0143-4179/90/0016-0163/$10.00
Neuropeptides (1990) 16,163-168 @ Longman Group UK Ltd 1990
Inhibition of Angiotensin III Formation by Thiol Derivatives of Acidic Amino Acids S. WILK and L. S. THLlRSTON* Department of Pharmacology, York, NY 10029, USA
Mount
Sinai School
of Medicine
of the City University
of New York, New
Abstract-Angiotensin
Ill is formed by removal of the N-terminal Asp residue of angiotbnsin II in a reaction catalyzed by glutamyl aminopeptidase (aminopeptidase A EC 3.4.11.7). Thiol derivatives of glutamate and aspartate in which the (Y-COOH group was replaced by -CH2SH were synthesized as inhibitors of glutamyl aminopeptidase. Glutamate thiol was a potent inhibitor of glutamyl aminopeptidase (Ki = 4 x lo-'M) but even more potently inhibited microsomal alanyl aminopeptidase (Ki = 2.5 x 10m7M). Aspartate thiol (p-homocysteinb) was a less potent but more selective inhibitor of glutamyl aminopeptidase (glutamyl aminopeptidase: Ki = 1.2 x 10m6M; microsomal alanyl aminopeptidase: Ki = 7.5 x 10’6M). Neither compound inhibited cytosolic leucyl aminopeptidase. Aspartate thiol blocked the conversion of angiotensin II to angiotensin III. These derivatives are more selective thajn amastatin and may be of value in studies probing the biological significance of angiotensin Ill.
Glutamyl aminopeptidase” (aminopeptidase A, EC 3.4.11.7) first partially purified from rat kidney, catalyzes the removal of N-terminal glutamyl and aspartyl residues from peptides (1). This enzyme is of particular interest since it converts angiotensin II (ang II) to angiotensin III (desAsp Ang II) (2). It is now well established that the brain contains all of the components of the renin-
Date received 21 December 1989 Date accepted 3 January 1990 *Lee S. Thurston current address: Dept. of Chemistry, University of Texas at Austin, Austin TX 78712-I 167, USA a) Enzyme nomenclature as recommended by J. K. McDonald and A. J. Barrett (1986) in: Mammalian Proteases, vol. 2 Exopeptidases, Academic Press, New York.
angiotensin system (3), and it has been suggested that within the central nervous system ang II must be converted to ang III to become active (4). Inhibitors of glutamyl aminopeptidase would therefore be of value in studies explqring the biological significance of ang III. Potent inhibitors of other aminopeptidases have been synthesized by replacement of the cw-carboxyl grqup of a structurally favourable amino acid with iCH$H. Thus derivatives of leucine (5) and phenylalanine (6) have been prepared as inhibitors of mierosomal alanyl aminopeptidase (aminopeptidase N, EC 3.4.11.2). A derivative of lysine synthesized as an inhibitor of soluble arginyl aminopeptidase (aminopeptidase B, EC 3.4.11.6) has a subnanomolar Ki (7). We have synthesized the corresponding
163
164
NEUROPEPTIDES
BH,-THF
CH+H
COOtBU-BOCN+CCOtB”
CH,OTs TosCl -BocN~CootB"-
Nal
CH,I BocN*
COOtBu
CH,SH NsSH -
BocN eCoolBum’
Fig. 1 Synthesis of glutamate thiol.
inhibitor of soluble arginyl aminopeptidase (aminopeptidase B, EC 3.4.11.6) has a subnanomolar Ki (7). We have synthesized the corresponding thiol derivatives of glutamate and aspartate and report on their properties as inhibitors of glutamyl aminopeptidase and on the conversion of Ang II to Ang III.
