HPLC purification and characterization of porcine muscle aminopeptidase B

Biochimie (1993) 75, 861-867

861

© Soci6t6 franqaise de biochimie et biologie mol6culaire / Elsevier, Paris

HPLC purification and characterization of porcine muscle aminopeptidase B M Flores, MC Aristoy, F Toldr~i* hlstituto de Agroqu{mica y Tecnoiog{a de Alimentos (CSIC), Jaime Roig 11. Valencia 46010. Spain (Received 10 July 1993; accepted 30 August 1993)

Summary m An aminopeptidase B from porcine skeletal muscle was successfully purified by ammonium sulphate fractionation and HPLC anion-exchange. The purified aminopeptidase B eluted at 0.18 M NaCl, had a relative molecular mass of 76000 Da and was markedly stimulated in the presence of 0.2 M chloride anion. The enzyme exhibited maximum activity for the hydrolysis of the arginine-aminoacyl bond at pH 6.5 and 37°C. Other substrates consisting of phenylalanine, proline and alanine-aminoacyl bonds were cleaved at 5.9, 5.1 and 2.5% of the maximum activity with the arginine-aminoacyl bond. The enzyme did not show endopeptidase activity and was very stable at pH above 6 and temperatures below 35°C. However, the enzyme inactivated very fast when incubated at pH 5 or at 50-65°C. Bestatin (50 laM) completely inhibited the aminopeptidase B activity while EDTA (5 mM) only inhibited 40% of its activity. However, 0.5 mM of E-64 did not cause any inhibition while 0.05 mM amastatin and 1 mM puromycin only inhibited 11% of the enzyme activity.

aminopeptidase / protease / purification / enzyme characterization

Introduction Aminopeptidases (l-aminoacylpeptide hydrolases, EC 3.4.1 I) are enzymes known to be present in skeletal muscle [1] and named according to their preference for a partict, lar N-terminal amino acid. Muscle aminopeptidases may play an important role on flavour development in meat and meat products. So, Okitani et al [2] and Nishimura et al [31 reported an increase in free amino acids during meat ageing which was attributed to these enzymes [4]. The high increase in the free amino acid concentration during the drycuring process [5] has also been attributed to muscle aminopeptidases [6]. In fact, alanyl, leucyi, arginyl, tyrosyi and pyroglutamyl hydrolyzing activity was determined on both raw and dry-cured ham showing a high stability even after 8 months [7]. The effect of curing agents and process parameters was also evaluated [8]. Arginyl aminopeptidase, also named aminopeptidasc B (EC 3.4.11.6), is one of the major aminopeptidoses existing in skeletal muscle [9-14] erythrocytes [15, 16] and organs such as the liver [17-20]. This enzyme is a chloride-activated aminopeptidase [12,

*Correspondence and reprints

21]. A better knowledge of porcine skeletal muscle aminopeptidase B would help to understand its possible role in processed meats and meat products. The isolation, purification, substrate specificity, inhibition and chemical properties of the porcine muscle aminopeptidase B are reported in this paper. Materials and methods MateriaL~ The aminoacyi-7-amino-4-methylcoumarin substrates and the inhibitors were obtained from Sigma (St Louis, MO). Bestatin was from Boehringer (Mannheim, Germany). Protein standards for electrophoresis were from BioRad (Richmond, VA). The anion-exchange HPLC column PL-1000 SAX (50 x 5 mm, 8 lam particle size) was purchased from Hewlett-Packard (Palo Alto, CA). Muscle biceps femoris from 6-month-old pigs was taken just after death and used as the enzyme source. The complete process (from raw muscle to the purified enzyme) was carried out within ! 6-20 h post-mortem.

Assay of aminopeptidase activit3." The standard assay for aminopeptidase B activity was performed at 37°C by using L-arginine 7-amido-4-methyl coumatin (Arg-MCA) as substrate in 50 mM phosphate buffer (pH 6.5), containing 0.2 M NaCl. The activity against AlaMCA was determined as described by Nishimura et al [41 using 100 mM Tris-HC! (pH 7.0), containing 2 mM dithio-

862 Table I. Purification of porcine muscle aminopeptidase B a

Protein

Crude extract Soluble fraction 40-60% (NH4).,SO4

(mg)

Total activity (U)

Specific activity (U/mg,)

Recovered activity (%)

