Abstract
Rat mast cells, harvested from the thoracic and peritoneal cavities and purified to at least 90% by density gradient centrifugation in 38% bovine serum albumin (BSA) were examined for cyclic nucleotide phosphodiesterase activities. The soluble fraction (200,000 x g) of disrupted mast cells was found to contain approximately 80% of the total activity at a substrate concentration of 0.13 µM or 6.0 µM cyclic AMP or 0.12 µM cyclic GMP. It could be separated by DEAE-Sephadex chromatography into two major fractions (IMC and IIIMC) and one minor fraction (IIMC). Fraction IMC contained phosphodiesterase activities with high apparent affinity for both cyclic GMP and cyclic AMP (app. Km values in the order of 0.1 to 0.2 µM), cyclic GMP being the preferred substrate. Fraction IIIMC hydrolyzed primarily cyclic AMP; the app. Km value was 0.4 µM. The particulate fraction (200,000 x g) of rat mast cells, which was found to contain approximately 20% of the total activity at the indicated substrate concentrations, hydrolyzed cyclic AMP and cyclic GMP with similar apparent Km values (∼0.5 µM) and maximum velocities. The phosphodiesterase activity pattern of supernatants from nonpurified thoracic and peritoneal cells was different from that of mast cells, indicating that contaminating cells are not the source of the two major phosphodiesterase fractions in mast cell supernatants. The antiallergic agents disodiumcromoglycate (DSCG) and 2-o-propoxyphenyl-8-azapurin-6-one (M&B 22,948) inhibited the activity of the separated mast cell cyclic AMP phosphodiesterase activities. The lowest I50 values observed were 250 µM and 10 µM, respectively, i.e., concentrations that exceed those sufficient for inhibition of antigen-induced histamine release from mast cells.
- Copyright © 1978 by Academic Press, Inc.
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