Materials and Methods N-a-t-Boc-glutamyl-y-t-butyl ester and cr-glutaobtained from myl+-naphthylamide were Bachem, Inc. Torrance, CA. Leucyl-p-nitroanilide, soluble leucyl aminopeptidase (EC 3.4.11.1), angiotensin II and angiotensin III were obtained from Sigma Chemical Co., St. Louis, MO. Sephadex G-200 DEAE-Sephadex and Phenyl Sepharose were products of Pharmacia, Inc. Piscataway, NJ. S-Benzyl cysteine, Silica gel (Merck) grade 60 and all other chemicals were obtained from the Aldrich Chemical Co., Milwaukee, WI. Proton NMR were obtained on a Varian FTSOA or Varian XL-300 in CDCl3, CD30D or in Dz,+ Synthesis of inhibitors Glutamate thiol. This compound was synthesized from N-a-t-Boc-glutamic acid-y-t-butyl ester (Fig. 1). The free ol-COOH group was reduced with BHs-THF to afford the alcohol in 81% yield. The alcohol was treated with tosyl chloride in pyridine, and the resulting tosylate was purified by silica gel chromatography [hexane/ethyl acetate 4:1] (60% yield). The tosylate was converted to the iodide by refluxing with NAI in acetone and purified by silica gel chromatography (hexane 95/ethyl acetate
5) in 56% yield. Conversion- to the thiol was effected by reaction with NaSH in absolute ethanol. The reaction mixture was taken to dryness and the product dissolved in ether. The ethereal filtrate was evaporated to dryness (91% yield). The residue was treated with trifluoracetic acid to remove the blocking groups. Trifluoro acetic acid was removed under high vacuum and the residue dissolved in water. Chromatography on an AG50 H+ column and elution with 1N NHbOH yielded glutamate thiol as the disulfide (73% yield). Anal: Calcd for Ci0HZON204S2: H, 6.76; C, 40.54; N, 9.46. Found: H, 7.39; c, 40.56; N, 8.90. ‘H NMR (300 MHz) in D20, 64.8 (s, HOD), 3.1 (dd, lH, J =14, 4Hz, HA of ABX system of B’-CHz), 2.9 (m, IH, c&H), 2.8 (dd, lH, J = 14,8Hz, Hn of ABX of B-‘CH& 2.32 (t, 2H, J = 17 Hz, y-CH2), 1.91 (m, 2H, B-CH2). The synthetic intermediates discussed above were monitored by proton NMR. Notably the relative absorbances of the B’ methylenic protons exhibited a distinct chemical shift depending on the substituent: alcohol, 83.6; tosylate, 64.0; iodide, 63.4; disulfide, 62.8-3.1. Aspartate thiol (P-homocysteine)
The procedure of Balenkovic and Fles (8) was used to synthesize S-benzyl+-homocysteine from Sbenzyl cysteine (Fig. 2). The benzyl group was removed by refluxing with Na in butanol as described by Patterson and du Vigneaud (9). B-Homocysteine was purified by reverse phase
-
H 2 NACHSBz-
2
am
sty -A
phthal~c anhydrIde
PthN
PthN C”z%
CH$Bz
PthN
A
I\
CEH
CH$Bz COCHN, CH,SBz
Fig. 2 Synthesis of aspartate thiol (P-homocysteine) scribed by Balenkovic and FIes (8).
as de-
INHIBITION OF ANGIOTENSIN III FORMATION BY THIOL DERIVATIVES OF ACIDIC AMINO ACIDS
HPLC followed by chromatography on an AG50 Hf column. ‘H NMR (300 MHz) in CDsOD, 63.45 (m, lH, a-CH), 3.3 (pentet, MeOD), 2.95 (dd, lH, J = 13.5,5Hz, H,, of B’-CH;?), 2.74 (dd, lH, J = 13.5,?.7 Hz, Ha of B’-CHZ), 2.48 (dd, lH, J = 15.7, 5 HZ, HA of B-CHZ), 2.30 (dd, lH, J = 15.7,8.3 HZ, Ha of P-CH;?). Measurement
of enzymatic activities
Glutamyl aminopeptidase. The incubation mixture in a final volume of 2.50~1 contained 0.05M Tris-HCl buffer pH 7.5, a-glutamyl P-naphthylamide (0,4mM), CaCl2 (4mM), and enzyme.
Microsomal alanyl aminopeptidase. The incubation mixture in a final volume of 250~1 contained 0.05M Tris-HCl buffer, pH 7.5, leucyl p-nitroanilide (final concentration of 0.4mM), and enzyme. Cytosolic feucyl aminopeptidase.
This enzyme was assayed exactly as described for microsomal alanyl aminopeptidase . All samples were incubated at 37” and reactions terminated by addition of 250~1 10% trichloroacetic acid. The concentration of aromatic amine released was measured spectrophotometrically (10,ll). The chromogen formed from 2-naphthylamine was measured at 580nm and the chromogen formed from p-nitroaniline measured at 540nm. A unit of enzymatic activity is defined as that amount of enzyme liberating 1 pmole aromatic amine per h. Enzyme purification
Glutamyl aminopeptidase and microsomal alanyl aminopeptidase were purified from rabbit kidney cortex. The enzymes were solubilized by autolysis at pH 3.8 for 18h. The supernatant of the autolysate was adjusted to pH7 and the aminopeptidases precipitated by addition of (NH&SO4 to 90% saturation. The dissolved precipitate was subjected to Sephadex G-200 chromatography. Fractions containing both aminopeptidases were pooled (the enzymes were not resolved at this step) and
165
chromatographed on a DEAE-Sephadex column at pH 8.4. The aminopeptidases were eluted with a linear NaCl gradient (O-400mM). Resolution of the aminopeptidases was not complete, but fractions enriched in each activity were sepatiately pooled and separately chromatographed on a Phenyl Sepharose column at a pH of 7.3. Elution was effected by a linear (NH&S04 gradiant in which the concentration of this salt was reduced from 25% to zero. This step afforded esseritially homogeneous microsomal alanyl aminopeptidase as judged by non-dissociating PAGE. Glutamyl aminopeptidase free of contaminating microsomal alanyl aminopeptidase was obtained by rechromatography on Phenyl Sepharose at pH 8.0 utillizing an identical (NH&SO4 gradient. The specific activity of microsomal alanyl aminopeptidas was 570/ml and that of glutamyl aminopeptidasle was 260 U/ml. Determination of inhibition constants
Since the thiols are isolated in the oxidized form, inhibition is totally dependent upon the preisence of a thiol reducing agent in the incubation miyture. Inhibitors were added to incubation containing substrate and dithiothreitol (2 mM, final cdncentration). Reactions were initiated by addition of enzyme. Inhibition constants were obtained graphically by the method of Dixon (12) usirig two substrate concentrations. HPLC analyses
The effect of p-homocysteine on the glytamyl aminopeptidase catalyzed conversion of ang II to ang III was studied by HPLC. Incubation mibrtures consisting of 2mM dithiothreitol, 1OmM (Cat+, 0.1 mM ang II, 0.4 units glutamyl aminopepiidase, 0.05 M Tris HCl pH 7.5 + 4Op.M P-homocysteine in a total volume of 250~1 were incubated for 30min at 37”. HPLC was performed on a 260mm X 4.6mM 5p. CB column (Ailtech). The column was equilibrated with a solvent prepared by addition of 5ml H3P04 + 170ml acetonitrile/l adjusted to pH 3.0 with triethylamine as descriued by Wright et al., (13). 100~1 was injected odto the column and chromatography was performtd isocratically at a flow rate of 0.8mYmin. Phptides were detected by their absorbance at 2lOnin.
