Brain Research, 406 (1987) 113-117 113 Elsevier BRE 12422 Dipeptidyl peptidase-II activity in cultured astroglial cells from neonatal rat brain Bruce R. Stevens, Mohan Raizada, Colin Sumners and Alarico Fernandez Department of Physiology, Collegeof Medicine, Universityof Florida, Gainesville, FL 32610 (U.S.A,) (Accepted 29 July 1986) Key words: Astroglial cell; Neonatal brain; Tissue culture; Dipeptidyl peptidase; Mercurial; Dipeptide Astrocytic glial cells in primary culture from neonatal rat brain possess prominent dipeptidyl peptidase-II activity. This enzyme has been previously isolated and purified from whole brain tissue. The glial enzyme characteristically hydrolyzed glycyl-proline-p-nitroa- nilide (GPN) substrate to release glycyl-proline dipeptide plus p-nitroaniline products. At the enzyme's optimal pH of 5.4, the activi- ties of other tested amino exopeptidases were virtually zero. At pH > 8, activity was less than 2% of the activity at pH 5.4, which sug- gested a paucity of the related enzyme, dipeptidyl peptidase-IV. No competitive inhibition was observed for glycine, proline nor their permuted dipeptides. Glial dipeptidyl peptidase-II activity was strongly inhibited by Hg2÷, while other redox sulfhydryl agents were ineffective. Tested cations did not affect activity, except K ÷ which was mildly inhibitory. Chelating agents were not inhibitory. Of the peptidase inhibitors tested, only phenylmethylsulfonyl fluoride and puromycin were partially inhibitory. We suggest that dipeptidyl peptidase-II may play a role in glial processing of brain peptides which possess an N-terminal penultimate proline residue. INTRODUCTION Dipeptidyl peptidase-II (DPP-II; EC 3.4.14.2) is an acid hydrolase found in a wide variety of tissues which process peptides; it is also present in the brain. This enzyme characteristically hydrolyzes the car- boxyl end of penultimate prolyl bonds to release the N-terminal glycyl-proline dipeptide residue from nat- ural oligopeptides (NH2-GIy-Pro-X~n)) ' or from the chromogenic substrate Gly-Pro-p-nitroanilide 7 at an optimal pH 5.4. In the rat brain, DPP-II occurs histologically in both glia and neurons (cf. refs. in ref. 4). DPP-II is believed to be associated with glial function, inas- much as kainic acid-lesioned brains stimulate DPP-II activity associated with a concomitant proliferation of glial cells 7. Physiologically, brain DPP-II possibly plays a role in neuropeptide regulation/degrada- tion4.7, 9. We report here the prominent activity of DPP-II in neonatal rat brain astrocytic glial cells in primary cul- ture. MATERIALS AND METHODS Astroglial cell cultures Astrocytic glial cells in primary culture were pre- pared as described in Chiu and Goldman 3 and Clark et al. 2. Briefly summarizing, brains from 1-day-old Sprague-Dawley rats were removed at the level of the medulla oblongata, and were placed in sterile iso- tonic phosphate-buffered saline containing antibiot- ics. The pia and blood vessels were removed, and the remaining brain material was minced. The cells were dissociated using a trypsin and deoxyribonuclease-II treatment, then were suspended in Dulbecco's mod- ified Eagle's medium (DMEM) containing t0% fetal bovine serum (FBS). The cells were washed, sus- pended in DMEM/FBS, and plated in Falcon tissue culture dishes precoated with poly-L-lysine. The dish- Correspondence.. B.R. Stevens, Department of Physiology, College of Medicine, University of Florida, Box J-274, Ganesville, FL 32610, U.S.A. 0006-8993/87/$03,50 (~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)