Prostaglandins 44:251-257, 1992
OPIOID PEPTIDES ARE SUBSTRATES FOR THE BIFUNCTIONAL LTA4 HYDROLASE/AMINOPEPTIDASE
ENZYME
K.J. Griffin', J. Gierse', Krivi2 and F.A. FitzpatrickE ,'3 1Department of Pharmacology C236, University of Colorado Health Science Ceqter, 4200 E. Ninth Avenue, Denver, CO 80262, Monsanto Corporate Research St. Louis, Missouri 63198
Abstract We determined if any naturally occurring peptides could act as substrates or inhibitors of the bifunctional, Zn*+ metalloenzyme LTA, hydrolase/aminopeptidase (E.C. 3.3.2.6). Several opioid peptides including met’-enkephalin, leu’-enkephalin, dynorphin,. 6, dynorphin,,, and dynorphin,, competitively inhibited the hydrolysis of L-proline-pnitroanilide by leukotriene A, hydrolasel aminopeptidase, consistent with an interaction at its active site. The enzyme catalyzed the N-terminal hydrolysis of tyrosine from met5enkephalin with K,,,=450 _f 58 PM and V, =4.9 + 0.6 nmol-hr-‘-ug“ and from ledenkephalin with I&=387 + 90 FM and V, =6.2 + 2.5 nmol-hr-‘-ug-‘. Bestatin, captopril and camosine inhibited the hydrolysis of the enkephalins. It is noteworthy that the bifunctional catalytic traits of this enzyme include generation of an hyperalgesic substance, LTB,, and inactivation of analgesic opioid peptides.
Introduction Leukotriene (LT)4A4 hydrolase (E.C. 3.3.2.6) is the rate-limiting enzyme for the formation of LTB,, a lipid mediator of inflammation and pain (l-4). This enzyme has been cloned (5,6) and its nucleotide sequence contains a ‘signature’ identifying it as a member of the Zn*+-metallohydrolase superfamily (7-l 1). Experiments have established that it is catalytically bifunctional, with an intrinsic peptidase activity that can hydrolyze L-amino acid amides of p-nitroaniline or /3-naphthylamine (12-14). Bestatin, an aminopeptidase inhibitor; captopril, an angiotensin-converting enzyme inhibitor; and thiorphan, a neutral endopeptidase (enkephalinase) inhibitor, are also inhibitors of aminopeptidase/LTA, hydrolase (14,15). These data suggest that it may have a role, distinct from LTB,, formation, such as processing or degradation of physiologically relevant peptides; however, no endogenous substrates have yet been identified. We report that certain opioid peptides including enkephalins and dynorphins can interact with LTA, hydrolase/aminopeptidase. Our results suggest that it may modulate pain and inflammation via two separate molecular pathways: formation of the hyperalgesic, inflammatory agent, LTB, (16,17) and degradation of analgesic enkephalins. 3To whom correspondence
should be addressed.
4 Abbreviations used: TFA, trifluoroacetic acid; LT, leukotriene; cr-MSH, hormone; RP-HPLC, reversed-phase high performance liquid chromatography.
