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Purification and properties of aminopeptidase C from porcine skeletal muscle
Comp. Biochem. Physiol. Vol. 102B, No. 1, pp. 129-135, 1992
0305-0491/92 $5.00 + 0.00 Pergamon Press Ltd
Printed in Great Britain
PURIFICATION A N D PROPERTIES OF AMINOPEPTIDASE C FROM PORCINE SKELETAL MUSCLE TOSHIHIDENISHIMURA,*t YUTAKAKATO,~ MEE RA RHYU,* AKIHIROOKITANI§ and HIROMICHIKATO* *Department of Agricultural Chemistry, The University of Tokyo, Tokyo 113, Japan; :~ResearchInstitute, Soda Aromatic Co., Ltd, Noda-shi, Chiba; and §Department of Food Science, Nippon Veterinary and Animal Science University, Musashino-shi, Japan (Received 7 August 1991)
Abstract--1. Aminopeptidase C was purified from porcine skeletal muscle. 2. The mol. wt of the enzyme was found to be 103,000 on both Sephadex G-200 column chromatography and SDS-PAGE. 3. The optimum pH for the hydrolysis of L-leucine p-nitroanilide was around 7.0. 4. The activity of this enzyme was strongly inhibited by EDTA, bestatin and puromycin. 5. The enzyme acted on the fl-naphthylamide derivatives of amino acids and oligopeptides.
INTRODUCTION
Aminopeptidases and endopeptidases are known to participate in the proteolytic system in the living cell. Aminopeptidases degrade the peptides produced from proteins through the action of endopeptidases to free amino acids. It was reported that the activities of intramuscular aminopeptidases were more markedly increased in dystrophic than normal mice (Aoyagi et al., 1981). A bestatin-sensitive aminopeptidase has been shown to be involved in the degradation of di- and tripeptides in mammalian cells (Noguchi et al., 1983; Takahashi et al., 1987; Botbol and Scornik, 1989). Recently, the neutral aminopeptidase activity located on the surface of T-cells has been shown to be associated with the mouse thymocyte-activating molecule (THAM) and to regulate THAM-mediated T-cell activation (Gorvel et al., 1990). Furthermore, some aminopeptidases have been reported to act on hormonally active peptides and to thereby inactivate them. The puromycin-sensitive aminopeptidase in rat brain (Dyer et al., 1990) and human placental aminopeptidase M (McLellan et aL, 1988) degrade enkephalin, and an aminopeptidase in human cerebrospinal fluid degrades the delta-sleep-inducing peptide (Nyberg et al., 1990). Although information on aminopeptidases in vitro or in vivo is gradually increasing, as described above, the actual functions of aminopeptidases and the mechanism of free amino acid production in vivo have not yet been completely elucidated. To answer these questions completely, it seems to be important to list and characterize major aminopeptidases. Neutral aminopeptidases of skeletal muscle that have been reported are leucine aminopeptidase (EC 3.4.11.1) (Joseph and Sanders, 1966), aminopeptidase B (EC 3.4.11.6) (Mantle et al., 1984, 1985: tAuthor to whom correspondence should be addressed. Abbreviations--LeuNap, L-leucine fl-naphthylamide; Nap,
/~-naphthylamide; PMSF, phenylmethylsulfonyl fluoride; MCE, 2-mercaptoethanol.
Ishiura et al., 1987), an aminopeptidase M-like enzyme (Ishiura et al., 1987), aminopeptidase C (Otsuka et al., 1976, 1980), the major aminopeptidase from human skeletal muscle (Mantle et al., 1983), aminopeptidase H (Okitani et al., 1980, 1981; Nishimura et al., 1983, 1988), pyroglutamyl aminopeptidase (EC 3.4.11.8) (Lauffart and Mantle, 1988), DAP III (Parsons and Pennington, 1976) and DAP IV (EC 3.4.21.26) (Kar and Pearson, 1978) and several dipeptidases (Smith, 1948 a-c). An aminopeptidase M-like enzyme (Ishiura et al., 1987) and the major aminopeptidase (Mantle et al., 1983) from human skeletal muscle and aminopeptidase C (Otsuka et al., 1976, 1980) from rabbit skeletal muscle have been shown to exhibit the highest activities against aminoacyl derivatives. The properties of aminopeptidase C are similar to those of the former two. Recently, an aminopeptidase C was found in chicken and porcine (Nishimura et al., 1990) skeletal muscles. In the present study, we purified aminopeptidase C from porcine skeletal muscle and clarified some of its properties. Furthermore, we proposed that the porcine aminopeptidase C and the major enzymes in skeletal muscle described above should be classified as one group and tentatively called collectively aminopeptidase C. MATERIALS AND METHODS
Materials
Porcine skeletal muscle (m. longissimus dorsi) was removed from the carcass immediately after slaughter, trimmed to remove fat and connective tissues, and then minced with a meat chopper. DEAE-cellulose (DE-52 preswollen) was purchased from Whatman (UK). Ultrogel AcA 34 was obtained from LKB (Sweden). Sephadex G-200 and hydroxylapatite were from Pharmacia Fine Chemicals (Sweden) and Bio-Rad, respectively. Ovalbumin, bovine serum albumin, aldose, catalase, ferritin and puromycin were purchased from Boehringer Mannheim GmbH (Germany). Pepstatin A and leupeptin were obtained from the Peptide Institute (Osaka 562,
129
TOSHImDE NISHIMURAet al.
130
Japan). SDS--6H (high mol. wt protein standard mixture for SDS-PAGE), PMSF and LeuNap were from Sigma Chemical Co. (USA). All other chemicals used were of reagent grade.
Enzyme assay The enzyme activities against the fl-naphthylamide derivatives of amino acids (amino acid Naps) were measured in the following way. After the enzyme had been incubated with 0.5 mM of each substrate in 50 mM Tris-HCl buffer (pH 7.2) at 37°C for 1-60 min, 0.4 ml of 0.23 N HCI in ethanol and 0.4 ml of 0.06% p-dimethylaminocinnamaldehyde in ethanol were added to stop the enzyme reaction. The red color that developed was measured at 540 nm and the flnaphthylamine released from the substrate was determined. The enzyme activity against Leu-p-nitroanilide was measured by the following method. After the enzyme had been incubated with 0.5 mM Leu-p-nitroanilide in 50 mM Mcllvain or ammonium buffer at various pHs at 37°C for 5 min, 0.8 ml of 0.23 N HCI in ethanol was added to stop the enzyme reaction. The p-nitroaniline released from the substrate was determined. The enzyme action towards di-, tri- and tetrapeptides was determined by measurement of the increase in ninhydrin positive materials during incubation. After the enzyme had been incubated with 0.5 mM of each peptide in 50 mM Tris-HCl buffer (pH 7.2) at 37°C for I hr, the incubation was stopped by heating at 100°C for 5 min.
Protein determination The absorbance at 280 nm was used to monitor the protein peaks on the column chromatographies. The concentrations of proteins were determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard.
