Peptides,Vol. 12, pp. 995-1000. ©PergamonPress plc, 1991. Printedin the U.S.A.

0196-9781/91 $3.00 + .00

Human Mast Cell Proteases Hydrolyze Neurotensin, Kinetensin and LeuS-Enkephalin I S A N F O R D M. G O L D S T E I N , 2 J A N E L E O N G A N D N I G E L W. B U N N E T r *

Departments of Dermatology and Physiology and *Surgery, University of California, San Francisco, CA Received 8 April 1991 GOLDSTEIN, S. M., J. LEONG AND N. W. BUNNETT. Human mast cell proteases hydrolyze neurotensin, kinetensin and LeuS-enkephalin. PEPTIDES 12(5) 995-1000, 1991.--Purified mast cell carboxypeptidase cleaved the C-terminal leucines from LeuS-enkephalin (Leu-ENK), neurotensin (NT), and kinetensin (KT), with Km values of 36, 16, and 15 p~M, and kcat values of 44, 51, and 53 s -1, respectively. To better predict potential in vivo hydrolysis products generated by mast cell proteases, these peptides were incubated with released skin mast cell supernatants. Leu5-enkephalin was hydrolyzed only by carboxypeptidase. Kinetensin was cleaved by tryptase, chymase, and carboxypeptidase to yield KT(1-3), KT(1-7), KT(1-8), KT(4-7), and KT(48), the last two peptides by the concerted action of two of the proteases. NT(1-11) and NT(1-12) were generated from neurotensin by chymase and carboxypeptidase, respectively. Skin mast cells LeuS-enkephalin

Mast cell proteases Kinetensin

Tryptase

Chymase

MAST cells and the peripheral nervous system are related anatomically and physiologically. Anatomically, mast cells exist in close approximation to nerves in a variety of tissues (32). Physiologically, histamine can stimulate antidromic axon reflexes (20), and neuropeptides, such as substance P, are capable of activating mast cells in vitro and in vivo (5, 7, 12, 18, 23, 33). Proteases released from neuropeptide-stimulated mast cells are, in turn, capable of degrading these neuropeptides (2, 3, 6, 34). Two mast cell neutral proteases, chymase, and tryptase, have been shown to hydrolyze several neuropeptides, including calcitonin gene-related peptide, peptide histidine methionine, and vasoactive intestinal peptide (6,34). Chymase also hydrolyzes substance P (6). Mast cell carboxypeptidase is the second most abundant mast cell protease in human mast cells (14,28). It is present only in the mast cell type MCTc that comprises 99% of skin mast cells (17). Thus far, angiotensin I is the only peptide with potential physiologic relevance identified as a substrate for the enzyme (14,15). Previous studies using synthetic tripeptides suggested that peptides with the carboxy-terminal hydrophobic residues tyrosine, phenylalanine or leucine would be potential substrates for this protease (13). Recent studies show that a variety of vasoactive peptides and neuropeptides with a C-terminal leucine residue are hydrolyzed by a carboxypeptidase from human stomach that appears to be identical to mast cell carboxypeptidase (3). Many of these peptides are in the neurotensin family, and their hydrolysis by mast cell proteases has not been previously described. We sought to investigate the kinetics of the hydrolysis of kinetensin (22), neurotensin (23), and LeuS-enkephalin (16) by purified mast cell carboxypeptidase. However, since mast cell proteases are released not as individual proteases, but as part of

Carboxypeptidase

Neurotensin

an enzyme-proteoglycan macromolecular complex, ionically linked to heparin proteoglycan (30), the in vitro use of all of the mast cell neutral proteases in this complexed form might better predict potential peptide hydrolysis products that might be generated in vivo. Therefore, we also evaluated the hydrolysis of neurotensin, kinetensin, and LeuS-enkephalin by the complexes in released skin mast cell supernatants. METHOD

Materials Neurotensin, LeuS-enkephalin, potato tuber carboxypeptidase inhibitor (PCI), tosyl-lysyl-chloromethyl ketone (TLCK) (Sigma Chemicals, St. Louis, MO); kinetensin (Peninsula Labs, Belmont, CA); angiotensin I (AI) (Star Biochemicals, Torrance, CA); trifluoroacetic acid (TFA), acetonitrile (CH3CN) (Fisher Scientific, San Francisco, CA); A23187, soybean trypsin inhibitor (SBTI) (Calbiochem, La Jolla, CA); tosyl-glycyl-L-prolyl-Llysyl-p-nitroanilide acetate (tosyl-Gly-Pro-Lys-pNa) (Boehringer Mannheim Biochemicals, Indianapolis, IN) were obtained as noted.

