Proc. Natl. Acad. Sci. USA Vol. 85, pp. 8326-8330, November 1988 Neurobiology A serine peptidase responsible for the inactivation of endogenous cholecystokinin in brain C. ROSE, A. CAMUS, AND J. C. SCHWARTZ Unitd de Neurobiologie et Pharmacologie (Unitd 109) de l'Institut National de la Santd et de la Recherche Mddicale, Centre Paul Broca, 2ter rue d'Aldsia, 75014 Paris, France Communicated by RolfLuft, July 26, 1988 (receivedfor review May 18, 1988)

ABSTRACT A serine endopeptidase was characterized as membranes, with cleavages occurring at various peptide a major inactivating enzyme for endogenous cholecystokinin bonds within the molecule. However, numerous peptidases (CCK) in brain. CCK-8 released by depolarization of slices of capable of hydrolyzing neuropeptides are present in cerebral rat cerebral cortex, as measured by its immunoreactivity membranes, and metabolic pathways of exogenous neuro- (CCK-ir), undergoes extensive degradation (=85% of the peptides may not reliably reflect those responsible for endog- amount released) before reaching the incubation medium. enous neuropeptide inactivation, as illustrated in the case of However, recovery of CCK-ir is enhanced up to 3-fold in the enkephalins' (1-5). In contrast, the study of the fate of presence of serine-alkylating reagents (i.e., phenylmethylsul- endogenous neuropeptides released by depolarization of fonyl fluoride) as well as selected active site-directed inactiva- brain slices allows the physiologically relevant peptidases tors (i.e., peptide chloromethyl ketones) or transition-state (20-22) and characteristic peptide fragments they produce inhibitors (i.e., peptide boronic acids) of serine peptidases. (23) to be identified. The main advantage of this system is Among these compounds, elastase inhibitors were the most that, by retaining much of the integrity of the native tissue, potent protecting agents, whereas trypsin or chymotrypsin the released peptide (in near-physiological concentrations) inhibitors were ineffective. HPLC analysis of endogenous comes into contact initially with physiologically relevant CCK-ir'recovered in media of depolarized slices indicated that peptidases presumably located close to the nerve endings endogenous CCK-5 [CCK-(29-33)-pentapeptidel was the most from which'the peptide originates. abundant fragment and that its formation was strongly de- This model, applied to CCK-8, has shown that only a small creased in the presence of an elastase inhibitor. HPLC analysis fraction (=-15%) of CCK-8-like immunoreactivity (CCK-8-ir) of fragments formed upon incubation of exogenous CCK-8 released by depolarization of cerebral slices is recovered as [CCK-(26-33)-octapeptide] with brain slices showed CCK-5, the intact (immunoreactive) peptide in the incubation me- Gly-Trp-Met, and Trp-Met to be major metabolites of CCK-8 dium (24). This suggested that endogenous CCK-8 is highly whose formation was prevented or at least diminished in the sensitive to hydrolysis by one or more unidentified tissue presence of the elastase inhibitor. It is concluded that there is peptidases, which seem(s) distinct from enkephalinase, since an elastase-like serine endopeptidase in brain that cleaves the the recovery was not significantly modified by an inhibitor of two peptide bonds of CCK-8 where the carboxyl group is this enzyme. donated by a methionine residue and constitutes a major We used the same model to identify the endogenous inactivation ectoenzyme for the neuropeptide. CCK-8-degrading enzymes and observed that a large variety of inhibitors of peptidases belonging to various catalytic The identification of the peptidases responsible for the