Gel electrophoresis SDS-PAGE was carried out by the method of Weber and Osborn (1969) using a 5% gel and Bromophenol Blue as the tracking dye. The proteins were stained with Coomassie Brilliant Blue R-250.
Molecular weight determination Gel filtration was performed on a Sephadex G-200 column (2.4 x 105 cm) equilibrated with 10 mM Tris-HC1
(pH 7.2) containing 0.1 M NaC1, 0.1% MCE and I mM NaN 3. The void volume was determined with Blue Dextran. Ferritin (M, = 450,000), catalase (240,000), aldolase (158,000), bovine serum albumin (68,000) and ovalbumin (45,000) were used as standard proteins. The mol. wt of the subunit of aminopeptidase C was determined by SDS-PAGE. Myosin (205,000), /~-galactosidase (116,000), phosphorylase b (97,400), bovine serum albumin (67,000), ovalbumin (45,000) and carbonic anhydrase (29,000) were used as standard proteins.
Amino acid analysis Free amino acids were analyzed and determined with an amino acid analyzer (Hitachi Model 835) after protein had been removed from the solution by ultrafiltration. RESULTS
Purification o f aminopeptidase C from porcine muscle A m i n o p e p t i d a s e C was purified from porcine muscle by four steps. All steps were carried o u t at 4°C.
Step 1. Extraction and ammonium sulfate fractionation. Minced muscle (508 g) was h o m o g e n i z e d with 3 vols of 40 m M T r i s - H C l buffer (pH 7.2) containing 0.1% M C E in a W a r i n g blender for 1 min. The h o m o g e n a t e was then centrifuged at 7 5 0 0 g for 15 m i n a n d the resultant s u p e r n a t a n t was filtered t h r o u g h four layers of gauze to remove floating fat. The filtrate (the crude extract) was subjected to a m m o n i u m sulfate fractionation. T h e precipitate o b t a i n e d between 45 a n d 6 5 % a m m o n i u m sulfate s a t u r a t i o n was collected a n d then dialyzed against 1 0 m M T r i s - H C l buffer (pH 7.2) containing 0.1 M NaC1, 0.1% M C E a n d 1 m M N a N 3 (Buffer A). The dialyzate was centrifuged at 7 4 0 0 g for 2 0 m i n to remove insoluble materials a n d the s u p e r n a t a n t was pooled.
Step 2. DEAE-cellulose column chromatography. The s u p e r n a t a n t (240 ml, 14.2 g) was p u t on a c o l u m n (3.0 x 27.0 cm) o f DEAE--cellulose equilibrated with Buffer A. After the u n b o u n d proteins h a d been
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Aminopeptidase C from porcine muscle
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Fraction n u m b e r Fig. 2. Rechromatography of porcine aminopeptidase C on a hydroxylapatite column. The active fractions (8.3 m g protein) obtained from the Ultrogel AcA 34 column were dialyzed against 10 m M K-phosphate buffer (pH 7.2) (Buffer B) and then applied onto a column (3.0 x 14.5 cm) of hydroxylapatite equilibrated with Buffer B. After the u n b o u n d protein had been washed out with the same buffer, the enzyme was eluted with 80 m M K-phosphate buffer (pH 7.2) and then with a linear gradient of K-phosphate (80-200 mM). Fractions o f 3 ml were collected ( A ) Protein; ( O ) L e u N a p hydrolyzing activity. The purified enzyme ( ~ ) was applied to a 5.0% polyacrylamide gel containing SDS.
completely removed from the column by washing with Buffer A, elution was started with a linear concentration gradient of NaCI in Buffer A. The activities against LeuNap were eluted at around 0.22 M NaCI. The active fractions, indicated by a bar in Fig. 1, were pooled. This step was very effective for the purification, since the bulk of the proteins did not bind to the DEAE-cellulose column. Step 3. Ultrogel AcA 34 column chromatography. The enzyme solution (15.5mg) from step 2 was concentrated by ultrafiltration using a Ultrafilter UK-10 membrane and then applied onto a column (2.5 x 103 cm) of Ultrogel AcA 34 equilibrated with Buffer A. The enzyme was eluted with the same buffer.
Step 4. Hydroxylapatite column chromatography. The enzyme solution (4.2mg) from step 3 was dialyzed against 10 mM potassium phosphate buffer (pH 7.2) containing 0.1% MCE. The dialyzate was put on a column (3.0 x 14.5 cm) equilibrated with the same buffer. After the unbound protein had been washed out with same buffer, the bound proteins were eluted with 80 mM K-phosphate buffer (pH 7.2)
and then a linear concentration gradient of Kphosphate buffer (pH 7.2), from 80 to 200 mM. The LeuNap hydrolyzing activity was eluted as two peaks, as shown in Fig. 2. The second peak was much larger than the first one. The second peak (indicated by a bar in Fig. 2) was collected and used as the purified aminopeptidase C to examine some of its properties, as described below. The results of the purification process are summarized in Table 1. Aminopeptidase C was purified 134-fold over the crude extract with a yield of 0.8%. Aminopeptidase C thus obtained gave a single band on S D S - P A G E (see Fig. 2).
Some properties of the purified aminopeptidase C Molecular weight. As shown in Figs 3 and 4, the mol. wt of aminopeptidase C was estimated to be 103,000 on both Sephadex G-200 column chromatography and SDS-PAGE. These results indicate that this enzyme is a single polypeptide.
Effect of pH on the enzyme's activity and stability. The enzyme activity against Leu-p-nitroanilide (Leu-pNA) was measured in 50mM Mcllvain or
Table 1. Purification steps for porcine aminopeptidase C Total protein Spec. act. Purity Recovery of Purification step (rag) (#mol min - l m g -1) (fold) activity (%) Crude extract 23,088 0.012 1.0 100 45-65%(NH4)2SO4Ppt 14,172 0.018 1.6 97.2 DEAE-cellulose 15.5 2.632 226.9 15.2 Ultrogei AcA 34 8.3 1.009 87.0 3.1 Hydroxylapatite 1.4 1.552 133.4 0.8 Aminopeptidase C was purified from 508 g of porcine muscle. The activity was measured using LeuNap as a substrate. The details of the assay are given in Materials and Methods.
TOSHIHIDE NISH]MURAel al.
