Vol. 105, No. 4, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS April 29, 1982 Pages 1482-1487

8-ENDORPHIN INHIBITS MET-ENKEPHALIN BREAKDOWN BY A BRAIN AMINOPEPTIDASE: STRUCTURE-ACTIVITY RELATIONSHIPS

Koon-Sea Hui, L&.zlA G&f*, and Abel Lajtha

Center for Neurochemistry, Rockland Research Institute Ward’s Island, New York 10035

Received February 12, 1982

SUMMARY: An aminopeptidase solubilized and isolated from rat brain membranes selectively splits the Tyrl-Gly2 peptide bond of Met-enkephalin. Oh-Endorphin, which is apparently resistant to the aminopeptidase, inhibited the action of this peptidase on Met-enkephalin degradation competitively; the Ki value was 11.5 FM. Arg6-6h-endorphin was found to be 10 times more potent than flh-endorphin. From further structure-activity data it is concluded that the N-terminal amino group and some residues within region 18-31 of the 8-endorphin structure are cooperatively involved in binding to the active site of the aminopeptidase.

INTRODUCTION

Several membrane-associated peptidases of the brain, such as aminopeptidase(s)

(1)) angiotensin-converting enzyme (2,3), and enkephalin dipeptidyl carboxy-

peptidase (4)) have been shown to degrade enkephalins in vitro. However, their -__

role in physiological inactivation of enkephalins is not yet clear (for review see

reference 5). Since enkephalins are metabolized most rapidly by the release of

the N-terminal tyrosine, which terminates their opioid effects (5), the membrane-

bound aminopeptidase is the focus of our interest. Recently, such an aminopeptidase

from rat brain membranes was solubilized and purified to homogeneity (6). This

enzyme rapidly removes the N-terminal Tyr from Met-enkephalin to yield Gly-Gly-

Phe-Met , but it is unable to degrade Y-endorphin ( 7 -EP+) and B-endorphin (6-EP)

under the same assay conditions (6). Similarly, aminopeptidase M has been reported

to act with decreasing velocity as the chain length of Met-enkephalin was increased

to give Y-EP and 6-EP (7). On the other hand, both Y- and 8-EP are potent in-

*Visiting scientist on leave from the Institute for Drug Research, Budapest, Hungary. +Abbreviations : Y-EP, Y-endorphin; Bh-EP, human 6-endorphin ; 6-LPH , O-liPOtrOPin; HPLC, high performance liquid chromatography.

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hibitors of the aminopeptidase from rat brain. The structural requirements for

the inhibitory effect of endorphins were investigated in the present study.

MATERIALS AND METHODS

All the synthetic peptides used in this study were obtained from Peninsula except des-Gly2-6,-EP and D-Leu5-Dh-EP, which were generously supplied by Dr. C. H. Li (University of California, San Francisco). Porcine 6-lipotropin ( BP-LPH) was prepared as described previously (8). Amino acyl-3-naphthylamides were obtained from Sigma.

The aminopeptidase was prepared by homogenization of rat brain with 9 volumes of 0.32 M sucrose. The homogenate was centrifuged at 800 g for 10 min, and the cell debris was discarded. After centrifugation at 30,000 g for 20 min the supernatant was aspirated, and the pellet, resuspended in 50 tiiM Tris-HCl buffer of pH 7.5, was centrifuged and washed two additional times asi described above. The membrane pellet was solubilized by resuspension in 20 volumes of 50 mM Tris-HCl buffer, pH 7.5, containing 1% (w/v) Triton X-100 and incubated at 37OC for 45 min. The solubilized enzyme, obtained after centrifugation at 30,000 g for 10 min, was further purified by successive chromatography on DEAE- Sephar@e, hydroxylapatite, and Sephadex G-200 columns. Details of the isolation procedure and the biochemical characterization of the enzyme are described else- where (6). Polyacrylamide gel electrophoresis revealed the presence of a single band with aminopeptidase activity, detected directly in the gel slab. The apparent molecular weight of the aminopeptidase was estimated to be 250,000 by exclusion chromatography on a Sephadex G-200 column. It was strongly inhibited by the known aminopeptidase inhibitors bacitracin, puromycin, bestatin, and amastatin (6).

