Human liver l -leucine aminopeptidase: Evidence for two forms compared to pig liver enzyme

BIOCHIMIE, 1983, 65, 397-404.

Human liver L-leucine aminopeptidase : Evidence for two forms compared to pig liver enzyme. Nelly L E D E M E , Odile VINCENT-FIQUET,

Ghislaine HENNON and Roger PLAQUET ~.

Laboratoire de Biochimie, Groupe de Recherche Enzymologie, Facultd de M~decine, 80036 Amiens Cedex (France).

Rdsum~.

Summary.

Nous avons mis en ~vidence deux formes de L-leucine aminopeptidase (E.C. 3.4.11.1) dans le foie humain. Ces deux formes de L-leucine aminopeptidase sp~cifiques du substrat z-leucine amide hydrolysent les z-leucylpeptides et sont sans action sur les substrats chromogknes : L-leucyl paranitroanilide et L-leucyl ~ naphtylamide. Nous avons pu distinguer les formes enzymatiques du foie humain et celle de l'enzyme du foie de pore, pr~c~demment d~crite, par chromatographie sur D E A E Sephacel et ~lectrophor~se sur feuilles d'ac~tate de cellulose.

Two forms of r-leucine aminopeptidase (E.C. 3.4.11.1) having a specific activity toward z-leucine amide and z,-leucylpeptides substrates but not toward chromogenic substrates : L-leucyl paranitroanilide or L-leucyl ~ naphthylamide have been evidenced from human liver. Human liver enzymes have been distinguished from pig liver enzyme by D E A E Sephacel chromatography and analytical electrophoresis on cellulose acetate strips.

Nous avons compar~ les caractkres physico-chimiques des ~leucine aminopeptidases du foie humain et du foie de pore : activation par les ions Mg ~+et M n ~÷et inhibition par les ions Z n ~+et Co ~+, E D T A et acide citrique. Les pH optimum sont de 10. Les enzymes du foie humain sont moins sensibles ~t la d~naturation thermique que renzyme du foie de pore. Mots-el6s : L-leueine aminopeptidase / p rot~ase / foie.

We compared enzymic properties of L-leucine aminopeptidases from human liver with pig liver enzyme : they were activated by M g ~+ and M n ~÷ and inhibited by Z n 2+ and Co ~÷, E D T A and citric acid. The optimum pH's were 10. Both human liver r-leucine aminopeptidases were less sensitive to heat elevation than pig liver enzyme.

Key-words : L-leueine aminopeptidase / protease / liver.

Introduction.

In a preceding article [1], we described, starting from pig liver, the first complete purification procedure of a LAP from hepatic tissue. It is a cytosol enzyme (EC 3.4.11.1) activated by Mg 2+ and Mn 2+ ions, able to split L-leucine amide and L-Ieucyl peptides but without significant activity toward L-leucyl-~-naphthylamide and L-leucyl paranitro

Abbreviation : LAP : L-leucine arninopeptidase (E.C. 3.4.11.1). (> To whom all correspondence should be addressed.

anilide chromogenic substrates. At this time, true human liver LAP had not yet been isolated. During the present experiment, we attempted to use the pig liver procedure as a model to study human liver LAP, but the results were different. Performing chromatography on D E A E Sephacel columns, we observed that two LAP activities could

398

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be separated from human liver, each of them being distinct from the pig liver enzyme. This had been observed neither by us in pig liver nor by authors who isolated and purified LAP from other animal organs, such as Spackman et al. [2], Smith and Hill [3], and Hanson et Hiitter [4] from pig kidney, and Hanson et al. [5] from bovine-eye lens tissue. In the present study we evidenced the two forms of human liver LAP, as compared to pig enzyme,

Materials and Methods. L-leucine amide hydrochloride (pure A R) was obtained from Koch Light, others amides, peptides and others chemicals from Fluka. L-leucyl ~ naphthylamide from Sigma and /-leucyl paranitro anilide from Boehringer.

