Properties of Carbonic Anhydrase Isozymes Isolated from Porcine Erythrocytes

Robert J Tanis,Richard E Tashian,Ya-Shiou L Yu

doi:10.1016/s0021-9258(18)62654-7

Robert J Tanis, Richard E Tashian + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)62654-7

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Abstract

Abstract Two major components of carbonic anhydrase were purified from porcine red cells by column chromatography and electrofocusing techniques. Both forms behaved as single components in sedimentation velocity experiments and during starch gel electrophoresis. The observed molecular weight of both forms was about 3 x 104. On the basis of their specific CO2 hydrase activities and amino acid compositions, these two carbonic anhydrase isozymes were designated as high activity (carbonic anhydrase C) or low activity (carbonic anhydrase B) forms which appear to be homologous to the high and low activity carbonic anhydrases, respectively, of other mammals. When these pig B and C isozymes were compared with the red cell carbonic anhydrases of other ungulates (cattle and horse), several interesting features were observed. In contrast to the electrophoretic gel patterns of the horse B and C isozymes in which the C form is markedly more basic than the B form, the high activity C form of pig was observed to be more acidic than the low activity B form. The tryptic peptide map of bovine carbonic anhydrase appears to be more similar to that of porcine carbonic anhydrase C than to B, indicating that they are probably homologous proteins. Neoprontosil binding by the pig and horse B isozymes give rise to essentially identical spectra in the 425- to 600-mµ region, whereas the C isozymes, from these two sources, generate quite different spectra.

Highlights

Carbonic anhydrase is known to occur in mammalian red blood cells as either one or two molecular forms; the enzyme showing the highest specific activity in the reversible hydration of COz is usually designated as carbonic anhydrase C or carbonic anhydrase II, and the low activity enzyme as carbonic anhydrase B or carbonic anhydrase I
Enzyme PurifLcation-The purification of porcine carbonic anhydrase was carried out using techniques which have been previously applied to isolation of this enzyme from other sources [1]
To separate the two major components from each other and from contaminating proteins, a DEAE-Sephadex column was employed in conjunction with a linear NaCl gradient

Summary

Methods

Carbonic anhydrase is known to occur in mammalian red blood cells as either one or two molecular forms; the enzyme showing the highest specific activity in the reversible hydration of COz is usually designated as carbonic anhydrase C or carbonic anhydrase II, and the low activity enzyme as carbonic anhydrase B or carbonic anhydrase I (for reviews see References l-3). A number of other isozymes have been observed, but recent genetic and chemical studies indicate that these represent alternate forms of either the B or C molecules and are not the products of additional genetic loci [4,5,6]. The two isozymes, carbonic anhydrase B and carbonic anhydrase. Enzyme Preparation-Porcine red blood cells were isolated from titrated whole blood obtained from Pentex Inc., Kankakee, Illinois. After concentrating the supernatant liquid, the material was passed over a column of DEAE-cellulose equilibrated with 0.003 M phosphate buffer, pH 7.1, and elution was effected with this same buffer

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