Unique properties of arginase purified from camel liver cytosol

International Journal of Biological Macromolecules 108 (2018) 88–97

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Unique properties of arginase purified from camel liver cytosol Tahany M. Maharem, Walid E. Zahran ∗ , Rasha E. Hassan, Mohamed M. Abdel Fattah Biochemistry Department, Faculty of Science, Ain Shams University, Cairo 11566, Egypt

a r t i c l e

i n f o

Article history: Received 26 July 2017 Accepted 21 November 2017 Available online 24 November 2017 Keywords: Camel Arginase Purification

a b s t r a c t Arginase (ARG) is an enzyme involved in urea cycle, where it catalyzes the hydrolysis of L-arginine into L-ornithine and urea. Since there is no information about the isolation and purification of ARG from camel liver, this investigation was designed to purify and characterize ARG from camel liver and compare its molecular and kinetic properties with that reported from other species. Camel liver arginase (CL-ARG) was purified to homogeneity using heat denaturation followed by ammonium sulphate precipitation with a combination of DEAE-cellulose, SP-Sepharose and Sephadex G 100-120 chromatography columns. The specific activity of CL-ARG was increased to 18,485 units/mg proteins with 23.5-fold purification over crude homogenate. It was observed that CL-ARG showed a similarity with other species such as behaviour on DEAE-cellulose column, kinetics of inhibition, necessity for metal ions as cofactor, and alkaline optimum pH. On the contrary, CL-ARG differed in its molecular weight (180 kDa), oligomeric protein structure, slightly neutral-alkaline pI value (7.7), Km value (7.1 mM), optimum pH (9, 10.7), and higher optimum temperature (70 ◦ C). In conclusion, this study investigated the properties of CL-ARG via a simple and reproducible purification procedure and provided valuable information for its production from available source in Egypt for medical and industrial purposes. © 2017 Elsevier B.V. All rights reserved.

1. Introduction L-arginase (E.C 3.5.3.1, L-arginine amidinohydrolase, ARG), one of the urea-cycle enzymes, is a binuclear manganese cluster metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea [1,2]. Arginase has roots in early life forms and is widely distributed in the five kingdoms of organisms as diverse as bacteria, yeasts, plants, invertebrates and vertebrates [3]. Arginases have been purified and characterized from a wide variety organisms [4]. Also, the crystal structure of arginases from many species has been solved, including those from Homo sapiens [5], Rattus norvegicus [6] and Bacillus caldovelox [7]. Mammalian arginase is active as a trimer, but some bacterial arginases are hexameric [8]. The enzyme requires a two-molecule metal cluster of manganese in order to maintain proper function. These Mn2+ ions coordinate with water, orienting and stabilizing the molecule by allowing water to act as a nucleophile and attack L-arginine, hydrolyzing it into ornithine and urea [9]. In most mammals, two isozymes of this enzyme exist: cytoplasmic urea cycle arginase I (ARG I) or liver arginase which is highly expressed in the liver primarily to carry out ureagenesis via ammonia detoxification [10,11] and a second mitochondrial isoen-

∗ Corresponding author. E-mail address: walid [email protected] (W.E. Zahran). https://doi.org/10.1016/j.ijbiomac.2017.11.141 0141-8130/© 2017 Elsevier B.V. All rights reserved.

zyme arginase II (ARG II) or nonhepatic arginase which is expressed in trace amounts in extra-hepatic tissues that lack a complete urea cycle, especially kidney, prostate gland, brain and lactating mammary gland [12] which is involved in L-arginine homeostasis [13] and regulating L-ornithine pools for subsequent biosynthetic transformations including the biosynthesis of polyamines, glutamate, proline [14] and controlling tissue level arginine for nitric oxide (NO) biosynthesis [15] by competing with inducible nitric oxide synthase (iNOS) for their common substrate, L-arginine, which is an important determinant of the inflammatory response in various organs and regulating nitric oxide-dependent apoptosis [14,16,17]. ARG I is one of the most important mammalian enzymes responsible for nitrogen metabolism since it comprises the main route for the elimination of excess nitrogen resulting from amino acid and nucleotide metabolism [18]. ARG I deficiency leads to hyperargininemia, characterized by progressive neurological and intellectual impairment, persistent growth retardation and infrequent episodes of hyperammonemia [19]. ARG I and ARG II possess the same catalytic function but differ with respect to tissue distribution, cellular localization, metabolic function, physicochemical and kinetic properties and immunological cross reactivity [4,20]. Arginases from eukaryotes have been under extensive study because they have important biological functions and are associated with a variety of diseases, such as diabetes [20], cardiovascular disorders and cancer progression [13,21]. Arginase, a powerful anti-