classes-i.e., metallopeptidase, thiol-endopeptidase, and physiological inactivation of cerebral neuropeptides released carboxypeptidase, had little or no effect on the recovery of into the extracellular space is a difficult but important task. CCK-8-ir. In contrast, serine-alkylating reagents such as It facilitates (i) the development of selective inhibitors diisopropyl fluorophosphate or phenylmethylsulfonyl fluo- protecting the'endogenous neuropeptides, thereby amplify- ride markedly protected CCK-8-ir, suggesting the involve- ing the biological responses they trigger; (ii) the rational ment of a serine peptidase. We have characterized this design of peptidase-resistant analogues able to enter the putative serine peptidase by establishing the relative poten- brain; and (iii) the identification of characteristic extracellu- cies of a series of inhibitors in protecting endogenous CCK-8 lar metabolites whose tissue levels may reflect the activity of and by identifying the peptide bonds within the CCK-8 corresponding peptidergic neurons. This is best illustrated in molecule that it hydrolyzes. the case of enkephalins (1-5). However, little is known ofthe mechanisms responsible for MATERIALS AND METHODS the inactivation of the cholecystokinin-(26-33) octapeptide (CCK-8), Preparation and Incubation of Brain Slices. Slices (250 .um thick) of rat cerebral cortex were repeatedly washed to free Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2,1 2 3 4 5 6 7 8 them from any detectable soluble CCK-hydrolyzing activity and then incubated in an oxygenated Krebs-Ringer medium an abundant neurotransmitter in brain (6-9), in contrast with (24). posttranslational enzymes involved in its biosynthesis, which After a 10-min preincubation at 370C with or without are being identified (10-12). Several studies have shown that peptidase inhibitors, the slice suspension (1.5-2 mg ofprotein ill-characterized acidic (13, 14) or neutral aminopeptidases in 0.8 ml) was incubated for 5 min in the presence of either (13), enkephalinase (EC 3.4.24.11) (15-17), a thiol-endo- peptidase (18) or a metallo-endopeptidase (19) may partici- Abbreviations: AAP-boro V, MeO-Suc-Ala-Ala-Pro-boro-Val; pate in the hydrolysis of exogenous CCK-8 by cerebral CCK, cholecystokinin; CCK-ir, CCK immunoreactivity; CCK-8, CCK-(26-33) octapeptide (amide sulfated); CCK-8(n.s.), nonsul- fated CCK-8; CCK-7, CCK-6, CCK-5, CCK-4, hepta-, hexa-, penta-, The publication costs of this article were defrayed in part by page charge and tetrapeptide derivatives of CCK-8; Tos-LysCH2Cl, N- payment. This article must therefore be hereby marked "advertisement" tosyllysine chloromethyl ketone; Tos-PheCH2CI, L-1-tosylamido-2- in accordance with 18 U.S.C. §1734 solely to indicate this fact. phenylmethyl chloromethyl ketone. 8326 Downloaded by guest on September 28, 2021 Neurobiology: Rose et al. Proc. Natl. Acad. Sci. USA 85 (1988) 8327

2 mM or 50 mM K +. After rapid centrifugation, the medium ._ MeOSuc-Ala-Ala-Pro-boro-Val was separated from the tissues, and the CCK-8 concentration L-Ala-Ala-Pro-VaICH2CI in both components was measured by RIA. For studies of I-- exogenous were L-Ala-Ala-Phe-CH2CI/ CCK hydrolysis, slices incubated for 15 min ) in the presence 2 mM of K+. 0 'aCa, Analysis of Endogenous CCK Peptides. or 0 Media tissue 0)~ 0D extracts were applied to small columns of polystyrene beads ._ from which CCK peptides were eluted with EtOH (24) [76- 90% recovery for CCK-(30-33)-tetrapeptide (CCK-4), CCK-

