Methionine aminopeptidase associated with liver mitochondria and microsomes

0020-71 IX:81/09+?91-07M2.00~0 Copyright 0 1981 Pergamon Press Ltd

In,. J. Bw&wt. Vol. 13. pp. 991 10 997. 1981 Prmted in Great Britain. All rlghls reserved

METHIONINE AMINOPEPTIDASE ASSOCIATED WITH LIVER MITOCHONDRIA AND MICROSOMES J. 0.

JR,’ C. TERMIGNONI,’ D. R. BORGES,’ C. A. M. SAMPAIO,’ J. L. PRADO’ and J. A. GIJIMAR~S~ Departments of ‘Biochemistry and 2Medicine. Institute of Pharmacology, Cx. Postal 20372, Escola Paulista de Medicina, 01000 Sio Paulo, SP-Brasil; and ‘Department of Physiology, Cx. Postal 183, Universidade Federal Fluminense, 24000 Niterbi, RJ-Brasil FREITAS

(Receioed 24 February 1981) Abstract-l.

A new, unstable, particle-bound (particularly mitochondria and microsomes) aminopeptidase, mol. wt 74,000, was partially purified from rat liver. 2. Among five aminoacyl+naphthylamides assayed, MetNA was the best substrate. The aminopeptidase releases NH,-terminal methionine from methionyllysylbradykinin. 3. Titration with p-hydroxymercuribenzoate indicated that this methionine aminopeptidase. contains cn five SH groups per mol.

INTRODUCTION

We have been able in recent years to prepare from human serum (Guimaries et al., 1973), human liver (Borges et al., 1974) and human urine (Brandi et al., 1976) aminopeptidases which split N-terminal residues from methionyllysylbradykinin (MLBK) or from lysylbradykinin (LBK) converting them into the limit nonapeptide bradykinin (BK), Arg-Pro-Pro-Gly-PheSer-Pro-Phe-Arg. A parallelism was found between this kinin-converting activity and the activity of these enzymes to hydrolyze /I?-naphthylamides (arylamidase activity) of neutral and basic amino acids (GuimarHes et al., 1973; Borges et nl., 1974; Brandi et al., 1976). Kazuyuki et al. (1980) recently used this finding to study the kinin-converting activity of dog plasma. However, the problem of identifying the kinin-converting activity with an arylamidase already known is not yet solved. Recently we found two arylamidases in human liver, only one of which had a kinin-converting activity (Freitas Jr et al., 1979); this kinin-converting arylaminopeptidase was tentatively identified by us with the homogeneous human liver arylamidase obtained by Behal et al. (1969). The kinin-converting activity of this enzyme is unknown but it catalyzes the hydrolysis of N-terminal amino-acid residues from peptides, amino-acid amides or certain chromogenic synthetic substrates; it prefers the L-alanine residue (Little et al., 1976). We knew that perfused rat liver is able to convert kinins (Prado et al., 1975) and we were also aware that rat liver was used as starting material to characterize a chloride dependent aminopeptidase B (EC 3.4.11.6) selective for N-terminal arginine and lysine residues (Hopsu et al., 1966a,b). It was claimed that this enzyme formed bradykinin from lysylbradykinin (Hopsu-Havu et al., 1966) but this activity was never assayed quantitatively. Further, in human liver the second arylamidase found, with a naphthylamidase activity similar to that of aminopeptidase B, had no kinin-converting activity (Freitas et al., 1979). We thought that it would be worthwhile trying an identi991

fication of the kinin-converting enzyme from rat liver. We knew that a kinin-converting arylamidase could be released from rat liver by adding 0.05% (v/v) Triton X-100 to the perfusing fluid (Termignoni et al., 1979), but in the present paper we started from liver homogenate. Unexpectedly, we found a particlebound rat liver arylamidase which prefers methionylB-naphthylamide, converts kinins, but which we were unable to identify with previously known arylamidases; this finding will be reported in the present paper. MATERIALS

