Carbonic anhydrase IV from human lung. Purification, characterization, and comparison with membrane carbonic anhydrase from human kidney.

X L Zhu,W S Sly

doi:10.1016/s0021-9258(19)38958-6

Abstract

We have purified carbonic anhydrase (CA) IV from human lung membranes to apparent homogeneity in a form which is catalytically active and stable to storage. It has an apparent molecular mass of 35 kDa, is insensitive to endoglycosidases, and seems to contain no N-linked or O-linked oligosaccharide chains. Reduction of disulfide linkages led to altered migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and loss of catalytic activity. CA IV resembles CA II in being a "high activity" isozyme, relatively resistant to inhibition by halide ions and sensitive to inhibition by sulfonamides. Application of this purification to human kidney membranes produced homogeneous enzyme with nearly identical properties. Amino acid compositions of both lung and kidney CA IV were similar, as were tryptic peptide patterns resolved on high performance liquid chromatography (HPLC). Amino-terminal sequences of native enzyme from lung and kidney were identical, as were amino-terminal sequences of the three major tryptic peptides resolved on reverse phase HPLC. Isoelectric focusing revealed microheterogeneity in enzyme from both sources. Antibody raised to human lung CA IV reacted equally strongly with CA IV from kidney, but very weakly or not at all with other CAs. Treatment of lung membranes and kidney membranes with phosphatidylinositol-specific phospholipase C released over half of the membrane-bound CA IV, suggesting that at least half of the CA IV in both organs is anchored to membranes by phosphatidylinositol-glycan linkages.

Highlights

Amino acid compositions of both lung and kidney carbonic anhydrase (CA) IV were similar, as were tryptic peptide patterns resolved on high performance liquid chromatography (HPLC)
We describe here the purification of CA IV from human lung to homogeneity by affinity chromatography using a procedure which provides milligram amounts of catalytically active enzyme in a form which is stable to storage
Unlike the soluble CAs, it is a very hydrophobic protein which is tightly anchored to membranes

Summary

AND COMPARISON WITH

We have purified carbonic anhydrase (CA) IV from human lung membranes to apparent homogeneity in a form which is catalytically active and stable to storage It has an apparent molecular mass of 35 kDa, is insensitive to endoglycosidases, and seems to contain no N-. A membrane CA had even earlier been purified to homogeneity from bovine lung microsomes by Whitney and Briggle [13] It was a cystine-containing glycoprotein with a molecular mass of 52 kDa on SDS-polyacrylamide gel electrophoresis. It was inactivated by treatment with reducing agents, suggesting that it was stabilized by one or more disulfide bonds This and other differences clearly distinguished the bovine membrane-associated enzyme from CA I, CA II, and CA III, the -known 29-kDa cytoplasmic CAs, and the bovine lung isozyme was provisionally called CA IV. Once purified, the bovine lung enzyme proved too unstable in SDScontaining buffers to permit extensive characterization

Carbonic Anhydrase IV from Human Lung and Kidney

DISCUSSION