Carboxyl‐Terminal Region of Human and Bovine Erythrocyte Carbonic Anhydrases

Abstract

1. Carboxyl-terminal sequences have been determined for forms B and C of the human erythrocyte carbonic anhydrase and form B of the bovine enzyme. The proteins were degraded with CNBr, and peptide fragments of 20 residues were isolated from human enzyme C and bovine enzyme B while the human B form gave a fragment consisting of only 19 residues. The sequences of these fragments were determined and by carboxypeptidase digestion of the whole proteins it was shown that they represent the carboxyl-terminal part of the proteins.2. Comparison of the sequences of the enzymes gives strong support for evolutionary homology. Greater similarities in sequence are found between bovine carbonic anhydrase and human carbonic anhydrase C than between the two forms of the human enzyme.3. All sequences cont,ain an Arg-Pro peptide bond. I n two of the sequences this bond was found to be hydrolyzable with trypsin.Several forms of carbonic anhydrase have been isolated from bovine and human erythrocytes [l -41. They have a molecular weight of about 30,000 [3,5,6] and contain in each molecule one zinc atom essential for catalytic activity [7-91. Of the bovine enzyme two electrophoretically different forms have been described. They appear to be identical in catalytic properties [l] and amino acid composition [5] and are named A and B (for nomenclature see [5]) the latter being the most abundant. Of the human carbonic anhydrases, forms B and C have becn the most studied. Form C bears resemblance to the bovine enzyme in catalytic properties [lo-121. The two human forms appear to have the same overall conformation [13,14] but differ markedly in several other respects. Besides considerable deviations in amino acid composition [3,5,15] there is evidence for structural dissimilarities in their active site regions as shown by differences in their properties as catalysts [12,16] MATERIALS AND METHODS Preparation of Carbonic AnhydrasesHuman erythrocyte carbonic anhydrases B and C were purified as described elsewhere [2]. The preparations were performed on a larger scale than earlier, starting from an amount of red cells corresponding to 20-25 liters of whole blood. The vast amount of human enzyme filtrate from the SE-Sephadex chromatography was concentrated either with Sephadex G-25 according to Flodin et al. [22] or by adsorption on a DEAE-Sephadex A-50 column in 5 mM borateNaOH buffer, pH 9.4, followed by elution with 0.25 M Tris-C1, pH 8.0, and subsequent dialysis.Bovine erythrocyte carbonic anhydrase U was prepared according to Lindskog [I] with chromatography on DEAE-cellulose as the final step. As a rule, the peak containing form B could be cut in such a way that there were no appreciable amounts of form A contaminating. Sometimes, when the separation was not satisfactory, a rechromatography on a longer DEAE-cellulose column was employed. Garboxypeptidase Digestion of Carbonic AnhydrasesPreliminary experiments showed a very slow release of ninhydrin-positive material from native human carbonic anhydrase B in the presence of carbox...

Molten-Globule State of Carbonic Anhydrase Binds to the Chaperone-like α-Crystallin

“…BCA, as supplied by Sigma, is a mixture of the isoforms BCA-A and BCA-B. The amount of the isoform A present in the mixture from bovine erythrocytes is negligible compared to the isoform B (26). The data reported in this paper were obtained using BCA (c-3934), and similar results were obtained with bovine carbonic anhydrase B (c-2522)).…”

The importance of being knotted: effects of the C‐terminal knot structure on enzymatic and mechanical properties of bovine carbonic anhydrase II1

“…Our hypothesis was supported when the reactivity of the two conformers towards CP‐Y was studied. It has been reported that wild‐type BCAII is poorly attacked by CP‐Y [33], however, when the C‐terminal tertiary structure is perturbed (as in the denatured state), it is readily hydrolyzed from the C‐terminus [8]. We therefore analyzed the susceptibility of the two conformers to CP‐Y by mass spectroscopy.…”

Carbon Dioxide Hydration Activity of Carbonic Anhydrase: Kinetics of Alkylated Anhydrases B and C from Humans

“…The B isoenzyme derivative studied is one prepared by carboxymethylation of a histidine at position 200 in the recent amino-acid sequence (5), the same histidine previously assigned (2,4) to position 204. The C isoenzyme, which is probably homologous to the B form (4)(5)(6), has a leucine replacement at the corresponding position 198 in its sequence (4,5). However, Gothe and Nyman (7) have been able to alkylate a histidine close to position 63 in the C isoenzyme * This is paper No.…”