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Fig. 3. Determination of the mol. wt of porcine aminopeptidase C on a Sephadex G-200 column. The porcine aminopeptidase C, and 5 mg each of ovalbumin, bovine serum albumin (BSA), aldolase, catalase and ferritin were put on a Sephadex G-200 column and eluted with Buffer A. The void volume, with Blue Dextran, is expressed as V0, and the volumes at which the aminopeptidase C and standard proteins were eluted as Ve. The details are given in Materials and Methods. a m m o n i u m buffer at various pHs. The o p t i m u m p H for the hydrolysis of L e u - p N A was a r o u n d 7.0 (Fig. 5). After the enzyme h a d been kept at various pHs in 5 0 m M M c l l v a i n or a m m o n i u m buffer at 37°C for 1 hr, the activity against L e u N a p was m e a s u r e d at p H 7.2. As shown in Fig. 6, the enzyme was stable in the p H range 6.0-8.0. Effect of heat on the enzyme's stability. After the enzyme h a d been kept at various temperatures in 5 0 m M Tris-HC1 buffer ( p H 7 . 2 ) for 10min, the activity against L e u N a p was measured. The enzyme
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10 6' 7t 8 ~ pH Fig. 5. Effect of pH on the activity of porcine aminopeptidase C. The activity was measured using 0.5 mM LeuNap as a substrate in K-phosphate (O) or ammonium (©) buffer. The details of the assay against Leu-p-nitroanilide are given in Materials and Methods. activity was stable up to a r o u n d 45°C, but m o s t activity was lost at 60°C (Fig. 7). Effects of inhibitors. As s h o w n in Table 2, the enzyme activity was inhibited strongly by E D T A , bestatin a n d puromycin, but was not affected so m u c h by m o n o i o d o a c e t i c acid, pepstatin or leupeptin. P M S F did n o t cause inhibition of the enzyme activity at all. Effects of metal ions. The enzyme activity was not affected so m u c h by Ca 2+, M g 2÷, Co 2+, M n 2÷ or Z n 2÷ at 0.005 or 0 . 0 5 m M . Co 2÷ a n d Z n 2÷ at 0 . 5 m M inhibited the enzyme activity strongly (Table 3).
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Fig. 4. Mol. wt determination of the subunit of porcine aminopeptidase C by SDS-PAGE. Aminopeptidase C, carbonic anhydrase, ovalbumin, bovine serum albumin (BSA), phosphorylase B, fl-galactosidase and myosin were applied to a 5.0% polyacrylamide gel containing SDS. The mobilities of the tracking dye and proteins are expressed as Ro and Re, respectively.
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Fig. 6. Effect of pH on the stability of porcine aminopeptidase C. After the enzyme had been kept at various pHs in 50 mM Mcllvain (O) or ammonium (O) buffer at 37°C for 1 hr, the activity against LeuNap was measured at pH 7.2. The details of the assay are given in Materials and Methods.
Aminopeptidase C from porcine muscle
133
Table 4. Hydrolytic activities of porcine aminopcptidase C towards fl-naphthylamide derivatives of amino acids (amino acid Naps)
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Fig. 7. Effect of heat on the stability of porcine a m i n o p e p tidase C. After the e n z y m e h a d been kept at v a r i o u s t e m p e r a t u r e in 50 m M T r i s - H C 1 buffer (pH 7.2) for 10 min, the activity a g a i n s t L e u N a p was measured. The details o f the assay are given in M a t e r i a l s a n d M e t h o d s .
Km and Vmax values. The Km and Vmaxvalues for the hydrolysis of LeuNap were 1.0 mM and 12.9 #mol rain- ~mg protein- 1, respectively. Substrate specificity. As shown in Table 4, this enzyme showed a broad substrate specificity for the fl-naphthylamide derivatives of amino acids. In particular, the enzyme exhibited high activities against Ala-, Lys-, Met- and LeuNap. The enzyme exhibited no activity against GluNap. The action of aminopeptidase C on 14 sorts of dipeptide which
Table 5. Hydrolytic activities of porcine aminopeptidase C towards peptides Peptide Dipeptides Lys-Gly Met-Gly Glu-Gly Leu-Gly
Phe-Gly Gly---Gly Val~;ly Ile431y Ala-Gly His431y Pro-Gly Ser431y Thr-Gly Trp-Gly
Relative activity (%) 234 100 82 67 43 35 11 4 0 0 0 0 0 0
Peptide
Relative activity
Tripeptides Leu-Gly-Gly Val-Tyr-Val
(%) 247 49
Tetrapeptides Phe-Gly-lle-Gly Lys~31y-Ile--Ala Glu-Gly-Ile-Ala Thr~31y-lle-Ala Glyq31y-lle--Ala lle431y-lle-Ala Ala-Gly-Phe-Ala Met-Gly-Phe-Ala Val~31y-Phe-Ala
908 243 177 45 26 19 339 196 32
After the enzyme had been incubated with 0.5 mM of each peptide in 50 mM Tris-HCI buffer (pH 7.2) at 37°C for l hr, the increase in ninhydrin positive materials was measured. The details of the assay are given in Materials and Methods.
Table 2. Effect of protease inhibitors on the activity of porcine aminopeptidase C Relative activity (%)
Inhibitor None PMSF (10 3M) Iodoacetic acid (10 -2 M) EDTA (10 -t M) Pepstatin (10 4M) Leupeptin (10 -4 M) Puromycin (10 4M) Bestatin (10 -4 M)
100 100 90.0 2.9 91.2 90.0 32.9 27.9
The activity was measured using LeuNap as a substrate in the absence or presence of each inhibitor. The details of the assay are given in Materials and Methods. The concentrations of inhibitors are indicated in parentheses. Table 3. Effect of metal ions on the activity of porcine aminopeptidase C Relative activity (%) Metal None Ca 2 + Mg 2 + Co s + Mn 2+ Zn 2+
0.005 mM
0,05 mM
0.5 mM
92 98 101 93 93
100 100 92 77 93 78
85 96 6 86 19
The enzyme activity was measured using LeuNap as a substrate in the absence or presence of each metal ion at three concentrations. The details of the assay are given in Materials and Methods.
have Gly at their C-terminal is shown in Table 5. The enzyme had high activities against Lys-, Met-, Gluand Leu-Gly, but no activity against Ala-, His-, Pro-, Ser-, Thr- and Trp-Gly. The hydrolytic activities against tri- and tetrapeptides were higher than those against dipeptides. The enzyme had particularly high activities against tri- and tetrapeptides which have Phe, Ala, Leu and Lys at their N-terminal. However, it could hardly act on tri- and tetrapeptides which have Ile, Gly, Val and Thr at their N-terminal. It was recognized that this enzyme released Leu or Met at first through its action towards Leu-Gly-Gly or Met-Gly-Ile-Ala, respectively. DISCUSSION
In this study, aminopeptidase C has been purified from porcine skeletal muscle and some of its properties have been clarified. Aminopeptidase C, of the neutral aminopeptidases in porcine skeletal muscle, showed the highest arylamidase activity. The major aminopeptidases in skeletal muscle of other species have been purified and characterized. The aminopeptidase C (Otsuka et al., 1976, 1980) purified from rabbit skeletal muscle, and the major aminopeptidase (Mantle et al., 1983) and an aminopeptidase M-like enzyme (Ishiura et aL, 1987) from human skeletal muscle are similar to the aminopeptidase C from
134
TOSHIHIDENISHIMURAet al.