2182.5

11.60

0.0053

100

1

466.8

9.99

0.021

86

3.9

99.8

4.67

0.047

40

8.9

3.02

7.94

26

Anion exchange

0.38

Purification (fold)

1498

aEnzyme activity in the crude extract, soluble extract and the dialyzed sample after ammonium sulphate fractionation was determined in the presence of 0.5 mM puromycin to inhibit the action of the alanyl aminopeptidase on the Arg-MCA substrate.

threitol (DTT) as reaction buffer while in the case of Leu-MCA the buffer was 50 mM Tris-HCl (pH 8.5), containing 5 mM MgCI, [14]. The reaction was incubated for 15 rain and stopped by the addition of 100 mM sodium acetate/chloroacetate buffer (pH 4.3). The fluorescence was measured at 360 nm and 440 nm as excitation and emission wavelenghts, respectively, in a Shimadzu RF-5000 spectrofluorophotometer. One unit of activity (U) was defined as the amount of enzyme hydrolysing I I.tmol of substrate per hour at 37°C. Four replicates (samples + controls) were measured for each experimental point. Optimal pH and temperature for aminopeptidase B were determined in the ranges 5.0-8.0 and 5-55°C, respectively. Optimal chloride concentration was determined by assaying the enzyme activity at 37°C and pH 6.5 with the addition of different NaC! concentrations (0-0,5 M).

Ett:yttw extraction The enzyme crude extracts were prepared as described by Lauffart and Mantle 1141 with slight modifications. Ten grams of muscle biceps t'emoris, with no visible fat or connective tissue, was homogenized in 50 ml of 50 mM phosphate buffer containing 5 mM ethylene glycol tetraacetic acid (EGTA), pH 7.5, by using a Polytron (three strokes, 10 s each at 27000 rpm with cooling in ice) homogenizer (Kinematica, Switzerland). The extract was centrifuged at 10000 g for 20 rain at 4°C and the supernatant, filtered through glass wool (soluble fraction), used for further purification.

Enzyme purification

The column was previously equilibrated by flowing through 5 mi of 10 mM Tris-HC! buffer (pH 7.0), containing 0.1 M sodium chloride, 0 . 1 % (v/v) ~-mercaptoethanol and 0.02 % (w/v) sodium azide [4]. The dialyzed sample was filtered through a 0.45 IJ.m nylon membrane filter and injected (250 l.tl) into the system. The column was eluted at 0.5 mi/min for 9 min with the equilibration butter, then, with a linear salt gradient (0.1-0.4 M NaCl) for 20 rain and, finally, with 0.4 M NaCI for 9 min. 34 fractions (0.5 ml each) were collected and assayed for aminopeptidase activity using different fluorescent aminoacyI-MCA substrates (Ala, Arg and Leu-MCA).

Substrate specificity The pt, ritied enzyme activity was measured against arginineMCA, alanine-MCA, leucine-MCA, tyrosine-MCA, phenylalanine-MCA, serine-MCA, glycine-MCA, proline-MCA, Z-arginine-arginine-MCA and N-CBZ-phenylalanine-arginine-MCA as substrates in 50 mM phosphate buffer (pH 6.5), containing 0.2 M NaCI.

Inhibition The effect of potential inhibitors was tested by incubating the enzyme in the presence of the following inhibitors and concentrations: amastatin (0-0.2 mM), bestatin (0-0.5 raM), E-64 (0-0.5 mM), ~-mercaptoethanol (0-0.1 mM), puromycin (02.0 mM), dithiothreitol (DTT, 0-2.0 mM) and EDTA (0-15 mM). Controls with no inhibitor addition were simultaneously ran.

Ammonium sulphatefractionation

Elet'trophoresis

Solid ammonium sulphate was added to the soluble fraction to give 40% saturation and left stirring for 30 rain at 4°C. Once centrifuged at 10000 g for 20 min, solid ammonium sulphate was added to the clear supematant to a final saturation of 60% and left to stand for 2 h. The solution was then centrifuged again and the precipitate dissolved in a minimum volume of 100 mM Tris-HCI buffer (pH 7.0), containing 0.02% (w/v) sodium azide and dialyzed against the same buffer.

The relative molecular mass and purity of the purified aminopeptidase B was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using 10% polyacrylamide gels and staining with Coomassie blue R-250 [22] or silver [231. Standard proteins were simultaneously ran for relative molecular mass identification.