Mast Cell Proteases Mast cell carboxypeptidase was purified from human skin extracts as described (14). Dispersed mast cells were prepared from the facial skin of patients undergoing cosmetic surgical procedures (14,28). Released mast cell supernatants were obtained by incubation of 5 x 105 dispersed mast cells of 13.2% purity in 2.5 ml PAGCM buffer (15) with calcium ionophore

1This work was supported by grants AM31901, DK39957, DK42341, RR01614 and RR04112 from the National Institutes of Health. 2Requests for reprints should be addressed to Sanford M. Goldstein, Dermatology Research, Box 0536, UCSF, San Francisco, CA 94143-0536.

995

996

A23187 (3 txM) at 37°C for 15 min. Mast cells were then removed by centrifugation at 225 x g for l0 min, and the supernatant was stored at - 7 0 ° C in aliquots. The net percent release of histamine, tryptase, chymase, and carboxypeptidase was determined, and a single preparation was used for all studies.

Enzyme and Mediator Assays Histamine was assayed using a radioimmunoassay kit (AMAC) according to the manufacturer's protocols. Mast cell tryptase activity was assayed as the hydrolysis of tosyl-Gly-L-Pro-L-LyspNa, in the presence of soybean trypsin inhibitor, monitored at 405 nm using a Varian DS spectrophotometer (28). Mast cell chymase and carboxypeptidase activities were assayed using angiotensin I. The hydrolysis of the Phe8-His9 bond of AI by chymase generating All, and the His9-Leu 1° bond by mast cell carboxypeptidase generating [des-Leul°]Al were analyzed by high pressure liquid chromatography (HPLC) (14, 15, 38). Lactate dehydrogenase was measured as described (1).

Hydrolysis of Neuropeptides Hydrolysis by purified skin mast cell carboxypeptidase. LeuS-enkephalin (20-100 ixM), kinetensin (5-50 IxM), and neurotensin (2.5-12.5 ixM) were incubated with 2-16 ng of purified mast cell carboxypeptidase at 37°C for up to 15 min to establish conditions under which the reactions exhibited first order kinetics. Reactions were stopped by the addition of four-fold excess HPLC buffer (0.1% TFA in H20). The hydrolysis products were characterized using HPLC (3), using a 250 × 4.5 mm C 18 Vydac column, 5 txm bead size, and a reverse phase gradient system of 0-80% v/v acetonitrile over 20 min in 0.1% v/v TFA at a flow rate of 1 ml/min. Peptides were analyzed at 214 nm. Once the number of products generated by purified mast cell carboxypeptidase from each peptide was determined, the kinetics of hydrolysis of NT, Leu-ENK, and KT by purified MCCP were investigated using an isocratic system of 25% v/v acetonitrile in 0.1% v/v TFA. Peptide hydrolysis was quantified by substrate degradation and by product formation. Retention times for neurotensin (NT), [des-Leul3]NT [NT(1-12)], Leu 5enkephalin (ENK), [des-LeuS]Leu-ENK [Leu-ENK(1-4)], kinetensin (KT) and [des-Leu9]KT [KT(1-8)] were 11.7, 7.8, 9.8, 6.8, 10.6, and 7.2 minutes, respectively. Conditions were chosen so that all assays were linear with respect to time and enzyme concentration. The kinetic constants Km, kcat, and kcat/Km were determined after 2-3 independent measurements, each using three concentrations of enzyme, by direct fit of 4-8 points using a computer software package (K-SOFT, BioMetallics, Inc.).