166
Fig. 3 Dixon plot of the inhibition of glutamyl aminopeptidase by glutamate thiol. Abscissa: inhibitor concentration. Substrate concentrations: -O- O.lmM; 0 0.4mM. Incubations conducted as described in methods.
Results and Discussion The thiol derivatives of glutamate and aspartate were isolated in the oxidized form. They were inactive in the absence of dithiothreitol demonstrating the requirement of a free thiol group for inhibitory activity. Glutamate thiol was a potent and reversible inhibitor of glutamyl aminopeptidase (Ki = 4 x 10W7) (Fig. 3). This compound however was an even more potent inhibitor of microsomal alanyl aminopeptidase (Ki= 2.5 x 10W7M). Aspartate thiol also inhibited glutamyl aminopeptidase but was not as potent as glutamate thiol (Ki = 1.2 x 10M6M)(Fig. 4). This is consistent with the preference of this enzyme for glutamyl over aspartyl residues (14). Aspartate thiol however was a more selective glutamyl aminopeptidase inhibitor since its Ki for inhibition of microsomal alanyl aminopeptidase was 7.5 x lO+jM. Both thiols tested at a concentration of 40Ol.~M failed to inhibit cytosolic leucyl aminopeptidase. Although it may seem surprising that glutamate thiol is a relatively strong inhibitor of microsomal alanyl aminopeptidase, it is in fact twenty times less potent than leucinthiol(5), and fifty times less potent than phethiol (6). Lysinethiol synthesized as an inhibitor of soluble arginyl aminopeptidase inhibits this enzyme with a Ki of 9.1 x lo-‘*M (7). By contrast glutamate and aspartate thiols are
NEUROPEPTIDES
much less potent inhibitors of their target enzyme. A distinguishing feature of glutamyl aminopeptidase is its stimulation by Ca++. Analysis of the pig kidney enzyme by atomic absorption spectrophotometry revealed 1 g atom Ca++/143OOOg protein (14). The moderate potencies of these inhibitors may be due to the fact that glutamyl aminopeptidase is a Ca++ rather than a Zn++ aminopeptidase. Alternatively a more extended compound capable of interacting with subsites on the enzyme may be a more favorable inhibitor. It is of interest to note that thiol derivatives of amino acids do not inhibit all Zn++ aminopeptidases. Thus, in common with the thiol derivatives of glutamate and aspartate, leucinthiol and lysinethiol do not inhibit cytosolic leucyl aminopeptidase (5, 7). Chung et al., (15) have synthesized and studied chloromethylketone and bromomethylketone derivatives of glutamate and aspartate as inhibitors of angiotensin III formation. a-L-Asp-CHzC1 (ICSo 3 x 10p6M) and a-L-Glu-CHzBr (I&, 3 X lo-‘M) have very similar potencies to the corresponding thiol derivatives. Their effect on microsoma1 leucyl aminopeptidase was not determined and it is not known whether they are more selective than the thiols. These inhibitors potentiated the blood pressure effects of Ang II. Glutamyl aminopeptidase catalyzes the conversion of ang II to ang III. HPLC analysis of an
60 Y
l/V
Fig.4 Dixon plot of the inhibition of glutamyl aminopeptidase by aspartate thiol @-homocysteine). Abscissa: inhibitor concentration. Substrate concentrations: + 0.1 mM; Q 0.4mM.
INHIBITION OF ANGIOTENSIN III FORMATION BY THIOL DERIVATIVES OF ACIDIC AMINO ACIDS
167
References A
C
B
1. Glenner, G. G., McMillan, P. J. and Folk, J.E. (1962). A mammalian peptidase specific for the hydrolysis of Nterminal a-L-glutamyl and aspartyl residues Nature (Lond.) 194: 867-868. 2. Nagatsu, I., Nagatsu, T., Yamamoto, T., Glenner, G. G. and Mehl, J. W. (1970). Purification of aminopeptidase A in human serum and degradation of angiotensin II by the purified enzyme. Biochim. Biophys. Acta 198: 255270.
