BIOCHEMICAL-CHARACTERIZATION OF PURIFIED CARBONIC-ANHYDRASE FROM THE OCTOCORAL LEPTOGORGIA-VIRGULATA

Carbonic anhydrase (CA) has been isolated and purified from the octoco ral Leptogorgia virgulata (Lamarck) in an effort to investigate its ro le in the mineralization and demineralization of spicules and other ca lcified hard tissues. Affinity-chromatography using Prontosil-derivati zed carboxymethylcellulose (CM) Bio-Gel A provided a one-step purifica tion for 30 kdalton polypeptides with carbonic anhydrase activity. Fou r distinct polypeptides (designated alpha, beta, gamma, and delta) are separated from one another at this molecular weight by two-dimensiona l gel electrophoresis. The specific activity of the L. virgulata CA is on average 57.5 +/- 1.5 U g(-1), is inhibitable by 10(-6) M acetazola mide, and is unaffected by 5 mM dithiotheitol. The amino acid composit ion of these polypeptides is similar to that of mammal, bird, reptile, fish and arthropod species. Antiserum made against the L. virgulata C A reacts specifically with the 30 kdalton polypeptides in western blot s, and crossreacts with human CA I and II. Antiserum against avian CA II crossreacts with the L. virgulata 30 kdalton polypeptides. This is the first report of the characterization of a purified CA from an octo coral, and production of a CA antiserum to a species in the phylum Cni daria.