Hydrolysis by released human skin mast cell supernatants. For analysis of the hydrolysis products of the neuropeptides by tryptase, chymase, and carboxypeptidase in mast cell supematants, kinetensin (50 IxM), neurotensin (12.5 ~xM), and LeuS-enkephalin (100 IxM) were incubated with 10 ~1 of supernatant, the equivalent of the total protease activity of - 6 0 0 - 1 2 0 0 mast cells. The activity of tryptase, chymase, and carboxypeptidase in the supernatants was independently confirmed using the assays described above. Conditions were chosen so that an equivalent amount of mast cell carboxypeptidase activity was used in these incubations as used previously in determining K m. To characterize the proteases that generated each specific hydrolysis product, mast cell supernatants were preincubated for 15 min in the presence or absence of the following individual protease inhibitors prior to initiation of the reaction with peptide substrate: tosyl-lysyl-chloromethyl ketone (TLCK) (2 mM), which inhibits tryptase, but not chymase or carboxypeptidase (31); soybean

GOLDSTEIN, LEONG AND BUNNET'I

TABLE 1 KINETICCONSTANTSFOR SUBSTRATEHYDROLYSISBY MCCP Substrate LeuS-enkephalin Neurotensin Kinetensin

kcat (s- l)

Km (l~m)

44 _ 3.5 51 --- 12 53 -+ 9.5

36 +__ 1 16 - 1 15 - 3

keat/Km (10 s- IM- 1

6)

1.2 3.2 3.5

Purified mast cell carboxypeptidase (MCCP) was incubated with four to five concentrations of each substrate. The rate of hydrolysis was measured by HPLC as described in the Method section. The values represent mean _ SE. trypsin inhibitor (SBTI) (10 IxM), which inhibits chymase, but not tryptase or carboxypeptidase (38); or potato tuber carboxypeptidase inhibitor (PCI) (1 I~M), which inhibits carboxypeptidase, but not tryptase or chymase (15). The reactions were analyzed by HPLC as described above, using a gradient of 0-80% v/v acetonitrile over 20 min for LeuS-enkephalin, kinetensin and neurotensin, and also a gradient ramp of 0-30% v/v acetonitrile over 1 h, 30-50% over the next 10 min, and 50-100% over the final 10 min for kinetensin. Peaks were identified by retention time on an HPLC system standardized by identification of peaks by amino acid analysis (3), and by collection of fractions and use of amino acid composition (14), or liquid secondary ion mass spectrometry (SIMS) (11). RESULTS

Hydrolysis of Peptides by Purified Mast Cell Carboxypeptidase Each substrate was hydrolyzed by mast cell carboxypeptidase and only one product was made from each peptide. In all circumstances these products had identical retention times to peptides previously identified by amino acid analysis as [des-Leu9]kinetensin, [des-LeuS]Leu-enkephalin, or [des-Leu 13] neurotensin (3) (not shown). Using the HPLC gradient system, no hydrolysis of the penultimate residues of kinetensin or Leu 5enkephalin was seen. kcat, Km, and kcat/Km values for LeuS-enkephalin, kinetensin, and neurotesin hydrolysis are shown in Table 1.

Effect of Mast Cell Released Supernatants on Leu-Enkephalin, Kinetensin, and Neurotensin The net percent release of histamine, tryptase, chymase, and carboxypeptidase in the supematants was 40%, 34%, 18%, and 17%, respectively. The release reaction was not cytotoxic as judged by lack of release of lactate dehydrogenase. The HPLC chromatograms of the reactions of each peptide with released mast cell supernatants are shown in Fig. 1A-C. The sites of cleavage by tryptase, chymase, and carboxypeptidase, and the identified peptide products are summarized in Fig. 2.

Leu5-Enkephalin As shown in Fig. 1A, only one product (peak A) was generated from LeuS-enkephalin by mast cell proteases. This product was identified by retention time as [des-LeuS]Leu-enkephalin, and was not seen when mast cell supernatants were preincubated with potato tuber carboxypeptidase inhibitor (not shown), confirming that this peak is the product of mast cell carboxypeptidase. SBTI had no effect on the formation of the peak. TLCK eluted in this area of the chromatogram, and therefore its effect

MAST CELLS HYDROLYZE NEUROTENSIN

A

L-ENK

997

B

C

NT

KT

B

E

L ,ifI,Ill. ,..~I!I'jl

........

