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).
Kit contents
The kit contains sufficient material for 96 assays. The following components are included:
- Reaction buffer
- ELIPA reagent 1 (MESG)
- ELIPA reagent 2 (PNP)
- Kinesin Heavy Chain motor domain protein (Cat. # KR01)
- Pre-formed microtubules (Cat. # MT002)
- Paclitaxel (Cat. # TXD01)
- Phosphate standard
- ELIPA reagent 1 resuspension buffer
- ATP stock (Cat. # BSA04)
- GTP stock (Cat. # BST06)
- Manual with detailed protocols and extensive troubleshooting guide
Equipment needed
- 96-well plate spectrophotometer capable at reading 360-370 nm. Monochromatic (360 nm) or filter based with narrow bandwith (370 nm filter with no more than 10 nm bandwith)
Example results
The microtubule activated ATPase activity of Kinesin Heavy Chain (Cat. # KR01), CENP-E (Cat. # CP01), Eg5 (Cat. # EG01) and Chromokinesin (Cat. CR01) was measured with BK060 (Fig 1).
Figure 1. Microtubule activated kinesin ATPase activity as measured with BK060. The ATPase activities of a selection of kinesin motors was measured. The Vmax for ATP hydrolysis ranges from 10,000 nmol ATP/min/mg for Kinesin HC to 470 nmol ATP/min/mg for Chromokinesin.
References
- Vale, R. D. and Fletterick, R. J. (1997). The design plan of kinesin motors. Ann. Rev. Cell Dev. Biol. 13: 745-777.
- Endow, S. A. (1999). Microtubule motors in spindle and chromosome motility. Eur. J. Biochem 262, 12-18.
- Johnson, K. A. and Gilbert, S. P. (1995). Pathway of the microtubule-kinesin ATPase. Biophys. J. 68, 173-179.
- Webb, M. R. (1992). A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc. Natl. Acad. Sci. USA 89, 4884-4887.
- Cheng Q., Wang Z-X., and Killilea S. D. (1997). A continuous spectrophotometric assay for protein phosphatases. Anal. Biochem. 226, 68-73.
For product Datasheets and MSDSs please click on the PDF links below. For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com
Question 1: Which kinesin motor proteins can be used in the Kinesin ELIPA Biochem Kit (Cat # BK060)?
Answer 1: The Kinesin ELIPA Biochem Kit (Cat. # BK060) can be used with any of the other kinesin motor proteins sold by Cytoskeleton, Inc. Please see the protein’s datasheet on our website (https://www.cytoskeleton.com/pdf-storage/datasheets/bk060.pdf) for representative data detailing each motor protein’s ATPase activity using either this kit (Cat. # BK053) or our Kinesin ELIPA Biochem Kit (Cat. # BK060).
Question 2: Are there specific requirements of the spectrophotometer that I will use to measure the inorganic phosphate production with the Kinesin ELIPA Biochem Kit (Cat # BK060)?
Answer 2: The assay is based upon an absorption shift from 340 nm to 360 nm. It is therefore very important to use a spectrophotometer that has a narrow bandwidth in order that the wavelength for reading the assay does not encroach upon the 340 nm range. It is recommended that a monochromatic spectrophotometer such as a SpectroMax 350 (Molecular Devices) be used when possible as the bandwidth in these machines is very narrow (2-5 nm). If a filter based system is being used then it is important to make sure that the filter bandwidth is 10 nm or less, if not the result will be a) significant background noise and b) greatly reduced sensitivity of the assay. The spectrophotometer should be at room temperature and set on kinetic mode and it is recommended to take a reading once every 30 seconds. There is no need to elect a blank well as the reaction minus motor will serve as a background control. Do not preread the microtiter plate. Start the kinetic readings at time zero.