porcine skeletal muscle in this study. Some properties of these enzymes are a little different. The aminopeptidases from human and porcine muscles are single polypeptides and their mol. wts (Mr) are about 100,000. On the other hand, the Mr of the rabbit aminopeptidase C has been shown to be 160,000 on gel filtration. The effect of a metal ion on the activities of these enzymes is different. The porcine aminopeptidase C was not affected by 0.5 mM Ca 2+. However, the aminopeptidase from human muscle was activated five times by 0.5mM Ca 2+ and that from rabbit muscle was strongly inhibited by 1 mM Ca 2+. Although some properties of these aminopeptidases show species differences between them as described above, others are almost the same. For example, these enzymes have been shown to possess the highest arylamidase activities among the neutral aminopeptidases in skeletal muscle. Their optimal pH has been shown to be around 7.0. These enzymes are stable in the neutral pH region and are inhibited by metal chelating reagents such as EDTA and o-phenanthroline. Accordingly, these enzymes should be classified as the same enzyme, so we tentatively recommend that these aminopeptidases in skeletal muscles should be collectively called aminopeptidase C. An aminopeptidase C may be similar to an aminopeptidase M, which is a membrane-bound aminopeptidase and localized in microsomes in kidney cells, as human aminopeptidase C has been called an aminopeptidase M-like enzyme. However, some properties, including the mol. wt, substrate specificity, and sensitivity to Co 2÷ and Tris, differ between the two enzymes. Also, an aminopeptidase C has been shown to be extracted by a buffer without a detergent, indicating that this enzyme is not a membrane-bound aminopeptidase. Accordingly, aminopeptidase C do not necessarily seem to be the same as aminopeptidases M. Clarification of the localization of aminopeptidase C and comparison of the immunological properties and sequences of aminopeptidases C and M will indicate whether aminopeptidase C is the same enzyme as aminopeptidase M or not. The porcine aminopeptidase C in this study was more active towards tri- and tetrapeptides than dipeptides. This enzyme did not hydrolyze Ala-Gly, whereas it showed high activities against Ala-Gly-Phe-Ala and AlaNap. Furthermore, it had no activity against GluNap, but had large activities against Glu-Gly and Glu-Gly-Ile-Ala. These results indicate that the subsite in the binding site of aminopeptidase C seems to be important for the hydrolysis of these substrates. It was reported that bestatin, an aminopeptidase inhibitor of bacterial origin, induced the accumulation of di- and tripeptide intermediates in the degradation of cellular proteins in mammalian cells (Noguchi et al., 1983; Takahashi et al., 1987; Botbol and Scornik, 1989). In muscle, aminopeptidases B and C are inhibited by bestatin, indicating that these enzymes are involved in the degradation of di- and tripeptides in mammalian cells. However, the latter has a much wider substrate specificity than the former, so aminopeptidase C seems to play a more important role in this degradation than aminopeptidase B. A puromycin-sensitive aminopeptidase has
been shown to degrade enkephalin (Dyer et al., 1990) and seems to play an important role in the degradation of active peptides. The aminopeptidase C in this study was also inhibited by puromycin. Clarification of the function of aminopeptidase C in the living cell seems to be an interesting problem. SUMMARY Aminopeptidase C was purified from fresh porcine skeletal muscle by ammonium sulfate fractionation and successive chromatographies on D E A E cellulose, Ultrogel AcA 34 and hydroxylapatite columns. The purified enzyme migrated as a single band on SDS-PAGE. Aminopeptidase C was purified about 134-fold over the crude extract with a yield of 0.8%. The mol. wt of the enzyme was found to be 103,000 on both Spehadex G-200 column chromatography and SDS-PAGE. The optimum pH for the hydrolysis of e-leucine p-nitroanilide was around 7.0. The enzyme was stable in the pH range 6.0-8.0. The activity of this enzyme was strongly inhibited by EDTA, bestatin and puromycin. Co 2÷ and Zn 2+ at 0.5 mM caused inhibition of the enzyme activity. The Km and Vmaxvalues for hydrolysis of LeuNap were 1.0mM and 12.9/~mol min -~ (mg protein- l). The enzyme showed high activities against the fl-naphthylamide derivatives of Ala, Lys, Leu and Met. The enzyme was more active towards tri- and tetrapeptides than dipeptides. REFERENCES
Aoyagi T., Wada T., Kojima F., Nagai M. and Umezawa H. (1981) Various enzyme activities in muscle and other organs of dystrophic mice. J. clin. Invest. 67, 51 59. Botbol V. and Scornik O. A. (1989) Role of bestatin-sensitive exopeptidases in the intracellular degradation of hepatic proteins. J. biol. Chem. 264, 13,504-13,509. Dyer S. H., Slaughter C. A., Orth K., Moomaw C. R. and Hersh L. B. (1990) Comparison of the soluble and membrane-bound forms of the puromycin-sensitive enkephalin-degrading aminopeptidases from rat. J. Neurochem. 54, 547-554. Gorvel J. P., Vivier I., Naquet P., Brekelmans P., Rigal A. and Pierres M. (1990) Characterization of the neutral aminopeptidase activity associated with the mouse thymocyte-activating molecule. J. Immun. 144, 2899-2907. Ishiura S., Yamamoto T., Yamamoto M., Nojima M., Aoyagi T. and Sugita H. (1987) Human skeletal muscle contains two major aminopeptidases: an anion-activated aminopeptidase B and an aminopeptidase M-like enzyme. J. Biochem. 102, I023-1031. Joseph R. L. and Sanders W. J. (1966) Leucin aminopeptidase in extracts of swine muscle. Biochem. J. 100, 827-832. Kar N. C. and Pearson C. M. (1978) Post-proline-cleaving enzyme in normal and dystrophic human muscle. Clin. chim. Acta III, 271-273. Lauffart B. and Mantle D. (1988) Rationalization of aminopeptidase activities in human skeletal muscle soluble extract. Biochim. biophys. Acta 956, 300-306. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent J. biol. Chem. 193, 265-275. Mantle D., Hardy M. F., Lauffart B., McDermott J. R., Smith A. I. and Pennington R. J. T. 0983) Purification and characterization of the major aminopeptidase from human skeletal muscle. Biochem. J. 211, 567-573.
