A number of elongation factor-2 kinase (eEF-2K)

No abstract

The interaction of fluorescent ATP analog 2'(3')-O-[N-[2-[3-(5-fluoresceinyl)thioureido]-ethyl]carbamoyl]adenosine 5'-triphosphate (FEDA-ATP) with rabbit skeletal myosin subfragment 1 (S1) and acto-S1 was studied. This and related ATP analogs are potentially useful for determination of the ATPase activity of single myosin filaments using fluorescence microscopy [Sowerby et al. (1993) J. Mol. Biol. 234, 114-123]. However, it is necessary that such analogs mimic ATP in their kinetics of turnover. The apparent second-order association rate constants for FEDA-ATP binding to S1 and for FEDA-ATP-induced dissociation of acto-S1 are about 4 times slower than those for ATP. As with ATP, the hydrolysis step is fast, so that the M.FEDA-ADP.P(i) complex is the major steady-state intermediate. The turnover rate is the same for the 2' and 3' FEDA-ATP derivatives and similar to that of ATP itself. The dissociation rate constant for FEDA-ADP from S1 is identical to that for ADP. Actin-activated turnover is comparable for both FEDA-ATP and ATP. The corresponding rhodamine and sulfoindocyanine, Cy3.18 (Cy3), derivatives also appear to be reasonable analogs. FEDA-ATP binding leads to a 25-40% reduction in fluorescein fluorescence. Spectral properties of the bound nucleotide were explored by trapping FEDA-ADP as its aluminum fluoride complex. The fluorescence quenching is a consequence of a reduction in both absorbance and excited-state lifetime, but there is little change in spectral shape.

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...