Anion exchange chromatography The chromatographic separation was carried out in a biocompatible (titanium) 1050 Hewlett-Packard liquid chromatt~graph equipped with a variable wavelength UV detector (280 nm).

Determination of protein concentration Protein concentration was determined by the method of Bradford [24] using bovine serum albumin as standard. The fractions eluted from the chromatographic system were also monitored at 280 nm.

863

Determination of the enzyme stability

200

100

0.5

III ~000

Thermal stability of the purified amlnopeptidase B was determined by incubation of the enzyme in a 100 mM phosphate buffer (pH 7.0), at the following temperatures: 15, 25, 35, 50 and 65°C.The effect of pH on the enzyme stability was determined by incubating the purified aminopeptidase B at 25°C in 100 mM phosphate buffer, pH 5.0, 6.0, 7.0 and 8.0.The activities were expressed as a percent of the activity remaining at each time interval using as control the value obtained at time 0 with the standard activity assay.

-

..

4oo i

i:~~ ....

The purity of the purified enzyme (peak I) was checked by SDS-PAGE. A single protein band was detected after silver staining (fig 1). This band corresponds to 76000 Da which is identical to the relative molecular mass reported by lshiura et al [13] for human skeletal muscle aminopeptidase B and very close to 72000 Da found by Mantle et al [12].

Effect of chloride ion The effect of sodium chloride on the purified enzyme is shown in figure 2. The activity of the enzyme increased with increase of the NaCI concentration and reached a maximum at a concentration of 0.15-0.2 M. Aminopeptidase B has been reported as a chloride-

50

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Purity a~d relative molecular mass

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Results

The results obtained through the purification process of aminopeptidase B from porcine skeletal muscle are summarized in table I. The anion-exchange chromatography of the dialyzed enzyme sample after ammonium sulphate fractionation (table I) gave three well-separated eluting peaks showing arginyl-MCA hydrolyzing activity (fig 1). Peak I, which eluted from the column at 0.18 M NaCI, hydrolyzed Arg-MCA exclusively. The second peak (II) eluted at 0.25 M NaC! and hydrolyzed Arg-MCA, AIa-MCA and a little of Leu-MCA. The last peak (III) eluted at 0.31 M NaC! and hydrolyzed AIa-MCA, Arg-MCA and Leu-MCA. This last enzyme, completely inhibited by the addition of 0.5 mM puromycin, corresponds to the major aminopeptidase described by Mantle et al [25]. The addition of 0.2 M NaCI, a clear activator of aminopeptidase B [13, 18], to all the fractions only showed a clear activation for peak I. This enzyme also showed a high specificity against Arg-MCA so that it can be considered aminopeptidase B and further research for characterization is reported. A summary of the aminopeptidase B purification is shown in table I. A 1498-fold purification was achieved.

0.4

0.3

0

Purijication of aminopeptidase B

._ . . . . . . .

• ..... .....

20

i

,

25

30

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number

Fig 1. Anion-exchange HPLC of dyalized sample after ammonium sulphate fractionation. Arginyl (e), leucyl ([]) and alanyl (V) aminopeptidase activity are reported as units of fluorescence/0.05 ml fraction. Protein is reported as absorbance (280 nm) x 10-3. Silver stained electropherogram of peak I is shown. activated arginyl aminopeptidase by many authors [ 12, 13, 18, 21 ]. The enzyme activity decreased at higher NaCI concentrations.

Effect of pH and temperature Optimum activity was found at pH 6.5 (data not shown) as also reported by Mantle et al [12]. This pH is very close to pH 7.0 reported by SOderling [21] and Ishiura et al I13]. The activity sharply decreased at

100

80 °~

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40 20

0

0.0

I

I

I

I

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0.1

0.2

0.3

0.4

0.5

0.6

NaCI Concentration (M)

Fig 2. Effect of sodium chloride on the activity of the purified aminopeptidase B.

864 Effect of various inhibitors

Table 11. Activity of the purified aminopeptidase B on various aminoacyl-MCA derivatives. Substrate

The effect of potential inhibitors on the activity of aminopeptidase B is shown in figure 3. The addition of E-64, a specific inhibitor of cathepsin H [26], did not inhibit aminopeptidase B (fig 3A). However, 0.05 mM of bestatin, a typical inhibitor of aminopeptidases [27-291, completely inhibited aminopeptidase B. 2-Mercaptoethanol (0.1 mM) also inhibited the enzyme (fig 3B). However, little inhibition was observed in the presence of amastatin, dithiothreitol or puromycin (fig 3B, D). The metal-chelating agent EDTA (15 mM) inhibited 75% of the enzyme activity (fig. 3C). Another substance with potential inhibitory action is ammonium sulphate (fig 4) which in very low amounts, such as 25 mM, gave a 75% inhibition of the aminopeptidase B.

