Purification and characterization of prolyl endopeptidase from rat skin

JOURNALOF

Journal of Dermatological Science 6 ( 1993) 138- I45

Dermatological Science

Purification and characterization of prolyl endopeptidase from rat skin Masahiro Kusuhara*, Hiroshi Hachisuka, Shunji Nakano, Yoichiro Sasai Culaneous Biology Unit, Department of Dermatology, Kurume University School of Medicine, Asahimachi 67. 830 Kurume, Japan

(Received 26 November 1992; revision received 3 March 1993; accepted 22 May 1993)

Abstract

An enzyme with the specificity of a prolyl endopeptidase was purified approximately 329-fold from rat skin. The enzyme has a molecular weight of 70 000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pH optimum of 5.8 as checked with 7-(Succinyl-Gly-Pro)-4-methylcoumarinamide (Sue-Gly-Pro-MCA) as the substrate. The optimal temperature for the enzyme activity was 40°C. The K, and V,,,,x values for Sue-Gly-Pro-MCA were 0.7 mM and 68 nmol/min per mg protein, respectively. The enzyme activity was markedly inhibited by diisopropyl fluorophosphate, p-chloromercuribenzoic acid, N-ethylmaleimide, Zn2+ and Cu’+, while it was partially inhibited by phenylmethanesulphonyl fluoride. The purified enzyme was shown to release the N-terminal tetrapeptide, Arg-ProLys-Pro, from substance P producing the C-terminal heptapeptide, Gln-Gln-Phe-Phe-Gly-Met-CONH,. In the skin, this enzyme might be related to the inactivation of substance P. Key words: Prolyl endopeptidase;

Rat skin; Substance P; Sue-Gly-Pro-MCA

1. Introduction

Prolyl

endopeptidase

(post-proline

cleaving

enzyme, EC 3.4.21.26) was found in human uterus by Walter et al. [ 11. The enzyme activity has been demonstrated

in

various

mammalian

organs

* Corresponding author. Tel.: 0942 35 3311; Fax: 0942 34 2620. Abbreviations:

AMC, 7-amine-4-methylcoumarin; DFP, diisopropyl fluorophosphate; DTT, dithiothreitol; EDTA, ethylendiamine tetra-acetate; HPLC, high-performance liquid chromatography; NEM, N-ethylmaleimide; PCMB, pchloromercuribenzoic acid; PMSF, phenylmethanesulphonyl fluoride; SBTI, soybean trypsin inhibitor; SDS, sodium dodecyl sulfate; Sue-Gly-Pro-MCA, 7-(Succinyl-Gly-Pro )-4methylcoumarinamide; TFA, trifluoroacetic acid.

[2-131, body fluids [14,15], plants [16] and Flavobacterium [17]. This endopeptidase is known to catalyze the cleavage of the Pro-X peptide bond and the hydrolysis ‘of several biologically active peptides, i.e. substance P, oxytocin, angiotensin II, thyrotropin releasing hormone, bradykinin, neurotensin and vasopressin [3-171. Substance P, which was isolated from equine brain and intestine [ 181,is distributed widely in the central and peripheral nervous systems [ 191. However, the metabolism of substance P, released from the peripheral nervous system by some forms of stimulation, is not clear. Prolyl endopeptidase of various mammalian organs can hydrolyze sub-

0923-181l/93806.00 0 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0923-1811(93)00222-M

