3 Results and Discussion
3.1 D ETERMINATION OF THE BASAL ATP ASE ACTIVITY IN MYOSIN VI
This chapter investigates the basal kinetics of myosin VI ATPase as a quality-check of the protein preparations used throughout the study with myosin II S1 as control. Two different methods for measuring the ATPase rates of the myosins are introduced.
3.1.1 Generating myosin II S1 constructs for standard measurements
The head or motor domain of myosin is a highly specialised ATPase which converts the energy derived from the hydrolysis of ATP into mechanical work. Since myosin II and its ATPase activity are well described, it was used in this thesis as a standard for verifying and validating measurements. Myosin II S1 constructs were produced by papain digestion of myosin II HMM (see 2.2.15). Figure 38 summarises the digestion process and shows corresponding SDS-PAGE pictures of the resulting constructs.
Figure 38: Enzymatic digestion of myosin II to HMM and S1 fragments. Essential (ELC) and regulatory (RLC) light chains are depicted in red and yellow respectively. Gel pictures show examples of correspondent constructs.
Modified after Lodish (2001).
3.1.2 Generation and purification of myosin VI full-length construct
For ATPase measurements of myosin VI a full length construct (VI FL) was expressed using the Sf21 baculoviral expression system (see 2.1.12). The VI FL splice variant from chicken used in this thesis contains the large insert (LI) with no small insert (see 1.4).
Figure 39: Generation and purification of myosin VI FL protein. (A): schematic of myosin VI FL, (B): SDS-PAGE of elution fractions corresponding to C (C): elution curve from FPLC (Äkta, GE). Number gives percentage of His-high buffer in the elution mix (see 2.2.6 for further information).
Figure 39 shows a typical purification profile of VI FL protein with a SDS-PAGE of the eluted fractions. The protein preparations of myosin VI used in this study were checked for their stability and monomeric state using size exclusion chromatography (SEC). As figure 40 shows myosin VI ran as a single peak. Myosin II HMM (350 kDa) which is a dimer consisting of two motor domains, four IQ domains with four light chains and a short tail elutes at ~ 9 ml elution volume whereas myosin II S1 (106 kDa) with its single motor domain and one IQ with one light chain bound elutes at 12 ml elution volume. This proved that myosin VI was indeed a monomeric protein and never showed a shift in elution. The Stokes radius or Stokes-Einstein radius of a solute is the radius of a hard sphere that diffuses at the same rate as that solute. It is closely related to solute mobility, factoring in not only size but also solvent effects. A smaller ion with stronger hydration, for example, may have a greater Stokes radius than a larger but weaker ion. The Stokes radius of myosin VI is 6 nm, as figure 40 shows.
Figure 40: FPLC Superdex-200 prep grade gel filtration of myosin VI. (A) Typical elution profile of myosin VI on a Superdex 200 column. (B) Calibration curve of the Superdex 200 gel filtration column using standard proteins of known Stokes radii: (Ch) chymotrypsinogen A, 2.1 nm; (O) ovalbumin, 3.1 nm; (B) BSA, 3.6 nm; (A) aldolase, 4.8 nm, (C) catalse, 5.2 nm; (F) ferritin, 6.1 nm; (T) thyroglobulin, 8.5 nm. The elution position of myosin VI is shown, giving it a Stokes radius of 6 nm (modified after: Lister et al. (2004)).
3.1.3 ATPase activity of myosin II S1 and myosin VI
Actin-activated ATPase activity of both myosin VI and S1 was determined with 100 nM motor protein (different preparations, in KMg50 buffer for myosin VI and PIPES buffer for myosin II S1) using 0 to 30 µM F-Actin and 2 mM Mg∙ATP in a HPLC based assay as well as a NADH-coupled assay (for further details, see 2.3.2).
Figure 41: Representation and schematic of the (A): principle of NADH-coupled assay and (B): example of HPLC based assay. The two peaks represent the elution peaks for ADP and ATP respectively.
The NADH-coupled assay measures the ATP hydrolysis as a linked process. The ATP hydrolysis is linked to the oxidation of NADH, which is measured spectroscopically at 340 nm by its decrease in concentration. The coupling takes place via phosphoenol pyruvic acid (PEP), pyruvic acid kinase (PK) and lactate dehydrogenase (LDH) (figure
41, A). The HPLC based ATPase assay (figure 41, B) only requires ATP, actin and myosin and is therefore a more direct way of measuring the ATPase activity compared to the NADH-coupled assay. In this assay myosin VI was mixed with actin and very pure ATP. The reaction was then stopped at different points in time of incubation with HCl acid. These samples were then centrifuged to rid the samples of the precipitated protein and measured on the HPLC where the ADP derived from ATP hydrolysis through myosin ATPase activity could be measured.