Relative activi~ (%)

Arg-MCA Phe-MCA Pro-MCA Ala-MCA Leu-MCA Ser-MCA Tyr-MCA Gly-MCA N-CBZ-Phe-Arg-MCA Z-Arg-Arg-MCA

100.0 5.9 5.1 2.5 0.2 0.0 0.0 0.0 0.0 0.0

•

o

Enzyme stability pH 5.5 down to 8% of the optimum activity and to negligible activity below pH 5.0. The optimum temperature for enzyme action was 37°C (data not shown).

The thermal stability of the enzyme was studied by measuring the activity remaining after incubation at various temperatures. The enzyme was relatively stable up to 35°C but completely inactivated in 22 h at 50°C and almost instantly at 65°C (fig 5). The enzyme showed high stability when maintained below 25°C. This enzyme from porcine muscle showed higher stability than the porcine liver aminopeptidase B studied by Kawata et al [18]. The dependence of the activity on pH was studied by incubating the enzyme with appropiate buffers between pH 5 and 8. The enzyme showed high stability at pH 8.0 and 7.0 but with lower stability at pH 6.0 (fig 6), However, the enzyme was quite unstable at pH 5.0. In fact, only 3% of the original activity was recovered after 16.5 h.

Substrate speciJicity The activity of the aminopeptidase B on a series of L-aminoacyi-MCA derivatives is shown in table II. Only arginyi derivative was sensibly hydrolyzed while phenylalanine-MCA, proline-MCA and alanineMCA derivatives were little cleaved. The enzyme did not show endopeptidase activity when using some Nterminus-blocked substrates as also reported by ishiura et al II 31.

B

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0

0,1

0.2

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5.0

10.0

15.0

0

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2.0

Concentration (raM) Fig 3. Effects of various inhibitors on the activity of the purified aminopeptidase B. The activity was determined by the standard assay. The activity with no inhibitor was taken as 100%. A. Bestatin (e) and E-64 (O). B. Amastatin (e) and 2-mercaptoethanol (O). C. EDTA (O). D. DTT (e) and puromycin (~).

865

100

v

80

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60

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ID

20 0

m

0.0

0.1

0.2

Ammonium

0.3

Sulphate

0.4

0.5

Concentration

0.6 (M)

Fig 4. Effect of ammonium sulphate on the activity of the purified aminopeptidase.

1000

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hydrolysing basic termini, is aminopeptidase B. The enzyme elutes in a one single peak at 0.18 M NaCI and only gives a single band when silver staining the SDS-PAGE electropherograms. The relative molecular mass of 76000 Da for porcine muscle aminopeptidase B is very similar to the 76000 and 72000 Da reported by lshiura et al [ 13] and Mantle et al [12], respectively, for human skeletal muscle but larger than 58000 Da reported by Kawata et al [18] for porcine liver and by MiJ.kinen and Makinen [15] for human erythrocytes. The addition of 0.2 M CI- results in an eight-fold increase in activity (fig 2). However, it is not clear yet if the presence of phosphate anion might have an additive effect [13] or simply no effect [21] on the final observed activation. Aminopeptidase B shows a high specificity against MCA-derivatives of L-arginine (table II) as expected [ 12, 13, 19, 20] but has no endopeptidase activity as also observed by Ishiura et al [ 13]. Bestatin is reported in the literature [27-29] as an effective inhibitor of aminopeptidases and, in fact, a concentration as low as 0.05 mM is enough for a complete inhibition of aminopeptidase B (fig 3A). Amastatin is a powerful inhibitor of leucyl aminopeptidase and aminopeptidase A [30] but little [31] or no inhibition [1] has been reported for aminopeptidase B which is in accordance with our results (fig 3B). Puromycin (1 mM) is another powerful inhibitor of the major aminopeptidase [251 or aminopeptidase C [32]. In our case, only 11% inhibition of aminopeptidase B is detected (fig 3D) which is close to the 15-20%

,D

1000 [

SO'C ( ,, )

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2

300

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65*C ( • )

p• 't,O ( v ), 8,0( o )

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0

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48

30

pH 6,0 ( O )

Time (hours)

Fig 5. Thermal stability of aminopeptidase B. The purified enzyme was incubated in 100 mM phosphate buffer (pH 7.0) at the temperatures shown.

