Purification and properties of an alanine aminopeptidase from camel liver

Article history: Received on: 03/10/2016 Accepted on: 20/01/2017 Available online: 30/05/2017 Alanine aminopeptidase is purified from camel liver to homogeneity and designated CLAAP. The purification procedure involved anion exchange chromatography on DEAE-cellulose column and gel filtration chromatography on Sephacryl S-300 column. The specific activity of CLAAP is increased to 9.9 folds over the crude extract with 25.3% yields. The purified CLAAP is homotrimmer protein with 180 kDa consists of three identical subunits of 60 kDa each. CLAAP displayed its optimum activity at pH 8.0 and the Km value is 0.083 mM alanine β-naphthylamide. The divalent cations CuCl2, MnCl2 and ZnCl2 inhibited CLAAP activity while CoCl2 and MgCl2 increased its activity. CLAAP was inhibited competitively with bestatin that has one binding site on the enzyme and Ki value of 14 μM. This report represents AAP purified from the camel liver as a safe source. Therefore, a task for the future will be the application of this purified CLAAP enzyme in the industry of meat and dairy products for flavor development.


INTRODUCTION
Aminopeptidases are exopeptidases proteins that remove the N-terminal amino acid of their protein substrates (Niven, 1995;Erbeznik and Hersh, 1997).These enzymes are widespread in prokaryotes, mammals and plants and their substrate specificities depending on their physiological roles (Barrett et al., 1998;Umetsu et al., 2003;Matsui et al., 2006;Bogra et al., 2009;Mane et al., 2010;Renwrantz and Lam, 2010).These proteins have substantial tasks in numerous physiological processes as it regulates certain pathways in metabolism, controls the cell cycle and selectively degrades certain proteins (Brownless and Williams, 1993).They can be classified into two main types, one hydrolyzes the amino acids with hydrophobic side chains from N-terminus of the protein, and the other removes other amino acid residues (Drag et al., 2005).Alanine aminopeptidases are present in almost all mammalian tissues including intestine, kidney, liver and pancreas (Mane et al., 2010).They are present in many human organs including; seminal plasma (Huang et al., 1997), liver (Yamamoto et al., 2000), placenta (Mizutani et al., 1993), pancreas (Sidorowicz et al., 1980), kidney (Mantle et al., 1990), gallbladder bile (Offner et al., 1994) and skeletal muscle (Mantle et al., 1983).They are present in other mammalian tissues as bovine skeletal muscle (Ye and Ng, 2011) and porcine kidney (Itoh and Nagamatsu, 1995).Mammalian aminopeptidases take part in metabolism of neurotransmitters and hormones (Hiroi et al., 1992;Umetsu et al., 2003), used as kidneys damage biomarker and used in diagnosis of certain kidney troubles as they are present at elevated urine percentages if there are kidney disorders (Flynn, 1990).For meat and dairy products industry, aminopeptidases share the flavor development as a direct action by removing bitter peptides which have high ratio of hydrophobic amino acids that are considered as perfect substrates for its action, or indirectly, where aminopeptidases may remove the aromatic amino acids that are main substrates of aromatic molecules present in cheese (Fernandez-Espla and Rul, 1999;Magboul and McSweeney, 1999;Martinez-Cuesta et al., 2001;Ye and Ng, 2011).This report represents purification and characterization of alanine aminopeptidase from camel liver as a safe source for the various industrial applications of aminopeptidases.

Liver tissues
Six fresh camel liver samples were obtained from different individuals in a local slaughter-house and stored at -40 ˚C.

Assay of alanine aminopeptidase activity
The alanine aminopeptidase activity assay reaction mixture contained in 1.5 ml of 0.1 M Tris-HCl buffer pH 8, 0.4 mM alanine-β-naphthylamide and the enzyme solution.Incubate for 1 hour at 37˚C; stop the reaction with 0.5 ml Fast Garnet GBC solution (1 mg / ml) in 1 M acetate buffer pH 4.2 containing 10% Tween 20 and read absorbance at 525 nm against control lacking enzyme.The concentration of the liberated β-naphthylamine was calculated from a standard curve of β-naphthylamine treated similarly.One unit of alanine aminopeptidase activity is the amount of enzyme which catalyzes the liberation of 1 nmol of βnaphthylamine per hour at 37 ˚C (Kawata et al., 1980).

Staining of alanine aminopeptidase activity on PAGE
After electrophoresis, the gel was incubated in 50 ml 0.1 M sodium phosphate buffer pH 5.8 containing 1 mM CoCl 2 , 0.06% alanine β-naphthylamide HCl and 0.06% Fast Garnet GBC salt until the develop of the red bands.The stained gel was washed with water and then fixed in 7% acetic acid (Chien et al., 2002).

Purification of camel liver alanine aminopeptidase Preparation of crude extract
All of the procedures were performed at 4˚C unless stated otherwise.10 grams of camel liver were homogenized in 0.02 M sodium phosphate buffer pH 7.4 using Omni mixer homogenizer.Cell debris and insoluble materials were removed by centrifugation at 5000 x g for 20 min and the supernatant was saved and designated as crude extract.