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Experimental Materials. L-proline-p-nitroanilide, leu5-enkephalin (H,N-tyr-gly-gly-phe-leu), met5enkephalin (H,N-tyr-gly-gly-phe-met), H,N-gly-gly-phe-met, H,N-gly-gly-phe-leu, tyrosine, bestatin [ (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine], substance P (H,N-arg-pro-lys-pro-gln-gln-phe-phe-gly-leu-met-N~, protease-free bovinealbumin, trifluoroacetic acid (Sigma Chemical Co.); dynorphin,, (H,N-tyr-gly-gly-phe-leu-arg ), dynorphin,, (H,-tyr-gly-gly-phe-leu-arg-arg), dynorphin,, (H,N-tyr-gly-gly-phe-leu-argarg-ile), dynorphin,, (H,N-tyr-gly-gly-phe-leu-arg-arg-ile-arg), a-endorphin (H,N-tyrgly-gly-phe-met-thr-ser-glu-lys-ser-gln-thr-pro-leu-v~-thr), /3-endorphin (H,N-tyr-glygly-phe-met-thr-ser-glu-lys-ser-gln-thr-pro-leu-v~-thr-leu-phe-lys-asn-~a-ile-ile-lys-asnala-tyr-lys-lys-gly-glu), N-acetyl-/3-endorphin, wMSH (N-acetyl-ser-tyr-ser-met-glu-hisphe-arg-trp-gly-lys-pro-val-NH,), neurotensin,, (pglu-leu-tyr-glu-asn-lys-pro-arg), neurotensin,.,, (pglu-leu-tyr-glu-asn-lys-pro-arg-arg-pro-tyr-ile-leu) (Bachem, Torrance CA); HPLC-grade acetonitrile and methanol (Fisher Scientific) were used. Recombinant human LTA, hydrolase/aminopeptidase was expressed in insect cells using a baculovirus expression system and purified to greater than 95% homogeneity as described (1,5). Aminopeptidase Assay. AminopeptidaselLTA, hydrolase (3-30 pg/ml) in 0.1 M Tris/HCl, pH 7, containing 0.2 M NaCl, 20 PM ZnSO, and 1 mg/ml of BSA was incubated with 400 PM L-proline p-nitroanilide at 22°C. Formation of p-nitroaniline was monitored at 405 nm, E = 10,800 M-‘cm-’ (14,15). Peptides were assessed as potential substrates by determining if they inhibited the hydrolysis of L-proline-p-nitroanilide. For instance, peptides typified by leus- enkephalin, met5-enkephalin, and dynorphins (o-1000 PM) were included in the incubation and the initial rates of p-nitroaniline formation were determined as above. Inhibition constants, &, were determined from Dixon plots of l/v versus peptide concentration, using 33, 100, and 333 PM proline p-nitroanilide. Values presented are mean + S.E. Hydrolysis of Enkephalins by LTA, Hydrolase/AminopeptidaseE. C.3.3.2.6. Enkephalins (O-500 PM) dissolved in 0.1 M pH 7.0 tris buffer with 1 mg/ml BSA were incubated with LTA, hydrolase/aminopeptidase (10 pg/ml) at 37°C. Aliquots (100 ~1) were removed at intervals from O-5 or O-24 hrs; quenched with mobile phase (400 ~1) containing bestatin (5 pg/ml) as an internal standard for quantitation. Intact enkephalins and their hydrolysis products were separated by HPLC on a Cl8 Dynamax 300A analytical column (Rainin Instruments) eluted at 1.O ml/min for 1 min with 97% solvent A (0.1% TFA in H,O) 3 % solvent B (0.1% TFA in acetonitrile) changing to 40% solvent B over 20 min and then to 100% solvent B over 3 min. Leu’-enkephalin eluted at approximately 14.6 min, met5-enkephalin at 13.8 min, tyrosine at 5.5 min, gly-glyphe-leu at 13.5 min, gly-gly-phe-met at 12.3 min and bestatin at 14.1 min. Compounds were detected by UV absorbance at 215 nm and quantitation was based on peak area ratios relative to the internal standard, bestatin. Peak identities were established by comparison with known standards. Kinetic Analyses. Kinetic constants and half-lives were estimated from regression analysis using the program InPlot (GraphPAD Software). K,,, and V,, were calculated from Lineweaver-Burk plots; Ki was determined from Dixon plots.
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Results Peptides which bind at the active site of aminopeptidase/LT& hydrolase (E.C. 3.3.2.6) with sufficient affinity should inhibit its hydrolysis of amino acid p-nitroanilides. This facilitates identification of potentially relevant, naturally-occurring substrates. For example, pentapeptide enkephahns inhibited the hydrolysis of L-proline-p-nitroanilide by aminopeptidase/LTA, hydrolase in a concentration dependent manner [ Figure 1 I. Dixon plots, l/v versus enkephalin concentration, were linear with inhibition constants Ki= 259 PM for met!-enkephalin [ Inset, Figure 1 ] and Ki = 798 PM for leu5-enkeph-
[Met
10
Enkephalin]
(fill)
A Leu Enkephal in 0 Met Enkephalin
1000
100 [Enkephalin]
(PM)
Figure 1: Effect of enkephalin concentration on inhibition of hydrolysis of proline p-nitroanilide by LTA, hydrolase. Enzyme was incubated with varying concentrations of enkephalins as described in methods. Hydrolysis of proline-p-nitroanilidewas monitored by absorbance at 415 nm. Inset: Dixon Plot for Met-Enkephalin inhibiting proline p-nitroanilide hydrolysis; Ki = 225 PM.
alin. Inhibition by enkephalins was maximal immediately after their addition to the enzyme; activity then gradually returned to control levels, suggesting that enkephalins RP-HPLC analysis were themselves degraded by aminopeptidase/LT& hydrolase. confirmed this: met?-enkephalin disappearance corresponded with the appearance of tyrosine and gly-gly-phe-met [ Figure 2 1. Results were similar for leu’-enkephalin.