Discussion We have been studying the proteolysis in the porcine skeletal muscle and have found an intense free amino acid generation [5] and an exopeptidase system active in the neutral pH region [7]. One of these enzymes,

pH 5,0( o )

I 0

I 24

1 48

I 72

I 96

Time (hours)

Fig 6. pH stability of aminopeptidase B. The purified enzyme was incubated at 25°C in 100 mM phosphate buffer at the pH shown.

866 reperted by Mantle et ai [ 12] and the absence of inhibition by Kawata et a l [ 19]. Inhibition of the enzyme by EDTA (fig 3C), as also reported by other authors [12, 13, 18, 31, 33], suggests that metal ions are essential to the activity. However, S6derling and Miikinen [ 16] considered that this enzyme can not be classified as metallopeptidase although further research is required to finally confirm whether this enzyme is a metalioenzyme or not. The significative inhibition by a m m o n i u m sulphate (fig 4) is quite important since an incomplete dialysis could affect the final measured activity due to the presence of low or trace amounts of a m m o n i u m sulphate in the reaction mixture. The purified aminopeptidase B differs from cathepsin H since it is not inhibited by E-64 (fig 3A) which is a typical inhibitor of cathepsin H [26], has no endopeptidase activity and its relative molecular mass is higher than the 27000 Da reported for cathepsin H [34]. Another Arg-MCA hydrolysing enzyme is aminopeptidase H [35] although its characteristics such as a molecular mass of 390000, exo- and endopeptidase activity, optimal pH around 8.0 and no inhibition by bestatin, completely differ from the aminopeptidase B purified in this work. Thus, the purified enzyme is characterized as an aminopeptidase B, a chloride-activated enzyme, specifically hydrolysing basic termini.

Acknowledgments This work has been tinantially supported by the Comisi6n Cientflica de Ciencia y Tecnologfa (CICYT, Spain), grant no ALi91.0752. The FPi scholarship to MF t'rom the Conselleria de Cultura, Educaci6 i Ci6ncia is also acknowledged.

References I

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Barrett AJ, Kirschke H (1981) Cathepsin B, cathepsin H and cathepsin L. h~: Methods ill en-vmology, Vol 80 (Lorand L, ed) Academic Press, New York, 535-561 27 Suda H, Aoyagui I", Takeuchi T, Umezawa H (1976) Inhibition of aminopeptidase B and ieucine aminopeptidase by bestatine and its stereoisomer. Arch Biochem Biophys 177, 196--200 28 Umezawa H, Aoyagui T, Suda H, Hamada M, Takeuchi T (1976) Bestatine, an inhibitor of aminopeptidase B, produced by actinomycetes. J Antibiotic 29, 97-99 29 Harbenson SL, Rich DH (1988) Inhibition of arginine aminopeptidase by bestatin and arphamenine analogues. Evidence for a new mode of binding to aminopeptidases. Biochem J 27, 7301-7310 30 Aoyagi T, Tobe H, Kojima F, Hamada M, Takeuchi T, Umezawa H (1978) Amastatin, an inhibitor of aminopeptidase A produced by actinomycetes. J Antibiot (Tokyo) 31, 636--638

31

McDermott JR, Mantle D, Lauffart B, Gibson AM (1988) Purification and characterization of two soluble Cl--activated argynil aminopeptidases from human brain apd their endopeptidase action on neuropeptides. J Neurochem 50,

32

Nishimura T, Kato Y, Okitani A, Kato H (1991) Purification and properties of aminopeptidase C from chicken skeletal muscle. Agric Bioi Chem 55, 17"/i-1778 Hopsu VK, M[ikinen KK, Glenner GG (1966) Characterization of aminopeptidase B: substrate specificity and affector studies. Arch Biochem Biophys 114, 567-575 Kirschke H, Barrett A (1987) Chemistry of lysosomal proteases. In: Lysosomes: Their role in protein breakdown (Glaumann H, Ballard FJ, eds) Academic Press, London, 193-238 Nishimura T, Rhyu MR, Kato H (1991) Purification and properties of aminopeptidase H from porcine skeletal muscle. Agric Bioi Chem 55, 1779- ! 786

176-182

33 34

35