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cancer enzyme, has been studied in vitro to treat several types of cancer, such as breast, rectal, and colon, to depletes blood L-arginine levels in order to starving cancer cells that are auxotrophic to L-arginine amino acid and argininosuccinate synthase-expressing tumors [22]. Many studies have shown that the increased stimulation of arginase expression in animal metabolism leads to the production of polyamines, which helps to stop tumor cell proliferation and wound healing [23]. Camel is one of the most common domestic mammals in Egypt, Arab world and the Middle East area, and arginase enzyme has never been purified from camel liver. Therefore, in this study, we attempted for the first time to set up a simple scheme to purify and characterize arginase from the camel liver cytosol to provide the optimal conditions for the production of arginase from camel liver as a locally available source for medical and industrial applications.

2. Materials and methods 2.1. Liver tissue Fresh liver samples of adult male camel Camelus dromedaries obtained from Cairo Slaughterhouse, were collected from at least ten different animals. Liver samples were obtained within an hour from sacrifice and washed with cold physiological isotonic saline to remove contaminating erythrocytes.

2.2. Reagents and chemicals Bovine serum albumin (BSA), L-arginine hydrochloride, thiosemicarbazide, diacetylmonoxime, urea, DEAE-cellulose (preswallen), gel filtration molecular weight marker protein kit (12.4–200 kDa), molecular weight SDS marker proteins (6.5–66 kDa) and isoelectric focusing protein marker kit (pI 3.29.3) were purchased from Sigma-Aldrich. Sephadex G 100-120 and Sulphopropyl Sepharose were purchased from Pharmacia. All other commercially available chemicals were of analytical grade and highest purity.

2.3. Assay of arginase activity Arginase activity was assayed by the colorimetric determination of urea released from the hydrolysis of L-arginine by arginase enzyme using diacetylmonoxime and thiosemicarbazide reagents according to Geyer and Dabich [24] as modified by Dabir et al. [25]. Arginase reaction started by adding the enzyme solution to a reaction mixture (1 ml) containing 50 mM carbonate-bicarbonate buffer (pH 9.5) containing 25 mM L-arginine and 6 mM MnCl2 . The assay reaction mixture was incubated at 37 ◦ C for 30 min then the reaction was stopped by adding 10% TCA. The mixture was centrifuged to remove the denatured protein and the produced urea was determined in the supernatant by adding 2 ml of the color reagent (3.6 mM diacetylmonoxime and 61.7 mM thiosemicarbazide) followed by 3 ml of acid solution (20% sulphuric acid, 56.7% o-phosphoric acid and 0.12 M ferric chloride) to 1 ml of the supernatant. The reaction mixture was placed in a boiling water bath for 10 min then ice cooled. The optical density of the solution was measured against a reagent blank at 520 nm. Urea standard curve was constructed using standard urea to cover the range from 500 to 5000 ␮M. The activity unit is defined as the amount of enzyme that catalyzes the release of 1 ␮mole of urea per minute at 37 ◦ C and pH 9.5.