DEAE-cellulose column Chromatography
The crude extract was chromatographed on DEAEcellulose column previously equilibrated with 0.02 M sodium Phosphate buffer pH 7.4.The proteins were eluted with stepwise NaCl gradient ranging from 0 to 1 M followed by 0.5% Triton X-100 prepared in the equilibration buffer.Fractions of 5 ml were collected at a flow rate of 60 ml/hour.Fractions containing alanine aminopeptidase activity were pooled and concentrated by lyophilization.

Sephacryl S-300 column Chromatography:
The concentrated solution containing the alanine aminopeptidase activity was applied onto a Sephacryl S-300 column (142 cm x 1.75 cm i.d.).The column was equilibrated and developed with 0.02 M sodium Phosphate buffer pH 7.4 at a flow rate of 30 ml / hour and 2 ml fractions were collected.

Electrophoretic analysis
Native gel electrophoresis was carried out with 7% PAGE (Smith, 1969).The subunit molecular weight of the purified enzyme was determined by 12% SDS-PAGE (Weber and Osborn, 1969;Laemmli, 1970).Coomassie brilliant blue R-250 was used to stain the proteins.

Protein determination
Protein content was determined by the dye binding assay method using BSA as a standard protein (Bradford, 1976).

Purification of camel liver alanine aminopeptidase
The purification of the major AAP from camel liver crude extract was monitored by ability of AAP to hydrolyze the substrate alanine-β-naphthylamide and the results of the purification is outlined in Table (1).The camel liver crude extract AAP specific activity was measured as 436 units/mg protein.Alanine aminopeptidase was purified with two chromatographic steps on DEAE-cellulose and Sephacryl S-300 columns.The elution profile of AAP on the anion exchange column showed one principal AAP activity peak (Fig. 1a) collected from the column with 0.5% Triton X-100 and designated as CLAAP and a minor AAP activity peak eluted with 0.2 M NaCl.The CLAAP peak was collected, lyophilized and further purified by a gel filtration Sephacryl S-300 column (Fig. 1b).After gel filtration chromatography, the CLAAP was purified with 4331 units/mg specific activity representing 9.9 folds and 25.3% yield.The CLAAP enzyme eluted from Sephacryl S-300 column with a native molecular mass of 180 kDa.

Analyses of CLAAP on polyacrylamide gel electrophoresis
The CLAAP enzyme purification cycles including liver extract, anion exchange fraction and gel filtration fraction were seen on 7 % native PAGE (Fig. 2a).The purified enzyme was seen as a single protein band corresponded with the enzyme activity band (Fig. 2b) demonstrating the tentative purity of the preparation.CLAAP enzyme was seen on SDS-PAGE as a single band of 60 kDa (Fig. 2c) which indicates the subunit molecular weight of the purified enzyme.
The Lineweaver-Burk plot calculates the Km value of the purified enzyme from (1/v) and (1/[S]) to be 0.083 mM alanine βnaphthylamide (Fig. 3b).   ) previously equilibrated with 0.02 M sodium Phosphate buffer pH 7.4.The proteins were eluted by a stepwise gradient of NaCl ranging from 0 to 1 M followed by 0.5% Triton X 100 in the equilibration buffer.5 ml fractions were collected at a flow rate of 60 ml / h.(b) A typical elution profile for the chromatography of CLAAP on Sephacryl S-300 column (142 cm x 2.4 cm i.d.) previously equilibrated with 0.02 M sodium Phosphate buffer pH 7.4.The proteins were elute d by the same buffer and 2 ml fractions were collected at a flow rate of 30 ml / h.

Effect of divalent cations and various inhibitors on CLAAP activity
The purified camel liver alanine aminopeptidase CLAAP was pre-coupled with two different concentrations of all divalent cations at 37˚C then test the enzyme activity.These measures were compared with a control test that has no cations.CuCl 2, MnCl 2 and ZnCl 2 inhibited the activity of CLAAP while CoCl 2 and MgCl 2 were found to be activators of the enzyme activity (Table 3).Furthermore, we preincubated the CLAAP with different inhibitors for 5 min at 37˚C then calculated the percent of inhibition in comparison with control with no inhibitors.Bestatin was the potent CLAAP enzyme activity inhibitor (Table 4).

Kinetics of CLAAP inhibition by Bestatin
The purified enzyme CLAAP activity was inhibited strongly with bestatin.The enzyme was subjected to inhibition with increasing concentrations of bestatin making a titration curve (Fig. 4a) demonstrating that 50% inhibition (I 50 ) of CLAAP activity was reached with 30 µM bestatin.Maximum enzyme inhibition of 97.8 % was reached by 100 µM bestatin.When the Hill plot for CLAAP inhibition by bestatin was constructed a linear relationship was obtained and the slope of the Hill plot was found 1.11 (Fig. 4b).The CLAAP activity inhibition by bestatin was a competitive type (Fig. 4c) with a Ki value of 14 µM (Fig. 4d).