3.1.4 Actin-dependent activation of myosin II S1
The actin-dependent ATPase rate is an essential value to assess the activity of myosins. In figure 42 a clear actin-activation of the myosin II S1 fragment could be seen for both assays. The ATPase rate increased with higher actin concentration. kcat measures the number of substrate molecules turned over by enzyme molecule per second. It is therefore called the turnover number. The basal rate without actin was 0.1 s-1. The measured maximum kcat was 4.2 s-1 (±0.7) in the NADH-coupled assay and 6.7 s-1 (± 0.5) with the HPLC method.
Figure 42: Actin-activated ATPase rate of myosin II S1. Filled circles represent average of 6 measurements with the NADH-coupled assay whereas empty circles represent average of 4 HPLC measurements. Both were performed with at least two different preps.
For the NADH-coupled assay that equalled a 50-fold increase and for the HPLC based assay even an 84-fold increase of ATPase rate. After fitting the curves with kinetic
hyperbola fits, the actin filament concentration at half-maximal activation of the steady-state ATPase was determined with KM-values of 29 µM (NADH-coupled) and 20.3 µM (HPLC based). Additionally vmax values of 8 and 13 s-1 were calculated respectively.
Although values for vmax were not reached in the experiments, the rates above 20 µM actin levelled off and higher concentrations did not change the maximal measured rates.
Due to the actin preparations available no higher concentrations than 30 µM could be tested.
3.1.5 Actin-dependent activation of myosin VI
The actin-dependent ATPase rates of myosin VI were used to measure the quality of myosin VI preparations. Figure 43 shows that the ATPase activity of myosin VI is actin-activated. A basal rate of 0.45 s-1 and maximum rates of 3.6 s-1 (± 0.04) (NADH-coupled) and 2.5 s-1 (± 0.4) (HPLC) respectively were measured which equalled an 8-fold increase for the NADH-coupled and a 5-fold increase for HPLC based measurements of ATPase rate.
Figure 43: Actin-activated ATPase rate of myosin VI. Filled circles represent average of 4 measurements with the NADH-coupled assay whereas empty circles represent average of 3 HPLC measurements. Both were performed with at least two different preps.
KM values of 25 (NADH coupled) and 29 µM (HPLC) were calculated as well as maximum velocities vmax of 6 s-1 for both assays. Again the rates levelled off at
concentrations higher than 20 µM actin and even higher concentrations did not change the maximal measured rates.
3.1.6 Discussion
The rates measured with the myosin-II S1 in this study are lower compared to the published rates, which show considerable variation ranging from 7.8 s-1 (Webb and Corrie 2001) over 18 s-1 (Kovács et al. 2004) to 29 s-1 (Trentham et al. 1976, Millar and Geeves 1983, Cremo and Hartshorne 2007). There are possible explanations to why the numbers show such variability: 1. used muscle types in preparations, 2. buffer conditions and 3. temperature. In previous publications the fast-twitch muscle (type II) m. psoas was used or a mixture of it and slow-twitch (type I) muscle m. soleus. We used a mixture of diverse muscles (e.g. m. quadriceps femoris, m. bizeps femoris, m. latissimus dorsae, m.
glutaeus maximus, m. longissimus, m. splenicus, m. trapezius), leaving out the above mentioned m.psoas and m. soleus. All muscle-types used are more or less a mixture of type I and type II fibres. Our muscle material came from an old male breeder in contrast to 1 year old males used in ATPase based studies in the literature which as well may have an influence on ATPase performance.
All assays were performed at room temperature, which had an influence on the ATPase rate as well. However, similar ATPase rates (kcat = 6 s-1) as Webb and Corrie (2001) (kcat=7.8 s-1) when using their buffer conditions (PIPES buffer) were obtained. This showed that differences in salt conditions and buffer substances as well as temperature have an influence on the obtainable maximum ATPase rates.
Calculated KM values of 20 µM to 29 µM were in the same range as published by Kovács et al. (2004) with 24 µM. Published ATPase rates for myosin VI range from 6 s-1 (De la Cruz, Ostap et al. 2001) to 8.3 s-1 (Buss and Kendrick-Jones 2007). KM values of
~18 µM (De la Cruz et al. 2001) were not reached in this study but it is most probable that the kcat of 3.6 s-1 obtained in this study were lower than the published rates, because of the lower temperatures used for our assays. All enzymes are sensitive to temperature, with different temperature optima. The optimal temperature for myosin VI seems to lie well above room temperature (21 °C), as will be shown later in chapter 3.3.7.2.