Product Uses Include
Kinetic measurement of microtubule activated kinesin ATPase activity
Discovery of kinesin inhibitors
Measurement of kinesin inhibitor IC50s
Introduction
Eukaryotic kinesin motor proteins orchestrate a wide range of kinetic events within a cell. They have been shown to move cargoes, such as chromosomes and vesicles, along microtubule tracks (1). They also play a major role in the organization of cytoskeletal architecture as evidenced in the establishment of the microtubule spindle during mitosis (2).
Kinesins operate by utilizing the energy of ATP hydrolysis to move along their microtubule (MT) substrates. Once a kinesin motor binds to its MT track, the ATPase rate of the motor is often enhanced several hundred to several thousand fold (3). MT activated kinesin ATPase is a major parameter in motor function and serves as a powerful method to monitor and study kinesin activity under various experimental conditions.
As part of its Cytoskeleton Motor Werks (CMW) line of research reagents, Cytoskeleton, Inc. has developed the Kinesin ELIPA™ (Enzyme Linked Inorganic Phosphate Assay) Biochem Kit. The assay is an adaptation of a method originally described by Webb for the measurement of glycerol kinase plus D-glyceraldehyde ATPase activity and for actin activated myosin ATPase (4). The assay is based upon an absorbance shift (330 - 360 nm) that occurs when 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) is catalytically converted to 2-amino-6-mercapto-7-methyl purine in the presence of inorganic phosphate (Pi). The reaction is catalyzed by purine nucleoside phosphorylase (PNP). One molecule of inorganic phosphate will yield one molecule of 2-amino-6-mercapto-7-methyl purine in an essentially irreversible reaction (5). Thus, the absorbance at 360 nm is directly proportional to the amount of Pi generated in the kinesin ATPase reaction.
End point assays for kinesin MT activated ATPase activity in high throughput format can be performed with Biochem Kit™ BK053. Cytoskeleton, Inc. also offers a wide range of kinesin motors (see the Cytoskeleton Motor Werks page).