Biol. Chem. Hoppe-Seyler Vol. 373. pp. 375-380, July 1992

Basic Amino Acids Preferring Broad Specificity Aminopeptidase from Human Erythrocytes MARIJA ABRAMIC AND LJUBINKA VITALE Dept. Organic Chemistry and Biochemistry, Rudjer Boskovi6 Institute, 41001 Zagreb, Croatia

Summary: An aminopeptidase hydrolyzing 2-naphthylamides of Lys, Arg, Leu, Met, Phe and Tyr, as well as different di- to tridecapeptides, was purified from the cytosol of human erythrocytes. The enzyme showed preference for Lys and Arg at N-terminus, as proline and D-amino acids were nonpermissive at P 1 ' site. Higher affinity for oligopeptides than for aminoacyl naphthylamides was observed. Among the substrates were Lys-bradykinin, angiotensin III, thymopentin and enkephalins. Aminopeptidase was shown to be a monomeric protein of Mr~110000 and of pl~4.8, activated by Co2* and inhibited by EDTA, pHMB, amastatin, bestatin and puromycin. The isolated enzyme could be classified as cytosolic, Lys(Arg) preferring, broad specificity aminopeptidase.

Introduction A lack of strict specificity of aminopeptidases for a particular amino acid, or at least for amino acids of the same type, hinder their identification. Therefore more extensive data on catalytic properties and specificity are needed to compare and classify these enzymes properly. Reports on several APs from blood cells, able to hydrolyze Arg-2NA [1-6], rose a question of their similarity and belonging to one of the already established groups of APs [ 7 ] , To clear dilemmas concerning AP from human red blood cells which we have previously described [ 2 ] , a new isolation procedure for this enzyme was developed and the study of its properties was extended. Materials and Methods Enzyme purification Fresh human erythrocytes were freed from other blood cells by filtration through Erypur filter, washed and lysed by the addition of 4 volumes of 1 mM EDTA solution. Hemolysate was centrifuged (50 min; 20000xg) to remove

Abbreviations: AP, aminopeptidase; 2NA, 2-naphthylamide; SDS, sodium dodecyl sulfate; pHMB, p-hydroxymercuribenzoate.

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stroma, adjusted to pH 6.7 and loaded on DEAE-cellulose (type 23SH, Serva) column (5x25 cm) equilibrated with 10 mM sodium-phosphate, 2 mM 2-mercaptoethanol, pH 6.7 b u f f e r . The column was rinsed with the starting buffer containing 0.12 M NaCl and then with linear 0.12-0.40 M NaCl gradient. Pooled active fractions eluted between 0 . 2 4 - 0 . 2 6 M NaCl were concentrated 90 fold by Ultrafiltration (Diaflo PM-10 membrane) and applied to the hydroxylapatite (Bio-Gel HTP, Βίο-Rad) column (0.9x18 cm) in 10 mM sodium-phosphate, 2 mM 2-mercaptoethanol, 0.01% Nonidet P40, pH 6.7 b u f f e r . Adsorbed material was eluted with 10-220 mM sodium-phosphate linear gradient. Fractions with AP activity, collected at 160-215 mM sodium-phosphate, were concentrated and loaded on the Sephacryl S-200, superfine, column (1.5x89 cm) in 50 mM Tris/ HC1, 0 . 2 M NaCl, pH 7 . 5 b u f f e r . Following equilibration of active fractions with 50 mM Tris/HCl pH 7.5, final purification was achieved by chromatography on Mono Q column (FPLC, Pharmacia) . Purified AP was stored in 20% glycerol at -10°C. Enzyme and protein assay AP activity was determined at 37°C in the presence of 0.088 mM Lys-2NA (or another amino acid-2NA) , 50 mM Tris/HCl, 0.1 mM CoCl 2 , pH 7.5, and an appropriate amount of enzyme. Liberated 2-naphthylamine was coupled to Fast-Blue B salt and Ac30nm was measured [ 6 ] . For kinetic constants determination 3 mM 2-mercaptoetnanol was added to the reaction mixture and liberation of 2naphthylamine was followed by Perkin Elmer luminescence spectrometer LS 50, using emission and excitation wavelength of 422 and 332 nm respectively. Constants were calculated according to Hanes and Lineweaver & Burk [ 8 ] , The unit of enzyme activity (U) was defined as the amount of AP which hydrolyses 1 μιηοΐ of substrate per minute. For peptide hydrolysis, mixtures containing 0.8 mM peptide, 5 mM Tris/HCl pH 7.2, and 1.1 μg/ml of purified enzyme were incubated for 2 hrs at 37°C and 10 μΐ aliquots were analyzed by thin-layer chromatography [ 5 ] . Proteins were determined by the method of Bradford [9] with bovine serum albumin as a standard. Determination of K, and IC50 Κ,- values were determined according to Chu and Orlowski [10] by treating peptides as alternate substrate inhibitors of 9 μΜ Lys-2NA hydrolysis. K i was calculated from the reaction rate at peptide concentration which caused inhibition close to 50%, and Km for Lys-2NA determined under the same conditions. In effector studies, metal ions, thiol compounds and organic solvents were preincubated with the enzyme for 3 min at 37°C, and inhibitors for 60 min at 25°C. IC50 were calculated according to Witwicki and Chidambaram [11]. Molecular mass and isoelectric point determination Relative molecular mass was estimated by gel filtration on Sephacryl S-200 superfine (1.5x80 cm) column in 50 mM Tris/HCl buffer pH 7.5 containing 0.2 M NaCl, at a flow rate of 10 m l / h . Mr was also determined by the native and SDS-polyacrylamide gel electrophoresis performed by PhastSystem on gradient and homogeneous 7.5 gels (Pharmacia). For SDS-PAGE proteins were treated with 2 . 5 % SDS and 5% 2-mercaptoethanol. Isoelectric focusing was performed by the same system on IEF 3-9 and 4-6.5 gels. As standards, protein calibration kits from Pharmacia were used.

