Active Carbonic Anhydrase VIII (CA8)

Carbonic Anhydrase (CA) catalyzes the reversible reaction of CO2  H2O = HCO3-  H , which is fundamental to many processes such as respiration, renal tubular acidification and bone resorption. CA8, also called CArelated protein (CARP), is a cytosolic protein without CA activity (i.e., the reversible hydration of CO2) due to point mutations in the zinc-binding site. CA8 is expressed exclusively in Purkinje cells of the cerebellum, where it binds inositol 1,4,5-triphosphate receptor type 1. CA8 overexpression in human colorectal cancer and non-small cell lung cancer indicates that it plays a role in the process of invasion in these types of malignancy. The activity of recombinant human CA8 was measured by its ability to hydrolyze 4-Nitrophenyl acetate (4-NPA) to 4-Nitrophenol. The reaction was performed in 12.5 mM Tris, 75 mM NaCl, pH 7.5 (assay buffer), initiated by addition 50 μL of various concentrations of CA8 (diluted by assay buffer) to 50 µL of 2 mM substrate 4-NPA (100 mM stock in Acetone, diluted by assay buffer). Incubated at 37℃ for 5min, then read at a wavelength of 400 nm.