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stance P to two fragments, the N-terminal tetrapeptide and the C-terminal heptapeptide. The Nterminal tetrapeptide fragment of substance P (Arg-Pro-Lys-Pro) and the C-terminal heptapeptide (Gln-Gln-Phe-Phe-Gly-Leu-Met-CONH2) are much less potent compared to intact substance P in histamine releasing properties as measured on rat peritoneal mast cells in vitro [20,21]. This observation raises the possibility that the prolyl endopeptidase plays a role in inactivating substance P in the skin. In this paper, we describe the purification and characterization of prolyl endopeptidase from rat skin, and show the hydrolysis of substance P by purified enzyme in vitro. 2. Materials and methods 2.1. Materials Substance P, substance P fragment l-4 (ArgPro-Lys-Pro), substance P fragment 5-l 1 (GlnGln-Phe-Phe-Gly-Leu-Met-NH3, diisopropyl fluorophosphate (DFP), phenylmethanesulphonyl fluoride (PMSF), p-chloromercuribenzoic acid (PCMB), N-ethylmaleimide (HEM) and soybean trypsin inhibitor (SBTI) were purchased from Sigma (St. Louis, MO). 7-(Succinyl-Gly-Pro)-4methylcoumarinamide (Sue-Gly-Pro-MCA) and Z-Gly-Pro-Leu-Gly-Pro - Hz0 +AcOEt were obtained from the Peptide Institute (Osaka, Japan). Sephacryl S-300, DEAE-Sephacel and AHSepharose 4B were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Reversed phase column (TSKgel ODS-120T) for high-performance liquid chromatography (HPLC) was purchased from Tosho (Tokyo, Japan). HPLC-grade acetonitrile and trifluoroacetic acid (TFA) were from Wako (Osaka, Japan). All other chemicals used were of analytical grade. The Wistar strain rats (150 g) were killed by exsanguination and the whole back skin was quickly removed, frozen, and stored at -80°C. 2.2. Assay of enzyme activity Prolyl endopeptidase activity was measured using Sue-Gly-Pro-MCA as substrate by the method described by Kato et al. [7] The reaction mixture (total vol. 100 ~1) contained 20 mM sodium phosphate buffer (pH 6.8) 0.5 mM sodium

139

ethylendiamine tetra-acetate (EDTA), 0.5 mM dithiothreitol (DTT), 0.5 mM Sue-Gly-Pro-MCA and the enzyme. They were incubated at 37°C for 10 min, and 1.0 ml of 1 M sodium acetate buffer (pH 4.2) was added to stop the enzyme reaction. The fluorescence of 7-amino-4-methylcoumarin (AMC) was measured in FS-8000 spectrophotofluorometer (Hitachi, Tokyo, Japan) with excitation at 380 nm and emission at 460 nm. 2.3. Purification of prolyl endopeptidase Since preliminary experiment revealed that the enzyme activity was equally present on the dermis and epidermis, prolyl endopeptidase was purified from the soluble fraction of rat whole skin. All of the following purification steps were performed at 0-4°C. The whole rat skin (about 40 g), cleaned of fat, was cut into small pieces and homogenized in 5 ~01s. of 25 mM sodium phosphate buffer (pH 6.8) containing 2 mM DTT and 0.5 mM EDTA. The homogenate was centrifuged at 100 000 x g for 90 min and the supematant was saved. Solid (NH&SO4 was added to 50% saturation. The suspension was stirred for 20 min and then centrifuged at 27 000 x g for 15 min. The precipitate was discarded, and solid (NH&SO4 was added to the supernatant to give 70% saturation. After mixing for 20 min, the solution was kept overnight and then centrifuged at 27 000 x g for 15 min. The supernatant was removed, and the precipitate was dissolved in 10 ml of 25 mM sodium phosphate buffer (pH 6.8) containing 1 mM DTT and 0.5 mM EDTA (buffer A). After centrifugation at 24 000 x g for 20 min, the supernatant was applied to a column (2.5 x 75 cm) of Sephacryl S300 equilibrated with buffer A. The column was eluted with the same buffer, and fractions of 6 ml were collected (Fig. 1). Protein concentration was monitored by measuring the absorbance at 280 nm. Fractions containing enzymatic activity were pooled and applied to a DEAE-Sephacel column (2.5 x 15 cm) equilibrated with buffer A. The enzyme was eluted with a linear gradient established between 180 ml of the starting buffer and 180 ml of the same buffer containing 0.3 M NaCl. Fractions of 3 ml were collected. The active fractions were pooled and concentrated by ultratiltration. The concentrated solution was dialyzed

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Fraction number Fig. I. Chromatography of the enzyme on the first column of Sephacryl S-300 (step 3 of the puritication procedure). Fractions of 6 ml were collected. Absorbance was measured at 280 nm (O---O). Enzyme activity was determined as described under Materials and Methods by using 0.5 mM of Sue-Gly-Pro-MCA as substrate (O- - -0). Fractions 51-56 were pooled. AMC; 7-amino4 methylcoumarin.