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Prostaglandins
O[Leu
Enkephalin]
0 [Gly-Gly-Phe-Leu] A [Tyrosine]
0
60
120
180
240
300
Time (min) Figure 2: Progress of enkephalin degradation reaction. Enkephalins were incubated with LTA, hydrolaselaminopeptidase as described in methods. Quenched samples were analyzed by RP-HPLC, and the peak areas used to determine the concentration of enkephalin at each time point. A typical timecourse for led-enkephalin is shown here. Initial velocities of degradation were used to calculate kinetic parameters with Lineweaver-Burk plots.
The specific activities for hydrolysis (V_) at pH 7 were 4.9 t 0.6 nmol met’enkephalin-h-l- pg-’ enzyme (mean + S.E., n=5 ) and 6.2 + 2.5 nmol let?-enkephalinh-t- pg-’ enzyme (mean _f S.E., n =4). Lineweaver-Burk plots [ Figure 3 I, showed that K,,, = 450 + 58 FM for met5-enkephalin and K,,, = 387 + 90 PM for leu5-enkephalin. Bestatin, captopril and thiorphan prevented enzymatic hydrolysis of the enkephalins by aminopeptidase/LTA, hydrolase as anticipated (14,15). LOO-
,?/ -.002
.,.,:,:,:,:( 0
,002
,004 ,006
,008 ,010 ,012
l/[Met Enkephalin] (PM-') Figure 3: Lineweaver-Burk plot: degradation of enkephalins by LTA, hydrolasel aminopeptidase. Enkephalins were incubated with enzyme, quenched, and analyzed as described in methods. A typical graph for metS-enkephalin is depicted here.
Prostaglandins
255
Among other opioid peptides investigated, few inhibited aminopeptidase/LTA, hydrolase [ Table 1 1. Dynorphin,, and ,_,, which contain the leu’-enkephalin sequence with one or two arginines added to the carboxy terminus (H,N-tyr-gly-gly-phe-leu-argarg), were exceptions with & values of approximately 60 ,uM [ Table 11. Met?enkephalin analogs of Dynorphin,, and r_, were also more potent inhibitors than met’enkephahn [ Table 11. Inhibitory potency declined by ten-fold or greater for dynorphin,_ 8 and 1.9. Table 1: Opioid peptides tested for inhibition of proline p-nitroanilide hydrolysis by LTA, hydrolasekuninopeptidase. Reactions were carried out as described in methods. K,‘s (mean 2 SEM) were calculated by Dixon analysis. Peptides with no inhibition at 300 FM are denoted with *** Peptide
Ki (rM)
Leu5-Enkephalin Dynorphin A,, Dynorphin A,., Dynorphin A,, Dynorphin A,,
780.0 60.4 53.6 654.0 761.0
+- 370 f. 0.6 ;t 2.9 + 107
Methionine Enkephalin Arg’-Met’-Eukephalin Met’-Enkephalin-A$ Met’-Enkephalin-Arg6-Arg’ d-Ala*-Met’-Enkephalin
259.2 143.1 114.2 51.3 310.6
+ 27.7 L 35.9 +. 18.2 +. 12.2 + 32.2
cr-Endorphin P-Endorphin N-Acetyl-P-Endorphin Substance P cr-MSH Neurotensin Neurotensin,.,
*** *** *** *** *** *** ***
Discussion Several different Zn*+-metallohydrolase enzymes inactivate enkephalins and related neuropeptides (18-21). Membrane associated neutral endopeptidase (EC 3.3.24.11) is a prominent, but not necessarily exclusive, source of their degradation. Aminopeptidases also contribute by eliminating the N-terminal tyrosine which is required for binding to opioid receptors (22). In fact, cleavage of the tyri-gly2 bond is the quantitatively dominant mode of enkephalin hydrolysis in cells, striatal slices, and