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 phenylmethanesulphonylfluoride, 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 x 10(-6) M 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 Km 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.

Protective Effect of Eurystatins A and B, New Prolyl Endopeptidase Inhibitors, on Scopolamine-Induced Amnesia in Rats

“…(2)(3)(4)(5), al though its physiological role still remains obscure. Since vasopressin, among these peptides, has been suggested to be deeply involved in the process of learning and memory in animal experiments (6-9), Yoshimoto et al synthesized a variety of PED inhibitors and demon strated a probable relationship between their PED in hibition in vitro and anti-amnesic activity in vivo (10).…”

Sequential Expression, Activity and Nuclear Localization of Prolyl Oligopeptidase Protein in the Developing Rat Brain

“…Previous studies have shown abundant fluctuation of enzyme activity and expression of POP during brain development [5,26,27]. High POP activities were measured particularly during early brain ontogeny.…”

Prolyl Endopeptidase Inhibitors from the Roots of Lindera strychnifolia F. VILL.

“…18) In addition, bacterial (Flavobacterium) and mammalian (bovine brain) enzyme genes were cloned and their amino acid sequences had 48% identity 24) ; we therefore investigated the inhibitory activities of six compounds by two different assays. One is the method of Yoshimoto et al, 9) which is the most popular Flavobacterium PEP inhibition assay, and the other is the method of Kato et al, 19) which measures inhibitory activity against PEP from rat brain supernatant. The concentrations of these compounds causing 50% inhibition are shown in Table 2, together with the positive controls, EGCG and Z-Pro-prolinal.…”

“…PEP Inhibition Assay (Rat Brain Supernatant) PEP activity was measured by a modification of the method of Kato et al 19) In brief, the brain of a rat was homogenized in 3 volumes of 0.1 M phosphate buffer (pH 7.0). After centrifugation at 10000ϫg for 20 min, the supernatant was used for the enzyme assay.…”