against a large volume of buffer A, and applied to a column (1 x 10 cm) of Z-Gly-Pro-Leu-Gly-Pro AH-Sepharose 4B (Z-GPLGP-AH-Sepharose), equilibrated with buffer A. The column was washed with 30 ml of buffer A and eluted with 75 ml of a linear gradient of NaCl (O-O.5 M) in the same buffer. Fractions of 3 ml were collected. The active fractions were collected and concentrated (Fig. 2). The enzyme solution was passed through the same column of Sephacryl S-300, and the active fractions were pooled. After concentrating by ultrafiltration, the solution was applied to a Z-GPLGP-AH Sepharose column (1 x 10 cm) equilibrated with buffer A. The column was eluted with a linear gradient described above. The active fractions were pooled and used for the following experiments. Protein concentration was measured by Bio-Rad Protein Assay (Bio-Rad Lab. Richmond, CA) with bovine serum albumin used as a standard. 2.4. Determination of enzyme properties The effect of pH on the activity of prolyl endopeptidase using Sue-Gly-Pro-MCA as a substrate was determined in a 0.1 -M sodium acetate

buffer between pH 4.0 and pH 7.0, and in a 50-mM Tris-maleate buffer between pH 5.8 and pH 8.0. The inhibitors used were pre-incubated with the enzyme at 37°C for 10 min. Residual activities were assayed as described above. 2.5. Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis was performed by the method of Laemmli [22] with the use of 12.5% sodium dodecyl sulfate (SDS)polyacrylamide gel. A total volume of 2 ~1containing 3 pg of protein was applied. The gel was stained for protein in 0.25% Coomassie brilliant blue. An electrophoresis calibration kit (Pharmacia) was used for molecular weight marking (94 OOO14 400). 2.6. Determination of activity with substance P The hydrolysis of substance P by purified enzyme was determined by reverse phase HPLC. Substance P (0.1 mM) was used as substrate instead of Sue-Gly-Pro-MCA in the incubation mixture described above. Incubation was carried out at 37°C for O-60 min, and the mixture was boiled at 95°C for 5 min. After centrifugation, the super-

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-14 0.16 -12

= -10

E f E

.8

% E E.

.6

P E

2

-2

Fraction number

Fig. 2. Chromatography of the enzyme on the first column of Z-Gly-Pro-Leu-Gly-Pro-NH-(CH&,-Sepharose 48 (step 5 of the puriftcation procedure). Each fraction of 3 ml was collected. Absorbance was measured at 280 nm (O---O). Enzyme activity was determined using 0.5 mM of Sue-Gly-Pro-MCA as substrate (O---O). Fractions 16-20 were pooled. The experimental details are described in Materials and Methods.

natant was filtered through a Millipore membrane filter (HV; pore size, 0.45 pm) and applied to a TSK-gel ODS-120T column (4.6 x 150 mm) equilibrated with 0.1% TFA. Elution was carried out at room temperature with a IO-min linear gradient of O-25% acetonitrile in 0.1% TFA and then a lo-min gradient of 25-30% acetonitrile at a flow rate of 1.0 ml/min. The peptides eluted were detected by monitoring the absorbance at 215 nm. The cleavage products were identified by comparing their retention times with those of authentic substance P fragments. 3. Results 3.1. Purification of prolyl endopeptidase from rat skin

The enzyme was purified from the supernatant of rat skin homogenate by ammonium sulfate fractionation and sequential chromatographies. The purification of the enzyme is summarized in Table 1. Finally the enzyme with prolyl endopeptidase activity was purified 329-fold from the soluble fraction of rat skin. In the purification procedure including gel filtration, ion-exchange chroma-

tography and affinity chromatography, the enzyme activity increased more than first total activity of homogenate supernatant, and then recovery exceeded 100°/o. 3.2. Enzymatic and physicochemical properties The purified enzyme from rat skin showed a pH optimum of 5.8 for the hydrolysis of Suc-Gly-ProMCA in sodium acetate and Tris-malate buffer. The optimal temperature for the enzyme activity was 40°C. About 50% of the original activity remained after incubation at 45°C at pH 6.8 for 20 min. The kinetic parameters of the purified enzyme toward Sue-Gly-Pro-MCA were determined by means of a Lineweaver Burk plot. The K,,, and I&, values were calculated to be 0.7 mM and 68 nmol/min per mg protein, respectively. The approximate molecular weight of the enzyme in SDS polyacrylamide gel electrophoresis was 70 000 (Fig. 3). 3.3. Effects of chemical reagents, inhibitors and heavy metals The enzyme activity was markedly inhibited by DFP, PCMB and NEM, and only partially in-