/',i

"~._.Jll .i,,..... :: "~jIl,~, ~

iI

,..t,f i

RT ~_/~,

RT .-~

RT.-~

FIG. 1. HPLC chromatograms of hydrolysis of neuropeptides by released human skin mast cell supernatants. The substrates investigated were: (A) LeuS-enkephalin; (B) kinetensin; (C) neurotensin. Peaks identified by letters are further identified under the Results section, in Table 2, and Fig. 2.

could not be assessed. The small peaks eluting before the product and substrate were seen in control reactions that lacked substrate. Kinetensin

Five major peaks are noted on the HPLC chromatogram of the hydrolysis products of kinetensin (Fig. 1B). Peaks A-C were identified by amino acid analysis (Table 2). The enzymes re-

CP

Tyr-Gly-Gly-Phe~Leu I R l

Leu e n k e p h o l i n

T CH CP I I I I I e-tim o-Arg-~Arg-H i s-Pro-Tyr-~Phet-Leu

K i net ensi n

A~I

B C I

I I

0

I

E CH NeuPotensin

Glu-Leu-Tyr-Glu-Rsn-Lys-Pro-Arg-RPg}

S

Po-Tyr-

CP e-Leu f

FIG. 2. The amino acid sequences of the neuropeptide substrates are shown, and the bonds hydrolyzed by tryptase (T), chymase (CH), and carboxypeptidase (CP) are indicated by arrows above each sequence. The identity of each HPLC peak from Fig. 1 is shown below the sequence of the parent neuropeptide.

sponsible for the generation of these peaks were identified by use of specific inhibitors. Peak A = Ile-Ala-Arg. Formation of this peak was abolished by TLCK, suggesting that it was generated by tryptase, consistent with the substrate preference of tryptase in cleaving the Arg3-Arg4 bond (35) (see the Discussion section). Peak B = Arg-His-Pro-Tyr. Formation of this peak was abolished when supernatants were preincubated either with TLCK or SBTI, suggesting that the sequential or concerted activity of two proteases is required for the generation of this product. This result is consistent with the action of tryptase on the Arg3-Arg4 bond and the cleavage of the TyrT-Phe8 bond by chymase (25). Peak C = Ile-Ala-Arg-Arg-His-Pro-Tyr. Formation of this peak was abolished after preincubation of supernatants with SBTI, which inhibits chymase cleavage of the TyrT-Phe8 bond. However, formation of peak C increased after preincubation with TLCK, which inhibits tryptase cleavage of the mrg3-Arg4 bond; such cleavage would yield products eluting as peaks A and B instead of peak C. Peak D = KT(4-8), Arg-His-Pro-Tyr-Phe, as determined by SIMS, with a mass determined at 719.2 A.M.U., compared to 718.9 predicted for this peptide. This HPLC peak was abolished by the preincubation of supernatants with PCI, and not with SBTI, demonstrating that this product was formed in part by mast cell carboxypeptidase. TLCK eluted in this area, and its effect could not be assessed. Peak E is KT(1-8) or [desLeu9]kinetensin based on SIMS, with a reported mass of 1059.4 A.M.U, identical to the predicted value for this peptide. This peak coeluted with [des-Leu9]KT produced by purified mast cell carboxypeptidase and was abolished after preincubation with PCI, but not SBTI. Neurotensin

The HPLC chromatogram of the hydrolysis products of neu-

998

GOLDSTEIN, LEONG AND BUNNET'I

TABLE 2 RELATIVEAMINOACIDCONTENTOF PEPTIDE HYDROLYSISFRAGMENTS(pmol) Peptide Fragment(HPLC peak) Kinetensin A lie (6209), Ala (6260), Arg (6084) B Arg (862), His (908), Pro (899), Tyr (852) C Ile (688), Ala (703), Arg (1028), His (517), Pro (540), Tyr (490) Neurotensin B Glu + Gin (1145), Leu (598), Tyr (1001), Asp +Asn (561), Lys (519), Pro (1064), Arg (1007), lie (552) Peak background contaminationwas 1691, 74, and 184, pmol for kinetensinfragmentsA (Leu), B (Gly), and C (Glu), respectively,and 338 and 248 pmol for neurotensinfragment B (Gly, Ser, respectively). Contamination from other amino acid residues was generally several-fold lower than these values.

