activity at an equivalent molecular sensitivity. Our initial studies have focused on myosin thick filaments, which contain several thousand myosin molecules (i.e. zeptomole levels), using an ATP derivative, FEDA-ATP, containing a fluorescein moiety attached to the ribose moiety by an ethylenediamine linker [2]. When single immobilized clam (Mercenuria) myosin filaments are perfused with 50 nM of FEDA-ATP, they fluoresce above the background because of the local high concentration of bound nucleotide and can be readily observed by epifluorescence microscopy. The nucleotide turnover rate can be measured by chasing with an excess of ATP. This method is suitable for determining the ATPase kinetics of myosin alone where the half times of displacement are several seconds or more, but it is insufficient to follow actin activation P I .To improve the time resolution of the chase we are using flash photolysis of caged ATP using a Xe flash lamp (Hi-Tech). To test for the generation of ATP under these conditions, we have initiated actin filament sliding in an in virro motility assay by this means. It appears that the unphotolysed caged ATP is a weak inhibitor of the ATPase [3], so that the velocity of sliding is about half that observed in a control assay with ATP. We are therefore maximising the percentage photolysis of the caged ATP by using a high NA condenser lens system for the photolysis light.were limited to low nucleotide concentrations (50 nM), comparable to the K, of the myosin ATPase, so as to minimise the background fluorescence [2]. Higher concentrations are desirable to near saturate the myosin sites and hence to maximise the signal, prior to the displacement by ATP. To reduce the fluorescence from the bulk solution we have turned to total internal reflectance fluorescence (TIRF) microscopy [4]. The FEDA-ATP bound to immobilised clam filaments is selectively excited using an argon ion laser @A88 nm) directed at a glancing angle to the cover slip. The beam is reflected at the glass-water interface so that only components within a few hundred nanometres of the interface are excited by the evanescent field.With this system, clam filaments are readily visualised even at 100 phi free FEDA-ATP (Fig. 1). However at these concentrations there may be a significant contribution from nonspecific binding as the polyphosphate moiety is known to interact with the filament backbone. Nevertheless, the method bodes well for displacement assays carried out at around 1 w concentrations of FEDA-ATP, which should be sufficient to saturate myosin filaments or heavy meromyosin tracks, prior to their interacting with actin. FEDA-ATP with rabbit skeletal myosin subfragments in solution[2] and shown that the ass...

Measurement of nucleotide exchange kinetics with isolated synthetic myosin filaments using flash photolysis

“…Conibeap; C.R. Bagshaw/FEBS Letters 380 (1996) [13][14][15][16] otides is likely to effect only the amplitude rather than the kinetics of observed intensity changes. The REDA-ADP-dissociation kinetics provide a test of the time resolution of the apparatus.…”

“…The finding that ATP can be released photolytically without damage to the catalytic activity of myosin is a necessary prerequisite for the general use of flash photolysis and bodes well for studies using caged fluorescent ATP analogs [14]. We have initiated the sliding of actin in in vitro motility assays with this apparatus [15], but this process is more susceptible to damage from the free radicals released during photolysis and inclusion of thiol reagents is essential. The main drawback to the use of caged-ATP is its competitive inhibition [13].…”

“…Further improvements could be achieved with the more favourable beam characteristics from a laser [10] and dichroic mirrors that reflect a maximal proportion of the incident light [4]. However, compared with the prism-based method [4,15], TIRF via the objective lens allows an optimal objective-to-specimen working distance as well as free access to the opposite surface for the photolysis beam.…”

“…This work is dedicated to the memory of Professor David Gingell who introduced us to total internal reflectance fluorescence microscopy [15]. mechanical disturbance occurring during flow and during a temporary pressure change in switching solutions.…”

Caged FEDA-ATP: a new tool in the measurement of ATP turnover during the in vitro motility assay

“…Total internal reflectance fluorescence microscopy offers one possibility around this problem [2]. An alternative strategy is to cage the fluorescein [ 31 to make a non-fluorescent derivative which can be photoactivated in localised regions containing the array of myosin heads.…”