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Table 1 Purification of prolyl endopeptidase from rat skin Step

Total protein (mg)

Specific activity (nmol/min/mg protein)

Recovery (Xl)

Purification

100 000 X g supernatant” (NH&so, @o-70%) 1st Sephacryl S-300 DEAE-Sephacel 1st AH-Sepharose 4Bb 2nd Sephacryl S-300 2nd AH-Sepharose 4Bb

1442 325.4 139.2 54.4 23.7 18.8 13.9

0.26 0.124 5.48 19.3 53.9 81.7 85.5

100 10 203 280 340 409 317

1.00 0.475 21.1 74.4 207.3 314.2 328.8

‘In this preparation, 40g of rat skin was used. b AH-Sepharose 4B; Z-Gly-Pro-Leu-Gly-Pro-NH-(CR&,-Sepharose

4B.

hibited by PMSF (Table 2). The enzyme was resistant to trypsin inhibitors from soybean and metal chelator, EDTA. No significant change in this pattern was observed even after prolonged preincubation (30 min, data not shown). The heavy metals Cu*+ and Zn*+ strongly inhibited the activity. Table 2 Effects of various inhibitors and metals on the enzyme activity

Enzyme

Addition?

Concentrations (mM)

Relative activity (%)

None DFP PMSF

-

100 0 91 53 0 4 93 92 91 95 150 0 137 145 0 197 113

+ 67,000

PCMB NEM SBTI EDTA CaCI,

-dye

front

1 2 Fig. 3. SDS-polyacrylamide gel electrophoretic patterns of the purified enzyme. 1, enzyme; 2, molecular weight standard proteins. Electrophoresis was carried out with 2-pl aliquots (1.5 mgAnl protein) at a constant current of 60 mA/gel.

CUCI, MgCIz ZnClz MnCIz

0.1 0.1 1.0 0.1 0.1 100 &ml 0.1 0.5 0.1 0.5 0.1 0.1 0.5 0.1 0.1 0.5

aThe enzyme was preincubated with respective inhibitor or metal for IO min at 37°C and the residual activities were assayed by the standard method.

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However, Ca*+, Mg*+ and Mn2+ caused activation at some concentrations. 3.4. Hydrolysis of substance P determined by HPLC

The HPLC pattern of the peptides produced from substance P through the action of the enzyme is shown in Fig. 4. Two newly formed peptidecontaining peaks (peaks 1 and 2) were detected, together with a peak of substance P. As the incubation time was prolonged, the two peaks became larger in parallel with a decrease in the

SP

(A) gradient

t a

w

_,I__

0

gradient

_

10

20

30

min Fig. 4. HPLC analysis of the products of cleavage of substance P by the puritied enzyme. The reaction mixture was incubated at 37°C for 0 (A), 10 (B), and 60 min (C). After boiling at 95°C for 5 min, 20-81aliquots of the mixture were analyzed by HPLC on a reversed-phase column (4.6 x 150 mm) of TSK-gel ODS120 T. The experimental details are described in Materials and Methods.