AND METHODS

The following materials were obtained from commercial ArgNA, the L-aminoacyl+naphthylamides, sources: LysNA, AlaNA, LeuNA and DL-MetNA (Sigma Chemical Co., U.S.A.); the peptides bradykinin, lysylbradykinin and methionyllysylbradykinin (Protein Research Foundation, Japan); the proteins bovine serum albumin, mol. wt 67,000 and bovine y-globulin, mol. wt 160,000 (Sigma Chemical Co., U.S.A.); ovoalbumin, twice crystallized, mol. wt 45,000 (Worthington Biochemical Co., U.S.A.); puromycin (Nutritional Biochemical Co., U.S.A.); Fast Garnet GBC salt, p-hydroxymercuribenzoate, Brij 35, Coomassie Brilliant Blue, Triton X-100 (Sigma Chemical Co., U.S.A.); DEAEcellulose (Cellex D), &mercaptoethanol, Acrylamide, Bisacrylamide, N,N,N’,N’-Tetramethylenediamine and Bio-Gel A,.,, (Bio-Rad, Lab. U.S.A.). Other reagents were obtained from the best commercially available sources. Protein determination

The absorbance at 280nm in a l.Ocm cuvette was used to measure protein concentration of solutions. Results are expressed as A2s0 U. The reaction with Coomassie Brilliant Blue, as described by Spector (1978). was also used having BSA as standard protein. Molecular

weight estimations

The purified enzyme preparation was chromatographed on two interligated Bio-Gel A,,,,,, columns (2 x 1OOcm) precalibrated with ovoalbumin (mol. wt 45,000). bovine serum albumin (mol. wt 67,000), bovine y-globulin (mol. wt 160,000). The molecular weight of arylamidase was estimated by the method of Andrews (1964).

992

.I. 0. FREITAS .IRet al. ogenized in a Potter-,EIvehjem homogenizer with 0.01 M sodium phosphate buffer, pH 6.0 containing O.l% (v/v) Triton X-100; a proportion of 4.0ml of buffer-Triton solution/g tissue was used. Liver tissue used for subcellular fractionation was homogenized in 0.01 M phosphate buffer, pH 7.0 containing 0.32 M sucrose.

_Ditute protein solutions were concentrated by pressure dialysis (nitrogen, 980g/cm*) in Visking tubes 8/32”, against distilled water at 4°C. Aryiamidase

assay

The hydrolysis of t.-aminoacyl-fi-naphthylamides was followed by the method of Hopsu et al. (1966a). The incubation mixture at 37°C contained 210nmol of the substrate, lO-208~1 of the aiiquot to be tested and 0.01 M sodium phosphate buffer, pH 7.9 containing, or not. 0.15 M NaC? to make up 3.0 ml. The hydrolysis was interrupted by adding 1.0 ml of freshly prepared Garnet Reagent (I mg Fast Garnet GBC salt in 0.2 M acetate bt&er, pH 4.2 containing 10% Y/Vof Tween 20). The absorbance was read 30 min later at 525 nm in a Beckman DW spectrophotometer and the liberated naphthylamine determined using a standard curve. The unit was defined as that amount of enzyme which hydrolyzes 1.0 pmol of substrate per minute under the conditions described. When the assay was made in the presence of b-mercaptoethanol the method of Barrett (1972) was used. In this case, the incubation mixture contained 105 nmol of the substrate, 5-100~1 aliquot of enzyme sample and the phosphate-saline buffer to make up 1.5 ml. The reaction was interrupted by adding 1.1 ml of a freshly prepared mixture (l:lf of Fast Garnet solution (0.5 mg/ml) contaming 4”1, (v/v) of Brij 35 and 10 mM p-hydroxymercuribenzoate containing 50 mM EDTA. Disc gel electrophoresis

Analytical polyacrylamide gel electrophoresis was carried out as described bv Gordon (1969) using 7.0% acrvlamide gels of 5 mm x 11 cm. Eiectrophore
Tabte t. distribution

Subcellular

fractionation

Subcellular fractions of rat liver were obtained essentially by the classical de Duve’s procedure (ef Mahler h Cordes, 1966). The homogenate was filtered through a double-layered tissue cloth and centrifuged at 700 r/l0 min at 4°C before starting the ~ract~ouation.-The fractions were kept in 0.01 M sodium vhosnhate buger-O.25 M sucrose. pH 7.0 and their purity assured by efectronic microscopy following inclusion in polylite resin (Weigl & Kisiehus, 1972). Aminopeptidase

assa)