rotensin is shown in Fig. 1C. Only two major peaks were seen; the other smaller peaks were found in control reactions without substrate, and did not change after preincubation with protease inhibitors. Peak A, pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-ProTyr [NT(1-11)], was identified by SIMS with a mass of 1446.8, compared to a predicted value of 1446.6. Formation of this peak was prevented only by preincubation of supernatants with SBTI, suggesting that it was generated by chymase hydrolysis of the Tyrll-Ilel2 bond. Peak B, [des-Leul3]neurotensin [NT(1-12)], was determined by amino acid analysis (Table 2), and by comparison to the retention time of the product formed by purified mast cell carboxypeptidase. This peak was eliminated by preincubation of supernatants with PCI. The use of TLCK had no effect on the chromatogram, suggesting that there was no TLCKinhibitable (tryptase) hydrolysis of NT. DISCUSSION If mast cells play a physiologic role in controlling neuropeptide activity in tissue, it is reasonable to speculate that proteolytic degradation by several enzymes of differing substrate specificity would be optimal to ensure the hydrolysis and inactivation of each neuropeptide, since these peptides have different amino acid sequences. Mast cell chymase and tryptase are thought of as the primary mast cell-derived inactivators of neuropeptides (6,34). The role of mast cell carboxypeptidase has not been defined. We investigated the hydrolysis of several neuropeptides with carboxy-terminal leucines, and demonstrated that these are substrates of potential physiologic importance for mast cell carboxypeptidase. Mast cell carboxypeptidase is of primary interest in the hydrolysis of LeuCenkephalin, because this peptide is not hydrolyzed by mast cell chymase or tryptase. We observed only one hydrolysis product, [des-Leu]Leu-~-enkephalin. However, studies with the synthetic tripeptide substrate Cbz-Gly-Gly-Phe suggest that the product generated from LeuS-enkephalin by mast cell carboxypeptidase, Tyr-Gly-Gly-Phe, should also be a substrate for carboxypeptidase, with a Km - 2 7 }xM and kcat/Km of - 9 x 105 s - l M -1 (13). Prolonged incubation of LeuCenkephalin with a carboxypeptidase purified from human gastric tissue demonstrated release of Phe residues from LeuCenkephalin generating Tyr-Gly-Gly (3). It is likely that we did not detect Tyr-GlyGly because the initial hydrolysis product of LeuCenkephalin, Tyr-Gly-Gly-Phe, only reached a maximum concentration of 16 laM in the reactions using 100 txM LeuCenkephalin with puri-

fied MCCP. Therefore, hydrolysis of Tyr-Gly-Gly-Phe may have been competitively inhibited, and/or the product of this reaction, Tyr-Gly-Gly, may have been below the level of detection in our assay. Considering the potential for diffusion of small peptides away from the mast cell, it is not clear how important this second reaction is. We felt that conditions of initial velocity might be more reflective of reactions in tissues, and we chose these for our experiments. In addition, the hydrolysis of the Gly3-Phe4 bond is probably not physiologically important, as removal of the C-terminal Leu eliminates the biologic activity of LeuS-enkephalin (3). Kinetensin was hydrolyzed by purified mast cell carboxypeptidase to yield a single product, KT(1-8) or [des-Leu9]kinetensin. Although prolonged incubation with mast cell carboxypeptidase derived from human stomach slowly hydrolyzed the TyrT-Phe8 bond of KT to yield KT(1-7) (3), we did not observe this under our conditions. As discussed above, it is possible that KT is an effective competitive inhibitor of this reaction, considering the low amounts of KT(1-8) generated and available for subsequent hydrolysis. However, judging from the hydrolysis of KT by mast cell supernatants, the cleavage of the TyrT-Phe8 bond by carboxypeptidase is likely to be of relatively little importance in vivo compared to the cleavage of the Tyr7-Phe8 bond by chymase that yielded KT(1-7) and KT(4-7) (Fig. 1B, peaks C and B; Fig. 2). The hydrolysis of kinetensin by mast cell proteases is most interesting for its demonstration of the concerted actions of mast cell chymase and tryptase in generating peak B, and carboxypeptidase and tryptase in generating peak D. The presence of multiple proteases in mast cells has induced speculation that these enzymes may act in a concerted or sequential fashion. However, most studies of these proteases have utilized single purified enzymes. Only recently have any peptide products generated by the concerted action of these enzymes been isolated from the hydrolysis of angiotensin I by rat chymase I and rat mast cell carboxypeptidase (9). Previously, rat mast cell chymase and rat mast cell carboxypeptidase were shown to act sequentially on apolipoprotein B to yield peptide fragments and free amino acids (19), and studies using separate purified components suggested that dog mast cell tryptase and dog chymase would also act together to generate several peptide fragments from vasoactire intestinal peptide (6). Our study demonstrated the concerted action of human mast cell neutral proteases for the first time. Mast cell tryptase in released supernatants did not hydrolyze neurotensin, despite the presence of the Arg8-Arg9 bond in the sequence Lys6-ProV-Arg8-Arg9-Pro m. Tryptase preferentially cleaves tripeptide chromogenic substrates carboxy-terminal to two adjacent basic residues, although it prefers Lys over Arg (35). Larger peptides are cleaved less predictably, between or carboxy-terminal to basic residues (10). Although the specific sequence Lys6-Pro 1° has not been extensively investigated, a similar sequence occurs in ACTH(1-39) (LyslS-Lys16-ArglvArglS-Pr019), and no hydrolysis was demonstrated carboxy-terminal to Arg Jv or Arg TM by purified pituitary tryptase (10). However, these sequences are not totally comparable, because in ACTH(1-39) the LyslCLys 16 residues create a favored site for typtase cleavage compared to Lys6-Prov in neurotensin. In addition, the catalytic efficiency of human skin tryptase is particularly decreased by a proline in the P3 position, as Prov is to the Arg9-Pro 1° bond in neurotensin (35). The extended binding site of tryptase has not yet been sufficiently characterized to permit accurate predictions of the cleavage of the ArgS-Arg9 bond of neurotensin based on the use of small synthetic tripeptides. The substrate specificity of tryptase has also not been sufficiently determined for the protease as part of a macromolecular complex with proteoglycans, even though there is evidence that