Aminopeptidase C from porcine muscle Mantle D., Lauffart B., McDermott J. R., Kidd A. M. and Pennington R. J. T. (1985) Purification and characterization of two C1--activated aminopeptidases hydrolysing basic termini from human skeletal muscle. Eur. J. Biochem. 147, 307-312. Mantle D., Lauffart B. and Pennington R. J. T. (1984) Purification and partial characterization of arginine aminopeptidase from human skeletal muscle. Biochem. Soc. Trans. 12, 826-827. McLellan S., Dyer S. H., Rodriguez G. and Hersh L. B. (1988) Studies on the tissue distribution of the puromycin-sensitive enkephalin-degrading aminopeptidases. J. Neurochem. 51, 1552-1559. Nishimura T., Okitani A., Katakai R. and Kato H. (1983) Mode of action towards oligopeptides and proteins of hydrolase H, a high-molecular-weight aminoendopeptidase from rabbit skeletal muscle. Eur. J. Biochem. 137, 23-27. Nishimura T., Okitani A. and Kato H. (1988) Identification of neutral aminopeptidases responsible for peptidolysis in postmortem rabbit skeletal muscle. Agric. Biol. Chem. 52, 2183-2190. Nishimura T., Okitani A., Rhyu M. R. and Kato H. (1990) Survey of neutral aminopeptidases in bovine, porcine and chicken skeletal muscles. Agric. Biol. Chem. 54, 2769-2775. Noguchi T., Sonaka I. and Naito H. (1983) Accumulation of acid soluble peptides in rat liver in vivo and after injection of bestatin. Agric. Biol. Chem. 47, 647~50. Nyberg F., Thornwall M. and Hetta J. (1990) Aminopeptidase in human CSF which degrade delta-sleeping inducing peptide (DSIP) Biochem. biophys. Res. Commun. 167, 1256-1262. Okitani A., Nishimura T. and Kato H. (1981) Characteriz-
CBPB 102/l--J
135
ation of hydrolase H, a new muscle protease possessing aminoendopeptidase activity. Fur. J. Biochem. 115, 269-274. Okitani A., Nishimura T., Otsuka Y., Matsukura U. and Kato H. (1980) Purification and properties of BANA hydrolase H of rabbit skeletal muscle, a new enzyme hydrolyzing ~-N-benzoyl-arginine-l/-naphthylamide. Agric. Biol. Chem. 44, 1705-1708. Otsuka Y., Okitani A., Katakai R. and Fujimaki M. (1976) Purification and properties of an aminopeptidase from rabbit skeletal muscle. Agric. Biol. Chem. 40, 2335-2342. Otsuka Y., Okitani A., Kondo Y., Kato H. and Fujimaki M. (1980) Further characterization of aminopeptidase of rabbit skeletal muscle. Agric. Biol. Chem. 44, 1617-1622. Parsons M. E. and Pennington R. J. T. (1976) Separation of rat muscle aminopeptidases. Biochem. J. 155, 375-381. Smith E. L. (1948a) The peptidases of skeletal, heart, and uterine muscle. J. biol. Chem. 173, 553-569. Smith E. L. (1948b) The glycylglycine dipeptidases of skeletal muscle and human uterus. J. biol. Chem. 173, 571-584. Smith E. L. (1948c) Studies on dipeptidases: II. Some properties of the glycyl-L-leucine dipeptidases of animal tissues. J. biol. Chem. 176, 9-19. Takahashi S., Kato H., Takahashi A., Noguchi T. and Naito H. (1987) Mode of action of bestatin and leupeptin to induce the accumulation of acid soluble peptides in rat liver in vivo and the properties of the accumulated peptides. The important role of bestatin- and leupeptinsensitive proteases in the protein degradation pathway in vivo. Int. J. Biochem. 19, 401~,12. Weber K. and Osborn M. (1969) The reliability of molecular weight determination by dodccyl sulfatc-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406-4412.
0305-0491/92 $5.00 + 0.00 Pergamon Press Ltd
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PURIFICATION A N D PROPERTIES OF AMINOPEPTIDASE C FROM PORCINE SKELETAL MUSCLE TOSHIHIDENISHIMURA,*t YUTAKAKATO,~ MEE RA RHYU,* AKIHIROOKITANI§ and HIROMICHIKATO* *Department of Agricultural Chemistry, The University of Tokyo, Tokyo 113, Japan; :~ResearchInstitute, Soda Aromatic Co., Ltd, Noda-shi, Chiba; and §Department of Food Science, Nippon Veterinary and Animal Science University, Musashino-shi, Japan (Received 7 August 1991)
Abstract--1. Aminopeptidase C was purified from porcine skeletal muscle. 2. The mol. wt of the enzyme was found to be 103,000 on both Sephadex G-200 column chromatography and SDS-PAGE. 3. The optimum pH for the hydrolysis of L-leucine p-nitroanilide was around 7.0. 4. The activity of this enzyme was strongly inhibited by EDTA, bestatin and puromycin. 5. The enzyme acted on the fl-naphthylamide derivatives of amino acids and oligopeptides.
INTRODUCTION
Aminopeptidases and endopeptidases are known to participate in the proteolytic system in the living cell. Aminopeptidases degrade the peptides produced from proteins through the action of endopeptidases to free amino acids. It was reported that the activities of intramuscular aminopeptidases were more markedly increased in dystrophic than normal mice (Aoyagi et al., 1981). A bestatin-sensitive aminopeptidase has been shown to be involved in the degradation of di- and tripeptides in mammalian cells (Noguchi et al., 1983; Takahashi et al., 1987; Botbol and Scornik, 1989). Recently, the neutral aminopeptidase activity located on the surface of T-cells has been shown to be associated with the mouse thymocyte-activating molecule (THAM) and to regulate THAM-mediated T-cell activation (Gorvel et al., 1990). Furthermore, some aminopeptidases have been reported to act on hormonally active peptides and to thereby inactivate them. The puromycin-sensitive aminopeptidase in rat brain (Dyer et al., 1990) and human placental aminopeptidase M (McLellan et aL, 1988) degrade enkephalin, and an aminopeptidase in human cerebrospinal fluid degrades the delta-sleep-inducing peptide (Nyberg et al., 1990). Although information on aminopeptidases in vitro or in vivo is gradually increasing, as described above, the actual functions of aminopeptidases and the mechanism of free amino acid production in vivo have not yet been completely elucidated. To answer these questions completely, it seems to be important to list and characterize major aminopeptidases. Neutral aminopeptidases of skeletal muscle that have been reported are leucine aminopeptidase (EC 3.4.11.1) (Joseph and Sanders, 1966), aminopeptidase B (EC 3.4.11.6) (Mantle et al., 1984, 1985: tAuthor to whom correspondence should be addressed. Abbreviations--LeuNap, L-leucine fl-naphthylamide; Nap,
/~-naphthylamide; PMSF, phenylmethylsulfonyl fluoride; MCE, 2-mercaptoethanol.
Ishiura et al., 1987), an aminopeptidase M-like enzyme (Ishiura et al., 1987), aminopeptidase C (Otsuka et al., 1976, 1980), the major aminopeptidase from human skeletal muscle (Mantle et al., 1983), aminopeptidase H (Okitani et al., 1980, 1981; Nishimura et al., 1983, 1988), pyroglutamyl aminopeptidase (EC 3.4.11.8) (Lauffart and Mantle, 1988), DAP III (Parsons and Pennington, 1976) and DAP IV (EC 3.4.21.26) (Kar and Pearson, 1978) and several dipeptidases (Smith, 1948 a-c). An aminopeptidase M-like enzyme (Ishiura et al., 1987) and the major aminopeptidase (Mantle et al., 1983) from human skeletal muscle and aminopeptidase C (Otsuka et al., 1976, 1980) from rabbit skeletal muscle have been shown to exhibit the highest activities against aminoacyl derivatives. The properties of aminopeptidase C are similar to those of the former two. Recently, an aminopeptidase C was found in chicken and porcine (Nishimura et al., 1990) skeletal muscles. In the present study, we purified aminopeptidase C from porcine skeletal muscle and clarified some of its properties. Furthermore, we proposed that the porcine aminopeptidase C and the major enzymes in skeletal muscle described above should be classified as one group and tentatively called collectively aminopeptidase C. MATERIALS AND METHODS
Materials
Porcine skeletal muscle (m. longissimus dorsi) was removed from the carcass immediately after slaughter, trimmed to remove fat and connective tissues, and then minced with a meat chopper. DEAE-cellulose (DE-52 preswollen) was purchased from Whatman (UK). Ultrogel AcA 34 was obtained from LKB (Sweden). Sephadex G-200 and hydroxylapatite were from Pharmacia Fine Chemicals (Sweden) and Bio-Rad, respectively. Ovalbumin, bovine serum albumin, aldose, catalase, ferritin and puromycin were purchased from Boehringer Mannheim GmbH (Germany). Pepstatin A and leupeptin were obtained from the Peptide Institute (Osaka 562,
129
TOSHImDE NISHIMURAet al.