soluble or particulate fractions of whole brain homogenates (23). Our data indicate that the bifunctional enzyme LTA, hydrolase/aminopeptidase can also catalyze the degradation of enkephalins. The K, values for this process are larger than values of 20-90 PM reported for neutral endopeptidase, a dipeptidyl peptidase, but similar in magnitude to values reported for other aminopeptidase enzymes including those found in human cerebrospinal fluid (24). LTA, The V,, values are also similar among the various aminopeptidases. hydrolase/aminopeptidase is found in the 100,000 x g supematant from human brain homogenates suggesting that it could participate in enkephalin deactivation (25). Using both conversion of LTA, to LTB., and immunochemical detection we have confirmed that
256
Prostaglandins
the 100,000 x g supematant from rat brain also contains this enzyme. The soluble fraction of whole brain homogenates also contains other aminopeptidases (23) and an endopeptidase (E.C. 3.3.24.15) which processes enkephalins and dynorphins (18). Thus, cytosolic localization may not necessarily restrict interactions between the enzyme and neuropeptides. Furthermore, conditions such as hemorrhagic stroke, head injury or cerebral inflammation could promote release of LTA, hydrolase/aminopeptidase from erythrocytes or neutrophils and infiltration of plasma which contains the enzyme (3,26). In addition to their roles as neurotransmitters or co-transmitters enkephalins and related endogenous opioids can act peripherally as mediators of inflammatory and immune responses (27,28). Their deactivation by a widely distributed aminopeptidases, including LTA4 hydrolase/aminopeptidase, is compatible with extraneuronal roles for enkephalins. It is interesting that this single, bifunctional enzyme is capable of generating an hyperalgesic substance and degrading endogenous analgesic substances. Acknowledgement USPHS grant NIH HL34303 awarded to F.A.F.
supported this investigation.
References
1. 2. 3. 4. 5. 6.
7. 8. 9.
10. 11. 12. 13.
14. 15.
Radmark, O., Shimizu, T., Jomvall, H., and Samuelsson, B. (1984) J. Biol. Chem. 259:12339-12345. Evans, J., Dupuis, P., and Ford-Hutchinson, A. W. (1985) Biochlm. Biophys. Acta 840:43-50. McGee, J. and Fitzpatrick, F.A. (1985) J. Biol. Chem. 260: 12832-12837. Samuelsson, B. and Funk, C. (1989) J. Biol. Chem. 264: 19469-19472. Funk, C., Radmark, O., Fu, J.-Y., Matsumoto, T., Jomvall, H., Shimizu, T., and Samuelsson, B. (1987) Proc. Natl. Acad. Sci. U.S.A. 84: 6677-6681. Minami, M., Ohno, S., Kawasaki, H., Radmark, 0.) Samuelsson, B., Jomvall, H., Shimizu, T., Seyama, Y., and Suzuki, K. (1987) J. Biol. Chem. 262: 1387313876. Jongeneel, C.V., Bouvier, J., Bairoch, A. (1989) FEBS L&t. 242: 211-214. Malfroy, B., Kado-Fong, H., Gros, C., Giros, B., Schwartz, J., and Helmiss, R. (1989) Biochem. Biophys. Res. Commun. 161: 236-241. Vallee, B. and Auld, D. (1990) Biochemistry 29: 5647-5659. Toh, H., Minami, M., and Shimizu, T. (1990) Biochem. Biophys. Res. Conunun. 171: 216-221. Haeggstrom, J., Wetterholm, A., Shapiro, R., Vallee, B., and Samuelsson, B. (1990) Biochem. Biophys. Res. Conunun. 172: 965-970. Haeggstrom, J., Wetterholm, A., Vallce, B., and Samuelsson, B. (1990) Biochem. Biophys. Res.Commun. 173: 431-437. Minami, H., Ohishi, N., Mutoh, H., Izumi, T., Bito, H., Wada, H., Seyama, Y., Toh, H., and Shimizu, T. (1990) Biochem. Biophys. Res. Commun. 173: 620626. Orning, L., Krivi, G., and Fitzpatrick, F. (1991) J. Biol. Chem. 266: 1375-1378. Oming, L., Krivi, G., Bild, G., Gierse, J., Aykent, S., and Fitzpatrick, F. (1991)