Enzyme assay. The LAP standard assay was performed by following the hydrolysis of L-leucine amide substrate, according to a method proposed by our laboratory for h u m a n sera [6] : 50 ~1 aliquots were mixed with 1 ml 10 m M L-leucine a m i d e / 1 0 m M sodium borate buffer (pH 10)/5 m M MgCI~ (for h u m a n liver) or 1.5 m M MgCI~ (for pig liver), and incubated for 15 rain at 37°C. Liberated ammonia was measured colorimetrically with phenol hypochlorite by the method of Berthelot [7] adapted to the Technicon I Auto Analyzer. Hydrolysis of the z-leucylql~-naphthylamide substrate was determined by the procedure of Arst et al. [8]. Hydrolysis of the L-leucyl paranitro anilide substrate was determined, according to Nagel et al. [9].

Protein determination. Proteins were determined by the method of Lowry et al. [10] with serum albumine as standard.

Metal activation. Metal ions Mg 2÷, M n 2+, Co 2+ and Z n 2+ were tested independently, according to our standard conditions, except that Mg 2+ was replaced by 1 m M bivalent metal.

Inhibitors. After mixing at various concentrations with the L-leucine amide substrate, L-leucine aminopeptidase activity was determined under our standard conditions.

Preparation o] crude homogenate. Liver tissue was obtained as soon as possible after death. It was divided into portions of 60 g each. Tissue could be stored at - - 20 ° C for several months without loss of enzymic activity. 50 g of liver were homogenized in 250 ml isotonic NaC1 in an Ultra Turrax homogenizer. The suspension was centrifuged at 23 000 × g for 15 rain at 4°C.

Acetone precipitation. 1) H u m a n liver treatment (~ H ~>solution) - - The whole supernatant (23 000 × g) was mixed with 0.5 volume of 60 per cent aqueous acetone solution at - - 2 0 ° C . The mixture was maintained for 15 rain at this temperature and centrifuged at 8 0 0 0 × g for 10 min at 4°C. One volume of the last supernatant was mixed with one volume of aqueous acetone solution, maintained for 30 min at - - 2 0 ° C and centrifuged at 8 0 0 0 × g. The pellet was resuspended in a volume of water corresponding to one third of the initial supernatant, homogenized and centrifuged at 8 000 × g for 10 min at 4°C. This supernatant, was called ~ H 7> solution. 2) Pig liver treatment (¢~ S >> solution) - - The 23 000 × g supernatant was directly precipitated by adding 1.5 volume 60 per cent aqueous acetone solution at - - 2 0 ° C , as described [1]. The pellet was resuspended in a volume of water corresponding to one fourth of the initial supernatant, homogenized and centrifuged at 8 000 × g. This supernatant was called ~¢S >> solution.

D E A E Sephacel column chromatography. The whole ~ H 7> or ~ S ~> solution, or a mixture of 25 ml ¢~H ~> + 35 ml ¢¢S >> solutions, were deposited on D E A E Sephacel column (13 × 5 cm). Elution was performed with 25 m M borate buffer (pH 8) and by increasing NaC1 molarity from 0 to 300 m M (fig. 1).

Preparative Cellogel block electrophoresis (Cellogel Chemetron Milano 4 × 17 cm). Electrophoresis was performed in a 80 m M veronal buffer (pH 8) for 5 h with a 20 m A constant current in each block. One block was stained with amidoblack in order to visualize the proteins. Other blocks were cut into several fractions whose elution was performed by extruding the liquid with a special press syringue. Cellulose acetate strip analytical electrophoresis was carried out at 200 V for 75 min in a 40 m M veronal buffer (pH 9.2). Strips were stained with Ponceau red dye.

Heat denaturation o] enzyme. Liver enzyme samples were submitted to preincubation at 5°C intervals from 50-80°C. Aliquots taken out after 1, 2, 3, 5, 10, 15 and 20 rain were analyzed for L-leucine aminopeptidase activity under standard conditions either with or without Mg 2÷.

K,,~ determination. Kinetics of L-leucine aminopeptidase activities were determined under standard conditions, the L-leucine amide concentration varying from 1 to 40 raM.

BIOCHIMIE, 1983, 65, n ° 7.