This activity was tested on the peptides methionyllysylbradykinin and lysylbradykinin. The aminopeptidase activity was followed either by measuring released metbionine and lysine or by bioassay of the incubated peptides. For the ammo acid analysis method, ahquots of the enzyme preparation were incubated with t25pM (final con#ntration~ of the peptides in l.Oml of 0.0%M sodium phosphate-O.15 M NaCf. pH 7.0, at 37’C: At indicated times aliquots (ZOO~l) of the incubation mixture were mixed with 300~1 of 0.001 N HCI and boiled for 1Omin. The samples were then diluted with 0.5 ml of 0.1 M sodium citrate buffer, pH 2.2 in IS% (v/v) polyethylene glycol-400. The analysis was performed by the method of Spackman st al. (1958) on an automatic instrument (Alonzo & Hirs. 1968). For the bioassay method, the aminopeptidase activity was followed by the differential sensitivity of the isolated guinea-pig ileum to the peptides (Schrser, 1970). if the substrates were converted to bradykinin (Arg-Pro-Pro~~y-Phe-~r_~~Phe~Arg) the formation of the fatter would be easily estimated (Cuimar~es er uf., 1973). The aliquots removed from the incubation mixture. after convenient dilution on 0.001 N HCI and boiling were bioassayed in the guinea-pig ileum preparation (Webster & Prado, 1970). RESULTS Aryiamiduse activity in exsu~~~j~ated rat tissues A widespread arylamidase activity, as measured on tive aminoa~l-8-naphthyfamidef, was found in nine-

of arylamidase activities* in exsan8uinated rat tissues and plasma Substrates

Homogenatest Liver Spleen Lung Kidney Brain Ileum Pancreas Muscle Heart Plasma

LysNA

ArgNA

LeuNA

MetNA

AlaNA

2.0 1.3 0.7 3.2 2.6 0.9

2.8 1.7 1.4 4.6 2.4

1.6 I.0 0.8 7.0 f.8

3.8 1.6 1.7 15.3 2.5

2.0 1.0 1.2 11.5 2.4

2.0

f.4

2.6

20

0.3

0.7

0.7

0.7 1.6 0.009

0.3 0.6 1.0 0.019

0.7 0.7 1.5 (I.038

0.4 0.7

13

0.006

A:;32

*Expressed as rmol oF naphthylamides hydrolyzed per minute per gram of tissue or per ml of plasma. tone gram of exsanguinated tissue in 4.0 ml of 0.01 M sodium phosphate buffer pH 6.0 containing O.iD/,(v/v) Triton X-100.

Methionine aminopeptidase

993

Table 2. Specific* and relativet arylamidase activities of exsangujnated rat organs homogenates~ and plasma Substrates LysNA

ArgNA

LeuNA

MetNA

AlaNA

Heart

3.6(1.0) 3.0(1.0) 2.6 (1.0) 12.4(1.0) 5.9 (1.0) 2.7ti.O) 1.2(1.0) 3.0(1.0) 5.5 (1.0)

5.1 (1.4) 4.0(1.3) 4.9 (1.9) 18.0 (1.5) 7.1 (1.2) 5.9 (2.2) 2.9 (2.4) 3.2(1.1) 6.4(1.2)

2.9 (0.8) 2.3 (0.8) 2.7 (1.0) 27.2 (2.2) 5.4 (0.9) 4.1(1.5) 1.3 (1.1) 2.8 (0.9) 3.6 (0.7)

6.8(1.9) 3.7 (1.2) 6.0 (2.3) 59.4 (4.8) 1.3 (1.2) 7.7 (2.9) 3.0 (2.5) 3.2 (1.1) 5.3 (1.0)

3.6(1.0) 2.3 (0.8) 4.3 (1.7) 44.6 (3.6) ‘7.2(1.2) 6.0 (2.2) 1.7(1.4) 3.1(1.0) 6.1(1.1)

Plasma

0.08 (1.0)

0.13 (1.6)

0.27 (3.4)

0.53 (6.6)

0.45 (5.6)

Homogenates Liver Spleen Lung Kidney Brain Beurn Muscle

*mU/Ata,; 1 mU = nmol hydrolyzed per minute under conditions described in Materials and Methods. STaking the hydrolysis rate of LysNA as 1.0, the ratios at which the other naphthy~amides were hydrolyzed are given in parentheses. iOne eram of exsanauinated tissue homoaenized in 4.0 ml of 0.01 M sodium phosphate buffer pH 6.0 containing 0.1% . _ (v/v) Triton X-100. rat tissue homogenates (Table 1). The highest specific activities were found in the kidney and the lowest in plasma (Table 2). Taking the specific activities of the homogenates on LysNA as 1.0, their relative specific activities on the other naphthylamides were calculated (Table 2). It was also found that the MetNAase activity was higher in all tissues except heart and spleen. A combination of some factors, including specific activity and organ size makes the liver a convenient enzyme source. It was observed that the hydrolysis of the basic naphthylamides by all homogenates and plasma was activated by chloride ions.