;
2
1
:_
_
L
Fig. 5 Effect of aspartate thiol (P-homocysteine) on the glutamyl aminopeptidase-catalyzed conversion of ang II to ang III. HPLC conditions as described in methods. Panel A: Separation of 1Oug each of ang III (peak 1) and ang II (peak 2). Panel B: Incubation mixture of ang II and glutamyl aminopeptidase. Panel C: As in panel B but in the presence of 4OpM B-homocysteine. Composition of incubation mixtures described in methods.
incubation mixture of purified glutamyl aminopeptidase and ang II demonstrated this conversion. This reaction could be blocked by addition of aspartate thiol to the incubation mixture (Fig. 5). Amastatin has been used in a number of studies probing the biological significance of Ang III. Although amastatin was first described as a potent inhibitor of glutamyl aminopeptidase (16), it is not a specific inhibitor of this enzyme. Tobe et al., report a Ki of 2.5 x lo-‘M for inhibition of glut amyl aminopeptidase (17), however amastatin is an even more potent inhibitor of microsomal alanyl aminopeptidase (Ki = 1.9 x 10d8M) (18). Moreover, unlike the thiols, amastatin also inhibits soluble leucyl aminopeptidase (Ki = 2.2 x lo-‘M) (18). Therefore glutamate thiol and especially aspartate thiol are more selective than amastatin and may be of value in studies exploring the biological significance of Ang III.
Acknowledgements Supported by NS-17392, a Research Scientist Award MH00350 to S.W. and a training grant ST3DA-07135 to L.S.T.
3. Ganten, D., Lang, R. E., Lehmann, E. and IJnger, T. (1984). Brain angiotensin: on the way to becoming a well-studied neuropeptide system. Biochem. Pharmacol. 33: 3523-3528. 4. Harding, J. W. and Felix, D. (1987). The effects of the aminopeptidase inhibitors amastatin and bestatin on angiotensin-evoked neuronal activity in rat brain. Brain Res. 424: 299-304. 5. Chan, W. W. C. (1983). L-Leucinthioi-a potent inhibitor of leucine aminopeptidase. Biochem. Biophys. Res. Corn. 116: 297-302. 6. Gros C., Giros B., Schwartz J. C., Vlaiculescu A., Costentin J. and Lecomte J. M. (1988). Potent it$ibition of cerebral aminopeptidases by carbaphethiol, a parenterally active compound. Neuropeptides 12: 111-118. 7. Ocain, T. D. and Rich, D. H. (1987). L-Lysinethiol: A subnanomolar inhibitor of aminopeptidase B.’ Biochem. Biophys. Res. Comm. 145: 1038-1042. 8. Balenkovic, K. and Fles, D. (1952). Synthesis of L-Samino-y-benzylthiobutyric acid. [L-B-amino&(benzyl) homocysteine]. Amino Acids VI J. Org. Chem. 17: 341-349. 9. Patterson, W. I. andduvigneaud, V. (1935). The synthesis of homocysteine. J. Biol. Chem. 111: 393-398. 10. Goldbarg, J. A. and Rutenberg, A. M. (8958). The calorimetric determination of leucine aminopeptidase in urine and serum of normal subjects and patients with cancer and other diseases. Cancer 11: 283-291, 11. Bratton, A. C. and Marshall A. K., Jr. (1939). A new coupling component for sulfanilamide determination. J. Biol. Chem. 128: 537-550. 12. Dixon, M. (1953). The determination of enzyme inhibitor constants. Biochem. J. 55: 170-171. 13. Wright, J. W., Sullivan, M. J., Bredl, C. R., Hanesworth, J. M., Cushing, L. L., and Harding, J. W. (1987). Delayed cerebroventricular metabolism of ]‘251]angiontensins in the spontaneously hypertensive rat. J. Neurochem. 49: 651-654. 14. Danielsen, E. M., Noren, 0.. Sjostrom, H., Ingram, J. and Kenny, A. J. (1980). Proteins of the kidney;microvillar membrane. Aspartate aminopeptidase: puri/ication by immunoadsorbent chromatography and properties of the detergent- and proteinase-solubilized forms. Biochem J. 189: 591-603. 1.5. Chung A., Ryan J. W. and Berryer, P (1983). Inhibition of the formation of angiotensin III. Adv. Exp. Med. Biol. 156: 693-701.
168 16. Aoyagi, T., Tobe, H., Kojima, F., Hamada, M,Takeuchi, T. and Umezawa, H. (1978). Amastatin, an inhibitor of aminopeptidase A, produced by actinomycetes. J. Antibiot. (Tokyo) Ser. A 31: 636-638. 17. Tobe H., Kojima F., Aoyagi, T. and Umezawa H. (1980). Purification by affinity chromatography using amastatin
NEUROPEPTIDES
and properties of aminopeptidase A from pig kidney. Biochim. Biophys Acta 613: 459-468. 18. Rich D. H., Moon B. J. and Harbeson S. (1984). Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effect of inhibitor structure on slow-binding processes. J. Med. Chem. 27: 417-422.
