Changes in Prolyl Endopeptidase During Maturation of Rat Brain and Hydrolysis of Substance P by the Purified Enzyme

Abstract

: We determined changes in prolyl endopeptidase activity in developing rat brain. A new and highly sensitive fluorogenic substrate, 7-(succinyl-Gly-Pro)-4-methylcoumarinamide, was used for determination of the enzyme activity. The enzyme activity per brain increased until 2 weeks of age, and then decreased during maturation. The enzyme was purified about 7800-fold from the brain of the rat at 2 or 3 weeks of age. The enzyme has a pH optimum of 5.8 to 6.5, and an approximate molecular weight of 70,000. The enzyme activity was completely inhibited by low concentrations of diisopropylfluorophosphate and partially inhibited by high concentrations of phenylmethanesul-phonylfluoride, which are potent serine protease inhibitors. Moreover, thiolblocking agents and some heavy metals also have a strong effect on the activity. Bacitracin was found to be a potent inhibitor, with an IC₅₀ value of 2.5 × 10⁻⁶m at 0.5 mm of the substrate. The enzyme was proved to hydrolyze the NH₂-terminal tetrapeptide, Arg¹-Pro²-Lys³-Pro⁴, from substance P to produce the heptapeptide, Gln⁵-Gln⁶-Phe⁷-Phe⁸-Gly⁹-Leu¹⁰-Met¹¹-CONH₂. The K_m value of the hydrolysis of substance P was 1.0 mm. This enzyme may be related to the regulation of substance P in the brain, and to the development of neurones by forming the tetrapeptide because the tetrapeptide has almost the same effect as substance P on the neurite extension of neuroblastoma.