Pmstaglandins
16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
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J. Biol. Chem. 266: 16507-16511. Goetzl, E., Payan, D., and Goldman, D. (1984) J. Clin. Immunol. 4: 79-84. Lewis, R., Austen, K.F., and Soberman, R. (1990) New Engl. J. Med. 323: 645655. Acker, G., Molineaux, C. and Orlowski, M. (1987) J. Neurochem. 48: 284-292. Matsas, R., Kenny, A.J. and Tumer,A.J. (1984) Biochem. J. 223: 433-440. Malfroy, B., Swerts, J.P., Guyon, A. , Roques, B.P. and Scwartz, J.C. (1978) Nature 276523-526. Roques, B.P., Foumie-Zaluski, M.C., Soroca, E., Lecomte, J.M., Malfroy, B., Llorens, C. and Schwartz, J.C. (1980) Nature 288: 286-288. Dua, A. K., Pinsky, C. and LaBella, F. S. (1985) Life Sci. 3: 985-992. Schwartz, J-C., Malfroy, B., and DeLa Baume, S. (1981) Life Sci. 29: 1715-1740. Benter, I., Hirsh, E., Tuchman, A., and Ward, P. (1985) Biochem. Pharmacol. 40: 465-472. Fu, J-Y., Haeggstrom, J., Collins, P., Meijer, J. and Radmark, 0. (1989) Biochim. Biophys. Acta 1006: 121-126. Fitzpatrick, F., Haeggstrom, J., Granstrom, E., and Samuelsson, B. (1983) Proc. Natl. Acad. Sci. USA 80: 5425-5429. Schipp, M.A., Stefano, G. B., D’Adamio,L., Switzer, S. N., Howard, F.D., Sinesterra, J., Scharrer, B. and Reinherz, E.L. (1990) Nature 347: 394-396. Carr, D. J. J. (1991) Proc. Sot. Exptl. Biol. and Med. 198: 710-720.
Editor:
P.W. Ranwell
Received:
6-23-92
Accepted:
6-30-92
OPIOID PEPTIDES ARE SUBSTRATES FOR THE BIFUNCTIONAL LTA4 HYDROLASE/AMINOPEPTIDASE
ENZYME
K.J. Griffin', J. Gierse', Krivi2 and F.A. FitzpatrickE ,'3 1Department of Pharmacology C236, University of Colorado Health Science Ceqter, 4200 E. Ninth Avenue, Denver, CO 80262, Monsanto Corporate Research St. Louis, Missouri 63198
Abstract We determined if any naturally occurring peptides could act as substrates or inhibitors of the bifunctional, Zn*+ metalloenzyme LTA, hydrolase/aminopeptidase (E.C. 3.3.2.6). Several opioid peptides including met’-enkephalin, leu’-enkephalin, dynorphin,. 6, dynorphin,,, and dynorphin,, competitively inhibited the hydrolysis of L-proline-pnitroanilide by leukotriene A, hydrolasel aminopeptidase, consistent with an interaction at its active site. The enzyme catalyzed the N-terminal hydrolysis of tyrosine from met5enkephalin with K,,,=450 _f 58 PM and V, =4.9 + 0.6 nmol-hr-‘-ug“ and from ledenkephalin with I&=387 + 90 FM and V, =6.2 + 2.5 nmol-hr-‘-ug-‘. Bestatin, captopril and camosine inhibited the hydrolysis of the enkephalins. It is noteworthy that the bifunctional catalytic traits of this enzyme include generation of an hyperalgesic substance, LTB,, and inactivation of analgesic opioid peptides.
