Heavy Meromyosin Protein (HMM Fragment)

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Measurement of microtubule-activated ATPase/GTPase assays
Identification/characterization of proteins or small molecules that affect motor ATPase/GTPase activity
Identification/characterization of proteins or small molecules that affect motor/microtubule interactions.
The conserved motor domain of human kinesin KIF7 was expressedin E.coli. The recombinant protein contains a 6 histidine tag at the amino terminal end and has a combined molecular weight of 44 kDa. The protein has been determined to be biologically active in a microtubule-activated ATPase/GTPase activity test (see below). The protein is supplied as a lyophilized powde

The reactions were conducted in 96 well plates (300 ul reaction volumes). Each reaction contains 5 ug of Kif7 protein (EG01), 0.7?M taxol stabilized microtubules (Cat. # MT001), 0.2 mM MESG, 0.3U PNP (purine nucleotide phosphorylase), 15 ?M taxol, 15 mM PIPES pH 7.0, 5 mM MgCl2, 0.6 mM ATP. Control reactions were carried out in the absence of KF51. These reactions gave readings of < 0.1 OD over 20 min. Reactions were measured in a SpectraMax 250 (Molecular Devices) set in kinetic mode and 360nm absorbance wavelength. Readings were taken at room temperature once every 30 seconds for a total reaction time of 20 minutes. Typical assay results are shown in Figure 2. The nmoles of Pi generated in a given time was determined by the use of a phosphate standard curve (not shown).
Kif7 ATPor GTPase activity was assayed in duplicate according to the method described. Control reactions without microtubules (open symbols) were compared to those with microtubules (closed symbols).

KIF7 ATPase and GTPase activities were measured by monitoring real time free phosphate generation using the Kinesin ELIPA Assay Kit (Cat. #BK060). The assay is based upon an absorbance shift (330 nm to 360 nm) that occursWhen 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) is catalytically converted to 2-amino-6-mercapto-7-methylpurine in the presence of inorganic phosphate (Pi). One molecule of Pi will yield one molecule of 2-amino-6-mercapto-7-methylpurine in an essentially irreversible reaction. Hence, the absorbance at 360 nm is directly proportional to the amount of Pi generated in the kinesin ATPase/GTPase reaction. Under the conditions outlined below, the Vmax for Kif7 microtubule-activated ATPase activity was 40 nmoles Pi generated per minute per mg of Kif7 and for the GTPase activity it was 230 nmoles Pi generated per min per mg (Figure 2).

Figure 2. KF51 microtubule-activated ATPase/GTPase activityusing the Kinesin ELIPA Assay Kit (Cat. # BK060). Kif7 ATPor GTPase activity was assayed in duplicate according to the method described. Control reactions without microtubules (open symbols) were compared to those with microtubules (closed symbols).