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2.4. Protein quantification The total protein concentration was determined according to Lowry et al. [26] as modified by Cooper [27], using bovine serum albumin (BSA) as a standard protein. 2.5. Purification of cytosolic camel liver arginase 2.5.1. Preparation of crude extract Twenty g of liver were homogenized on ice in 100 ml of 100 mM Tris-HCl buffer (pH 7.5) containing 5 mM MnCl2 and 100 mM KCl. The liver homogenates were centrifuged at 4 ◦ C at 15,000 xg for 30 min to collect a clear supernatant (cytosol). The clear supernatants containing most of the enzyme activity were saved and the pellets were discarded. 2.5.2. Heat treatment Liver crude extract was incubated at 60 ◦ C for 20 min then ice cooled. The denatured proteins were removed by centrifuging the protein solutions at 5000×g for 10 min and the collected supernatants were designated as heat-treated enzyme solutions. 2.5.3. Ammonium sulphate fractionation The supernatants of heat-treated sample was adjusted to 35% saturation with ammonium sulphate by the addition of solid ammonium sulphate in a 4 ◦ C ice bath with continuous stirring using magnetic stirrer for 30 min and the mixtures formed were centrifuged at 5000×g for 10 min at 4 ◦ C. The precipitate was discarded and the collected supernatant was raised to 75% saturation with ammonium sulphate. After one hour, the mixture was centrifuged at 10,000×g for 10 min at 4 ◦ C to collect the precipitated proteins. The precipitate was completely dissolved in 15 mM Tris-HCl buffer (pH 8) containing 5 mM MnCl2 and 1 mM ␤-mercaptoethanol, using a magnetic stirrer for 15 min in a 4 ◦ C ice bath. The supernatant was collected after centrifugation of the dissolved protein solutions at 4 ◦ C for 5 min at 5000×g. The collected supernatant was dialyzed overnight against 15 mM Tris-HCl buffer (pH 8) containing 5 mM MnCl2 and 1 mM ␤-mercaptoethanol at 4 ◦ C using dialysis cellophane bag. The dialyzed protein solution was centrifuged at 3000×g for 5 min at 4 ◦ C. This protein solution was designated as dialyzed ammonium sulphate fraction. 2.5.4. Diethylaminoethyl (DEAE) cellulose column chromatography The dialyzed ammonium sulphate fraction was mounted on the top of a DEAE-cellulose column previously equilibrated with 15 mM Tris-HCl buffer (pH 8) containing 5 mM MnCl2 and 1 mM ␤-mercaptoethanol. After washing the column, the adsorbed proteins were eluted using an equilibration buffer containing 0–1 M sodium chloride gradient at a flow rate of 30 ml/h. Fractions containing most of the enzyme activities were pooled (DEAE-cellulose pooled fraction) and dialyzed overnight at 4 ◦ C against 15 mM Tris-HCl buffer (pH 5.6) containing 5 mM MnCl2 and 1 mM ␤mercaptoethanol. The dialyzed DEAE-cellulose pooled fractions were concentrated using Amicon ultrafiltration apparatus YM10 membrane. 2.5.5. Sulphopropyl (SP) sepharose column chromatography The concentrated dialyzed DEAE-cellulose fraction was mounted on the top of SP-Sepharose column, which was previously equilibrated with 15 mM Tris-HCl buffer (pH 5.6) containing 5 mM MnCl2 and 1 mM ␤-mercaptoethanol. After washing the column, the adsorbed proteins were eluted using an equilibration buffer containing 0–1 M sodium chloride gradient at a flow rate of 20 ml/h. Fractions containing most of the enzymes activities were

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Fig. 1. DEAE-cellulose column chromatography of camel liver ammonium sulphate fractions. A stepwise gradient of 0-1 M NaCl in equilibration buffer was used to elute fractions 1-160 at a flow rate 30 ml/h and 5 ml/fraction [-䊉- OD at 280 nm & -о- arginase activity (units/ml)].

pooled and dialyzed overnight against 15 mM Tris-HCl buffer (pH 7.5) containing 5 mM MnCl2 and 1 mM ␤-mercaptoethanol. 2.5.6. Sephadex G 100-120 gel filtration chromatography The dialyzed pooled SP-Sepharose sample was mounted on the top of previously equilibrated Sephadex G 100-120. The column was washed with the equilibration buffer at a flow rate 20 ml/h and 2 ml/fraction. Protein elution was monitored by reading the absorbance at 280 nm and the enzyme activity was assayed in the collected fractions.

2.9. Determination of manganese content The manganese content of the purified CL-ARG was determined in the Central Laboratory at the Faculty of Science (Ain Shams University) using PERKIN Elmer 3100 Atomic Absorption Spectrometer.

3. Results 3.1. Purification of CL-ARG

2.6. Molecular weight determination The molecular weight of CL-ARG was determined by gel filtration chromatography according to the method of Andrews [28] using Sephadex G 100-120 column. The column was equilibrated with 50 mM Tris-HCl buffer (pH 7.5) containing 100 mM KCl. The void volume (Vo ) was determined with blue dextran (2000 kDa). The column was calibrated with standard molecular weight protein markers and washed with the equilibration buffer. Protein fractions were collected in a 2 ml volume at a flow rate of 20 ml/h. The plots of log molecular weight of the protein markers versus the elution volume/void volume (Ve /Vo ) were constructed.