Introduction Leukotriene (LT)4A4 hydrolase (E.C. 3.3.2.6) is the rate-limiting enzyme for the formation of LTB,, a lipid mediator of inflammation and pain (l-4). This enzyme has been cloned (5,6) and its nucleotide sequence contains a ‘signature’ identifying it as a member of the Zn*+-metallohydrolase superfamily (7-l 1). Experiments have established that it is catalytically bifunctional, with an intrinsic peptidase activity that can hydrolyze L-amino acid amides of p-nitroaniline or /3-naphthylamine (12-14). Bestatin, an aminopeptidase inhibitor; captopril, an angiotensin-converting enzyme inhibitor; and thiorphan, a neutral endopeptidase (enkephalinase) inhibitor, are also inhibitors of aminopeptidase/LTA, hydrolase (14,15). These data suggest that it may have a role, distinct from LTB,, formation, such as processing or degradation of physiologically relevant peptides; however, no endogenous substrates have yet been identified. We report that certain opioid peptides including enkephalins and dynorphins can interact with LTA, hydrolase/aminopeptidase. Our results suggest that it may modulate pain and inflammation via two separate molecular pathways: formation of the hyperalgesic, inflammatory agent, LTB, (16,17) and degradation of analgesic enkephalins. 3To whom correspondence
should be addressed.
4 Abbreviations used: TFA, trifluoroacetic acid; LT, leukotriene; cr-MSH, hormone; RP-HPLC, reversed-phase high performance liquid chromatography.
Copyright
0 1992 Butterworth-Heinemann
a-melanocyte
stimulating
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Experimental Materials. L-proline-p-nitroanilide, leu5-enkephalin (H,N-tyr-gly-gly-phe-leu), met5enkephalin (H,N-tyr-gly-gly-phe-met), H,N-gly-gly-phe-met, H,N-gly-gly-phe-leu, tyrosine, bestatin [ (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine], substance P (H,N-arg-pro-lys-pro-gln-gln-phe-phe-gly-leu-met-N~, protease-free bovinealbumin, trifluoroacetic acid (Sigma Chemical Co.); dynorphin,, (H,N-tyr-gly-gly-phe-leu-arg ), dynorphin,, (H,-tyr-gly-gly-phe-leu-arg-arg), dynorphin,, (H,N-tyr-gly-gly-phe-leu-argarg-ile), dynorphin,, (H,N-tyr-gly-gly-phe-leu-arg-arg-ile-arg), a-endorphin (H,N-tyrgly-gly-phe-met-thr-ser-glu-lys-ser-gln-thr-pro-leu-v~-thr), /3-endorphin (H,N-tyr-glygly-phe-met-thr-ser-glu-lys-ser-gln-thr-pro-leu-v~-thr-leu-phe-lys-asn-~a-ile-ile-lys-asnala-tyr-lys-lys-gly-glu), N-acetyl-/3-endorphin, wMSH (N-acetyl-ser-tyr-ser-met-glu-hisphe-arg-trp-gly-lys-pro-val-NH,), neurotensin,, (pglu-leu-tyr-glu-asn-lys-pro-arg), neurotensin,.,, (pglu-leu-tyr-glu-asn-lys-pro-arg-arg-pro-tyr-ile-leu) (Bachem, Torrance CA); HPLC-grade acetonitrile and methanol (Fisher Scientific) were used. Recombinant human LTA, hydrolase/aminopeptidase was expressed in insect cells using a baculovirus expression system and purified to greater than 95% homogeneity as described (1,5). Aminopeptidase Assay. AminopeptidaselLTA, hydrolase (3-30 pg/ml) in 0.1 M Tris/HCl, pH 7, containing 0.2 M NaCl, 20 PM ZnSO, and 1 mg/ml of BSA was incubated with 400 PM L-proline p-nitroanilide at 22°C. Formation of p-nitroaniline was monitored at 405 nm, E = 10,800 M-‘cm-’ (14,15). Peptides were assessed as potential substrates by determining if they inhibited the hydrolysis of L-proline-p-nitroanilide. For instance, peptides typified by leus- enkephalin, met5-enkephalin, and dynorphins (o-1000 PM) were included in the incubation and the initial rates of p-nitroaniline formation were determined as above. Inhibition constants, &, were determined from Dixon plots of l/v versus peptide concentration, using 33, 100, and 333 PM proline p-nitroanilide. Values presented are mean + S.E. Hydrolysis of Enkephalins by LTA, Hydrolase/AminopeptidaseE. C.3.3.2.6. Enkephalins (O-500 PM) dissolved in 0.1 M pH 7.0 tris buffer with 1 mg/ml BSA were incubated with LTA, hydrolase/aminopeptidase (10 pg/ml) at 37°C. Aliquots (100 ~1) were removed at intervals from O-5 or O-24 hrs; quenched with mobile phase (400 ~1) containing bestatin (5 pg/ml) as an internal standard for quantitation. Intact enkephalins and their hydrolysis products were separated by HPLC on a Cl8 Dynamax 300A analytical column (Rainin Instruments) eluted at 1.O ml/min for 1 min with 97% solvent A (0.1% TFA in H,O) 3 % solvent B (0.1% TFA in acetonitrile) changing to 40% solvent B over 20 min and then to 100% solvent B over 3 min. Leu’-enkephalin eluted at approximately 14.6 min, met5-enkephalin at 13.8 min, tyrosine at 5.5 min, gly-glyphe-leu at 13.5 min, gly-gly-phe-met at 12.3 min and bestatin at 14.1 min. Compounds were detected by UV absorbance at 215 nm and quantitation was based on peak area ratios relative to the internal standard, bestatin. Peak identities were established by comparison with known standards. Kinetic Analyses. Kinetic constants and half-lives were estimated from regression analysis using the program InPlot (GraphPAD Software). K,,, and V,, were calculated from Lineweaver-Burk plots; Ki was determined from Dixon plots.