exsanguinated

Su~cellulur distribution We found an uneven distribution of the arylamidase specific activities in homogeneous organelles of rat liver cells. Nuclear and lysosomal fractions had no activity under the assay conditions used. In the other fractions we found distributions (Table 3) which may perhaps indicate that three different arylamidases occur in rat liver: (a) an arylamidase activity on basic na~hthylamide~ 85% of which was found in the solubte fraction; (b) the arylamidase activity on LeuNA and AlaNA which was more evenly distributed between soluble (60%) and particulate (40%) fractions; (c) a MetNAase activity, 69% of which was found in the particulate fractions, particularly (64%) in mitochondria and microsomes. If the distribution in the homogenate and subcellular fractions is represented in relation to the respective specific activity on

LysNA (Table 4), the activity on MetNA in mitechondria and mi~rosomes becomes even more conspicuous. Preparation of purified methionine aminopeptidase from rat liver

Rat livers from 19 animals (200-3OOg) were exsanguinated in situ (Borges et al., 1976) but with perfusing Tyrode solution without atropine, diphenhydramine and glucose. From now on, all steps were carried out at 4°C. The livers (173 g) were minced and homogenized in an Omni mixer with 865 ml of 0.01 M sodium phosphate buffer-&l% (v/v) Triton X-100, pH 6.0: three cycles of 30-set periods of homogenization followed by 1 min refrigeration in an ice bath were used. The homogenate at this step could be kept frozen at -20°C for periods up to 6 months without losing activities. A sample of 855 ml of frozen homogenate was melted and centrifuged at 10,000 g for 60 min in a refrigerated Sorvall centrifuge. The pellet was suspended in the same buffer mixture and centrifuged again. To 11 of combined supernatants soIid am-’ monium sulfate was gently added under stirring to make up a 0.35 salt saturation; the precipitate formed in 30 min was separated by 10 min centrifugation at 22.0009. Ammonium sulfate was added to the supernatant as before, to make up a 0.60 saturation; the precipitate obtained after centrifugation was dissolved in 0.02 M sodium phosphate buffer, pH 7.0 (conductivity at 25”C, 1.6mS). This solution was dialyzed

Table 3. Subcellular djstribut~on of aryiamidase activities* in rat liver Substrates Subcellular fractions Mitochondria Microsomes Ribosomes Soluble fraction

LysNA

ArgNA

272 (4.3) 360 (5.7) 445 (7.1)

262 (2.6) 561 (5.6) 508 (5.0)

[ “5186 (82.9)

8755 (86.8)

*Measured on five aminoacyi-~-naphthylamides

LeuNA

AlaNA

661 (13.6) 1176 (24.3) 296 (6.1)

330 (6.9) 996 (20.8) 392 (8.2)

“2713 (56.0)

3063 (64.1)

MetNA m] 508 (4.9) 3158 (30.6)

and expressed both as nmol hydrolyzed x.minute and, in parentheses, of

as percent of the respective tota1. The rectangies a, b and c show different quantitative subcellular distributions arylamidase activities.

994

J. 0.

FREITAS JR et al.

Table 4. Relative activity* of rat liver arylamidase in the homogenate and subcellular fractions Relative activities Fraction

LysNA

ArgNA

LeuNA

MetNA

AlaNA

Homogenate Mitochondria Microsome Ribosome Soluble

1.0 1.0 1.0 1.0 1.0

1.4 1.0 1.6 1.2 1.7

0.8 2.5 3.4 0.7 0.5

1.9 11.9 10.2 1.2 0.6

1.0 1.3 2.8 0.9 0.6

*Specific activities on LysNA were taken as 1.0; the specific activities of each fraction on the other substrates are relative to that on LysNA. against the same buffer reaching a final volume of 310 ml. It was then diluted to 2 1 with 0.02 M sodium phosphate buffer, pH 7.0 and filtered through a bed (4.5 x 26 cm) of DEAE-cellulose equilibrated with the sample buffer. About 30”/, of the total MetNAase activity was not retained by the gel, being then easily separated from the other activities, which will be studied in a separate paper. The nonadsorbed arylamidase was concentrated by pressure dialysis and gel filtered on Bio-Gel A0.5m. Figure 1 shows that only one peak of arylamidase activity was obtained, corresponding to a molecular weight of 74,000 daltons. In this paper this preparation will be referred to as purified aminopeptidase. Regular disc gel electrophoresis on 7% polyacrylamide showed one main protein band and the presence of two small contaminants. The MetNAase ac-

tivity was found only in the region corresponding the main protein band.

