Purification of cochlear carbonic anhydrase

Abstract

Carbonic anhydrases appear to function in the excretion of CO2 and secretion of K*. To characterize cochlear carbonic anhydrase at the molecular level, I have purified this enzyme from membranous lateral wall (stria vascularis, spiral ligament, spiral prominence, and part of the outer sulcus) of the inner ear of guinea pig. To my knowledge, this represents the first purification of a cochlear enzyme. Membranous lateral walls from 60 cochleae were dissected at 2°C in 100 mM Na2SO4 + 10 mM TRIS sulfate pH 7.4, homogenized, centrifuged, and the supernatant was applied to a column of Bio-Rad P-100 resin. Since the cochlear enzyme appears to be inhibited by chloride, sulfate was used throughout. The initial gel filtration showed that the enzyme had a molecular weight of about 30 000 and that at least 98% was not associated with larger macro-molecules. Absorbance at 410 nm indicated that contamination by blood accounted for less than 4% of the enzyme activity. Further purification by DEAE agarose chromatography and gel filtration with Bio-Rad P-60 resin resulted in one active peak, suggesting one major charged species. This study indicated that 2% (2–3 μg/dissected cochlea) of the protein of the membranous lateral wall is carbonic anhydrase. [Supported by Intramural Research Program, NIH]