Isolation, purification and characterization of an insect carbonic anhydrase
1992
Burt, E. | Darlington, M.V. | Graf, G. | Meyer, H.J.
Carbonic anhydrase (carbonate hydro-lyase: EC4. 2. 1. 1) was purified and partially characterized from post-feeding larvae of the face fly, Musca autumnalis DeGeer. Analytical polyacrylamide gel electrophoresis (PAGE) of the enzyme purified by affinity chromatography showed the presence of three bands. Band-B appeared as a minor component which was lost during subsequent purification steps. Band-A and band-C appeared as major proteins and were separated using PAGE. Isoelectric points of 6.4 and 5.4 were estimated for band-A and band-C, respectively, by chromatofocusing. SDS-PAGE of the purified proteins produced single bands each having a mol. wt of approx. 32,000. The native molecular weight of these bands, estimated by gel filtration chromatography, was 31,000. The absence of inhibition by omicron-iodosobenzoate and rho-chloromercuribenzoate suggested that reduced sulfhydryl groups were not essential to maintain high activity. A Km of 13.1 mM was determined for the dehydration reaction and is of the same order of magnitude as K(m)s reported for the high activity forms studied in mammals. The amino acid composition of the enzyme was determined by reversed phase HPLC. Zn content was determined by atomic absorption spectroscopy.
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