1. INTRODUCTION
Motor proteins participate in various processes in the cell such as: transporting organelles, chromosome segregation, and muscle fiber contraction. Motor proteins bind to the microtubules filaments and walk along it using the energy from Adenosine triphosphate (ATP) hydrolysis. One of the motor proteins is called kinesin. Kinesin is a motor protein with two heads (motor domain), α-helical coiled coil that bind to the cargo. A single kinesin molecule moves along a single profilament of a microtubule at a velocity proportional to the ATP concentration [1]. At low ATP concentration kinesin molecules travel sufficiently slowly to observe individual steps. Each step is 8nm length.
Kinesin motion is highly processive. The heads of kinesin can only attach to the microtubules in a specific way- from the minus to the plus ends, and walk in this direction. Single-molecule measurement allows measuring processive motion. It takes a single kinesin molecule 100 or more steps toward the plus end of the microtubule [2]. It is not yet clear if this holds for fluorescent ATP. Accordingly we study the following questions :
- Checking if kinesin can hydrolyze fluorescent ATP
- Studying if the speed of kinesin is dependant on the ATP concentration, and if we can determine difference between normal and fluorescent ATP.
In this report we observe gliding assay with kinesin and fluorescent ATP.
1.1 ATP ATP is the major energy of the cell providing energy for most of the energy-consuming activities of the cell. It plays a central role in the energetic of the cells in both the transduction mechanisms and the metabolic pathways. Motor proteins bind to the filaments inside the cells and walk along them using the energy derived from ATP (Adenosine triphosphate).
1.2 FLUORESCENT ATP Fluorescent ATP is labeled by Cy3-EDA fluorophor. This structure of fluorophor is connected to the phosphate group of ATP structure.
1.3 ATP REGENERATION SYSTEM We are using a regeneration system for low ATP concentrations because it regenerates ATP from ADP and keeps ATP concentration constant. ATP regeneration system contents two enzymes: phospocreatine and creatine kinase. The phosphate group in creatine phosphate is attached by a "high-energy" bond in the same way as it is done in ATP. Creatine phosphate derives its high-energy phosphate from ATP and can donate it back to ADP to form ATP. Creatine phosphate + ADP --> creatine + ATP In our experiments we use the following two enzymes: Phospocreatine 10 mM = 500 mM stock in PEM12
Creatine kinase 50µg/ml =2.5 mg/ml in PEM12
It is necessary to use oxygen scavenger system for fluorescence assay the necessary.
1.4 OXYGEN SCAVENGER SYSTEM We use assay oxygen scavenger to limit photodemage of fluorescent. The most convenient method for that is to use catalase, glucose, and glucose oxidase.
1.5 MICHAELIS MENTEN EQUATION Michaelis and Menten [4] proposed a scheme for enzyme that catalyzes the S substrate to P product P from S that is widely used in modeling biological behavior. Enzyme E binds S to form enzyme-substrate complex ES [4]. The ES complex has two has two possible fates. It can dissociate to E and S with rate k-1, or it can proceed to form product P, with rate constant k2, The terms k1, k-1 and k2 are rate constants for the association of substrate and enzyme. The extent of product formation is determined as a function of time for a series of substrate concentration [2].
In our project enzymes correspond to the proteins that bind to the substrate that corresponds to ATP. Then the ES complex catalyzes the kinesin hydrolyze ATP reaction. The Michaelis-Menten equation can be solved with the following solution.
V = Vmax[S] / (Km + [S])
The equation told us that at very low substrate concentration when S is much less than Km. V is equal to (Vmax/Km)/[S], and these rate is proportional to the substrate concentration. At high concentration of S, V is equal to the Vmax ,rate is maximal, independent of substrate concentration. When [S]=Km, than V=Vmax/2 . That way Km is equal to the substrate concentration at which the rate is half its maximal value. Km is an important characteristic of enzyme-catalyzed reaction and is significant for is biological function [2].