Phosphatidate bilayers composed of dilauroylphosphatidate, dimyristoylphosphatidate, dipalmitoylphosphatidate and dioleoylphosphatidate were prepared. Their interaction with AMP deaminase isolated from pig heart was investigated. Dioleoylphosphatidate bilayers were found to exert non-competitive inhibition on the AMP deaminase with a K1 of 15 x 10-6 M. This inhibition is three orders of magnitude stronger than that exerted by orthophosphate. The phosphatidate species containing saturated fatty acids were either non-inhibitory or inhibited enzyme activity rather poorly. However, alkalinization of the medium from pH 6.5 to pH 7.9 led to the inhibition of pig heart AMP deaminase by dilauroylphosphatidate bilayers. This was accompanied by the fluidization of the saturated phosphatidate species, i.e. the lowering of their phase transition temperature in alkaline pH, as measured by light-scattering and fluorescence scans. The possible significance of these findings for the regulation of AMP deaminase activity in vivo by natural membranes is discussed.

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The interaction of pig heart AMP deaminase with different chemical species of phosphatidylcholine and with natural plasma membranes has been investigated. Phospholipids added to the system either as natural biological membranes (plasma membrane vesicles) or in the form of liposomes containing unsaturated phosphatidylcholine considerably enhanced AMP deaminase activity. The secondary structure of pig heart AMP deaminase in the absence and in the presence of dioleoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine liposomes was investigated by Fourier-transform infrared spectroscopy. Quantitative analysis of the amide I band showed that the enzyme contains 45% beta-sheets, 28% alpha-helix, 16% turns and 11% non-ordered structure. In the presence of dioleoyl phosphatidylcholine liposomes, the beta/alpha content ratio decreased; this decrease was dependent on the amount of lipid added. This phenomenon was not observed in the case of dipalmitoyl phosphatidylcholine liposomes. These data suggest a possible role for membrane phospholipids in the regulation of AMP deaminase activity.

The purpose of this study was to verify whether injection of tert-butyl hydroperoxide (Bu(t)OOH, a well-known prooxidant agent) into the bile-pancreatic duct can induce acute pancreatitis. A rapid blockade of the secretion was observed in the majority of the animals after 3 hours of observation. After 6 hours, the secretion reached a very low level, significantly different compared with controls. In groups of rats injected with Bu(t)OOH, pancreatic weight gain was observed compared with the rats injected with physiologic saline. Histology of pancreata removed 3 hours after injection of Bu(t)OOH showed acinar cell vacuolization, interstitial edema, focal necrosis of pancreatic acini, fat-tissue necrosis, and leukocyte infiltration of the organ. These changes were considerably greater after the 6-hour observation period. Electron-microscopic inspection revealed profound morphologic changes 3 hours after Bu(t)OOH injection. The control rats receiving physiologic saline alone had well-preserved pancreatic tissue structure. In conclusion, injection of the prooxidant agent, tert-butyl hydroperoxide, into common bile-pancreatic duct induces acute necrotizing pancreatitis, which indicates the crucial role of free radical reactions in pathogenesis of this disease.