2.7. Electrophoretic analyses The subunit molecular weights of the purified CL-ARG was determined according to Weber and Osborn [29] using 15% SDS-PAGE as described by Laemmli [30] from a standard curve constructed from molecular weight protein markers. CL-ARG isoelectric point (pI) determined according to O’Farrell [31] using 5% polyacrylamide gel as described by Ubuka et al. [32] from a calibration curve of protein markers. Proteins were stained with 0.25% coomassie brilliant blue R-250.

The typical purification scheme of CL-ARG is summarized in Table 1. The specific activity of CL-ARG in the crude extracts was estimated to be 786 units/mg protein. The dialyzed ammonium sulphate precipitation protein solution of camel extract had a specific activity of 3596.5 units/mg protein with an increased fold purification of 4.58 over the crude homogenate. Fig. 1 shows that arginase from camel liver was unadsorbed on DEAE-cellulose and eluted in the equilibration buffer just after the void volume in a single sharp peak. The concentrated dialyzed pooled DEAE-cellulose enzyme solution of camel extract had a specific activity of about 6470.6 units/mg protein with 8.23-fold purification. SP-Sepharose elution profile of CL-ARG, Fig. 2, shows that the enzyme was completely adsorbed on the top of the matrix and eluted in a single sharp peak in 0.1 M NaCl. The dialyzed SP-Sepharose pooled fractions of the adsorbed CL-ARG had specific activity of 17949 units/mg protein and an increased fold of purification up to 22.8 over the crude homogenate. The obtained profile of Sephadex G 100-120 column, Fig. 3, shows that CL-ARG was eluted as a single peak after the void volume of the column and had an increase in specific activity up to 18485 units/mg protein with a raised purification up to 23.5-fold and a 6.1% recovery.

3.2. Purity and homogeneity 2.8. Determination of carbohydrate content The carbohydrate content of CL-ARG was determined using the anthrone method according to Plummer [33] by constructing a glucose standard curve in the range of 2–20 ␮g glucose, then the hexose content in the purified enzyme as g hexose/100 g protein was elucidated.

SDS-PAGE was carried out to examine the homogeneity of different purification steps. It appears from Fig. 4 that Sephadex G 100-120 enzyme sample of camel liver was separated from large amounts of contaminated proteins during the purification process and shows a single band which indicates that the enzyme preparation after this step of purification was homogenous.

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Table 1 A typical purification scheme of CL-ARG.

*

Steps of purification

Total activity (unit)*

Total protein (mg)

Specific activity**

Fold purification

Yield (%)

1 2 3 4 5 6 7

1,670000 1,566000 1,060000 984,000 264,000 113,898 101,667

2120 870 288 273.6 40.8 6.63 5.5

786 1800 3703.7 3596.5 6470.6 17949 18485

– 2.29 4.71 4.58 8.23 22.8 23.5

100 93.8 63.9 58.9 15.8 6.8 6.1

20% Crude extract Heat treatment Ammonium sulphate fraction (35-75%) Dialyzed ammonium sulphate fraction DEAE-cellulose column (pooled fraction) SP-Sepharose column (adsorbed fraction) Sephadex G 100-120 column (pooled fraction)

Arginase unit is expressed as the amount of enzyme that catalyzes the release of 1 ␮mole urea per minute at 37 ◦ C and pH 9.5. Specific activity expressed as units/mg protein.

**

Fig. 2. SP-Sepharose column chromatography of concentrated dialyzed DEAE-cellulose pooled fractions. A stepwise gradient of 0-1 M NaCl in equilibration buffer was used to elute fractions 1-80 at a flow rate 20 ml/h and 1 ml/fraction [-䊉- OD at 280 nm & -о- arginase activity (units/ml)].

Fig. 3. Gel filtration of dialyzed SP-Sepharose adsorbed fraction on Sephadex G 100–120 column at a flow rate 20 ml/h and 2 ml/fraction [-䊉- OD at 280 nm & -о- arginase activity (units/ml)].