MAST CELLS HYDROLYZE NEUROTENSIN

999

the substrate specificity of tryptase may be altered by its binding to heparin (27,29). We demonstrated that mast cell carboxypeptidase hydrolyzes several neuropeptides with KmS between 15 and 36 ttM. The concentration of released neuropeptides in tissue is not known. However, it is likely that mast cell proteases, particularly mast cell chymase and carboxypeptidase, are most active in close proximity to mast cells and that the concentrations of substrate and enzymes in specific microenvironments in tissue are the most relevant to these potential reactions. The amounts of enzyme we used are the equivalent of the activity contained in roughly 0.1 mm 3 of skin (21), although it is unlikely that all of this activity would be released from mast cells in vivo. The K m and kcat/Km values of mast cell carboxypeptidase for the neuropeptides are close to that reported for human mast cell tryptase and the neuropeptides CGRP, peptide histidine methionine, and vasoactive intestinal peptide, supporting the possibility that these reactions are sufficiently rapid to have potential physiologic relevance (34). The generation of multiple peptides from the concerted action of several mast cell proteases on kinetensin, and the hydrolysis of neurotensin may yield biologically active smaller peptides. While kinetensin is a secretagogue for rat peritoneal mast cells (33), the activity of small fragments of kinetensin has not been reported. Cleavage of the carboxy-terminal residues from neurotensin appears to greatly reduce the biologic activity of the peptide (17), but a recent preliminary report suggested that NT(1-

12) may still be able to activate rat mast ceils and increase vasopermeability (8). The biologic effect of NT(1-12) on human mast cells has not been determined (7). The reactions of mast cell proteases and these three peptides will be physiologically relevant only if mast cell proteases have access to the peptides. Kinetensin, neurotensin, and LeuS-en kephalin, unlike substance P, CGRP, and VIP, have not, to date, been localized to nerves innervating mast cells. The distribution of LeuS-enkephalin-containing nerves in relation to mast cells has not been investigated, nor to our knowledge has the anatomic relationship of neurotensin-containing nerves to chymaseand carboxypeptidase-containing mast cells (MCTc) in the human gastrointestinal tract (17), although both neuropeptides have been identified in the gut (26). Reports of immunoreactive neurotensin in skin have been conflicting (24, 36, 37). We speculate that neurotensin and kinetensin might become available to skin or gut mast ceils through leakage of plasma into sites of inflammation and increased vasopermeability (4,36). It has also been proposed that neurotensin is potentially available in cutaneous nerves after in situ generation or uptake from plasma (36). ACKNOWLEDGEMENTS The authors gratefully acknowledge Drs. Kathryn Ivanetich and Ralph C. Reid at the Biomolecular Resource Center at U.C.S.F. for amino acid analyses, Dr. David Maltby at the U.C.S.F. Mass Spectrometry Facility for LSIMS, and Patricia Roloff for preparation of the manuscript.

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25.

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Human mast cell proteases hydrolyze neurotensin, kinetensin and Leu5-enkephalin.

Purified mast cell carboxypeptidase cleaved the C-terminal leucines from Leu5-enkephalin (Leu-ENK), neurotensin (NT), and kinetensin (KT), with Km val...
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