Active RAB7A, Member RAS Oncogene Family (RAB7A)

RAB7A is a ubiquitous small GTPase, which controls transport to late endocytic compartments. Silencing or overexpression of wild type RAB7A changed the soluble/insoluble rate of peripherin indicating that RAB7A is important for peripherin organization and function. Besides, disease-causing RAB7A mutant proteins bind more strongly to peripherin and their expression causes a significant increase in the amount of soluble peripherin. The altered interaction between disease-causing RAB7A mutants and peripherin could play an important role in CMT2B neuropathy. The activity of recombinant human RAB7A was measured by its ability to hydrolyze the substrate GTP to phosphate which was detected by the malachite green phosphate detection Kit (Beyotime # S0196M). The reaction was performed in the assay buffer 20 mM tris, 200 mM NaCl and 10% glycerol, pH 7.5, initiated by addition 100 μL of various concentrations of RAB7A (diluted by assay buffer) to 100 µL of 50 uM GTP. The final well serves as a negative control with no RAB7A, replaced with 100 μL assay buffer. Incubated at 37℃ for 5min, then add 70 ul phosphate detection agent and incubated at room temperature for 30min, read at a wavelength of 630 nm. The specific activity of recombinant human RAB7A is > 800 pmol/min/µg.