Aminopeptidase activity was assayed using reverse-phase high performance liquid chromatography (HPLC) by measuring the disappearance of Met-enkephalin and the generation of tetrapeptide Gly-Gly-Phe-Met. The HPLC analysis was carried out on a Waters high performance liquid chromatograph equipped with a Radial-Pak C8 column (100X5 mm) with particle size of 10 km. The samples were eluted isocratically at ambient temperature. The solvent systems (acetonitrile- 0.1 M phosphate buffer, pH 3.0) for Met-enkephalin and Gly-Gly-Phe-Met were 20: 80 and 10: 90 respectively. The peptides were monitored by UV absorbance at 280 and/or 205 nm.

In a typical experiment, 75 FM Met-enkephalin, 20 ng aminopeptidase, and the inhibitor were incubated in a final volume of 150 ~1 of 25 mM Tris-HCl buffer, pH 7.5, containing 2 mM [ethylenebis(oxyethylenenitrilo)] - tetraacetic acid and 0.2 mM dithiothreitol at 37OC for 30 min. The reaction was terminated by adding 20 @ of 20% trichloroacetic acid. As internal standard, 50 ~1 of 225 PM Leu- enkephalin was added, and 30 pl of the sample was submitted to HPLC analysis.

The aminopeptidase activity was also assayed on arginyl- and tyrosyl-6- naphthylamides by measuring the released 6-naphthylamine with an Aminco spec- trofluorometer (excitation at 360 nm and emission at 450 nm). The reaction was performed in a 3-ml cuvette of l-cm width, in 0.2 M phosphate buffer of pH 7.5 containing 0.2 M dithiothreitol.

RESULTS AND DISCUSSION

Some kinetic constants for the aminopeptidase isolated from rat brain mem-

branes (6) are given in Table 1. In its low affinity for Met-enkephalin and its

relatively high affinity for Arg-9-naphthylamide, the enzyme resembles a previously

isolated membrane-bound aminopeptidase designated as MI (9).

The inhibitory effects of a series of f3-EP analogs on Met-enkephahn break-

down are compared in Table 2. Two 6-EP analogs, des-Gly’-Dc-EP and D-Leu5-

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TABLE 1. Kinetic constants of the aminopeptidase solubilized and isolated from rat brain membranes

Substrate

Met-enkephalin

Km

(1O-5 M)

27.8

V max

K catiKm

(Fmol/min/mg) (M-lx s-l x 10m5)*

45.0 6.7

Arg- 8-naphthylamide 5.0 27.5 22.9

Tyr-8-naphthylamide 3.1 3.3 4.4

* The molecular weight of the purified enzyme used for the calculation was 250,000.

6h-EP, which have no significant opiate-like biological properties (lo), showed

inhibitory properties comparable to that of Bh-EP. In addition, three ligands

for opiate receptors, D-Ala2-enkephalinamide, morphine, and naloxone, had no

effect on the aminopeptidase activity (Table 2). Thus, it is clear that the

structural features of fi-EP required for opiate receptor and aminopeptidase bind-

ing are not coincident.

TABLE 2. Percent inhibition of Met-enkephaIin* breakdown by the aminopeptidase

PeptidelInhibitor Concentration (@I) Inhibition (%)

Oh-EP 8 43

des-Gly2-Dc-EP 8 45

D-Leu’-Oh-EP 8 44

des-Tyrl-Dh-EP 8 34

Bh-EP-(6-31) 8 15

N-acetyl-Bh-EP 8 15

Y-EP 32 43

des-Tyr’-T-EP 32 0

Dp-LPH 10 0

D-Ala2-enkephalinamide 60 0

morphine 60 0

naloxone 60 0

* The concentration of Met-enkephalin was 75 @M. For the assay conditions see MATERIALS AND METHODS.

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100 r

IO -6 -5

10 -4

10

Concentration , M

Fig. 1. Inhibition of the aminopeptidase degradation of Met -enkephalin by different peptides. W, T-EP ; 0, des-Tyrl-JIh-BP ; 0, Dh-EP ; . , Argo-Ph-EP.

The removal of Tyr’ from Bh-EP slightly decreased the inhibitory potency

of the polypeptide (Table 2); furthermore, Bh-EP-(6-31) and N-acetyl-Bh-EP

displayed some activity. These data, indicating that the N-terminal tyrosine

’ s I , mM -1

Fig. 2. Lineweaver-Burk plot of hydrolysis of Met-enkephalin with the aminopeptidase (0) and the inhibition of Met-enkephalin breakdown by 1 pM (A> and 3 PM Argo-Dh-EP (A ).