130
Japan). SDS--6H (high mol. wt protein standard mixture for SDS-PAGE), PMSF and LeuNap were from Sigma Chemical Co. (USA). All other chemicals used were of reagent grade.
Enzyme assay The enzyme activities against the fl-naphthylamide derivatives of amino acids (amino acid Naps) were measured in the following way. After the enzyme had been incubated with 0.5 mM of each substrate in 50 mM Tris-HCl buffer (pH 7.2) at 37°C for 1-60 min, 0.4 ml of 0.23 N HCI in ethanol and 0.4 ml of 0.06% p-dimethylaminocinnamaldehyde in ethanol were added to stop the enzyme reaction. The red color that developed was measured at 540 nm and the flnaphthylamine released from the substrate was determined. The enzyme activity against Leu-p-nitroanilide was measured by the following method. After the enzyme had been incubated with 0.5 mM Leu-p-nitroanilide in 50 mM Mcllvain or ammonium buffer at various pHs at 37°C for 5 min, 0.8 ml of 0.23 N HCI in ethanol was added to stop the enzyme reaction. The p-nitroaniline released from the substrate was determined. The enzyme action towards di-, tri- and tetrapeptides was determined by measurement of the increase in ninhydrin positive materials during incubation. After the enzyme had been incubated with 0.5 mM of each peptide in 50 mM Tris-HCl buffer (pH 7.2) at 37°C for I hr, the incubation was stopped by heating at 100°C for 5 min.
Protein determination The absorbance at 280 nm was used to monitor the protein peaks on the column chromatographies. The concentrations of proteins were determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard.
Gel electrophoresis SDS-PAGE was carried out by the method of Weber and Osborn (1969) using a 5% gel and Bromophenol Blue as the tracking dye. The proteins were stained with Coomassie Brilliant Blue R-250.
Molecular weight determination Gel filtration was performed on a Sephadex G-200 column (2.4 x 105 cm) equilibrated with 10 mM Tris-HC1
(pH 7.2) containing 0.1 M NaC1, 0.1% MCE and I mM NaN 3. The void volume was determined with Blue Dextran. Ferritin (M, = 450,000), catalase (240,000), aldolase (158,000), bovine serum albumin (68,000) and ovalbumin (45,000) were used as standard proteins. The mol. wt of the subunit of aminopeptidase C was determined by SDS-PAGE. Myosin (205,000), /~-galactosidase (116,000), phosphorylase b (97,400), bovine serum albumin (67,000), ovalbumin (45,000) and carbonic anhydrase (29,000) were used as standard proteins.
Amino acid analysis Free amino acids were analyzed and determined with an amino acid analyzer (Hitachi Model 835) after protein had been removed from the solution by ultrafiltration. RESULTS
Purification o f aminopeptidase C from porcine muscle A m i n o p e p t i d a s e C was purified from porcine muscle by four steps. All steps were carried o u t at 4°C.
Step 1. Extraction and ammonium sulfate fractionation. Minced muscle (508 g) was h o m o g e n i z e d with 3 vols of 40 m M T r i s - H C l buffer (pH 7.2) containing 0.1% M C E in a W a r i n g blender for 1 min. The h o m o g e n a t e was then centrifuged at 7 5 0 0 g for 15 m i n a n d the resultant s u p e r n a t a n t was filtered t h r o u g h four layers of gauze to remove floating fat. The filtrate (the crude extract) was subjected to a m m o n i u m sulfate fractionation. T h e precipitate o b t a i n e d between 45 a n d 6 5 % a m m o n i u m sulfate s a t u r a t i o n was collected a n d then dialyzed against 1 0 m M T r i s - H C l buffer (pH 7.2) containing 0.1 M NaC1, 0.1% M C E a n d 1 m M N a N 3 (Buffer A). The dialyzate was centrifuged at 7 4 0 0 g for 2 0 m i n to remove insoluble materials a n d the s u p e r n a t a n t was pooled.
Step 2. DEAE-cellulose column chromatography. The s u p e r n a t a n t (240 ml, 14.2 g) was p u t on a c o l u m n (3.0 x 27.0 cm) o f DEAE--cellulose equilibrated with Buffer A. After the u n b o u n d proteins h a d been
(51.4)
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Fraction number Fig. 1. DEAE-cellulose column chromatography of porcine aminopeptidase C. The protein solution obtained between 45 and 65% ammonium sulfate saturation (14.2 g protein) was put on a column (3.0 x 27 cm) equilibrated with 10 mM Tris-HCl (pH 7.2) containing 0.1 M NaCI, 1 mM NaN 3 and 0. 1% MCE (Buffer A). After unbound protein had been washed out with the same buffer, the bound protein was eluted with a gradient of NaCI (---). The active fractions indicated by the bar ([---~)were pooled for the next step. Fractions of 20 ml were collected. (A) Protein; (O) LeuNap hydrolyzing activity. The concentration of the protein peak is indicated in parentheses.
Aminopeptidase C from porcine muscle
131
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Fraction n u m b e r Fig. 2. Rechromatography of porcine aminopeptidase C on a hydroxylapatite column. The active fractions (8.3 m g protein) obtained from the Ultrogel AcA 34 column were dialyzed against 10 m M K-phosphate buffer (pH 7.2) (Buffer B) and then applied onto a column (3.0 x 14.5 cm) of hydroxylapatite equilibrated with Buffer B. After the u n b o u n d protein had been washed out with the same buffer, the enzyme was eluted with 80 m M K-phosphate buffer (pH 7.2) and then with a linear gradient of K-phosphate (80-200 mM). Fractions o f 3 ml were collected ( A ) Protein; ( O ) L e u N a p hydrolyzing activity. The purified enzyme ( ~ ) was applied to a 5.0% polyacrylamide gel containing SDS.
completely removed from the column by washing with Buffer A, elution was started with a linear concentration gradient of NaCI in Buffer A. The activities against LeuNap were eluted at around 0.22 M NaCI. The active fractions, indicated by a bar in Fig. 1, were pooled. This step was very effective for the purification, since the bulk of the proteins did not bind to the DEAE-cellulose column. Step 3. Ultrogel AcA 34 column chromatography. The enzyme solution (15.5mg) from step 2 was concentrated by ultrafiltration using a Ultrafilter UK-10 membrane and then applied onto a column (2.5 x 103 cm) of Ultrogel AcA 34 equilibrated with Buffer A. The enzyme was eluted with the same buffer.