peak of substance P. Control incubations, in which enzyme and substrate were omitted, respectively, revealed that no UV-absorbing materials were released from substance P and the purified enzyme during the incubation period and boiling. The retention times of peaks 1 and 2 were corresponded with those of authentic substance P fragment l-4 and 5- 11, respectively. 4. Discussion Prolyl endopeptidase has been demonstrated in various mammalian tissues, and purified from brain [4-91, kidney [12], liver [lO,ll], pituitary [ 131and testis [3]. We detected the prolyl endopeptidase activity in soluble fraction of rat skin and have succeeded in purifying the enzyme to 329fold. The enzyme activity was, however, much lower than that of other tissues [7,10,1 l] using the same substrate, Sue-Gly-Pro-MCA. In the purification procedure, for the steps of column chromatography, the yield of activity exceeded 100%. Previously, the presence of endogenous prolyl endopeptidase inhibitor in hog pancreas [23], rat brain [24] and liver [25] and sperm of the ascidian [15] were reported. It has been pointed out that the inhibitor is widely distributed in several mammalian tissues and body fluids. From this it may be inferred that the endogenous inhibitor(s) of rat skin prolyl endopeptidase were removed by the steps of column chromatography. The inhibitor(s) seem to have been completely removed from the enzyme fraction by the second gel liltration using Sephacryl S-300. Otherwise, the possibility cannot be ruled out that the precursor of this enzyme was activated during the purilication procedure. Prolyl endopeptidase of rat skin was strongly inhibited by a serine protease inhibitor, DFP, and sulfhydryl-blocking reagents, PCMB and NEM, but it was only slightly affected by PMSF. This property is similar to the enzyme from other sources [3,5-7,9,10]. It has been previously indicated that the prolyl endopeptidase belongs to the serine proteinases [26], and that one or more sulfhydryl residue seems to be located close to the active site [5,7,11]. The same is true for the enzyme of rat skin. Furthermore, the enzymes from plants

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and mammals are very sensitive to PCMB as well as DFP, while the microbial enzymes are insensitive to PCMB [3,17]. Cu*+ and Zn*+ were potent inhibitors of the enzyme from rat skin. There is no doubt that prolyl endopeptidase can hydrolyze substance P regardless of their source. In the present study, it was also recognized that prolyl endopeptidase from rat skin can cleave substance P to N-terminal tetrapeptide and Cterminal heptapeptide. With regard to histamine releasing property of substance P, the N-terminal tetrapeptide is much less potent than intact substance P either on its own or when added together with the C-terminal heptapeptide [20]. On the basis of these observations, it is assumed that hydrolyzing substance P between Pro and Gln residues is most effective to inactivate the histamine releasing property of substance P. The mechanism involved in the inactivation of substance P is unknown, although a number of enzymes capable of degrading this peptide have been reported [27-301. We suppose that skin prolyl endopeptidase plays an important role in regulating histamine release from peripheral mast cells by substance P. However, this enzyme was observed in the soluble fraction, and it is unclear whether the enzyme is located intra- or extracellularly. Further studies may provide relevant information of the localization of the enzyme in the skin and the inactivation of substance P released from peripheral nerves.

6

7

8

9

IO

II

12

13

14

I5

16

5. References Walter R, Shlank H, Glass JD, Schwartz IL, Kerenyi TD: Leucylglycinamide released from oxytosin by human uterine enzyme. Science 173: 827-829, 1971. Yoshimoto T, Ogita K, Walter R, Koida M, Tsuru D: Post-proline cleaving enzyme: Synthesis of a new fluorogenic substrate and distribution of the endopeptidase in rat tissues and body fluids of man. Biochim Biophys Acta 569: 184-192, 1979. Yoshimoto T, Oyama H, Koriyama N, Tsuru D: Prolyl Purification, endopeptidase from bovine testis: characterization and comparison with the enzymes from other tissues. Chem Pharm Bull 36: 1456-1462, 1988. Blumberg S, Teichberg VI, Charli JL, Hersh LB, Mckelvy JF: Cleavage of substance P to an N-teminal tetrapeptide and a C-terminal heptapeptide by a post-proline cleaving enzyme from bovine brain. Brain Res 192: 447-486, 1980. Yoshimoto T, Nishimura T, Kita T, Tsuru D: Post-

