Apparent inactivation of alpha 1-antiproteinase by sulphur-containing radicals derived from penicillamine

alpha 1-Antiproteinase is the major inhibitor of proteolytic enzymes, such as elastase, in human plasma. Its elastase-inhibitory capacity can be inactivated by exposure to hydroxyl radicals (.OH) generated either by pulse radiolysis or by an Fe3+-EDTA/H2O2/ascorbic acid system. Inactivation of alpha 1-antiproteinase by radiolytically-generated .OH under anoxic conditions was decreased by adding a range of anti-inflammatory drugs to the reaction mixtures, including the thiol compound penicillamine. However, under conditions favouring formation of oxysulphur radicals, protection by thiols such as penicillamine was much decreased. It is proposed that sulphur-containing radicals resulting from attack of biologically-produced oxidants upon penicillamine in the presence of O2 can themselves inactivate alpha 1-antiproteinase, and that such radicals might contribute to the side-effects produced by penicillamine or gold thiol therapy in rheumatoid arthritis.     

Identification and selective inhibition of four distinct soluble forms of cyclic nucleotide phosphodiesterase activity from kidney

Homogenization of rat kidney under isotonic conditions and in the presence of protease inhibitors showed that some 92% of the cyclic AMP phosphodiesterase activity and some 83% of the cyclic GMP phosphodiesterase activity was released into the soluble fraction. Analysis of soluble phosphodiesterase activity by FPLC on a Mono-Q column resolved four distinct fractions expressing cyclic nucleotide phosphodiesterase activity. Lineweaver-Burk plots for the hydrolysis of both cyclic GMP and cyclic AMP yielded linear results. The first two peaks (KPDE-MQ-II, KPDE-MQ-III) showed higher activities towards cyclic GMP than cyclic AMP with the ratio of their Vmax values for the hydrolysis of cyclic AMP/cyclic GMP being 0.66 and 0.16, respectively. For the second two peaks (KPDE-MQ-IV, KPDE-MQ-V) the Vmax ratios for the hydrolysis of cyclic AMP/cyclic GMP were 6.4 and 16.7, respectively. All enzymes exhibited similar low Km values for both cyclic AMP and cyclic GMP but had very different Vmax values. KPDE-MQ-II was activated by Ca2+/calmodulin. The cyclic AMP phosphodiesterase activity of KPDE-MQ-III was augmented by the presence of low concentrations of cyclic GMP. Thermal denaturation studies showed that the phosphodiesterase activity of each fraction decayed as a single exponential indicating that each phosphodiesterase fraction contained but a single phosphodiesterase activity. The inhibitors IBMX, zaprinast, milrinone, amrinone, buquineran, carbazeran, ICI 118233, ICI 63197 exerted selective effects on the activities of these enzymes. We compared the action of these compounds on cyclic GMP phosphodiesterases from bovine retina. Over the concentration ranges used, the bovine retinal enzyme was only inhibited by IBMX, zaprinast and carbazeran. The cytosolic isoenzymes of cyclic AMP phosphodiesterases play a much more important role in metabolizing cyclic AMP in kidney compared with liver, where the activity of membrane-bound isoenzymes predominate.