Step 4. Hydroxylapatite column chromatography. The enzyme solution (4.2mg) from step 3 was dialyzed against 10 mM potassium phosphate buffer (pH 7.2) containing 0.1% MCE. The dialyzate was put on a column (3.0 x 14.5 cm) equilibrated with the same buffer. After the unbound protein had been washed out with same buffer, the bound proteins were eluted with 80 mM K-phosphate buffer (pH 7.2)
and then a linear concentration gradient of Kphosphate buffer (pH 7.2), from 80 to 200 mM. The LeuNap hydrolyzing activity was eluted as two peaks, as shown in Fig. 2. The second peak was much larger than the first one. The second peak (indicated by a bar in Fig. 2) was collected and used as the purified aminopeptidase C to examine some of its properties, as described below. The results of the purification process are summarized in Table 1. Aminopeptidase C was purified 134-fold over the crude extract with a yield of 0.8%. Aminopeptidase C thus obtained gave a single band on S D S - P A G E (see Fig. 2).
Some properties of the purified aminopeptidase C Molecular weight. As shown in Figs 3 and 4, the mol. wt of aminopeptidase C was estimated to be 103,000 on both Sephadex G-200 column chromatography and SDS-PAGE. These results indicate that this enzyme is a single polypeptide.
Effect of pH on the enzyme's activity and stability. The enzyme activity against Leu-p-nitroanilide (Leu-pNA) was measured in 50mM Mcllvain or
Table 1. Purification steps for porcine aminopeptidase C Total protein Spec. act. Purity Recovery of Purification step (rag) (#mol min - l m g -1) (fold) activity (%) Crude extract 23,088 0.012 1.0 100 45-65%(NH4)2SO4Ppt 14,172 0.018 1.6 97.2 DEAE-cellulose 15.5 2.632 226.9 15.2 Ultrogei AcA 34 8.3 1.009 87.0 3.1 Hydroxylapatite 1.4 1.552 133.4 0.8 Aminopeptidase C was purified from 508 g of porcine muscle. The activity was measured using LeuNap as a substrate. The details of the assay are given in Materials and Methods.
TOSHIHIDE NISH]MURAel al.
132
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Fig. 3. Determination of the mol. wt of porcine aminopeptidase C on a Sephadex G-200 column. The porcine aminopeptidase C, and 5 mg each of ovalbumin, bovine serum albumin (BSA), aldolase, catalase and ferritin were put on a Sephadex G-200 column and eluted with Buffer A. The void volume, with Blue Dextran, is expressed as V0, and the volumes at which the aminopeptidase C and standard proteins were eluted as Ve. The details are given in Materials and Methods. a m m o n i u m buffer at various pHs. The o p t i m u m p H for the hydrolysis of L e u - p N A was a r o u n d 7.0 (Fig. 5). After the enzyme h a d been kept at various pHs in 5 0 m M M c l l v a i n or a m m o n i u m buffer at 37°C for 1 hr, the activity against L e u N a p was m e a s u r e d at p H 7.2. As shown in Fig. 6, the enzyme was stable in the p H range 6.0-8.0. Effect of heat on the enzyme's stability. After the enzyme h a d been kept at various temperatures in 5 0 m M Tris-HC1 buffer ( p H 7 . 2 ) for 10min, the activity against L e u N a p was measured. The enzyme
0ID L 3 4
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i 5
10 6' 7t 8 ~ pH Fig. 5. Effect of pH on the activity of porcine aminopeptidase C. The activity was measured using 0.5 mM LeuNap as a substrate in K-phosphate (O) or ammonium (©) buffer. The details of the assay against Leu-p-nitroanilide are given in Materials and Methods. activity was stable up to a r o u n d 45°C, but m o s t activity was lost at 60°C (Fig. 7). Effects of inhibitors. As s h o w n in Table 2, the enzyme activity was inhibited strongly by E D T A , bestatin a n d puromycin, but was not affected so m u c h by m o n o i o d o a c e t i c acid, pepstatin or leupeptin. P M S F did n o t cause inhibition of the enzyme activity at all. Effects of metal ions. The enzyme activity was not affected so m u c h by Ca 2+, M g 2÷, Co 2+, M n 2÷ or Z n 2÷ at 0.005 or 0 . 0 5 m M . Co 2÷ a n d Z n 2÷ at 0 . 5 m M inhibited the enzyme activity strongly (Table 3).
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Fig. 4. Mol. wt determination of the subunit of porcine aminopeptidase C by SDS-PAGE. Aminopeptidase C, carbonic anhydrase, ovalbumin, bovine serum albumin (BSA), phosphorylase B, fl-galactosidase and myosin were applied to a 5.0% polyacrylamide gel containing SDS. The mobilities of the tracking dye and proteins are expressed as Ro and Re, respectively.
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Fig. 6. Effect of pH on the stability of porcine aminopeptidase C. After the enzyme had been kept at various pHs in 50 mM Mcllvain (O) or ammonium (O) buffer at 37°C for 1 hr, the activity against LeuNap was measured at pH 7.2. The details of the assay are given in Materials and Methods.
Aminopeptidase C from porcine muscle
133
Table 4. Hydrolytic activities of porcine aminopcptidase C towards fl-naphthylamide derivatives of amino acids (amino acid Naps)
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70
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Fig. 7. Effect of heat on the stability of porcine a m i n o p e p tidase C. After the e n z y m e h a d been kept at v a r i o u s t e m p e r a t u r e in 50 m M T r i s - H C 1 buffer (pH 7.2) for 10 min, the activity a g a i n s t L e u N a p was measured. The details o f the assay are given in M a t e r i a l s a n d M e t h o d s .
Km and Vmax values. The Km and Vmaxvalues for the hydrolysis of LeuNap were 1.0 mM and 12.9 #mol rain- ~mg protein- 1, respectively. Substrate specificity. As shown in Table 4, this enzyme showed a broad substrate specificity for the fl-naphthylamide derivatives of amino acids. In particular, the enzyme exhibited high activities against Ala-, Lys-, Met- and LeuNap. The enzyme exhibited no activity against GluNap. The action of aminopeptidase C on 14 sorts of dipeptide which
Table 5. Hydrolytic activities of porcine aminopeptidase C towards peptides Peptide Dipeptides Lys-Gly Met-Gly Glu-Gly Leu-Gly
Phe-Gly Gly---Gly Val~;ly Ile431y Ala-Gly His431y Pro-Gly Ser431y Thr-Gly Trp-Gly
Relative activity (%) 234 100 82 67 43 35 11 4 0 0 0 0 0 0
Peptide
Relative activity
Tripeptides Leu-Gly-Gly Val-Tyr-Val
(%) 247 49
Tetrapeptides Phe-Gly-lle-Gly Lys~31y-Ile--Ala Glu-Gly-Ile-Ala Thr~31y-lle-Ala Glyq31y-lle--Ala lle431y-lle-Ala Ala-Gly-Phe-Ala Met-Gly-Phe-Ala Val~31y-Phe-Ala
908 243 177 45 26 19 339 196 32
After the enzyme had been incubated with 0.5 mM of each peptide in 50 mM Tris-HCI buffer (pH 7.2) at 37°C for l hr, the increase in ninhydrin positive materials was measured. The details of the assay are given in Materials and Methods.