Results Enzyme isolation The purification of Lys-2NA hydrolyzing AP from human erythrocytes cytosol is summarized in Table 1. DEAE-cellulose chromatography separated the enzyme from the other two APs which degrade the same substrate. After three additional steps, a preparation homogeneous by electrophoretic criteria, was

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Aminopeptidase from Human Erythrocytes


obtained. The enzyme recovery was virtually higher than 2 . 3 % , since cytosol activity encompassed the action of all

Lys-2NA splitting enzymes.

Table l. Purification of Lys-2NA hydrolyzing aminopeptidase from human erythrocytes. Start: 180 ml of erythrocytes. Volume (ml)

Purification step

Protein (mg/ml)

40.0 5.61


Cytosol DEAE-cellulose Hydroxylapatite Sephacryl S-200 Mono Q


4.50 0.215

2.6 7.3 1.5


Spec. act. (U/mg)


0.0032 0.214 0.550 1.890 16.205


(%) 14.8

6.4 2.9 2.3

Enzyme properties Relative molecular mass of AP from human erythrocytes was determined by three methods to be about 110000. Its the

optimal pH, which was between pH 6 and 7, addition of

raised AP activity up to up

isoelectric point was at



0 . 5 mM Co2* ions

30% regardless of 2NA substrate used, whereas Ca2*

10 mM was without effect.



pH 4 . 8 . At


ions did









starting from 1% in the reaction mixture inhibited AP. The activity of


purified AP was rapidly lost even at -20°C in 40% glycerol. The presence of 1 mM 2-mercaptoethanol or the reduced form of glutathione had a protective effect.











activate the enzyme. Susceptibility

of isolated AP to inhibitors is illustrated in Table 2.

Table 2. Effect of inhibitors on human erythrocyte aminopeptidase. *2-mercaptoethanol absent Inhibitor

IC50 (M)

EDTA pHMB* Amastatin Bestatin Leupeptin Puromycin

9.0 9.1 9.0 1.0 2.3 2.5

X X X X X χ

ΙΟ' 7 ΙΟ' 5 10'8 ΙΟ' 6 ΙΟ'* 10'6

Inhibition by chelating and sulphydryl reagents, as well as its

response to

Co2* ions, indicate that the enzyme belongs to metallo-, thiol dependent APs. It is also puromycin-sensitive, whereas amastatin is more potent inhibitor than bestatin. Substrate specificity Purified human erythrocyte AP hydrolyzed 2NAs of various amino acids,


little or no hydrolysis of Pro-, Val-, lie- and His-2NA was observed.