(29-33)-pentapeptide (CCK-5), CCK-(28-33)-hexapeptide 0 (CCK-6), and CCK-(27-33)-heptapeptide (CCK-7)]. Eluates 0) were dried (Speed Vac, Savant), the residues were redis- E solved in H20 and either directly radioimmunoassayed or 0 0 injected into a C18 ABondapak HPLC column (Waters). Isocratic elution was performed at 1.5 ml/min with 0.1% CF3COOH (adjusted to pH 4 with diethylamine) mixed with 24% CH3CN. Recovered fractions (1.5 ml) were lyophilized -7 -6 -5 -4 -3 and subjected to'RIA (24). Immunoreactive'peaks 'were INHIBITORS LOG M identified by comparing their retention times with those of authentic peptides. Recovery of total injected immunoreac- FIG. 1. Effect of various serine peptidase inhibitors on the recovery of endogenous CCK-ir released from depolarized slices of tivity was 78 ± 4%. rat cerebral cortex. Slices were preincubated for 15 min in the Analysis of Exogenous CCK Peptides. Media from incuba- presence of inhibitors and depolarized by addition of 50 mM K + for tions in the presence of exogenous CCK-8 were subjected to 5 min, and the CCK-ir was assayed in tissues and medium. The isocratic HPLC on C18 ,uBondapak column eluted with K '-evoked release of CCK-ir, expressed as CCK-8, was calculated CH3CN/0.1% or 1% CF3COOH buffered 'to pH 2.5 or 4 with as the difference between tissue levels (pmol/mg of protein) in the diethylamine (31:69, 30:70, 18:82, or 7:93). Peaks were presence of2 mM K+ (2.49 + 0.08) and 50 mM K+ (1.87 ± 0.14) and detected (by UV absorbance at 254 nm) and quantified by equalled 0.62 ± 0.15. CCK-ir release was not significantly modified comparison with corresponding standards. Substances char- in the presence of any of the inhibitors. CCK-ir recovered in the acterized by the peaks were identified by (i) their retention medium was calculated as the difference between its levels in media time in at two systems, containing 50 mM K + and 2 mM K -i.e., 61 4 and 5 ± 1 fmol/mg least distinct HPLC (ii) fluorescence of protein, respectively. Recovery was expressed either in fmol/mg ofphenylalanine, tyrosine, or tryptophan, and (iii) amino acid of protein (left ordinate) or as the percentage of released CCK-ir analysis (25). Assignment of unknown peaks (e.g., the peak (right ordinate). Data are means ± SEM oftwo or three experiments characteristic of Gly-Trp-Met) was later confirmed when the with quadruplicate determination for each inhibitor. PMSF, phenyl- authentic peptide was obtained by custom synthesis and its methylsulfonyl fluoride. HPLC retention time was determined. Materials. CCK-8 and its C-terminal and N-terminal frag- diisopropyl fluorophosphate (2 mM) had a similar effect (not ments as well as the endopeptides Gly-Trp, Trp-Met, and the shown). Similar protective effects were observed with se- amino acid tryptophan were obtained from Bachem. Gly- lected (but not all) serine peptidase inhibitors belonging to Trp-Met has been synthesized by Novabiochem (Laufel- either the chloromethyl ketone or the boronic acid families finger, Switzerland). Thiorphan '(DL-3-mercapto-2-benzyl- (Fig. 1 and Table 1). propanoylglycine) was obtained from Bioprojet (Paris), and One of the most potent compounds, MeO-Suc-Ala-Ala- bestatin was from Laboratoire Roger Bellon (Paris). Pro-boro-Val (AAP-boro-V), was used at a concentration Peptide chloromethyl ketones were obtained from Bachem corresponding to near maximum effect to investigate the except for N"-tosyllysine chloromethyl ketone (Tos- changes elicited in the pattern ofCCK-ir peptides. Incubation LysCH2Cl) and L-1-tosylamido-2-phenylmethyl chloro- media were analyzed by HPLC in an isocratic system methyl ketone (Tos-PheCH2CI) which were obtained from allowing clear-cut separation of short CCK peptides except Sigma. Diisopropyl fluorophosphate and phenylmethylsul- CCK-6 and CCK-7, which displayed similar'retention times fonyl fluoride