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proline cleaving enzyme (Prolyl endopeptdase) from bovine brain. J Biochem 94: 1179-I 190, 1983. Orlowski M, Wilk E, Pearce S, Wilk S: Purification and properties of a prolyl endopeptidase from rabbit brain. J Neurochem 33: 461-469, 1979. Kato T, Nakano T, Kojima K, Nagatsu T, Sakakibara S: Changes in prolyl endopeptidase during maturation of rat brain and hydolysis of substance P by the purified enzyme. J Neurochem 35: 527-535, 1980. Taylor WL, Dixon JE: Catabolism of neuropeptides by a brain proline endopeptidase. Biochem Biophys Res Commun 94: 9-15, 1980. Yoshimoto T, Simmons WH, Kita T, Tsuru D: Postproline cleaving enzyme from lamb brain. J Biochem 90: 325-334, 1981. Moriyama A, Sasaki M: Porcine liver succinyltrialaninepnitroanilide hydrolytic enzyme. Its purification and characterization as a post-proline cleaving enzyme. J Biochem 94: 1387-1397, 1983. Yamakawa N, Soeda S, Shimeno H, Nagamatsu A: Purification and characterization of proline endopeptidase from rat liver. Chem Pharm Bull 34: 256-263, 1986. Koida M, Walter R: Post-proline cleaving enzyme: Puritication of this endopeptidase by affinity chromatography. J Biol Chem 251: 7593-7599, 1976. Knisatschek H, Bauer K: Characterization of “Thyroliberin-deamidating enzyme” as a post-proline cleaving enzyme. J Biol Chem 254: 10936-10943, 1979. Soeda S, Ohyama M, Yamakawa N, Shimeno H, Nagamatsu A: Two molecular species of proline endopeptidase in human plasma: isolation and characterization, Chem Pharm Bull 32: 4061-4069, 1984. Yokosawa H, Miyata M, Sawada H, Ishii S: Isolation and characterization of a post-proline cleaving enzyme and its inhibitor from sperm of the Asicdian, Halocynthia roretzi. J Biochem 94: 1067-1076, 1983. Yoshimoto T, Sattar AKMA, Hirose W, Tsuru D: Studies on prolyl endopeptidase from carrot (Daucus carota): purification and enzymatic properties. Biochim Biophys Acta 916: 29-37, 1987. Yoshimoto T, Walter R, Tsuru D: Proline-specific endopeptidase from Flavobacterium. J Biol Chem 255: 4786-4792, 1980. Von Euler US, Gaddum JH: An unidentiried depressor substance in certain tissue extracts. J Physiol (London) 72 192: 74-87, 1931. Hiikfelt T, Kellerth J-O, Nilsson G, Pernow B: Substance P: Localization in the central nervous system and in some primary sensory neurons. Science 190: 889-890, 1975. Mazurek N, Pecht I, Teichberg VI, Blumberg S: The role of the N-terminal tetrapeptide in the histamine releasing P. Neuropharmacology 20: action of substance 1025-1027, 1981. Renner H, Foreman JC, Jordan CC, Oehme P: Histamine release from mast cells by substance P and substance P sequenscs. Pharmazie 37: 866, 1982. Laemmli UK: Cleavage of structural proteins during the

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assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970. 23 Yoshimoto T, Tsukumo K, Takatsuka N, Tsuru D: An inhibitor for post-proline cleaving enzyme; distribution and partial purification from porcine pancreas. J Pharm Dyn 5: 734-740, 1982. 24 Soeda S, Yamakawa N, Ohyama M, Shimeno H, Nagamatsu A: An inhibitor of proline endopeptidase: purification from rat brain and characterization. Chem Pharm Bull 33: 2445-2451, 1985. 25 Yamakawa N, Shimeno H, Soeda S, Nagamatsu A: Inhibition of proline endopeptidase activity by acylcoenzyme A esters. Biochim Biophys Acta 1037: 302-306. 1990. 26 Yoshimoto T, Orlowski RC, Walter R: Postproline cleaving enzyme: identification as serine protease using active site specitic inhibitors. Biochemistry 16: 2942-2948, 1977.

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Skidgel RA, Engelbrecht S, Johnson AR, Erdos EG: Hydrolysis of substance P and neurotensin by converting enzyme and neutral endopeptidase. Peptides 5: 769-776, 1984. 28 Matsas R, Rattray M, Kenny AJ, Turner AJ: The metabolism of neuropeptides. Endopeptidase-24.11 in human syaptic membrane preparations hydrolyses substance P. Biochem J 228: 487-492, 1985. 29 Lee C, Sandberg BEB, Hanley MR, Iversen LL: Puritication and characterisation of a membrane-bound substance-P-degrading enzyme from human brain. Em J Biochem 114: 315-327, 1981. 30 Endo S, Yokosawa H, Ishii S: Purifiation and characterization of a substacne P-degrading endopeptidase from rat brain. J Biochem 104: 999-1006. 1988.