Neuropeptides (1990) 16,163-168 @ Longman Group UK Ltd 1990
Inhibition of Angiotensin III Formation by Thiol Derivatives of Acidic Amino Acids S. WILK and L. S. THLlRSTON* Department of Pharmacology, York, NY 10029, USA
Mount
Sinai School
of Medicine
of the City University
of New York, New
Abstract-Angiotensin
Ill is formed by removal of the N-terminal Asp residue of angiotbnsin II in a reaction catalyzed by glutamyl aminopeptidase (aminopeptidase A EC 3.4.11.7). Thiol derivatives of glutamate and aspartate in which the (Y-COOH group was replaced by -CH2SH were synthesized as inhibitors of glutamyl aminopeptidase. Glutamate thiol was a potent inhibitor of glutamyl aminopeptidase (Ki = 4 x lo-'M) but even more potently inhibited microsomal alanyl aminopeptidase (Ki = 2.5 x 10m7M). Aspartate thiol (p-homocysteinb) was a less potent but more selective inhibitor of glutamyl aminopeptidase (glutamyl aminopeptidase: Ki = 1.2 x 10m6M; microsomal alanyl aminopeptidase: Ki = 7.5 x 10’6M). Neither compound inhibited cytosolic leucyl aminopeptidase. Aspartate thiol blocked the conversion of angiotensin II to angiotensin III. These derivatives are more selective thajn amastatin and may be of value in studies probing the biological significance of angiotensin Ill.
Glutamyl aminopeptidase” (aminopeptidase A, EC 3.4.11.7) first partially purified from rat kidney, catalyzes the removal of N-terminal glutamyl and aspartyl residues from peptides (1). This enzyme is of particular interest since it converts angiotensin II (ang II) to angiotensin III (desAsp Ang II) (2). It is now well established that the brain contains all of the components of the renin-
Date received 21 December 1989 Date accepted 3 January 1990 *Lee S. Thurston current address: Dept. of Chemistry, University of Texas at Austin, Austin TX 78712-I 167, USA a) Enzyme nomenclature as recommended by J. K. McDonald and A. J. Barrett (1986) in: Mammalian Proteases, vol. 2 Exopeptidases, Academic Press, New York.
angiotensin system (3), and it has been suggested that within the central nervous system ang II must be converted to ang III to become active (4). Inhibitors of glutamyl aminopeptidase would therefore be of value in studies explqring the biological significance of ang III. Potent inhibitors of other aminopeptidases have been synthesized by replacement of the cw-carboxyl grqup of a structurally favourable amino acid with iCH$H. Thus derivatives of leucine (5) and phenylalanine (6) have been prepared as inhibitors of mierosomal alanyl aminopeptidase (aminopeptidase N, EC 3.4.11.2). A derivative of lysine synthesized as an inhibitor of soluble arginyl aminopeptidase (aminopeptidase B, EC 3.4.11.6) has a subnanomolar Ki (7). We have synthesized the corresponding
163
164
NEUROPEPTIDES
BH,-THF
CH+H
COOtBU-BOCN+CCOtB”
CH,OTs TosCl -BocN~CootB"-
Nal
CH,I BocN*
COOtBu
CH,SH NsSH -
BocN eCoolBum’
Fig. 1 Synthesis of glutamate thiol.
inhibitor of soluble arginyl aminopeptidase (aminopeptidase B, EC 3.4.11.6) has a subnanomolar Ki (7). We have synthesized the corresponding thiol derivatives of glutamate and aspartate and report on their properties as inhibitors of glutamyl aminopeptidase and on the conversion of Ang II to Ang III.