to

Hydrolysis rates of some naphthylamides

The specific activities (mU/A,,,) of the purified aminopeptidase were: 0.44 on LysNA; 0.76 on ArgNA; 1.60 on AlaNA; 6.20 on LeuNA and 10.80 on MetNA. Taking as one the hydrolysis rate of LysNa, the rates at which the other naphthylamides were split by the purified aminopeptidase were: ArgNA 1.7; AlaNA 1.5; LeuNA 14.3 and MetNa 24.5. None of these activities were activated by chloride ions. Optimum pH In phosphate buffer the optimum pH for hydrolysis of MetNA was situated in the range of 6.5-7.5.

.5

z ‘0 .o

E T L .f 6 rt

I.5

Effluent volume, ml Fig. 1. Gel filtration of a rat liver aminopeptidase preparation on Bio-Gel A,,,,. The MetNAase preparation separated by ammonium sulfate between 0.35-0.60 saturation, and not retained by DEAEcellulose column fractionation (see text), was used. An aliquot of 5.0 ml equivalent to a protein content of 237 A2s0 U was applied to the interligated columns (2 x KlOcm x 2 cm) packed with Bio-Gel A,,s, and equilibrated with 0.02 M sodium phosphate buffer, pH 7.0. The proteins were eluted with the same buffer in 4.5 ml fractions at a flow rate of 11.0 ml/hr. The protein content, MetNAase and LeuNAase activities were assayed as mentioned (see Materials and Methods). e-0, proteins, A,,,/ml; A-A, MetNAase activity: W-D, LeuNAase activity.

995

Methionine aminopeptidase Table 5. Effect of some substances and metallic ions on the MetNAase activity of the purified aminopeptidase

Concentration (mM)

Inhibitors 1. l,lO-Phenanthroline 2. o-Hvdroxvmercuribenzoate 3. PuromycB 4. Methionine 5. cu ‘+, Zn’+. Nit* 6. Co” 7. Mn” 8. Cazf . M g‘+

3.0 0.001 1.8 5.0 1.0 10.0 10.0

10.0

Inhibitory effect (%I 61 73 50 45 90 60 37

0

Substances 1-4 were preincubated for 1 hr at 4°C with the enzyme solution. Metallic ions were tested following overnight dialysis of the enzyme solution against 5 mM EDTA and then against 10mM sodium phosphate buffer pH 7.0 until complete removal of EDTA. The metallic ions tried were incubated for 6 hr, at 4°C with the dialyzed enzyme solution.

Kinetic parameters The kinetic constants for hydrolysis of MetNA (substrate range were: K, = 6.1 x lo-‘M 0.105-4.20 mM) and V,,, = 20 nmol/min/A~s~.

It was found that the purified aminopeptidase would continuously lose its activity on MetNA when kept at 20,4 or - 20°C. At - 20°C for instance, it lost 25% of its activity in 7 days. These losses were unaffected in the presence of 2.2 M glycerol or 2.0 mM fi-mercaptoethanol. Inhibitors Table 5 shows the effect of some substances and metallic ions on the MetNAase activity of the enzyme. It can be seen that l,I~Qhenantroline, Quromy~n, methionine and some divalent cations inhibited the enzyme in the mM concentration range. The most efficient inhibitor was p-hydroxymercuribenzoate, which produced a 73% inhibition even at 1 PM concentration. Titration of the purified enzyme with this protein-SH-group reagent is shown in Fig. 2. It was

calculated that total inhibition could be obtained at a I/E molar ratio of 4.6. This result indicates that probably five SH-groups are present in the enzyme molecule, thus explaining its high degree of instability. Ami~pept~~e activity This activity was tested on the peptides MLBK and LBK as substrates, once verified that the purified aminopeptidase was not contaminated with kinases (kinin-inactivating enzymes). Figure 3 shows the removal of NH,-terminal methionine. It was calculated that methionine was released in this experiment at the rate of 25 nmol/min/Azsr, U. No lysine was released up to 15-min incubation. In a similar experiment using lysylbradykinin as the starting substrate, lysine was released at the much slower rate of 2 nmol/min/Azs* U. Using excess enzyme and the bioassay method, a typical kinin-converting activity was also found; there was, indeed, a more than 2-fold increase of the guinea-pig ileum contracting activity within 5 min of incubation (Fig. 3, insert).

