Membrane-associated carbonic anhydrase purified from bovine lung.

P L Whitney,T V Briggle

doi:10.1016/s0021-9258(18)33676-7

Abstract

We found carbonic anhydrase activity associated with particulate fractions of homogenates of rat, rabbit, human, and bovine lungs. These membrane-associated carbonic anhydrases were remarkably stable in solutions containing sodium dodecyl sulfate (SDS). The bovine enzyme was dissolved with SDS and purified by affinity chromatography and gel filtration. The purified enzyme contains glucosamine, galactose, and sialic acid; it is at least 20% carbohydrate. The apparent molecular weight by SDS-polyacrylamide gel electrophoresis (52,000) may be higher than the actual molecular weight due to the presence of carbohydrate. The enzyme contains cystine, an amino acid that is absent in bovine erythrocyte carbonic anhydrase. Dithiothreitol greatly accelerated the rate of inactivation of the membrane-associated enzyme in SDS, so disulfide bonds appear to stabilize this enzyme. The specific CO2-hydrating activity was about half that of the erythrocyte enzyme. Acetazolamide inhibits the membrane-associated enzyme (Ki = 10 nM) nearly as well as the erythrocyte enzyme (Ki = 3 nM). Antibody to bovine erythrocyte carbonic anhydrase did not inhibit the membrane-associated enzyme. Other investigators have accumulated a good deal of evidence for carbonic anhydrase on the luminal surface of pulmonary capillaries. The enzyme described here appears to be a new isozyme whose properties are consistent with such a localization.

Highlights

Antiserum to the erythrocyte enzyme completely anhydrase on the luminal surface of pulmonary capil- inhibited the activity of the erythrocyte enzyme but did not laries
Studies on the rate of CO, hydration and HC03- dehydration in lung capiUaries have pointed to the presence of carbonic anhydrase on the luminal surface of the pulmonary endothelial cells (1-3)
If carbonic anhydrase is located on the luminal surface of the endothelial ceUs of lung capillaries, itmust be bound antibody molecules could bind to the membrane-associated enzyme without inhibiting its activity, especially with a small substrate like CO

Summary

Introduction

It is known that the erythrocyte carbonic bonds appeatro stabilize this enzyme. The specCifoicr- anhydrase must have Zn2’ in the active site topromote strong hydrating activity was about half thatof the erythro- binding of acetazolamide (10).Since the membrane-associated cyte enzyme. Antiserum to the erythrocyte enzyme completely anhydrase on the luminal surface of pulmonary capil- inhibited the activity of the erythrocyte enzyme but did not laries.

Results

Conclusion