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‘- lo-

/

m c .- E

5- _

I 1 -4 -2 2 6 10 14

’ I s, rnht-’ Fig. 3. Lineweaver-Burk plot of hydrolysis of Met-enkephalin with the

aminopeptidase ( l ) and the inhibition of the breakdown by 8 PM (0 ) and 24 PM Dh-EP (B).

is not essential for the interaction of the polypeptide with the aminopeptidase,

together with the relatively high proteolytic coefficient (Kcat/Km) for Arg-8-

naphthylamide (Table l), prompted us to test Argo-Bh-EP. This 6-EP analog

was found to be about 10 times more potent as an inhibitor of Met-enkephalin

breakdown than oh-EP (Figures 1 and 2). According to the results of amino

acid analysis, no arginine was released from Argo-Bh-EP by the enzyme. The

kinetics of the inhibition of Met-enkephalin breakdown with lIh-EP (Figure 3)

and Argo-8 h -EP (Figure 2) showed that both polypeptides were competitive

inhibitors of the aminopeptidase, with Ki values of 11.5 PM and 1.0 FM, respec-

tively .

Whereas modification of the Dh-EP N-terminus by either shortening [des-

Tyrl-Bh-EP, Bh-EP-(6-31)] or extending (Argo-8,-EP) the peptide chain did not

result in a loss of the inhibitory potency, the removal of Tyr’ from Y-EP abolished

its activity, which was 25% of that of Sh-EP (Table 2, Figure 1). From these

structure-activity data it appears that the N-terminal amino group and some

residues within sequence region 18-31 (missing from Y-EP) are cooperatively

involved in binding to the active site of the aminopeptidase, thus preventing

enkephalin degradation. The decrease or loss of the susceptibility of the

N-terminal tyrosine of endorphins to aminopeptidase action (7,11,12) might

be due to the very same interactions between the peptides and the enzyme.

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Previously, three other neuropeptides , somatostatin, Substance P, and

angiotensin-II, were reported to inhibit membrane-associated (1) and soluble

(13) aminopeptidases of rat brain. Though no direct evidence supports the

possible physiological significance of the above findings and our present

findings, such inhibitory mechanisms could theoretically serve as an efficient

means of regulating enkephalin (and other neuropeptide) levels at the func-

tional receptor sites in the brain. Also, some pharmacological effects of 8-EP

may be due to its inhibitory action on aminopeptidases.

ACKNOWLEDGEMENTS

The work was partly supported by NSF Grant 7826164. We thank Dr. C .H. Li for suppl ‘ng us with two synthetic 6-endorphin analogs, des- Gly2-8 -EP and D-Leu -Oh-EP, and Dr. M. Banay-Schwartz for the amino acid r analys& .

1. 2.

3.

4.

5.

6.

7.

8.

9. 10.

11.

12. 13.

REFERENCES

Knight, M. and Klee, W.A. (1978) J. Biol. Chem. 253, 3843-3847. Erdos, E.G., Johnson, A .R. and Boyden, N .T . (1978) Biochem. Pharmacol. 27, 843-846. Benuck, M. and Marks, N. (1979) Biochem. Biophys. Res. Commun. 88, 215-221. Malfroy, B., Swerts, J.P., Guyon, A., Roques, B .P. and Schwartz, J.C. (1978) Nature 276, 523-526. Schwartz, J.C., Malfroy, B. and De La Baume, S. (1981) Life Sci. 29, 1715-1740. Hui, K.-S., Wang, Y.-J. and Lajtha, A. (1981) Proc. Natl. Acad. Sci. USA (in press). Graf, L., Cseh, G., Barat, E., Ronai, A.Z., Szekely, J.I., Kenessey, A. and Bajusz, S. (1977) Ann. N.Y. Acad. Sci. 297, 63-83. Graf, L. and Cseh, G. (1968) Acta Biochim . Biophys. Acad. Sci. Hung. 3, 175-177. Hersh, L.B. (1981) Biochemistry 20, 2345-2350. Li , C. H. (1981) in “Hormonal Proteins and Peptides” (ed C . H . Li) , Vol. 10, Academic Press, New York, pp. 3-34. Austen, B.M. and Smyth, D.G. (1977) Biochem. Biophys. Res. Commun. 76, 477-482. Hersh, L.B., Smith, T.E. and McKelvy, J.F. (1980) Nature 286, 160-162. Barclay, R.K. and Phillipps, M.A. (1980) Biochem. Biophys. Res. Commun. 96, 1732-1738.

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