DISCUSSION

OL 0

0.25

I

L

0.5

075

, 1.0

A_ , I.25

poHMB, Mx166

Fig. 2. Titration curve of the purified aminopeptidase preparation with ~hydroxymercuri~~oate CpOHMB). The enzyme aliquots (60 pig each) prepared for arylamidase assay (see Materials and Methods), were preincubated for 30 min at 25°C in 3.0 ml incubation mixture with the indicated concentrations of pOHMB. They were then assayed on MetNA.

The finding that rat tissues exhibited different specific arylamidase activities measured on five aminoacyl-~-naph~yl~ides, suggested that they might contain different proportions of more than one arylamidase. We were attracted by the fact that the specific activity on MetNA was the highest in most tissues and the subcellular distribution in liver tissue revealed three different arylamidase distributions. Further, about 700/, of the MetNAase activity in liver tissue was particle-bound, mostly in mitochondria and microsomes. Although the finding of about five SHgroups per enzyme molecule was important, we were unable to overcome the great enzyme instability, It is probably for this reason that the three steps purification procedure adopted in the present report supplied MetNAase preparations of low specific activity; however, they contained repr~ucibly the MetNAase activity in one main protein band when subjected to disc electrophoresis on 7% polyacrylamide gel.

996

J. 0.

FREITAS JR ef ul.

incuba&,

-

min

03510-

-lOolooK)(31oO

mixture, pi

- -

IO

Time, min Fig. 3. Release of methionine from methionyllysylbradykinin by purified aminopeptidase. An aliquot (200~1)of purified aminopeptidase equivalent to a protein content of 0.226A280U was incubated with 125PM (final concentration) of methionyllysylbradykinin (MLBK) in 1.0 ml of 0.01 M sodium phosphate buffer415 M NaCt, pH 7.0 at 37°C. At the indicated incubation times aliquots were removed and prepared for methionine determination in an amino acid analyzer (see Materials and Methods). Insert: kin~n-converting activity of purified amino~ptida~ as measured by bioassay. The enzyme sample (0.370 A280 U) was incubated at 37°C with 20 pg MLBK in 1.0 ml of buffer. At indicated times, 200 ~1 of the mixture were removed, diluted to 2.0ml. boiled and bioassayed on the isolated guinea-pig ileum preparation.

The methionine aminopeptidase from rat liver is chloride independent and has a molecular weight estimated by gel filtration as 74,000. The relative hydrolysis rates of the five aminoacyi-/3-naphthyiamides used show that it prefers clearly MetNA and LeuNA; it is thus different from the two aryiamidases described in human liver (Freitas Jr et al., 1979). These two aryiamidases were (a) the basic, which hydrolyses exciusiveiy ArgNA and LysNA and does not convert kinins; (b) the neutral, which prefers AiaNA, has kinin-converting activity and seems to be similar if not identical with the homogeneous aryiamidase from human liver (EC 3.4.11.14) obtained by Behai et al. (1969). The methionine aminopeptidase differs also from rat liver aminopeptidase B (EC 3.4.11.6), which does not hydrolyze neutral aminoacyl-fl-naphthylamides. In this laboratory Termignoni (1980) separated from rat liver perfusates both a methionine aminopeptidase and an aminopeptidase 3; he observed several differences between these two enzymes and found also that while methionine amino~ptid~e converted MLBK and LBK into BK, amino~ptidase B had no such action. We compared the properties of the rat liver methionine aminopeptidase with those of the known aminoaianine peptidases, including the particle-bound aminopeptidase (EC 3.4.11.2), and concluded that we may be dealing with a new, unstable enzyme. Probably the kinin-converting activity of liver methionine aminopeptidase is a secondary action of this enzyme. It seems worth mentioning however, that Camargo et al. (1972) observed that an aryiamidase may be si~i~~t in the brain metabolism of peptides and Kerwar et at. (1971) found an aminopeptidase associated with brain ribosomes which shows a higher activity towards methionyl-~-naphthyiamide

