A novel kinin-generating protease (kininogenase) in the porcine anterior pituitary

The purpose of the present study was to determine whether porcine pituitary tissue contains kininogenase activity. Porcine anterior and posterior pituitaries were homogenized in 0.25 M sucrose containing 10 mM Tris/HCl (pH 7.5) and the homogenate subjected to differential centrifugation (10,000 x g for 20 min, then 105,000 x g for 60 min). Fractions were examined for kininogenase activity using canine plasma kininogen as the substrate; kinins were measured by bioassay or radioimmunoassay. The anterior pituitary was found to have kininogenase activity highly concentrated in the 105,000 x g particulate pellet (376 ng of bradykinin generated/mg of protein/h) with little or no activity detectable in other fractions. The porcine posterior pituitary 105,000 x g pellet contained 22% of the kininogenase activity of the anterior pituitary. Anterior pituitary kininogenase activity had a broad pH optimum (pH 7.5-9.0), was heat-sensitive, was inhibited by dithiothreitol, soybean trypsin inhibitor, aprotinin, and diisopropyl fluorophosphate, and was resistant to lima bean trypsin inhibitor, pepstatin, and hirudin. Bradykinin was found to be the principal kinin generated by the anterior pituitary kininogenase as determined by high performance liquid chromatography. The anterior pituitary kininogenase differed from other kininogenases in its sensitivity to inhibitors and substrate specificity.