Results. Cellular location o[ liver L A P . Pig and human liver were fractionated into various cellular components according to Appelm a n s et al. [11]. T h e r e s u l t s d e m o n s t r a t e t h a t in h u m a n as w e l l as i n p i g liver, L A P a c t i v i t y is e s s e n tially located in the 105 000 × g supernatant fraction corresponding to cytosol.

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Fro. 1. - - I o n exchange c h r o m a t o g r a p h y on D E A E Sephacel. Elution was performed at 4°C with a 25 m M sodium borate buffer (pH 8), then by increasing NaC1 molarity (×--×--×) from 0 to 75 m M over a period of 16 h and then maintaining NaC1 molarity at 75 m M for 24 h. Elution was continued during a 24 h period with a NaC1 linear gradient 75-150 raM, a concentration plateau for 8 h and finally applied to a 150-300 m M NaC1 linear gradient for 16 h. Three fractions were collected per hour. In each fraction (4.6 ml) the level of protein ( A - - A - - A ) ant the splitting activities toward L-leucine amide ( O - - O - - O ) , L-leucyl paranitro anilide (. . . . . ), and z-leucyl [5 naphthylamide ( O - - © - - © ) substrates were determined. FIG. 1 A : I o n e x c h a n g e c h r o m a t o g r a p h y on D E A E ~ H ~> solution. Fro. 1 B : l o n exchange (~ S >>sohttion. BIOCHIMIE,

1983, 65, n ° 7.

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DEAE Sephacel chromatography. 1) Human liver precipitated enzyme, <>solution, was deposited on a DEAE Sephacel chromatography column and eluted by NaC1 gradient. The elution profile showed two peaks (Fractions I and II) (fig. 1 A) splitting L-leucine amide but not chromogenic substrates. Fractions I and II were eluted, at 75 mM concentration and with 75150 mM NaC1 linear gradient, respectively. In the chromatography described here, fraction I had a higher activity than fraction II (spec. act. 16.26, 5.40, respectively - - table IA) but in experiments performed with other liver samples, the fraction I/fraction II activities ratios were rather variable and sometimes inferior to 1. 2) Pig liver LAP partially purified by acetone treatment, ¢ S >>solution, was submitted to DEAE Sephacel chromatography using the same NaC1 elution gradient procedure. As was reported in a precedent article [1] the elution pattern showed a single LAP peak. It was eluted by a 150-300 mM NaC1 linear gradient (fig. 1 B) and exhibited a specific activity of 20.58 (table I B). 3) A third experiment using a mixture of <~H >> and <~S >> solutions enabled us to distinctly separate three active peaks able to split r-leucine amide corresponding, to human fractions I and II and to pig liver enzyme respectively, (fig. 1 C) and exhibiting the following specific activities : 5.49, 5.96, 9.92, respectively (table I). BIOCHIMIE,

1983,

65, n ° 7.

Electrophoretic mobility. Human liver fractions I and II and pig liver active fraction collected from D E A E Sephacel chromatography were separately submitted to preparative block electrophoresis. Each fraction, containing LAP activity, thus recovered, was deposited on cellulose acetate strips (fig. 2). Both LAP activities from human liver migrated nearly at the same rate and were hard to separate ; fraction I still contained a protein contaminant. LAP activity from pig liver was more rapid and was found at the front of human liver enzymes.

Enzymic properties. 1) Substrate specificity : both human and pig LAP were highly specific for L-leucine amide with the exception of some aromatic amino acids amides : z.-tyrosine amide and z,-phenylalanine amide which were slightly hydrolyzed. Among peptidic substrates, L-leucyl-glycine was split into L-leucine and glycine ; r-leucine-glycine-glycine into L-leucine and glycyl-glycine ; reduced glutathione remained unmodified. Pig liver enzyme has a similar behaviour. Meucyl ~ naphthylamide and L-leucyl paranitro anilide were not attacked by human liver fractions I and II and weakly by purified pig liver LAP (table II). 2) Metal activation : both human liver LAP activities were enhanced by 1 mM Mg 2÷ or Mn 2+ and inhibited by 1 mM Zn 2+ or Co 2+. This should

Human liver L-leucine aminopeptidase.