3.3. Native molecular weight determination For native molecular weight determination of CL-ARG, Sephadex G-100-120 gel filtration column was used. One milliliter of the purified CL-ARG (20,333 units, 1.1 mg protein) was applied on the top of a Sephadex G 100-120 column (100 × 1.6 cm), previously equilibrated with 50 mM Tris-HCl buffer (pH 7.5) containing 100 mM KCl

and the proteins were eluted with the equilibration buffer. Fractions were collected in a volume of 2 ml at a flow rate of 20 ml/h. The absorbance at 280 nm was detected in the collected fractions and the elution volume (Ve ) was determined. Knowing the void volume of the column using dextran blue (Vo ), then Ve /Vo can be calculated for each marker of standard proteins with known molecular weights. Ve /Vo of CL-ARG was calculated to be 1.1 giving a

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T.M. Maharem et al. / International Journal of Biological Macromolecules 108 (2018) 88–97 Table 2 Effect of metal ions on purified CL-ARG. Metal Ion (6 mM)

Remaining Activity (%)

Before Dialysis After Dialysis Mn+2 Co+2 Fe+3 Ni+2 Mg+2 Sr+3 Zn+2

100 38.7 96 95 93.5 53 48.3 24 21

ted against 1/[S]. The Km value for CL-ARG was calculated to be 7.1 mM. 3.8. Determination of optimum temperature Fig. 4. SDS-PAGE purification steps pattern of camel liver samples. (1) crude extract, (2) heat treatment, (3) ammonium sulphate fraction, (4) DEAE-cellulose pooled fraction, (5) SP-Sepharose adsorbed fraction, and (6) Sephadex G 100–120. Proteins stained with Coomassie Brilliant Blue R-250 stain.

molecular weight of about 180 kDa calculated from the calibration curve of standard protein markers.

To assess the effect of incubation temperature on the activity of CL-ARG, the enzyme assay method was used with varying incubation temperatures (10–90 ◦ C). It can be seen from Fig. 7 that CL-ARG activity was increased as the temperature increased giving an optimum temperature at 70 ◦ C followed by a sharp decrease in the activity at 80 ◦ C and 90 ◦ C.

3.4. Electrophoretic analyses

3.9. Effect of pH

PAGE under denatured condition was carried out to determine the molecular weight of the purified CL-ARG using standard protein markers [␣-lactalbumin (14.2 kDa), trypsin inhibitor (20.1 kDa), tripsinogen (24 kDa), glyceraldehydes-3-phosphate (36 kDa), egg albumin (45 kDa) and bovine serum albumin (66 kDa)](Fig. 5a). The relative mobility (Rf ) for CL-ARG was calculated to be 0.4. This Rf value corresponds to an apparent molecular weights of 35 kDa derived from the calibration curve of the standard proteins. Also, native IEF was used to determine pI of CL-ARG. Trypsinogen (pI 9.3), lectin (pI 8.8, 8.6, 8.2), myoglobin (pI 7.2, 6.8), carbonic anhydrase I (pI 6.6), carbonic anhydrase II (pI 5.9), ␤-lactoglobulin (pI 5.1), trypsin inhibitor (pI 4.6) and amyloglucosidase (pI 3.6) were used as standard proteins. Fig. 5b shows that purified CL-ARG was separated according to its pI into a single band with Rf value of 0.33. This Rf value corresponds to pI value of 7.7 derived from the calibration curve of the standard proteins. Thus, it can be concluded that CL-ARG has a slightly basic pI.

The effect of pH on CL-ARG activity was studied in the pH range 3.6–10.7 using 50 mM sodium acetate buffer (pH range 3.6–5.6), 50 mM potassium phosphate buffer (pH range 6–8), 50 mM Tris-HCl buffer (pH range 7.2–9) and 50 mM carbonate bicarbonate buffer (pH range 9.3–10.7). Arginase activity was assayed at 37 ◦ C using the enzyme assay method in various pH buffer solutions. It can be seen from Fig. 8 that CL-ARG activity as a function of pH was increased as the pH increased up to pH 9 and pH 10.7 of 50 mM TrisHCl buffer and 50 mM carbonate bicarbonate buffer, respectively. In the pH range (6–8) using 50 mM potassium phosphate buffer, the enzyme activity was increased as the pH increased up to pH 8, while in the pH range (3.6–5.6) using 50 mM sodium acetate buffer, CL-ARG did not exhibit any activity.