Materials and Methods N-a-t-Boc-glutamyl-y-t-butyl ester and cr-glutaobtained from myl+-naphthylamide were Bachem, Inc. Torrance, CA. Leucyl-p-nitroanilide, soluble leucyl aminopeptidase (EC 3.4.11.1), angiotensin II and angiotensin III were obtained from Sigma Chemical Co., St. Louis, MO. Sephadex G-200 DEAE-Sephadex and Phenyl Sepharose were products of Pharmacia, Inc. Piscataway, NJ. S-Benzyl cysteine, Silica gel (Merck) grade 60 and all other chemicals were obtained from the Aldrich Chemical Co., Milwaukee, WI. Proton NMR were obtained on a Varian FTSOA or Varian XL-300 in CDCl3, CD30D or in Dz,+ Synthesis of inhibitors Glutamate thiol. This compound was synthesized from N-a-t-Boc-glutamic acid-y-t-butyl ester (Fig. 1). The free ol-COOH group was reduced with BHs-THF to afford the alcohol in 81% yield. The alcohol was treated with tosyl chloride in pyridine, and the resulting tosylate was purified by silica gel chromatography [hexane/ethyl acetate 4:1] (60% yield). The tosylate was converted to the iodide by refluxing with NAI in acetone and purified by silica gel chromatography (hexane 95/ethyl acetate
5) in 56% yield. Conversion- to the thiol was effected by reaction with NaSH in absolute ethanol. The reaction mixture was taken to dryness and the product dissolved in ether. The ethereal filtrate was evaporated to dryness (91% yield). The residue was treated with trifluoracetic acid to remove the blocking groups. Trifluoro acetic acid was removed under high vacuum and the residue dissolved in water. Chromatography on an AG50 H+ column and elution with 1N NHbOH yielded glutamate thiol as the disulfide (73% yield). Anal: Calcd for Ci0HZON204S2: H, 6.76; C, 40.54; N, 9.46. Found: H, 7.39; c, 40.56; N, 8.90. ‘H NMR (300 MHz) in D20, 64.8 (s, HOD), 3.1 (dd, lH, J =14, 4Hz, HA of ABX system of B’-CHz), 2.9 (m, IH, c&H), 2.8 (dd, lH, J = 14,8Hz, Hn of ABX of B-‘CH& 2.32 (t, 2H, J = 17 Hz, y-CH2), 1.91 (m, 2H, B-CH2). The synthetic intermediates discussed above were monitored by proton NMR. Notably the relative absorbances of the B’ methylenic protons exhibited a distinct chemical shift depending on the substituent: alcohol, 83.6; tosylate, 64.0; iodide, 63.4; disulfide, 62.8-3.1. Aspartate thiol (P-homocysteine)
The procedure of Balenkovic and Fles (8) was used to synthesize S-benzyl+-homocysteine from Sbenzyl cysteine (Fig. 2). The benzyl group was removed by refluxing with Na in butanol as described by Patterson and du Vigneaud (9). B-Homocysteine was purified by reverse phase
-
H 2 NACHSBz-
2
am
sty -A
phthal~c anhydrIde
PthN
PthN C”z%
CH$Bz
PthN
A
I\
CEH
CH$Bz COCHN, CH,SBz
Fig. 2 Synthesis of aspartate thiol (P-homocysteine) scribed by Balenkovic and FIes (8).
as de-
INHIBITION OF ANGIOTENSIN III FORMATION BY THIOL DERIVATIVES OF ACIDIC AMINO ACIDS
HPLC followed by chromatography on an AG50 Hf column. ‘H NMR (300 MHz) in CDsOD, 63.45 (m, lH, a-CH), 3.3 (pentet, MeOD), 2.95 (dd, lH, J = 13.5,5Hz, H,, of B’-CH;?), 2.74 (dd, lH, J = 13.5,?.7 Hz, Ha of B’-CHZ), 2.48 (dd, lH, J = 15.7, 5 HZ, HA of B-CHZ), 2.30 (dd, lH, J = 15.7,8.3 HZ, Ha of P-CH;?). Measurement
of enzymatic activities
Glutamyl aminopeptidase. The incubation mixture in a final volume of 2.50~1 contained 0.05M Tris-HCl buffer pH 7.5, a-glutamyl P-naphthylamide (0,4mM), CaCl2 (4mM), and enzyme.
Microsomal alanyl aminopeptidase. The incubation mixture in a final volume of 250~1 contained 0.05M Tris-HCl buffer, pH 7.5, leucyl p-nitroanilide (final concentration of 0.4mM), and enzyme. Cytosolic feucyl aminopeptidase.
This enzyme was assayed exactly as described for microsomal alanyl aminopeptidase . All samples were incubated at 37” and reactions terminated by addition of 250~1 10% trichloroacetic acid. The concentration of aromatic amine released was measured spectrophotometrically (10,ll). The chromogen formed from 2-naphthylamine was measured at 580nm and the chromogen formed from p-nitroaniline measured at 540nm. A unit of enzymatic activity is defined as that amount of enzyme liberating 1 pmole aromatic amine per h. Enzyme purification
Glutamyl aminopeptidase and microsomal alanyl aminopeptidase were purified from rabbit kidney cortex. The enzymes were solubilized by autolysis at pH 3.8 for 18h. The supernatant of the autolysate was adjusted to pH7 and the aminopeptidases precipitated by addition of (NH&SO4 to 90% saturation. The dissolved precipitate was subjected to Sephadex G-200 chromatography. Fractions containing both aminopeptidases were pooled (the enzymes were not resolved at this step) and
165
chromatographed on a DEAE-Sephadex column at pH 8.4. The aminopeptidases were eluted with a linear NaCl gradient (O-400mM). Resolution of the aminopeptidases was not complete, but fractions enriched in each activity were sepatiately pooled and separately chromatographed on a Phenyl Sepharose column at a pH of 7.3. Elution was effected by a linear (NH&S04 gradiant in which the concentration of this salt was reduced from 25% to zero. This step afforded esseritially homogeneous microsomal alanyl aminopeptidase as judged by non-dissociating PAGE. Glutamyl aminopeptidase free of contaminating microsomal alanyl aminopeptidase was obtained by rechromatography on Phenyl Sepharose at pH 8.0 utillizing an identical (NH&SO4 gradient. The specific activity of microsomal alanyl aminopeptidas was 570/ml and that of glutamyl aminopeptidasle was 260 U/ml. Determination of inhibition constants
Since the thiols are isolated in the oxidized form, inhibition is totally dependent upon the preisence of a thiol reducing agent in the incubation miyture. Inhibitors were added to incubation containing substrate and dithiothreitol (2 mM, final cdncentration). Reactions were initiated by addition of enzyme. Inhibition constants were obtained graphically by the method of Dixon (12) usirig two substrate concentrations. HPLC analyses
The effect of p-homocysteine on the glytamyl aminopeptidase catalyzed conversion of ang II to ang III was studied by HPLC. Incubation mibrtures consisting of 2mM dithiothreitol, 1OmM (Cat+, 0.1 mM ang II, 0.4 units glutamyl aminopepiidase, 0.05 M Tris HCl pH 7.5 + 4Op.M P-homocysteine in a total volume of 250~1 were incubated for 30min at 37”. HPLC was performed on a 260mm X 4.6mM 5p. CB column (Ailtech). The column was equilibrated with a solvent prepared by addition of 5ml H3P04 + 170ml acetonitrile/l adjusted to pH 3.0 with triethylamine as descriued by Wright et al., (13). 100~1 was injected odto the column and chromatography was performtd isocratically at a flow rate of 0.8mYmin. Phptides were detected by their absorbance at 2lOnin.