Prostaglandins
253
Results Peptides which bind at the active site of aminopeptidase/LT& hydrolase (E.C. 3.3.2.6) with sufficient affinity should inhibit its hydrolysis of amino acid p-nitroanilides. This facilitates identification of potentially relevant, naturally-occurring substrates. For example, pentapeptide enkephahns inhibited the hydrolysis of L-proline-p-nitroanilide by aminopeptidase/LTA, hydrolase in a concentration dependent manner [ Figure 1 I. Dixon plots, l/v versus enkephalin concentration, were linear with inhibition constants Ki= 259 PM for met!-enkephalin [ Inset, Figure 1 ] and Ki = 798 PM for leu5-enkeph-
[Met
10
Enkephalin]
(fill)
A Leu Enkephal in 0 Met Enkephalin
1000
100 [Enkephalin]
(PM)
Figure 1: Effect of enkephalin concentration on inhibition of hydrolysis of proline p-nitroanilide by LTA, hydrolase. Enzyme was incubated with varying concentrations of enkephalins as described in methods. Hydrolysis of proline-p-nitroanilidewas monitored by absorbance at 415 nm. Inset: Dixon Plot for Met-Enkephalin inhibiting proline p-nitroanilide hydrolysis; Ki = 225 PM.
alin. Inhibition by enkephalins was maximal immediately after their addition to the enzyme; activity then gradually returned to control levels, suggesting that enkephalins RP-HPLC analysis were themselves degraded by aminopeptidase/LT& hydrolase. confirmed this: met?-enkephalin disappearance corresponded with the appearance of tyrosine and gly-gly-phe-met [ Figure 2 1. Results were similar for leu’-enkephalin.
254
Prostaglandins
O[Leu
Enkephalin]
0 [Gly-Gly-Phe-Leu] A [Tyrosine]
0
60
120
180
240
300
Time (min) Figure 2: Progress of enkephalin degradation reaction. Enkephalins were incubated with LTA, hydrolaselaminopeptidase as described in methods. Quenched samples were analyzed by RP-HPLC, and the peak areas used to determine the concentration of enkephalin at each time point. A typical timecourse for led-enkephalin is shown here. Initial velocities of degradation were used to calculate kinetic parameters with Lineweaver-Burk plots.
The specific activities for hydrolysis (V_) at pH 7 were 4.9 t 0.6 nmol met’enkephalin-h-l- pg-’ enzyme (mean + S.E., n=5 ) and 6.2 + 2.5 nmol let?-enkephalinh-t- pg-’ enzyme (mean _f S.E., n =4). Lineweaver-Burk plots [ Figure 3 I, showed that K,,, = 450 + 58 FM for met5-enkephalin and K,,, = 387 + 90 PM for leu5-enkephalin. Bestatin, captopril and thiorphan prevented enzymatic hydrolysis of the enkephalins by aminopeptidase/LTA, hydrolase as anticipated (14,15). LOO-
,?/ -.002
.,.,:,:,:,:( 0
,002
,004 ,006
,008 ,010 ,012
l/[Met Enkephalin] (PM-') Figure 3: Lineweaver-Burk plot: degradation of enkephalins by LTA, hydrolasel aminopeptidase. Enkephalins were incubated with enzyme, quenched, and analyzed as described in methods. A typical graph for metS-enkephalin is depicted here.