3.5. Carbohydrate content To investigate whether arginase might be a glycoprotein, the anthrone method was carried out to determine the carbohydrate content in the purified enzyme. The results indicate that CL-ARG contains about 2.7 g hexose/100 g protein. 3.6. Manganese (Mn+2 ) content Atomic absorption spectrographic data obtained for purified CLARG revealed the presence of 3.5 ␮g Mn+2 /mg protein. 3.7. Michaelis-Menten constant (Km) value To study the effect of substrate concentration on CL-ARG reaction velocity, arginine concentration in the range (10–50 mM) was used and the enzyme assay method as mentioned was performed. Fig. 6 (a and b) reveals that CL-ARG exhibited a typical Michaelian behaviour in the range of arginine concentration described for each reaction and a linear relationship was obtained when 1/V was plot-

3.10. Effect of metal ions To assess the effect of some metal ions on the purified CLARG activity, enzyme samples were dialyzed for two days against distilled water to remove the loosely bound Mn+2 ions. CL-ARG dialyzed portions were treated before the assay with different metal ions (Mn+2 , Mg+2 , Co+2 , Ni+2 , Zn+2 , Sr+3 and Fe+3 ) in the concentration of 6 mM for each metal ion. Table 2 shows that CL-ARG retained 38.7% of its original activity after dialysis. In the presence of Mn+2 ion as a cofactor, CL-ARG regained 96% of its initial activity. It was found that CL-ARG regained 95% of its original activity in the presence of Co+2 and Fe+3 , respectively as cofactors, indicating that they act as good activators for the enzyme. On the contrary, it was observed that Sr+3 and Zn+2 act as potent inhibitors for CL-ARG. 3.11. Kinetics of inhibition The effect of selected amino acids on the activity of the purified CL-ARG was performed. The results reveal that as the amino acid concentration increased in the assay reaction mixture the remaining activity decreased (not shown). The inhibitory effect of these amino acids (L-ornithine, L-lysine, L-valine, L-leucine, and DL-isoleucine) towards CL-ARG was investigated via constructing Lineweaver-Burk plots of the reciprocal of initial reaction velocity against reciprocal of arginine concentration in the presence of vari-

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Fig. 5. Electrophoretic analysis of (a) 15% SDS-PAGE and (b) 5% IEF gel for CL-ARG (C) with respect to standard protein markers (M). Proteins stained with Coomassie Brilliant Blue R-250 stain.

Fig. 6. (a) Michaelis-Menten plot of purified CL-ARG as a function of arginine concentration. (b) Lineweaver-Burk plot relating purified CL-ARG to arginine concentration. The enzyme assay method used with varied arginine concentration (10–50 mM).

Fig. 7. Effect of temperature on the purified CL-ARG.

ous concentrations of amino acids (Table 3 & Fig. 9). It was observed that these amino acids act as competitive inhibitors for CL-ARG. Also, the IC50 (the concentration of inhibitor required to produce

50% inhibition of the enzyme activity) value for each amino acid was determined by measuring the activity of the arginase enzyme in the presence of varying concentrations of amino acids and plotting the

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Fig. 8. Effect of pH on the activity of purified CL-ARG using 50 mM sodium acetate buffer pH (3.6-5.6), 50 mM potassium phosphate buffer pH (6–8), 50 mM Tris-HCl buffer (7.2-9) and 50 mM carbonate-bicarbonate buffer pH (9.3-10.7).

Table 3 IC50, Ki and inhibition type of amino acids on purified CL-ARG. Amino acid

Inhibition Type

IC50 (mM)

Ki (mM)

L-ornithine l-lysine L-valine L-leucine DL-isoleucine

competitive competitive competitive competitive competitive

24 22 13 9 9.5

30 8 5 10 17.5

remaining activity percent values versus Log amino acid concentration (not shown). The concentrations of L-ornithine causing 50% inhibition for the activity of CL-ARG was calculated to be 24 mM (Table 3). It was observed that 22 mM of l-lysine caused 50% inhibition to CL-ARG activity while that of L-valine equals 13 Mm. On the other hand, 9 and 9.5 mM of L-leucine and DL-isoleucine reduced the initial activity of CL-ARG to 50%, respectively (Table 3). It could be concluded that L-leucine is the most potent inhibitor for CLARG followed by DL-isoleucine, L-valine, l-lysine and L-ornithine. The Ki values (inhibitor constant) for these amino acids were determined by plotting 1/Vi versus amino acid concentrations at varying concentrations of L-arginine (Table 3). 4. Discussion Arginases are ubiquitous in nature, having been found in bacteria, fungi, plants, and mammals [4]. In mammals, arginase has been detected in many different tissues having complete and incomplete urea cycle, such as liver, kidney and mammary gland [34]. Aminlari and Vaseghi [35] reported that the liver was the richest source of arginase in most of domestic animals. However, there is no information about the purification and the properties of this enzyme in camel, the current study was designed to set up a simple scheme to purify arginase from camel liver cytosol and compare its molecular and kinetic properties with that were reported from other species. In the present investigation, the purification procedure permitted us to obtain a highly purified enzyme preparation. The overall yield of purified CL-ARG being almost 6.1% with a final specific activity of 18485 units/mg protein. ␤-Mercaptoethanol was used in all the purification steps to prevent the oxidation of the native enzyme during purification and thus decreasing the formation of multiple active forms [36]. Harell and Sokolovsky [37] purified the beef liver arginase to a homogenous state of 10.2% yield with a specific activity of 790 units/mg protein using heat treatment, acetone