166
Fig. 3 Dixon plot of the inhibition of glutamyl aminopeptidase by glutamate thiol. Abscissa: inhibitor concentration. Substrate concentrations: -O- O.lmM; 0 0.4mM. Incubations conducted as described in methods.
Results and Discussion The thiol derivatives of glutamate and aspartate were isolated in the oxidized form. They were inactive in the absence of dithiothreitol demonstrating the requirement of a free thiol group for inhibitory activity. Glutamate thiol was a potent and reversible inhibitor of glutamyl aminopeptidase (Ki = 4 x 10W7) (Fig. 3). This compound however was an even more potent inhibitor of microsomal alanyl aminopeptidase (Ki= 2.5 x 10W7M). Aspartate thiol also inhibited glutamyl aminopeptidase but was not as potent as glutamate thiol (Ki = 1.2 x 10M6M)(Fig. 4). This is consistent with the preference of this enzyme for glutamyl over aspartyl residues (14). Aspartate thiol however was a more selective glutamyl aminopeptidase inhibitor since its Ki for inhibition of microsomal alanyl aminopeptidase was 7.5 x lO+jM. Both thiols tested at a concentration of 40Ol.~M failed to inhibit cytosolic leucyl aminopeptidase. Although it may seem surprising that glutamate thiol is a relatively strong inhibitor of microsomal alanyl aminopeptidase, it is in fact twenty times less potent than leucinthiol(5), and fifty times less potent than phethiol (6). Lysinethiol synthesized as an inhibitor of soluble arginyl aminopeptidase inhibits this enzyme with a Ki of 9.1 x lo-‘*M (7). By contrast glutamate and aspartate thiols are
NEUROPEPTIDES
much less potent inhibitors of their target enzyme. A distinguishing feature of glutamyl aminopeptidase is its stimulation by Ca++. Analysis of the pig kidney enzyme by atomic absorption spectrophotometry revealed 1 g atom Ca++/143OOOg protein (14). The moderate potencies of these inhibitors may be due to the fact that glutamyl aminopeptidase is a Ca++ rather than a Zn++ aminopeptidase. Alternatively a more extended compound capable of interacting with subsites on the enzyme may be a more favorable inhibitor. It is of interest to note that thiol derivatives of amino acids do not inhibit all Zn++ aminopeptidases. Thus, in common with the thiol derivatives of glutamate and aspartate, leucinthiol and lysinethiol do not inhibit cytosolic leucyl aminopeptidase (5, 7). Chung et al., (15) have synthesized and studied chloromethylketone and bromomethylketone derivatives of glutamate and aspartate as inhibitors of angiotensin III formation. a-L-Asp-CHzC1 (ICSo 3 x 10p6M) and a-L-Glu-CHzBr (I&, 3 X lo-‘M) have very similar potencies to the corresponding thiol derivatives. Their effect on microsoma1 leucyl aminopeptidase was not determined and it is not known whether they are more selective than the thiols. These inhibitors potentiated the blood pressure effects of Ang II. Glutamyl aminopeptidase catalyzes the conversion of ang II to ang III. HPLC analysis of an
60 Y
l/V
Fig.4 Dixon plot of the inhibition of glutamyl aminopeptidase by aspartate thiol @-homocysteine). Abscissa: inhibitor concentration. Substrate concentrations: + 0.1 mM; Q 0.4mM.
INHIBITION OF ANGIOTENSIN III FORMATION BY THIOL DERIVATIVES OF ACIDIC AMINO ACIDS
167
References A
C
B
1. Glenner, G. G., McMillan, P. J. and Folk, J.E. (1962). A mammalian peptidase specific for the hydrolysis of Nterminal a-L-glutamyl and aspartyl residues Nature (Lond.) 194: 867-868. 2. Nagatsu, I., Nagatsu, T., Yamamoto, T., Glenner, G. G. and Mehl, J. W. (1970). Purification of aminopeptidase A in human serum and degradation of angiotensin II by the purified enzyme. Biochim. Biophys. Acta 198: 255270.