Table 2. Effect of protease inhibitors on the activity of porcine aminopeptidase C Relative activity (%)
Inhibitor None PMSF (10 3M) Iodoacetic acid (10 -2 M) EDTA (10 -t M) Pepstatin (10 4M) Leupeptin (10 -4 M) Puromycin (10 4M) Bestatin (10 -4 M)
100 100 90.0 2.9 91.2 90.0 32.9 27.9
The activity was measured using LeuNap as a substrate in the absence or presence of each inhibitor. The details of the assay are given in Materials and Methods. The concentrations of inhibitors are indicated in parentheses. Table 3. Effect of metal ions on the activity of porcine aminopeptidase C Relative activity (%) Metal None Ca 2 + Mg 2 + Co s + Mn 2+ Zn 2+
0.005 mM
0,05 mM
0.5 mM
92 98 101 93 93
100 100 92 77 93 78
85 96 6 86 19
The enzyme activity was measured using LeuNap as a substrate in the absence or presence of each metal ion at three concentrations. The details of the assay are given in Materials and Methods.
have Gly at their C-terminal is shown in Table 5. The enzyme had high activities against Lys-, Met-, Gluand Leu-Gly, but no activity against Ala-, His-, Pro-, Ser-, Thr- and Trp-Gly. The hydrolytic activities against tri- and tetrapeptides were higher than those against dipeptides. The enzyme had particularly high activities against tri- and tetrapeptides which have Phe, Ala, Leu and Lys at their N-terminal. However, it could hardly act on tri- and tetrapeptides which have Ile, Gly, Val and Thr at their N-terminal. It was recognized that this enzyme released Leu or Met at first through its action towards Leu-Gly-Gly or Met-Gly-Ile-Ala, respectively. DISCUSSION
In this study, aminopeptidase C has been purified from porcine skeletal muscle and some of its properties have been clarified. Aminopeptidase C, of the neutral aminopeptidases in porcine skeletal muscle, showed the highest arylamidase activity. The major aminopeptidases in skeletal muscle of other species have been purified and characterized. The aminopeptidase C (Otsuka et al., 1976, 1980) purified from rabbit skeletal muscle, and the major aminopeptidase (Mantle et al., 1983) and an aminopeptidase M-like enzyme (Ishiura et aL, 1987) from human skeletal muscle are similar to the aminopeptidase C from
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TOSHIHIDENISHIMURAet al.
porcine skeletal muscle in this study. Some properties of these enzymes are a little different. The aminopeptidases from human and porcine muscles are single polypeptides and their mol. wts (Mr) are about 100,000. On the other hand, the Mr of the rabbit aminopeptidase C has been shown to be 160,000 on gel filtration. The effect of a metal ion on the activities of these enzymes is different. The porcine aminopeptidase C was not affected by 0.5 mM Ca 2+. However, the aminopeptidase from human muscle was activated five times by 0.5mM Ca 2+ and that from rabbit muscle was strongly inhibited by 1 mM Ca 2+. Although some properties of these aminopeptidases show species differences between them as described above, others are almost the same. For example, these enzymes have been shown to possess the highest arylamidase activities among the neutral aminopeptidases in skeletal muscle. Their optimal pH has been shown to be around 7.0. These enzymes are stable in the neutral pH region and are inhibited by metal chelating reagents such as EDTA and o-phenanthroline. Accordingly, these enzymes should be classified as the same enzyme, so we tentatively recommend that these aminopeptidases in skeletal muscles should be collectively called aminopeptidase C. An aminopeptidase C may be similar to an aminopeptidase M, which is a membrane-bound aminopeptidase and localized in microsomes in kidney cells, as human aminopeptidase C has been called an aminopeptidase M-like enzyme. However, some properties, including the mol. wt, substrate specificity, and sensitivity to Co 2÷ and Tris, differ between the two enzymes. Also, an aminopeptidase C has been shown to be extracted by a buffer without a detergent, indicating that this enzyme is not a membrane-bound aminopeptidase. Accordingly, aminopeptidase C do not necessarily seem to be the same as aminopeptidases M. Clarification of the localization of aminopeptidase C and comparison of the immunological properties and sequences of aminopeptidases C and M will indicate whether aminopeptidase C is the same enzyme as aminopeptidase M or not. The porcine aminopeptidase C in this study was more active towards tri- and tetrapeptides than dipeptides. This enzyme did not hydrolyze Ala-Gly, whereas it showed high activities against Ala-Gly-Phe-Ala and AlaNap. Furthermore, it had no activity against GluNap, but had large activities against Glu-Gly and Glu-Gly-Ile-Ala. These results indicate that the subsite in the binding site of aminopeptidase C seems to be important for the hydrolysis of these substrates. It was reported that bestatin, an aminopeptidase inhibitor of bacterial origin, induced the accumulation of di- and tripeptide intermediates in the degradation of cellular proteins in mammalian cells (Noguchi et al., 1983; Takahashi et al., 1987; Botbol and Scornik, 1989). In muscle, aminopeptidases B and C are inhibited by bestatin, indicating that these enzymes are involved in the degradation of di- and tripeptides in mammalian cells. However, the latter has a much wider substrate specificity than the former, so aminopeptidase C seems to play a more important role in this degradation than aminopeptidase B. A puromycin-sensitive aminopeptidase has
been shown to degrade enkephalin (Dyer et al., 1990) and seems to play an important role in the degradation of active peptides. The aminopeptidase C in this study was also inhibited by puromycin. Clarification of the function of aminopeptidase C in the living cell seems to be an interesting problem. SUMMARY Aminopeptidase C was purified from fresh porcine skeletal muscle by ammonium sulfate fractionation and successive chromatographies on D E A E cellulose, Ultrogel AcA 34 and hydroxylapatite columns. The purified enzyme migrated as a single band on SDS-PAGE. Aminopeptidase C was purified about 134-fold over the crude extract with a yield of 0.8%. The mol. wt of the enzyme was found to be 103,000 on both Spehadex G-200 column chromatography and SDS-PAGE. The optimum pH for the hydrolysis of e-leucine p-nitroanilide was around 7.0. The enzyme was stable in the pH range 6.0-8.0. The activity of this enzyme was strongly inhibited by EDTA, bestatin and puromycin. Co 2÷ and Zn 2+ at 0.5 mM caused inhibition of the enzyme activity. The Km and Vmaxvalues for hydrolysis of LeuNap were 1.0mM and 12.9/~mol min -~ (mg protein- l). The enzyme showed high activities against the fl-naphthylamide derivatives of Ala, Lys, Leu and Met. The enzyme was more active towards tri- and tetrapeptides than dipeptides. REFERENCES
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