fractionation, DEAE-cellulose column chromatography, molecular sieving and isoelectric focusing. Also, Dabir et al. [25] purified buffalo liver arginase to a homogenous state with a yield of 18.3% using heat treatment, ammonium sulphate fractionation, DEAEcellulose column chromatography, molecular sieving and affinity chromatography. The purified CL-ARG preparation gave a single diffuse protein band that migrated towards the anode throughout native PAGE at pH 8.8. The anionic behaviour in electrophoresis relates the camel liver enzyme to human, rabbit, horse, calf and pig liver arginases [36]. The native molecular weight of purified CL-ARG was determined by size-sieving chromatography. Sephadex G 100-120 gel filtration column revealed that CL-ARG exhibited a molecular weight about 180 kDa. The sedimentation and diffusion studies carried out by Hirsch-kolb et al. [38] with high purified beef liver arginase suggested a molecular weight between 120 and 140 kDa. Molecular weights in the range from 120 to 160 kDa obtained for ureotelic liver arginases differ remarkably from the molecular weights recorded for uricotelic and fungal arginases (lizard liver arginase molecular weight equals 276 kDa and arginase from Neurospora crassa has a molecular weight of 278 kDa), which seems to be twice higher [39,40]. Using SDS-PAGE, resolution of purified CLARG exhibited a single band corresponding to apparent molecular weights of 35 kDa, indicating its oligomeric structure. Jenkinson et al. [3] showed that the subunit molecular masses for mammalian arginases vary between about 30 kDa and 40 kDa. Arginase from lizard and chicken livers has a molecular weight of about 280 kDa which represents an association of two tetramers to form an octamer [40]. The molecular weight of earth warm gut arginase was estimated to be 27 kDa, which represents a monomer arginase [41]. The isoelectric point of CL-ARG was determined by native IEF at a pH gradient in the range (3–10) using standard protein markers with known isoelectric points. Native IEF reveals the presence of only one major band for CL-ARG with a slightly neutral-basic pI value of about 7.7. The calculated pI value for the purified CL-ARG coincides with the behaviour of the enzyme on the anion and cation exchanger columns chromatography. Mammalian liver arginases tend to have basic pI values of about 8.8-9.4 as rat liver arginase (9.4) and human liver arginase (9), while pI values closer to a neutral pH exist in beef liver arginase (5.9), pig liver arginase (6.9 and 8.8), rabbit liver arginase (6.5–7.2) and monkey liver arginase (6.8–7.5) [3]. Türkoglu and Özer [42] separated bovine liver arginase into three distinct peaks by chromatofocusing in the pH range 4–7 since they

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Fig. 9. Lineweaver-Burk plot relating purified CL-ARG reaction velocity to arginine concentration in absence and presence of different L-ornithine, l-lysine, L-valine, L-leucine and L-isoleucine concentrations with varied concentrations of L-arginine and constant concentration of CL-ARG (0.036 mg protein/assay).

concluded that the enzyme was homogenous towards subunit size and kinetic behaviour but heterogeneous via its molecular charge. By investigating the enzyme composition, sample of the purified CL-ARG was assayed with anthrone to detect the carbohydrate content. The assays yielded a hexose content of 2.7%. Mammalian liver arginase contains little or no bound carbohydrate. Hexose sugar content for rat and beef liver arginases has been estimated to be 1–3% and 3–5%, respectively [37,43], while there was no carbohydrate detected in human liver arginase [44].