401

sence of M g > ions were 20, 23 and 12 for h u m a n liver fractions I and I I and pig liver e n z y m e respectively).

be c o m p a r e d to purified pig liver L A P activity [1]. M o r e o v e r L-leucine amide hydrolysis velocity study, according to Mg 2+ concentration, showed that the highest velocity was obtained for M g > concentrations superior to 3 mM.

4) p H activity studies : under our standard conditions, m a x i m u m velocity was observed at p H 10 for both fractions I and I I f r o m h u m a n liver, as well as for pig liver enzyme (fig. 3).

3) Inhibitors : The metal chelators E D T A and citric acid were inhibitors of h u m a n and pig liver enzymes (relative activities for h u m a n liver fractions I and I I and pig liver enzyme were, respectively : 30, 35.8, 25 in the presence of 100 m M E D T A ; 24.5, 21, 31 in the presence of 100 m M

5) H e a t denaturation of enzyme : H u m a n liver enzymes are less sensitive to heat denaturation than pig liver enzyme : they were significantly denaturated at 6 5 ° C and 70°C. A f t e r a 20 min of

TABLE 1.

Purification of L-leucine aminopeptidases jrom human and pig livers. Enzymatic Activity (I.U./ml)

Protein (mg/ml)

Specific Activity (I.U./mg)

Purification

Yield (per cent)

A . HUMAN LIVER

Homogenate Supernatant 23 000 X g for 15 mill Acetone precipitation DEAE Sephacel chromatography Fraction I Fraction II B. PIG LIVER Homogenate Supernatant 23 000 × g for 15 rain Acetone precipitation DEAE Sephacel chromatography

10.73

30.8

0.35

1

13.88 27.63

23.1 12.5

0.60 2.21

1.71 6.31

71.52

16.26 5.40

46.46 15.43

17.58 13.30

5.04 2.58

0.31 0.478

100

21.28

29.79

0.71

1

100

23.66 98.75

23.72 14.75

1.00 6.69

1.41 9.42

84.13

26.55

1.29

20.58

28.99

36.81

11.42 25.2

24.93 26.5

0.46 0.95

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11.8 24.29

18.55 20.7

0.64 1.17

1.39 1.23

22.62 94.5

12.6 18.9

1.80 5.00

3.91 5.26

77.86

5.49 5.96 9.92

11.93 12.96 10.44

17.47 20.58 32.35

C. MIXTURE OF HUMAN AND PIG LIVER

Homogenate Human Pig Supernatant 23 000 × g for 15 min Human pig Acetone precipitation Human Pig DEAE Sephacel chronaatography Human Fraction I Human Fraction II Pig

1.18 1.64 9.94

citric acid). Diisopropyl fluorophosphate did not inhibit the enzymes nor did monoiodacetic acid. H o w e v e r 1 m M p-chloromercuribenzoate produced strong inhibition (relative activities in the pre-

BIOCHIMIE, 1983, 65, n ° 7.

0.215 0.275 1.002

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preincubation at 70°C, only 22 per cent of the initial activities were recovered. U n d e r the same conditions no activity was recovered for the pig liver enzyme,

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H u m a n and pig liver aminopeptidase specific activities toward L-leucine amide and chromogenic substrates ( I . U . / m g ) . L-leucyl 15 naphthylamide

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B. PIG LIVER Homogenate Supernatant 23 000 × g for 15 min Acetone precipitation D E A E Sephacel chromatography

BIOCHIMIE, 1983, 65, n ° 7.

403

H u m a n liver L-leucine aminopeptidase.