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Vol. 373 (1992)

Catalytic constants (Table 3) reveal that the best substrates were Lys- and Arg-2NA, followed by Met-, Leu-, Phe- and Tyr-2NA. Ala-2NA was also significantly hydrolyzed. Na-Bz-L-Arg-2NA was not a substrate of this AP. Table 3. Kinetic constants for the hydrolysis of aminoacyl-2-naphthylamides by human erythrocyte aminopeptidase Substrate


Lys-2NA Arg-2NA Leu-2NA Met-2NA Phe-2NA Tyr-2NA

2.8 3.3 9.3 10.0 21.9 22.9

Above 0.1 mM the


cat( s ~ 1 ) 25.9 23.0 19.6 16.9 21.0 10.0

* 9250 6970 2108 1690 959 437

inhibition of AP with aminoacyl-2NAs was observed.

Specificity of human erythrocyte aminopeptidase was further investigated by its ability to hydrolyze different peptides (Table 4 ) . Table 4. Interaction of aminopeptidase from human erythrocytes with different peptides. +++ ~50%, ++ ~10-30%, + up to 10% hydrolyzed, - not hydrolyzed. (*) Substrate gradually added to the reaction mixture to prevent inhibition. Peptide


Κ,. (μΜ)

Arg-Arg-Leu-Ile-Glu-Asp-. . tridecapeptide Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Arg-Val-Tyr-Ile-His-Pro-Phe Lys-Phe-I le-Gly-Leu-MetNH2 Arg-Gly-Pro-Phe-Pro-Ile Met-Gly-Trp-Met-Asp-PheNH2 Lys-Val-Ile-Leu-Phe Arg-Tyr-Leu-Pro-Thr Leu-Trp-Met-Arg-Phe Arg-Lys-Asp-Val-Tyr Tyr-Gly-Gly-Phe-Met

+ + +(*) + ++ ++ +++ +++ + (*) +++ ++

-1.4 0.22 0.65 0.017 1.80 -3.0

Arg-Gly-Phe-Phe Leu-Trp-Met-Arg

+ (* ) +

0.36 0.82

Arg-Phe-Ala Lys-Phe-Lys Leu-Trp-Met Met-Arg-Phe Phe-Gly-Gly

+++ +++ ++ + +

2.10 2.30 2.40 -3.0 >100

Arg-Phe Lys-Phe Lys-Lys Phe-Phe His-Phe; Met-Phe; Leu-Val

++ +++ +++ +++ +

1.70 4.00 7.50 18.50 n.d.

0.11 0.18 0.30 5.10 31.00

Ala-Ala-Ala Glu-Ala-Ala Arg-Pro-Pro-Gly-Phe Tyr-D-Ala-Gly-Phe-Leu

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Aminopeptidase from Human Erythrocytes


Isolated AP hydrolyzed di- to oligopeptides, exclusively in an exopeptidase manner, with P1 specificity comparable to that observed with 2NA substrates. Comparison of K,. for peptides having identical starting sequence revealed the increase of affinity with peptide length, and illustrated the extended active site, as well as the importance of interactions at P,' , P 2 ' , P 3 ' subsites. The enzyme had especially high affinity for Lys-Phe-Ile-Gly-Leu-MetNH2. Among good substrates were proctolin, kallidin, angiotensin III, thymopentin and Met-enkephalin. Bradykinin(l-S) (Arg-Pro-Pro-Gly-Phe) , Tyr-D-Ala-Gly-Phe-Leu and aprotinin (Arg-Pro-Asp-Phe-Cys-Leu...50 amino acids) were neither among substrates nor inhibitors, indicating that proline and D-amino acids might be nonpermissive at P 1 ' position. Discussion Chromatographie behavior on a DEAE-cellulose column and a lack of activation by Cl"

ions distinguishes

isolated human erythrocytes AP from

the Cl~-

activated arginyl and Cl'-activated alanyl AP from the same source or from the other mammalian tissues [3,5,12]. Characteristics of its specificity are preference for Lys- and Arg-2NA but also an acceptance of Met-, Leu- and Phe2NA as substrate. The presence of a broad specificity AP preferring basic amino acids in human erythrocytes was suggested by Mäkinen & Mäkinen [1] , but the enzyme was not further studied. Broad specificity,