were also from Sigma. (Fig. 2). Whereas CCK-ir from 2 mM K+ incubations was The peptide boronic acids MeO-Suc-Ala-Ala-Pro-boro-P- distributed throughout the HPLC profile, CCK-ir of media he, MeO-Suc-Ala-Ala-Pro-boro-Val, MeO-Suc-Ala-Ala-Pro- from depolarized slices was characterized by several peaks boro-Ile, and MeO-Suc-Ala-Ala-Pro-boro-Ala were gifts of comigrating with authentic CCK-4, CCK-5, CCK-6 (and/or C. Kettner (E. 1. duPont de Nemours). Table 1. Protection of endogenous CCK-ir released from brain RESULTS slices: comparative potencies of various serine peptidase inhibitors Effects of Serine Peptidase Inhibitors on the Recovery of Endogenous CCK Released from Depolarized Brain Slices. Inhibitors IC50, AM When slices of rat cerebral cortex were depolarized for 5 min Ala-Ala-Pro-Val-CH2CI 0.21 ± 0.11 with 50 mM K+, the tissue CCK-ir contents, essentially Ala-Ala-Phe-CH2CI 5.4 ± 1.3 CCK-8 (26, 27), were consistently reduced by 15-20%. D-Phe-Pro-A2rg-CH2Cl -1000 Without any peptidase inhibitor, the K+ stimulus increased Tos-Phe-CH2Cl >1000 the CCK-ir in the incubation medium by about 6-fold; but this Tos-Lys-CH2Cl >1000 increase represented only 13 ± 1% of the amount released. MeO-Suc-Ala-Ala-Pro-boro-Phe 0.29 ± 0.11 With inhibitors of various classes of peptidases-i.e., me- MeO-Suc-Ala-Ala-Pro-boro-Val 0.16 ± 0.05 tallopeptidases (bestatin, amastatin, puromycin, thiorphan, MeO-Suc-Ala-Ala-Pro-boro-Ile 0.09 ± 0.02 captopril, or o-phenantroline), thiol-endopeptidases (leu- MeO-Suc-Ala-Ala-Pro-boro-Ala 0.46 ± 0.27 peptin, antipain) or carboxypeptidases (pepstatin)-the re- Data of Fig. 1 or similar experiments were analyzed by an iterative covery was not significantly improved. In contrast, addition computer program based upon the least-squares method. The IC50 of phenylmethylsulfonyl fluoride increased the recovery by values (± SEM) refer to compounds preincubated for 15 min before 2- to 3-fold (Fig. 1), and the other serine-alkylating reagent, a 5-min release period. Downloaded by guest on September 28, 2021 8328 Neurobiology: Rose et al. Proc. Natl. Acad. Sci. USA 85 (1988)

Table 2. Effects of AAP-boro-V, a serine peptidase inhibitor, on the recovery of CCK-ir peptides in the incubation medium of depolarized slices of cerebral cortex Recovery in medium C ~~~r1 (% of CCK-ir released from slices) o 8 CCK-8 0. Peptide ir Control AAP-boro-V 0) CCK-4-ir 23 ± 9 28 ± 8 E 15 _ CCK-5-ir 14 ± 3 4 ± 3* ( -72%) co CCK-6-ir and/or CCK-7-ir 2 ± 0.3 13 ± 2t (+550%) CCK-6 CCK-8-ir 8 ± 1 23 ± 4O (+187%) o and CCK-7 CCK-8(n.s.)-ir 1 ± 0.3 1 ± 0.7 Total 48 ± 3 70 ± 7* CT~ ~ ~ Values for each HPLC immunoreactive peak were calculated by 10-010_ ~~~~~~I~ l dividing their level, expressed in CCK-8 equivalents, by the corre- o CCK-4 sponding RIA cross-reactivity factor, [i.e., 1.00 for CCK-8 or E CCK-8(n.s.), 0.29 for CCK-6 (and/or CCK-7), 0.10 for CCK-5, and 0.04 for CCK-4]. The cross-reactivity factors for C-terminal frag- CC K-5 ments smaller than CCK-4 and for N-terminal or internal fragments ~~~ ~ ~ ~ ~~~I CCK-8(n.s.) of the CCK-8 molecule were <0.01. The recovery in medium was 05 expressed as a percentage of CCK-8-ir released from tissues by the K + stimulus (546 ± 72 fmol/mg of protein). Data are means ± SEM of five experiments. II *p < 0.05. tp < 0.001. tP < 0.01. CCK-7, CCK-8, and nonsulfated CCK-8 [CCK-8(n.s.)]