The differentiation of the A-type esterases in sheep serum.

The differentiation of paraoxonase (an A-type esterase in serum) from related enzymes has been complicated by overlapping substrate specificities and by the complex nature of these enzymes. Rabbit-serum paraoxonase has been differentiated from hog-kidney diisopropyl phosphorofluoridatase by Mounter (1954). He confirmed the observation of Aldridge (1953b) that one enzyme in rabbit serum hydrolyses p-nitrophenyl acetate and paraoxon (diethyl-p-nitrophenyl phosphate) and extended the observation to include tetraethyl pyrophosphate and diisopropyl phosphoroflucridate. The hog-kidney enzyme was activated by manganous ions (Mounter & Chanutin, 1953) but did not hydrolyse paraoxon (Mounter, 1956), whereas rabbit-serum paraoxonase was inhibited by manganese and hydrolysed paraoxon. Bergmann, Segal & Rimon (1957) have shown that diisopropyl phosphorofluoridatase does not hydrolyse p-nitrophenyl acetate. In addition they have demonstrated a new C-esterase hydrolysing this substance in purified preparations of the hogkidney enzyme. According to the usage of Aldridge (1954), C-esterase is an A-type esterase. The ability of rabbit-serum paraoxonase to hydrolyse p-nitrophenyl acetate could also be used to differentiate paraoxonase and diisopropyl phosphorofluoridatase. The paraoxonases of rabbit, rat and horse sera hydrolysed paraoxon and p-nitrophenyl acetate but the ratio of hydrolysis of p-nitrophenyl acetate to hydrolysis of paraoxon varied from 4 in rabbit to 18 in horse. From summation experiments Aldridge concluded that one enzyme in these sera was responsible for this hydrolysis and attributed the varying ratios to species differences. However, when the ratios for other sera are calculated, in many cases they are greater. For instance, from Aldridge's results the ratio for ferret serum was 130, for sheep 50, for goat 170 and for human 48. As observed in the present work, the ratio for hog serum was 5700. The possibility that A-type esterases, other than paraoxonase, hydrolysing p-nitrophenyl acetate, occur in some sera cannot be excluded. Mounter& Whittaker (1953) suggested that serum arylesterase and paraoxon-hydrolysing A-esterase were identical. * Present address: Research Group, Occupational Health Division, 45 Spencer Street, Ottawa, Ontario, Canada. Although hog-kidney diisopropyl phosphorofluoridatase and rabbit-serum paraoxonase can be clearly differentiated, the situation becomes more confused when other tissues, sera and species are examined. Mounter (1955) studied the diisopropyl phosphorofluoridatase activities in rat and hog liver and kidney fractions and observed that, unlike the enzyme of the soluble fraction, the enzymes of insoluble fractions of liver were inhibited by manganese and activated by calcium when di-nbutyl phosphorofluoridate was the substrate. The distribution and nature of diisopropyl phosphorofluoridatases have been studied in some detail by Mounter, Dien & Chanutin (1955). After comparing these enzymes in the tissues of various species they concluded that a number of diisopropyl phosphorofluoridatases exist. Augustinsson & Heimburger (1954) have reported evidence suggesting that serum and kidney tabunase are not identical and also that rabbit-serum tabunase and A-esterase are not identical. On the basis of summation experiments, Cohen & Warringa (1957) were unable to conclude that their highly purified hog-kidney B-fraction, derived from most purified A-2 fraction of Mounter, Floyd & Chanutin (1953), contained only one enzyme hydrolysing diisopropyl phosphorofluoridate and tabun (ethyl-Ndimethylphosphoramidocyanate). In the present work a substrate-specificity study with purified paraoxonase preparations from sheep serum (Main, 1960) has been made for the purpose of further differentiating these various activities. Data from other experiments with rabbit, horse, sheep and pig sera have also been employed.