than to the other aminoacyl+naphthyiamides tried. Recently Suda et al. (1980) isolated from rat liver an enzyme which hydrolyzes N-formyimethionine-pnaphthylamide and which is distinct from a MetNAase activity also present in the liver homogenate. Previously Yoshida & Lin (1972), working with rabbit reticuiocytes, found two different amino~ptida~s which specifically hydrolyze peptides containing N-terminal methionine or ~-formyimethionine. They suggested that their enzymes were related to the removal of methionine from nascent peptides. These authors have also found similar enzymes in the supernatant of 10,OOOgfrom liver and other homogenates. Since this supernatant contains microsomes and ribosomes, the possibility of any similarity between the presently described rat liver methionine aminopeptidase and the methionine aminopeptidase found by Yoshida & Lin (1972) should be investigated. SUMMARY

A wide distribution of arylamidase activity, on five aminoacyl-~-naphthyiamides measured (LysNA, ArgNA, L.euNA, AlaNA and MetNA), was found in several exsanguinated rat tissue homogenates; the highest activity found in most homogenates was on MetNA. In liver tissue, nuclear and iysosomai fractions had no activity; in the other subcellular fractions three distinct arylamidase distributions were found. The MetNAase activity was mainly (69%) a particulate activity, being found particularly in mitochondria and microsomes. This activity was purified starting with a liver homogenate in 0.1 M sodium phosphate buffer pH 6.0 containing O.i% Triton X-100. Following ammonium sulfate fractionation

997

Methionine aminopeptidase

between 0.354.60, the arylamidase activity was separated on a DEAE-cellulose column. About 30”/, of the total MetNAase activity was not absorbed and was further purified on a Bio-gel A,,, m column giving one active peak which showed only one active band on 7”/;, polyacrylamide disc gel electrophoresis. Taking the specific activity of the purified MetNAase on LysNA as 1.0 the rates for the other naphthylamides were: ArgNA 1.7, AlaNA 1.5, LeuNA 14.3 and MetNA 24.5. The enzyme activity does not depend on chloride ions and the most efficient inhibitor found was p-hydroxymercuribenzoate. Titration with this inhibitor showed that about five SH groups are present in each mol which probably explains the enzyme instability even at -20°C. A typical aminopeptidase activity was found using methionyllysylbradykinin as substrate and measuring released methionine in an amino acid analyzer; under the same conditions lysine was released from lysylbradykinin at a 12-fold lower rate. This methionine aminopeptidase seems to be a new enzyme. Aclinowledyemenrs-This work was supported by grants from Financiadora de Estudos e Proietos (FINEP). Rio and Fundacio de Amparo B Pesquisa do Estado de SBo Paulo (FAPESP). SBo Paulo. J. 0. Freitas Jr. C. A. M. Sampaio, J. L. Piado and J. A. Guimaries received fellowships from CNPq and C. Termignoni received a PICD fellowship from CAPES-UFRGS.

REFERENCES

ALONZON. & HIRS C. H. (1968) Automation of sample application in amino acid analyzers. Analyt. Chem. 23, 272-288.

ANDREWSP. (1964) Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem. .I. 91, 222-233. BARRETTA. J. (1972) A new assay for cathepsin Bl and other thiol proteinases. Analyt. Biochem. 47, 28&293. BEHALF. J., LITTLEG. H. & KLEIN R. A. (1969) Arylamidase of human liver. Biochim. hiophys. Acta 178, 118-127. BORGESD. R.. PRADO J. L. & GUIMAR~TES J. A. (1974) Characterization of a kinin-converting arylaminopeptidase from human liver. Naun?n-Schmiedeberg’s Archs Pharmac.

281,

403-414.

BORGESD. R., LIMBOSE. A. & PRADOJ. L. (1976) Catabolism of vasoactive polypeptides by perfused rat liver. Naun.vn-Schmiedeberg’s

Archs

Pharmac.

2M,

33-40.

BRANDIC. M. W.. PRADO E. S.. PRADO M. J. B. A. & PRADO J. L. (1976) Kinin-converting aminopeptidase from human urine partial purification and properties. Inr.

J. Biochem.

7, 335-341.