Prostaglandins
255
Among other opioid peptides investigated, few inhibited aminopeptidase/LTA, hydrolase [ Table 1 1. Dynorphin,, and ,_,, which contain the leu’-enkephalin sequence with one or two arginines added to the carboxy terminus (H,N-tyr-gly-gly-phe-leu-argarg), were exceptions with & values of approximately 60 ,uM [ Table 11. Met?enkephalin analogs of Dynorphin,, and r_, were also more potent inhibitors than met’enkephahn [ Table 11. Inhibitory potency declined by ten-fold or greater for dynorphin,_ 8 and 1.9. Table 1: Opioid peptides tested for inhibition of proline p-nitroanilide hydrolysis by LTA, hydrolasekuninopeptidase. Reactions were carried out as described in methods. K,‘s (mean 2 SEM) were calculated by Dixon analysis. Peptides with no inhibition at 300 FM are denoted with *** Peptide
Ki (rM)
Leu5-Enkephalin Dynorphin A,, Dynorphin A,., Dynorphin A,, Dynorphin A,,
780.0 60.4 53.6 654.0 761.0
+- 370 f. 0.6 ;t 2.9 + 107
Methionine Enkephalin Arg’-Met’-Eukephalin Met’-Enkephalin-A$ Met’-Enkephalin-Arg6-Arg’ d-Ala*-Met’-Enkephalin
259.2 143.1 114.2 51.3 310.6
+ 27.7 L 35.9 +. 18.2 +. 12.2 + 32.2
cr-Endorphin P-Endorphin N-Acetyl-P-Endorphin Substance P cr-MSH Neurotensin Neurotensin,.,
*** *** *** *** *** *** ***
Discussion Several different Zn*+-metallohydrolase enzymes inactivate enkephalins and related neuropeptides (18-21). Membrane associated neutral endopeptidase (EC 3.3.24.11) is a prominent, but not necessarily exclusive, source of their degradation. Aminopeptidases also contribute by eliminating the N-terminal tyrosine which is required for binding to opioid receptors (22). In fact, cleavage of the tyri-gly2 bond is the quantitatively dominant mode of enkephalin hydrolysis in cells, striatal slices, and soluble or particulate fractions of whole brain homogenates (23). Our data indicate that the bifunctional enzyme LTA, hydrolase/aminopeptidase can also catalyze the degradation of enkephalins. The K, values for this process are larger than values of 20-90 PM reported for neutral endopeptidase, a dipeptidyl peptidase, but similar in magnitude to values reported for other aminopeptidase enzymes including those found in human cerebrospinal fluid (24). LTA, The V,, values are also similar among the various aminopeptidases. hydrolase/aminopeptidase is found in the 100,000 x g supematant from human brain homogenates suggesting that it could participate in enkephalin deactivation (25). Using both conversion of LTA, to LTB., and immunochemical detection we have confirmed that
256
Prostaglandins
the 100,000 x g supematant from rat brain also contains this enzyme. The soluble fraction of whole brain homogenates also contains other aminopeptidases (23) and an endopeptidase (E.C. 3.3.24.15) which processes enkephalins and dynorphins (18). Thus, cytosolic localization may not necessarily restrict interactions between the enzyme and neuropeptides. Furthermore, conditions such as hemorrhagic stroke, head injury or cerebral inflammation could promote release of LTA, hydrolase/aminopeptidase from erythrocytes or neutrophils and infiltration of plasma which contains the enzyme (3,26). In addition to their roles as neurotransmitters or co-transmitters enkephalins and related endogenous opioids can act peripherally as mediators of inflammatory and immune responses (27,28). Their deactivation by a widely distributed aminopeptidases, including LTA4 hydrolase/aminopeptidase, is compatible with extraneuronal roles for enkephalins. It is interesting that this single, bifunctional enzyme is capable of generating an hyperalgesic substance and degrading endogenous analgesic substances. Acknowledgement USPHS grant NIH HL34303 awarded to F.A.F.
supported this investigation.
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Editor:
P.W. Ranwell
Received:
6-23-92
Accepted:
6-30-92