DISCUSSION
Alanine aminopeptidases are widespread in mammalian tissues and body fluids.They have an important role in liberation of the neutral amino acids of proteins as Alanine, Leucine, Methionine and Tyrosine (Mane et al., 2010).In this study, the camel liver AAP is purified by a simple and reproducible method consists only of two column chromatographic steps which were found to be convenient to set a homogenous form of the enzyme.Separation of the camel liver crude extract on DEAE-cellulose column revealed the existence of one major form of camel liver alanine aminopeptidase termed CLAAP and another minor form (Fig. 1a).CLAAP eluted from the Sephacryl S-300 column as one peak of AAP activity with a native molecular weight of 180 kDa (Fig. 1b) while its subunit molecular weight is estimated at 60 kDa by SDS-PAGE indicating that the enzyme is homotrimmeric protein consists of three identical subunits (Fig. 2c).The molecular mass of CLAAP is similar to that of 180 kDa alanine aminopeptidase of chicken intestine (Mane et al., 2010) and higher than that of 160 kDa chicken intestine (Jamadar et al., 2003), 153 kDa human seminal plasma (Huang et al., 1997) and 60 kDa bovine skeletal muscle (Ye and Ng, 2011).The specific activity of the prepared CLAAP isoenzyme is increased to 4331 U/mg protein with 25.3% recovery and 9.9 folds purification (Table 1).Very low recovery of 3.43% was reported for chicken intestine alanine aminopeptidase (Mane et al., 2010).CLAAP homogeneity is proved by native polyacrylamide gel with a single band for the purified protein which coincided with the band of enzyme activity (Fig. 2a and 2b).
In this study, the effect of various inhibitors on the purified camel liver AAP revealed that, CLAAP was resistant to DTT, N-Ethylmalimide, β-Mercaptoethanol and TPCK.It was inhibited with bestatin, glutathione, 1,10 phenanthroline, PMSF, puromycin, soya bean trypsin inhibitor and TLCK.The purified CLAAP is suggested to have a cysteine protease activity due to its sensitivity to the cysteinyl protease inhibitor pchloromercuribenzoic acid.It also suggested having a serine protease activity due to the sensitivity to PMSF and soya bean trypsin inhibitor suggesting the presence of serine residue in the enzyme active site.The CLAAP inhibition with 1, 10 phenanthroline indicates that CLAAP is a metalloenzyme in similarity with porcine liver (Imamura et al., 1983) and rabbit kidney aminopeptidases (Oliveira et al., 1999).Bestatin is reported as a specific inhibitor of alanyl aminopeptidases (Tieku and Hooper, 1992).Here, bestatin inhibited CLAAP vigorously since 100 µM bestatin inhibited the enzyme completely (Table 4) confirming that it is an alanine aminopeptidase.A straight line was obtained when drawing the Hill plot for the bestatin inhibition of the purified CLAAP.The Hill plot has a slope of 1.11 indicating that there is one binding site for bestatin on CLAAP molecule (Fig. 4b).Bestatin inhibited CLAAP competitively since the bestatin existence did not alter the Vmax value and increased the Km value (Fig. 4c).To determine the Ki value, we plotted slopes of the reciprocal plots lines against the bestatin concentrations.The CLAAP bestatin inhibition Ki value was found to be 14 µM directly from the plot X axis intercept (Fig. 4d).The low Ki value proves that the bestatin is a potent inhibitor of alanine aminopeptidases.In conclusion, this report presents a simple, convenient and reproducible method for purification of alanine aminopeptidase from the camel liver as a safe, locally available and rich source.Large scale production of this well characterized enzyme will allow its using in different applications as in meat and dairy products industry in removing pitter peptides and in examination of the protein primary structure.

Fig. 3 :
Fig. 3: (a) Effect of pH on the purified CLAAP using 0.05 M potassium phosphate buffer, pH (7.0 -9.0).(b) Lineweaver-Burk plot relating the reciprocal of the reaction velocity of the purified CLAAP to alanine βnaphthylamide concentration in mM.
(a) A typical elution profile for the chromatography of camel liver crude extract on DEAE-cellulose column (6 cm x 2.4 cm i.d.

*
These values represent % of the control and the means of triplicate experiments.
(a) Inhibition of CLAAP by varying concentrations of Bestatin.(b) Hill plot for inhibition of CLAAP by Bestatin.(c) Lineweaver-Burk plots showing the type of inhibition of CLAAP by Bestatin.(d) Determination of the inhibition constant (Ki) value for the inhibition of the CLAAP by Bestatin.

Table 3 :
Effect of divalent cations on the purified camel liver alanine aminopeptidase CLAAP.

Table 4 :
Effect of inhibitors on the purified camel liver alanine aminopeptidase (CLAAP).