;
2
1
:_
_
L
Fig. 5 Effect of aspartate thiol (P-homocysteine) on the glutamyl aminopeptidase-catalyzed conversion of ang II to ang III. HPLC conditions as described in methods. Panel A: Separation of 1Oug each of ang III (peak 1) and ang II (peak 2). Panel B: Incubation mixture of ang II and glutamyl aminopeptidase. Panel C: As in panel B but in the presence of 4OpM B-homocysteine. Composition of incubation mixtures described in methods.
incubation mixture of purified glutamyl aminopeptidase and ang II demonstrated this conversion. This reaction could be blocked by addition of aspartate thiol to the incubation mixture (Fig. 5). Amastatin has been used in a number of studies probing the biological significance of Ang III. Although amastatin was first described as a potent inhibitor of glutamyl aminopeptidase (16), it is not a specific inhibitor of this enzyme. Tobe et al., report a Ki of 2.5 x lo-‘M for inhibition of glut amyl aminopeptidase (17), however amastatin is an even more potent inhibitor of microsomal alanyl aminopeptidase (Ki = 1.9 x 10d8M) (18). Moreover, unlike the thiols, amastatin also inhibits soluble leucyl aminopeptidase (Ki = 2.2 x lo-‘M) (18). Therefore glutamate thiol and especially aspartate thiol are more selective than amastatin and may be of value in studies exploring the biological significance of Ang III.
Acknowledgements Supported by NS-17392, a Research Scientist Award MH00350 to S.W. and a training grant ST3DA-07135 to L.S.T.
3. Ganten, D., Lang, R. E., Lehmann, E. and IJnger, T. (1984). Brain angiotensin: on the way to becoming a well-studied neuropeptide system. Biochem. Pharmacol. 33: 3523-3528. 4. Harding, J. W. and Felix, D. (1987). The effects of the aminopeptidase inhibitors amastatin and bestatin on angiotensin-evoked neuronal activity in rat brain. Brain Res. 424: 299-304. 5. Chan, W. W. C. (1983). L-Leucinthioi-a potent inhibitor of leucine aminopeptidase. Biochem. Biophys. Res. Corn. 116: 297-302. 6. Gros C., Giros B., Schwartz J. C., Vlaiculescu A., Costentin J. and Lecomte J. M. (1988). Potent it$ibition of cerebral aminopeptidases by carbaphethiol, a parenterally active compound. Neuropeptides 12: 111-118. 7. Ocain, T. D. and Rich, D. H. (1987). L-Lysinethiol: A subnanomolar inhibitor of aminopeptidase B.’ Biochem. Biophys. Res. Comm. 145: 1038-1042. 8. Balenkovic, K. and Fles, D. (1952). Synthesis of L-Samino-y-benzylthiobutyric acid. [L-B-amino&(benzyl) homocysteine]. Amino Acids VI J. Org. Chem. 17: 341-349. 9. Patterson, W. I. andduvigneaud, V. (1935). The synthesis of homocysteine. J. Biol. Chem. 111: 393-398. 10. Goldbarg, J. A. and Rutenberg, A. M. (8958). The calorimetric determination of leucine aminopeptidase in urine and serum of normal subjects and patients with cancer and other diseases. Cancer 11: 283-291, 11. Bratton, A. C. and Marshall A. K., Jr. (1939). A new coupling component for sulfanilamide determination. J. Biol. Chem. 128: 537-550. 12. Dixon, M. (1953). The determination of enzyme inhibitor constants. Biochem. J. 55: 170-171. 13. Wright, J. W., Sullivan, M. J., Bredl, C. R., Hanesworth, J. M., Cushing, L. L., and Harding, J. W. (1987). Delayed cerebroventricular metabolism of ]‘251]angiontensins in the spontaneously hypertensive rat. J. Neurochem. 49: 651-654. 14. Danielsen, E. M., Noren, 0.. Sjostrom, H., Ingram, J. and Kenny, A. J. (1980). Proteins of the kidney;microvillar membrane. Aspartate aminopeptidase: puri/ication by immunoadsorbent chromatography and properties of the detergent- and proteinase-solubilized forms. Biochem J. 189: 591-603. 1.5. Chung A., Ryan J. W. and Berryer, P (1983). Inhibition of the formation of angiotensin III. Adv. Exp. Med. Biol. 156: 693-701.
168 16. Aoyagi, T., Tobe, H., Kojima, F., Hamada, M,Takeuchi, T. and Umezawa, H. (1978). Amastatin, an inhibitor of aminopeptidase A, produced by actinomycetes. J. Antibiot. (Tokyo) Ser. A 31: 636-638. 17. Tobe H., Kojima F., Aoyagi, T. and Umezawa H. (1980). Purification by affinity chromatography using amastatin
NEUROPEPTIDES
and properties of aminopeptidase A from pig kidney. Biochim. Biophys Acta 613: 459-468. 18. Rich D. H., Moon B. J. and Harbeson S. (1984). Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effect of inhibitor structure on slow-binding processes. J. Med. Chem. 27: 417-422.