. Lri L When their levels were calculated by taking into account their respective cross-reactivities in the RIA, the most abundant 10 20 30 40 components were CCK-4-ir, CCK-5-ir, and CCK-8-ir, and RETENTION TIME (min) the total recovery was equivalent to 48% of released CCK- FIG. 2. HPLC analysis of the endogenous CCK-ir peptides 8-ir. In the presence of the serine peptidase inhibitor, CCK- recovered in the incubation medium of cortical slices after K+- 8-ir and CCK-6-ir (and/or CCK-7-ir) were substantially evoked depolarization: effects of AAP-boro-V, a serine peptidase increased, whereas CCK-5-ir was reduced by 72%, and the inhibitor. Incubation media corresponding to 15 mg of tissue protein total recovery was 70% of released CCK-8-ir (Table 2). were purified on polystyrene bead columns and extracts were Effects of AAP-boro-V, a Serine Peptidase Inhibitor, on the injected into a C18 ,Bondapak column. Eluted fractions were Hydrolysis of Exogenous CCK-8 by Brain Slices. Cortical lyophilized and subjected to RIA. Arrows indicate the retention time slices hydrolyzed CCK-8 at a linear rate up to 45 min (data not of authentic peptides. Results are expressed in fmol of "equivalent shown). After 15 min in the absence of any <20% CCK-8" released by the K+ stimulus-i.e., values obtained after inhibitor, subtraction of the corresponding 2 mM K+ value but without of the substrate was hydrolyzed, the main fragment being correction for the different cross-reactivities ofthe various molecular tryptophan, which represented 60% of the total hydrolysis products (Table In the forms of CCK. -, Control; ---, incubation in the presence of 10 ,uM 3). presence ofthiorphan and bestatin, AAP-boro-V. The figure shows a single representative experiment, CCK-8 hydrolysis was not significantly modified, whereas which was replicated five times (see Table 2). the levels of intermediate fragments (CCK-4, Gly-Trp-Met,

Table 3. Effects of AAP-boro-V on the formation of hydrolysis fragments of exogenous CCK-8 by slices of cerebral cortex Fragment formation, nmol/mg of protein Without Thiorphan + Thiorphan + bestatin inhibitor bestatin + AAP-boro-V Total* 8.2 ± 0.9 6.6 ± 0.6 4.0 ± 0.5t (-39%) CCK-6/CCK-7 0.30 ± 0.02 0.33 ± 0.01 0.44 ± 0.04t (+33%) CCK-5 1.4 ± 0.2 1.8 ± 0.2 1.3 ± 0.1t (-30%) CCK-4 0.8 ± 0.1 1.5 ± 0.1 1.2 ± 0.2 NS Gly-Trp-Met 0.10 ± 0.02 1.1 ± 0.1 0t (-100) Trp-Met 0.04 ± 0.02 0.40 ± 0.02 0.12 ± 0.02§ (-70%) Trp 5.0 ± 0.6 0.78 ± 0.04 0.46 ± 0.02 NS Slices (2-3 mg of protein) were preincubated for 10 min in the presence of inhibitors (0.1 ,uM thiorphan, 0.1 mM bestatin, and 10 ,uM AAP-boro-V) and incubated for 15 min in the presence of 0.4 mM CCK-8. Peptides were analyzed by HPLC using (i) 31% CH3CN in pH 4 buffer for separation of CCK-8 and CCK-6 (and CCK-7), with retention times of 5.8 and 8.4 min, respectively; (ii) 30%o CH3CN in pH 2.5 buffer for separation of CCK-5 and CCK-4, with retention times of 5.4 and 4.6 min, respectively; (iii) 18% CH3CN in pH 2.5 buffer for Gly-Trp, Trp-Met, and Gly-Trp-Met, with retention times of 3.4, 5.6, and 7.1 min, respectively; (iv) 7% CH3CN in pH 2.5 buffer for tryptophan (7.3 min). Column effluents were continuously monitored by UV absorbance (254 nm; Waters spectrometer 440) by using a computing integrator [Spectra Physics 4200 (Santa Clara, CA)]. Levels of identified peptides were calculated by comparison with the height of the corresponding external standards. Percentages in parentheses show changes as compared with thiorphan + bestatin. NS, not significant. *Total fragment formation was calculated as the difference between the amounts of CCK-8 introduced as substrate and recovered in intact form at the end of incubations. tp < 0.05. tP < 0.001 as compared with thiorphan plus bestatin. §P < 0.01. Downloaded by guest on September 28, 2021 Neurobiology: Rose et al. Proc. Natl. Acad. Sci. USA 85 (1988) 8329 and Trp-Met) were increased at the expense of tryptophan, have resulted from cleavage of the Met-Asp bond alone, has