Membrane-associated carbonic anhydrase from rat lung. Purification, characterization, tissue distribution, and comparison with carbonic anhydrase IVs of other mammals.

A Waheed,X.L Zhu,W.S Sly

doi:10.1016/s0021-9258(19)50732-3

Abstract

Carbonic anhydrase (CA) IV was purified to homogeneity from rat lung microsomal and plasma membranes. The single N-terminal amino acid sequence showed 55% similarity to that reported for human CA IV. A monospecific antibody to the 39-kDa rat enzyme that cross-reacts on Western blots with CA IVs from other mammalian species was produced in rabbits. Digestion of rat lung enzyme with endoglycosidase (peptide-N-glycosidase F) reduced the Mr to 36,000, suggesting that rat CA contains one N-linked oligosaccharide chain. All of eight additional mammalian CA IVs that were examined also contained oligosaccharide chains, as evidenced by reduction in Mr from 52,000 (cow, sheep, and rabbit), 42,000 (pig, guinea pig, and dog), and 39,000 (mouse and hamster) to 36,000 after treatment of the respective lung microsomal membranes with peptide-N-glycosidase F. The 36-kDa human enzyme showed no change in molecular mass with this treatment. Thus, the human CA IV is the exceptional one in lacking carbohydrate. Rat lung CA IV was found to be relatively resistant to sodium dodecyl sulfate and to be anchored to membranes by a phosphatidylinositol-glycan linkage; both properties were found to be shared by other mammalian CA IVs. Western blot analysis indicated distribution of CA IV in rat tissues other than kidney and lung where it was previously known to be present. CA IV was particularly abundant in rat brain, muscle, heart, and liver, all locations where the CA IV enzyme was not known to be present previously. None was detected in rat skin or spleen.

Highlights

Paradoxito sodium dodecylsulfate andto be anchored to mem- cally, antiserum raised against SDS-treated34.4-kDa enzyme branes by a phosphatidylinositol-glycanlinkage; both reacted only with a polypeptide of 55 kDa, which was found properties were found to be shared by other mamma- in many tissuesand was attributed toCA IV,despite the lian Carbonic anhydrase (CA) IVs
The successfulapplication of the purification procedure we reported for CA IV from human lung to CA IV from rat lungs demonstrates the versatility of this procedure for producing relatively large amounts of homogeneous enzyme in a form that is active and stableto storage
The same purification has been applied successfully to CA IV from bovine lungs, porcine lungs, and rabbit lungs.3The success of this procedure relies on one property that all the CA IVs appear to have in common, namely relative stability in1-5% sodium dodecyl sulfate

Summary

The carbonic anhydrase isozymes are zinc metalloenzymes

+ + that catalyze the reversible hydration of carbon dioxide (COz H 2 0 $ HCO: H+).They vary in their subcellular localization, with cytoplasmic (CA I, CA 11, CA 111, and CA VII)l [1, 2], cell surface membrane (CA IV) [3,4,5], mitochondrial (CA V) [6], and secretory (CA VI) [7,8,9] forms having been described. The interesting similaritiesand differences between bovine lung CA IV [3] and CA IV from human lung and kidney [5] led us to purify the membrane-associated CA IVfrom rat lung. We report immunologic studies with antibody raised to the purified rat lung CA IV that show that these two properties are shared by CA IVs from nine different species. These studies show that the large molecular weight differences between the human CA IV andthe CA IVs in various other mammalian species are almost entirelyexplained by differences in carbohydrate content. We present the first evidence for the tissue distribution of CA IV in rattissues other thankidney

DISCUSSION

Rat and Other MammaliaCnarbonic Anhydrase IVs

Human Rat

Bovine Sheep