The effect of substrate concentration on the reaction velocity of purified CL-ARG was examined using arginine concentration in the range (10–50 mM). The results reveal the enzymatic hyperbolic dependence of hydrolysis rate on substrate concentration and a linear relationship was obtained when 1/v was plotted against 1/[S]. The Michaelis constant (Km ) of CL-ARG was calculated to be 7.1 mM. Beef liver arginase has a Km value of 14 mM [45], the Km value of buffalo liver arginase was 2 mM [25], that of human liver arginase

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was 5.4 mM [46], the Km values recorded for rabbit, monkey and horse were 1.4 mM, 6.5 mM and 4.6 mM, respectively [38]. The major variable in the kinetics of the arginase reaction is the pH determinant, which reflects the amphoteric character of both the substrate (L-arginine) and the enzyme. Another factor is the effect of metal ion cofactor on the dissociation of ionizable groups of the catalytically active center of the enzyme [47]. CLARG activity as a function of pH increased giving two optimum pH at 9 and 10.7 using 50 mM Tris-HCl buffer and 50 mM carbonatebicarbonate buffer, respectively. The optimum pH of buffalo liver arginase is 9.2 [25], human liver arginase has an optimum pH of 9.3 [36]. The pH optima of rabbit liver arginase is 10, that of monkey is 9.5-10.5 while that of horse is 10.2 [38]. Gasiorowska et al. [48] found four differently charged isozymes of arginase in different rat tissues one of which had a pH optimum of 7.5 also a minor arginase component was reported by Robbins and Shields [49] in beef liver with an optimal pH 7. The variation of activity with pH suggests titration of an ionizable group that may function at the catalytic site. The basic pH-activity profile may reflect ionization of manganese-bound water [3]. Also, our study showed that CL-ARG has an optimum temperature at 70 ◦ C. It was found that arginase from buffalo liver showed maximum activity at 42 ◦ C [25]. A common feature of arginases, whether of eukaryotic or prokaryotic origin, is a requirement of divalent cations for activity. Mn+2 is the physiologic activator, although the divalent cation requirement for some arginases has been reported to be satisfied by Co+2 and Ni+2 and in some instance by Fe+2 and Cd+2 . It was shown that fully Mn+2 -activated arginase contains 2 Mn+2 /subunit and these Mn+2 ions form electron spin-coupled binuclear centers [50]. In the present study, Fe+3 and Co+2 were found to act as good activators while Sr+3 and Zn+2 act as inhibitors for CL-ARG. Dabir et al. [25] observed the activation of buffalo liver arginase with Mg+2 , Ca+2 , Co+2 , Ni+2 , Cd+2 and its inhibition by Zn+2 . Arginases from several species and tissues have been found to be inhibited by amino acids [51]. In the present study, monocarboxyilic amino acids with five or more carbon atoms such as ornithine, lysine, valine, leucine and isoleucine inhibited CL-ARG. The results revealed that as the amino acid concentration increased in the assay reaction mixture the remaining enzyme activity decreased. The Linweaver-Burk plots revealed that all the tested amino acids were competitive inhibitors for CL-ARG. The competitive inhibition would be explained by a conformational change induced by the substrate arginine which prevents the binding of the inhibitor to the enzyme. The existence of allosteric sites on arginase molecule has been suggested for the enzyme from bovine liver [52], human liver [53], rat kidney [54] and rat liver [55]. In parallel with arginase buffalo liver [25], ornithine and lysine caused competitive inhibition like CL-ARG. Finally, this work represents a reproducible and simple method for CL-ARG purification from the camel liver cytosol as a locally available rich source in Egypt for medical and industrial purposes. Cheng et al. [56] and Lam et al. [57] showed that the application of pegylated recombinant human arginase I (rhArg-PEG) either alone or in combination with chemotherapeutic drugs might represent a specific and effective therapeutic strategy against advanced hepatocellular carcinoma (HCC). They suggest that rhArg-PEG is a good candidate for the treatment of HCC, whose anti-cancer activity is mediated by the depletion of arginine, resulting in a cell cycle arrest in HCC cells. They developed a recombinant form of human ARG I, covalently modified with polyethylene glycol (PEG) via a succinimidyl propionate (SPA) linker. Pegylation greatly increased the enzyme’s half-life without affecting its enzymatic activity. The prepared rhArg-PEG inhibited the proliferation of HCC cell lines (HepG2, Hep3B, PLC/PRF/5, Huh7 and SK-HEP-1). Towards industrial applications, a great demand exists for L-ornithine production

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