and CCK-5 became the major fragment. Simultaneous addi- never been detected, and since Gly-Trp-Met formation by the tion of AAP-boro-V with thiorphan and bestatin decreased serine peptidase occurred at a rate 30-times higher with both CCK-8 hydrolysis and the appearance of CCK-5 and CCK-5 as the substrate instead ofCCK-8 (see Results). Since suppressed Gly-Trp-Met production completely and Trp-Met Gly-Trp-Met and Trp-Met (which may be formed by hydrol- formation almost completely (Table 3). ysis of the tripeptide by a bestatin-insensitive aminopepti- When 0.4 mM CCK-6 was used as a substrate, it was dase) are nonimmunoreactive fragments, they may account hydrolyzed at a higher rate-i.e., 12.0 ± 1.3 nmol/mg of to a great extent for the incomplete recovery of endogenous protein per 15 min. Moreover, CCK-5 formation was signif- CCK-ir. icantly increased by the serine peptidase inhibitors (2.9 ± Since CCK-5 retains <0.01% of CCK-8 biological activity 0.2) instead of 2.1 ± 0.2 nmol/mg of protein per 15 min), in pancreas (31) and 2-10% in binding tests with cerebral whereas Gly-Trp-Met formation was abolished. membranes (32, 33), its formation corresponds to an inacti- When 0.4 mM CCK-5 was used as a substrate, it was vation process, particularly since it is itselfhydrolyzed by the hydrolyzed at a rate of 18.6 ± 2.4 nmol/mg of protein per 15 serine peptidase activity at a much higher rate than CCK-8 min (i.e., much higher than CCK-8), the major peptide (see Results). metabolite being Gly-Trp-Met, whose formation occurred at Although formation of Gly-Trp-Met has never been re- a rate of2.7 ± 0.3 nmol/mg ofprotein per 15 min and was also ported, that of CCK-5 has been detected in some studies with suppressed by serine peptidase inhibitors. exogenous CCK-8 and synaptic membranes [but not in others (17)] and was attributed to thiol- (18) or metallo-endopeptidase DISCUSSION activities (19), in view of the inhibitions elicited by organo- mercurial and agents, In contrast, We have characterized a serine endopeptidase as a major chelating respectively. inactivating enzyme for endogenous CCK-8 in brain slices by various serine peptidase inhibitors were ineffective (18, 34). using a three-stage strategy. This consisted of (i) identifying, Although the role for an endogenous serine peptidase in a large series of peptidase inhibitors, those able to protect inhibitor in the pathophysiology of Alzheimer disease was depolarization-released CCK-ir and establishing their rela- recently proposed, few serine peptidases have yet been tive potencies; (ii) determining by HPLC the pattern of CCK characterized in brain (35-38). The CCK-inactivating en- immunoreactive peptides originating from the tissues and the zyme identified in the present study does not appear to effect of the most potent inhibitor identified at the preceding correspond to any known cerebral peptidase. However, this stage; and (iii) confirming and extending these data by study provides it with an "identification card" that should establishing the pattern of exogenous CCK hydrolysis in the allow it to be monitored in the course of purification. The same preparation and studying its modification by the same hydrolysis of exogenous CCK-8 by brain slices indicates that inhibitor. This last stage was necessary in view of the it is an ectoenzyme-i.e., a membrane-bound enzyme with limitations of the approach based upon endogenous CCK-ir its active site directed towards the extracellular space. It is an analysis, in which short fragments with low (if any) immu- endopeptidase rather than an exopeptidase because it does noreactivity cannot be reliably if at all detected. not cleave the