CAMARGOA. C. M.. RAMALHO-PINTO F. J. & GREENEL. J. (1972) Brain peptidases: conversion and inactivation of kinin hormones. J. Neurochem. 19, 37-49. FREITASJ. 0. JR. GUIMAR~ESJ. A., BORGESD. R. & PRADO J. L. (1979) Two arylamidases from human liver and

their kinin-converting

activity.

Int.

J.

Biochem.

IO,

81-89.

GORDONA. H. (1969) wry/amide

and Starch

Electrophoresis of Proteins in PolyGels. North-Holland, Amsterdam.

GUIMAR~~ES J. A., BORGESD. R., PRADOE. S. & PRADOJ. L. (1973) Kinin-converting aminopeptidase from human serum. Biochem. Pharmac. 22, 3157-3172. HOPSUV. K., M~~KINEN K. K. & GLENNERG. G. (1966a) Purification of a mammalian peptidase selective for N-terminal arginine and lysine residues: aminopeptidase B. Archs Biochem. Biophys. 114, 557-566. HOPSUV. K., M~~KINEN K. K. 8~ GLENNERG. G. (1966b) Characterization of aminopeptidase B: substrate specificity and affector studies. Archs Biochem. Biophys. 114, 567-575.

HOPSU-HAVUV. K., M~~KINENK. K. & GLENNERG. G. (1966) Formation of bradykinin from kallidin-10 by aminopeptidase B. Nature 212, 1271-1272. KAZUYUKIK., MORIWAKIC. & MORIYAH. (1980) Kininconverting activity in the dsg pseudoglobulin fraction from heated plasma and kinin liberation from dog kininogen by guinea-pig coagulating gland kallikrein (CGK). Chem.

pharm.

Bull.,

Tokyo

28, 42-48.

KERWARS. S., WEISSBACH, H. & GLENNERG. G. (1971) An amino-peptidase activity associated with brain ribosomes. Archs Biochem. 143, 336337. LITTLEG. H., STARNESW. L. & BEHALF. J. (1976) Human liver aminopeptidase. In Methpds in Enzymology, Vol. 45, part B, pp. 495-503. Academic Press, New York. MAHLERH. A. 8~ CORDESE. H. (1966) Biological Chemistry. pp. 393-394. Harper & Row, New York. PRAW J. L., LIM;~OSE. A., ROBLEROJ., FREITASJ. 0. JR, PRADO E. S. & PAIVA A. C. M. (1975) Recovery and conversion of kinins in exsanguinated rat preparations. Naunyn-Schmiedeberg’s ArchsPhormac. 290, 141-205. SCHROEDER E. (1970) Structure-activity relationship of kinins. In Handbook of Experimental ~harmacology,~ Vol. XXV. pp. 324-350. Springer, Berlin. SPACKMAND. H., STEINW. R. & MOORES. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Ana/yr. Chem. 30, 1l9%1206. SPECTORT. (1978) Refinement of the Coomassie blue method of a protein quantitation. A simple and linear spectrophotometric assay for 10.5 to 5Opg of protein. Analyr.

Biochem.

86,

144146.

SUDAH., YAMAMOTO K.. AOYAGIT. & UMEZAWAH. (1980) Purification and properties of N-formylmethionine aminopeptidase from rat liver. Biochim. biophys. Acra 616,

6&67.

TERMIGNONI C., FREI~ASJ. 0. JR, GUIMAR~~ES J. A., BORGES D. R. & PRADOJ. L. (1979) Methionyl-aminopeptidase from rat liver. An. Acad. Ci&c., Brasil 51, 4. TERMIGNONIC. (1980) Perfuslo do figado de rato corn Triton X100: remocio e caracterizacio de uma aminopeptidase cinino-conversora e de uma arilamidase. Thesis, Escola Paulista de Medicina, Sio Paulo, Brasil. YOSHIDAA. & LIN M. (1972) NH,-terminal formylmethionine- and NH,-terminal methionine-cleaving enzymes in rabbits. J. biol. Chem. 247, 952-957. WEBSTERM. E. & PRADOE. S. (1970) Glandular kallikrein from horse and from hog pancreas. In Methods in Enzymology, Vol. 19. pp. 681-699. Academic Press, New York. WEIGLD. R. & KISIELIUSJ. J. (1972) A resina polylite na microscopia eletrhnica. Ci&. e Cult., S Paula, Suppl. 24, 212.