Co2*-activated APs

having similar Mr, pH optimum and patterns of sensitivity toward inhibitors and specificity toward aminoacyl-2NAs (estimated on the basis of V|nax/Km ratios) were isolated from the cytosol of bovine leukocytes [ 4 ] , porcine liver [13] and skeletal muscle [14]. However, there are no data on oligopeptides hydrolysis by these enzymes. By degradation of Met-enkephalin and 2-naphthylamides, and by its pronounced susceptibility to puromycin, human erythrocyte AP bears resemblance to puromycin-sensitive AP purified from bovine, rat and human brain [15,16,17]. Even all cited broad specificity APs preferring


amino acids

(described under different




necessarily identical, it seems that they all belong to the same type of AP. Broad organ distribution [18], and preservation during vertebrate evolution as shown for the brain enzyme [19], point to its biological importance. At the same time cytosolic nature and other properties imply that the main role of these APs would be intracellular protein catabolism. This, however, does not exclude the processing of biologically active peptides, particularly those having Arg or Lys at N-terminus. Since isolated human erythrocytes AP differs from AP types recognized by McDonald and Barrett [ 7 ] , we propose for this enzyme to be classified as soluble, L-Lys(L-Arg) preferring, broad specificity aminopeptidase.

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Vol. 373 (1992)

Acknowledgements: We are grateful to Mrs Ljerka DolovCak for the excellent technical assistance. This work was financially supported by the Ministry of Science of the Republic Croatia.

References 1. Mäkinen, K . K . & Mäkinen, P.-L. (1978) Biochem.J. 175, 1051-1067. 2. Vitale, L j . , Zubanovic, M. & Abramic, M. (1981) Acta 40 1489-1495. 3. Söderling, E. (1983) Arch.Biochem.Biophys. 2 2 0 , 1-10. 4. Aratani, H . , Kawata, S., Tsuruyama, S., Yoshida, N. & Makisumi, S. (1984) J.Biochem. 96, 107-115. 5. Abramic, M. & Vitale, Lj. (1989) FEES Lett. 253, 79-82. 6. Grdisa, M. & Vitale L j . (1991) Int.J.Biochem. 23, 339-345. 7. McDonald, J.K. & Barrett, A.J. (1986) Mammalian Proteases; A Glossary and Bibliography. Vol.2: Exopeptidases, Academic Press, London. 8. Cornish-Bowden, A. (1979) Fundamentals of Enzyme Kinetics, Butterworths London 9. Bradford, M . M . (1976) Anal.Biochem. 72, 248-254. 10. Chu, T . G . & Orlowski, M. (1985) Endocrinology 116 1418-1425. 11. Witwicki, J. & Chidambaram, M . V . (1983) Enzyme 29 131-132. 12. Kawata, S., Takayama, S . , Ninomiya, K. & Makisumi, S (1980) J.Biochem. 88, 1601-1605. 13. Kawata, S., Imamura, T . , Ninomiya, K. & Makisumi, S. (1982) J.Biochem 92, 1093-1101. 14. Ishiura, S . , Yamamoto, T . , Yamamoto, M . , Nojima, M . , Aoyagi, T. & Sugita, H. (1987) J.Biochem. 102, 1023-1031. 15. Hersh, L.B. & McKelvy, J.F. (1981) J.Neurochem. 3 6 , 171-178. 16. Johnson, G . D . & Hersh, J.B. (1990) Arch.Biochem.Biophys. 276, 305-309 17. Duarte, G . I . B . P . , Souza, F . G . M . , Bruno. J . A . , Camargo, A . C . M . & Carvalho, K . M . (1988) Neuropeptides 12, 67-73. 18. McLellan, S., Dyer, S . H . , Rodriguez, G. & Hersh, L.B. (1988) J.Neurochem. 51, 1552-1559. 19. Coelho de Souza, A . N . , Bruno, J.A. & Carvalho, K . M . (1991) Comp.Biochem Physiol. 99c, 363-367.

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Basic amino acids preferring broad specificity aminopeptidase from human erythrocytes.

An aminopeptidase hydrolyzing 2-naphthylamides of Lys, Arg, Leu, Met, Phe and Tyr, as well as different di- to tridecapeptides, was purified from the ...
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