Met-Gly bond of CCK-6 (i.e., Met-Gly-Trp- The involvement of a serine peptidase in endogenous CCK Met-Asp-Phe-NH2). As an endopeptidase it appears to have hydrolysis was shown by the concentration-dependent pro- preference for bonds where the carboxyl group is donated by tection provided by selected (but not all) inhibitors belonging a methionine residue. From the marked differences in po- to three distinct classes of compounds-i.e., serine-alkylat- tencies of the various inhibitors, particularly among the ing reagents (phenylmethylsulfonyl fluoride and diisopropyl chloromethyl ketones-Tos-LysCH2Cl and Tos-PheCH2CI fluorophosphate), active site-directed inactivators known to being inactive and various elastase inhibitors being quite alkylate the histidine residue involved in catalysis (peptide potent-this serine peptidase does not appear to be either a chloromethyl ketones), and transition-state inhibitors be- trypsin-like or a chymotrypsin-like but rather an "elastase- lieved to form a nonproductive tetrahedral complex with the like" serine peptidase. active-site serine (peptide boronic acids) (28-30). In addition, less well characterized peptidases may be With AAP-boro-V, one of the most potent inhibitors, the responsible for alternative degradation pathways for CCK, protection of CCK-ir corresponded to an increase of about particularly when the serine peptidase pathway is inhibited. 200% in the recovery of CCK-8-ir itself, indicating that the These pathways are responsible for the formation of both putative seine peptidase is a primary hydrolyzing enzyme. CCK-4 and CCK-6, of which the former was previously CCK-8-ir protection was accompanied by a marked impair- detected with exogenous CCK-8 as substrate (34). Eventu- ment in the formation of CCK-5-ir, the most abundant ally, taking into account the low level of CCK-8(n.s.)-ir immunoreactive fragment (not including CCK-4-ir, whose detected, it appears that CCK-8 desulfation, which corre- identity remains conjectural in view of its low immunoreac- sponds to a loss ofbiological activity (39), does not constitute tivity and the possibility of contamination by oxidized CCK- a major inactivation pathway. of CCK-8 the at the amide 8). Cleavage by serine peptidase 1. Schwartz, J. C., Malfroy, B. & De La Baume, S. (1981) Life Sci. bond between Met-3 and Gly-4 was confirmed in studies with 29, 1715-1740. exogenous CCK-8, in which the formation of CCK-5 (i.e., 2. Schwartz, J. C. (1983) Trends NeuroSci. 6, 45-48. Gly-Trp-Met-Asp-Phe-NH2), again a major hydrolysis prod- 3. Llorens-Cortes, C., Gros, C. & Schwartz, J. C. (1986) Proc. uct, was significantly reduced by AAP-boro-V (Table 3). Nati. Acad. Sci. USA 83, 6226-6230. Interestingly the nonimmunoreactive "endopeptide" Gly- 4. Lynch, D. R. & Snyder, S. H. (1986) Annu. Rev. Biochem. 55, Trp-Met, resulting from the cleavages ofthe two amide bonds 773-799. where the carboxyl group is donated by a methionine residue, 5. McKelvy, J. F. & Blumberg, S. (1986) Annu. Rev. Neurosci. 9, was another major metabolite of exogenous CCK-8, and its 415-434. became when its 6. Dockray, G. J. (1983) in Brain Peptides, eds. Krieger, D. T., formation, which apparent secondary deg- Brownstein, M. J. & Martin, J. B. (Wiley, New York), pp. 851- radation into tryptophan was prevented, was completely 869. blocked by AAP-boro-V. It seems likely that cleavage of the 7. Morley, J. E. (1982) Life Sci. 30, 479-493. bond between Met-6 and Asp-7 occurs after cleavage of the 8. Beinfeld, M. C. (1983) Neuropeptides 3, 411-427. bond between Met-3 and Gly-4 [i.e., when CCK-5 has been 9. Rehfeld, J. F. (1985) J. 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