Various Ways to Explore Processivity of Eg5 Motor Protein cd.

2. MATERIALS AND METHODS

2.1 DIC Microscopy

We use the DIC Microscopy to visualize the microtubules moving on the protein. In the Microscopy the light from the lamp passes through a polarizer, and then the light beam is split into two polarized rays by a Wollaston prism, and as a result the two beams are traveling in different directions. The prism consists of two halves parts with orthogonal optical axes (named ordinary and extraordinary wave fronts). The beams vibrate perpendicular to each other that prevents them from interfering. Than the separated beams pass through the condenser, the sample, and the objective. When the two beams pass through the sample their paths are altered due to the sample's varying thickness, slopes, and refractive indices (that induce a phase offset between the two rays) [4]. After the objective, the two beams are focused in another Wollaston prism. It removes the path difference between the two beams. As a result the two previously orthogonally polarized rays interfere by projecting their polarizations onto the original direction in analyzer. DIC technique is capable of imaging an object that is as small as the microtubules by enhancing the contrasts to detect separate motor step.

2.2 In Vitro Motility Assays

The study of motor protein was revolutionized by the development of in vitro motility assay in which the motility of purified motor proteins along purified cytoskeletal filaments is reconstituted in the cell free conditions [2]. There are two geometries used in in vitro motility assay. First the bead assay and gliding assays. At these work gliding assays were performed with Differential Interference Contrast microscopy.

In gliding assays, the motor themselves are fixed to the substrate, and the filaments are observed to diffuse down from solution, attach to, and glide along motor-coated surface [3].

2.3 Velocity Measurement

Eg5 velocity was measured with the surface motility method. The DIC Microscopy was connected to a camera and a computer that record the images of moving microtubules as bitmaps of 740 x 576 pixels with the time marked on them. These pictures allow calculating velocity of the microtubules. The method was to measure the vertical and horizontal position of microtubule's end. For these calculations Pythagoras law was used:

x2-x1 – distance between microtubule’s end in two pictures in axis scale
y2-y1 – distance between microtubule’s end in two pictures in vertical scale


where:
z - distance that microtubule move
t - time of movement, taken form the pictures
The distances were measured in pixels in Corel Photo Paint 9, then scaling μm with using calibration factor. The error of measuring the end of microtubule was estimated. This error it's partly because of little nonlinear movement of microtubules, partly because of the difficulties in finding the exact place of the microtubule's end.

2.3 Michaelis-Menten Equation

The biomolecular equation is very important in biochemistry. In enzyme kinetics we can notice that


E - enzyme
S - substrate
ES - intermediate state that breaks down into enzyme plus product
The steady state of product formation is:


This is the Michaelis-Menten equation and KM is the Michaelis-Menten constant. kcat is the maximum rate per enzyme molecule and KM is the concentration of substrate for which the rate is half maximal. If ES is in equilibrium with E and S. (k-1>>k2), than the reaction is said to follow a Michaelis-Menten kinetic mechanism [3].