Purification and Characterization of a Polymorphic Equine Muscle Carbonic Anhydrase

Abstract

A polymorphic form of the equine muscle carbonic anhydrase (CA-III) was isolated from two kinds of horses, characterized and designated as CA-III_a. Immunologic reactivity, amino acid composition and molecular weight of CA-III_a were similar to those of CA-III. However, CA-III_a was immunochemically distinct from CA-1 or CA-II of equine blood carbonic anhydrase. The major differences between CA-III_a and CA-III were their electrophoretic mobilities, isoelectric points and specific activities. When CA-III_a was alkylated with iodoacetamide under non-denaturing condition, 2 mol of carboxymethylcysteine (CMC) was formed from one mol of the enzyme, suggesting that two cysteine residues is located on the surface of the molecule. The titrations of the enzymes with DTNB under denaturing condition showed that one mol of CA-III and CA-III_a contained 1.7 and 0.9 mol, respectively, of sulfhydryl group. Thg acidic components released from the purified CA-III_a had the amino acid composition of cysteic acid (0.63 nmol), glycine (0.85 nmol), and glutamic acid (1.56 nmol). The electrophoretic mobility of CA-III was not changed by mixing with reduced glutathione in vitro, suggesting that CA-III binds with glutathione in vivo and is converted into acidic form designated as CA-III_a.