6) Influence of L-leucine amide substrate concentration : The Michaelis constant calculated from Lineweaver-Burk reciprocal plot was established to be : 0.77 × 10 -2 M for human liver fraction I, 1.05 × 10 2 M for human liver fraction II, and 1.54 × 10 -2 M for pig liver, in the presence of Mg 2+ at 37°C. Without Mg 2÷ addition, the Michaelis constant was 1.97 × 10 -2 M for both human fractions and 2.85 × 10 -2 M for pig liver.

fails to promote any hydrolysis of z-leucine amide and z-leucyl-glycine. Sugiura et al. [14] purified from human liver, a peptidase that easily split L-leucyl-leucine dipeptide, but barely hydrolyzed L-leucyl-glycine and z--leucyl-glycine-glycine and was devoided of any activity toward c-leucine amide substrate. Its optimum pH was 8, and its activity was not enhanced by Mg 2+ ions. Thus it is clearly different from the human liver LAP that we describe. The separation into two components in DEAE Sephacel chromatography seems to be

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Discussion. In human as well as in pig liver tissue, our results clearly demonstrate that LAP activity is essentially located in the 105 000 × g supernatant, close to LAP from pig kidney described by Smith and Hill [3] and from bovine eye lens purified by Hanson et al. {5], contrasting with microsomal pig kidney aminopeptidase studied by Pfleiderer and Celliers [12]. Human and pig liver LAP hydrolyse L-leucine amide and L-leucine-glycine dipeptide, usually recognized as the best substrates for enzymes of the EC. 3.4.11.1 group. The LAP from human liver studied here, is apparently unrelated to the amino acid naphthylamidase partially purified by Smith et al. [13], these authors using the substrate r-leucyl ~ naphthylamide which BIOCHIMIE, 1983, 65, n ° 7.

an original characteristic of the human liver enzyme, which has been described neither in pig liver, nor in kidney or in eye lens purified true LAP. Finally human liver LAP activities can be easily separated from pig liver LAP, in DEAE Sephacel chromatography as well as in cellulose acetate electrophoresis, indicating different isoelectric points. Human liver LAP chemical properties described here offer real similarities with human serum alcaline leucine amidase activity which we have previously described [6]. Moreover, accounting for serum z-leucine amidase activity that is absent or very low in normal subjects, but very high after hepatitis (often higher than transaminase activity), it should be postulated that during hepatic cytolysis, high level of alcaline leucine amidase activity in serum is essentially issued from liver LAP.

N. L e d e m e and coil,

404 REFERENCES.

1. Ledem6, N., Hennon, G., Vincent-Fiquet, O. e Plaquet, R. (1981) Biochim. Biophys. Acta, 660, 262-270. 2. Spackman, D. H., Smith, E. L. e Brown, D. M. (1955) J. Biol. Chem., 212, 217-299. 3. Smith, E. L. ,~ Hill, R. L. (1960) Enzymes, 4, 37-62. 4, Hanson, H. e Hiitter, H, J. (1966) Z. Physiol. Chem., 347, 118-126. 5. Hanson, H., Gliisser, D. e Kirschke, H. (1965) Z. Physiol. Chem., 34'0, 107-125. 6. Plaquet, R., Ledem6, N., Vincent-Fiquet, O. e Biserte, G. (1973) Clin. Chim. Acta, 46, 91-103. 7, Berthelot, M. (1859) Coloration du ph6nol ammoniacal par le chlorure de chaux (1858-1859). Soci~t~ Chimique de Paris. R~pertoire de Chimie Pure et Appliqu~e, 1, 284.

BIOCH1MIE, 1983, 65, n ° 7,

8. Arst, H. E., Manning, R. T. e Malhon Delp., M. D. (1959) Am. J. Med. Sci., 120/598, 131/609. 9. Nagel, W., Willig, F. ~ Schmidt, F. H. (1964) Klin. Wschr., 42, 447-449. 10. Lowry, O. H., Rosebrough, N. J., Farr, A. L. Randal, R. J. (1951) J. Biol. Chem., 194, 265-275. 11. Appelmans, F., Wattiaux, R. e De Duve, C. (1955) Biochem. J., 59, 438-445. 12. Pfleiderer, G. ~ Celliers, P. G. (1963) Biochem. Z., 339, 186-189. 13. Smith, E. E., Kaufman, J. T. e Rutenberg, M. (1965) J. Biol. Chem., 240, 1718-1721. 14. Sugiura, M., ito, Y., Hirano, K. e Sawaki, S. (1977) Biochim. Biophys. Acta, 481, 578-585.