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This study, “CHARACTERIZATION OF ARGINASE FROM THE LIVER OF MANGO TILAPIA (Sarotherodongalilaeus)” contains concise information that will serve as a framework or guide for your project work. The project study is well-researched for academic purposes and are usually provided in complete chapters with adequate References.





Arginase is a detoxifying enzyme that catalyses the hydrolysis of arginine into ornithine and urea, the last step of urea cycle- a process through which the body disposes off harmful ammonia. This research was carried out to determine the characteristics of liver arginase of mango tilapia (Sarotherodongalilaeus) in Opa river, Osun state.

The enzyme ‘arginase’ was isolated from the liver of mango tilapia through the process of homogenization and centrifugation which was done at 4000rev/min.The protein concentration was determined using Bradford method and the arginase assay was determined by Kaysen and Strecker method.

The kinetic study shows that mango tilapia of liver arginase has a Km value of 0.2M and a Vmax value of 166.7µmol/ml/min. The effect of temperature on arginase activity was tested and the optimum temperature for mango tilapia liver arginase is 50°C at activity of 63.44µmol/ml/min. The effect of pH was also investigated and optimum pH is 8.0 at activity of 165.1µmol/ml/min. Inhibition study was also carried out and it was observed that calcium (51.4±1.13) and zinc (51.5±4.27) strongly inhibit arginase while mercury, magnesium and sodium have little or no inhibitory effect on arginase. Also from the result, it can be deduce that citrate (20.8±4.67) and glutathione (28.6±2.53) and ethylenediamineacetic acid (EDTA) slightly inhibit arginase while urea has les inhibitory effect on arginase. The result for the effect of amino acids on enzyme activity shows that liver arginase of mango tilapia to be in this order: arginine > valine> aspartate > cysteine > lysine with residual activity of 107.2%, 86.6%, 73.4%, 62.1% and 58.7% respectively.

Mango tilapia liver arginase belongs to the ureotelic class of arginases according to Mora J et al classification (1965). It has a Km value of 0.2M and aVmax value of 166.7µmol/ml/min.


1.1                                          INTRODUCTION

Arginase is the fifth and last step of the urea cycle, a series of biochemical reactions through which the body disposes off harmful ammonia. It catalyses the hydrolysis of L-arginine into ornithine and urea; a highly active and specific enzyme, catalyzing only canavanine and L-arginine and not D-arginine or other guanido compounds (de Grutyter, 1975).

Arginase is the enzyme responsible for the hydrolysis of arginine to ornithine and urea (Kreb and Greenberg, 1960). Arginase was first detected in mammalian liver as terminal enzyme of urea cycle.

The structure of the native enzyme, arginase, was determined from the murine liver enzyme and its structure has been used to explore the biology and chemistry of binuclear manganese cluster (Christianson, 2000). The three dimensional structure of arginase is consistent with a metal-activated hydroxide mechanism of arginase hydrolysis, where both manganese ions serve to activate the catalytic nucleophile.


Mora et al (1965) classified liver arginases into two types, theureotelic and uricotelic. These arginases have been shown to possess different properties with respectto Km values, inhibition by excess of substrate and antigenicity; they also have different molecular weights.

The ureotelic arginases have molecular weight of approximately 13000 and a Km for arginine that lower than 10Mm; the uricotelicarginaseshave molecular weights of approximately 280000 and a Km for the substrate that is of the order of 100Mm. This large difference in the Km for the substrate must have a metabolic significance, since it may affect the efficiency of the enzyme in the hydrolysis of endogenous arginine. In fact, ureotelic arginases have their main function in the urea cycle, whereas the function of uricotelic is unknown and their efficiency in hydrolyzing endogenous arginine is unknown. They have been considered as vestigial stage in the development of uricotelic animals (Mora et al, 1965). Uricotelicarginase seems to be involved in nitric oxide metabolism, thus indirectly it can affect blood pressure regulation, cytostatic response, phagocytosis, platelet aggregation and many other processes (Davis BJ, 1964).

Arginase occurs as two distinct isoenzymes in mammals. The type 1 arginases are cytosolic and are primarily in the liver where they function as the final enzyme in the urea cycle (Haraguchi et al, 1987; Jenkinson et al, 1996). In contrast, the second isoenzyme type 11 arginases are mitochondrial, extrahepatic in location and are responsible for the net synthesis of ornithine for the production of polyamines, spermine and spermidine, glutamate and proline (Tabor and Tabor, 1981; Morris, 1987; Ash, 2001). Both isoforms are believed to participate in the regulation of nitric oxide (NO) biosynthesis by competing with NO production, although arginase 11 probably plays a more important role in this regard (Kanyo et al, 1996; Cox et al, 2001). Another form of difference between the two isoenzymes is in term of their catalytic, molecular, immunological properties and amino acid sequence (Haraguchi et al, 1987; Cox et al, 2001). These distinctions result in isoenzyme selective amino acid inhibitors for example, L-ornithine is an inhibitor of the type 1 isoenzyme with a Ki value of 1Mm, while it is a poor inhibitor of the type 11 isoenzyme where it has a Ki value greater than 10Mm (Colleluori and Ash,2001).


Arginase as an enzyme that catalyses the hydrolysis of L-arginine into ornithine and urea has been found to occur in almost all living organisms. Human arginase has been reported by Carvajal et al (1971); Yip and Knox (1972); Kaysan and Strecker (1973); Porembska and Kedra (1975). Rabbit liver arginase by Trainelleet al (1973), Grazi and Magri (1972).

In mammalian liver, arginase completes the urea cycle unlike other urea cycle enzymes. Arginase is widely distributed amongst tissues including brain, kidney, heart, lung, prostrate, small intestine, mammary gland. It is also present in blood serum (Baranczyk-Kuzma, 1981). Although activity is found in the liver (Herzfeld A and Raper S.M, 1976), the enzyme is also found in non-hepatic tissues such as red blood cells (Kedra-Luboiska et al, 1988); lactating mammary gland (Yip M.C.M and Knox W.E, 1972) and kidney (Herzfeld A and Raper S.M, 1976). The function of arginase in those tissues that lack complete urea cycle is thought to be the production of L-ornithine, which serves as a biosynthetic precursor of L-proline (Yip M. C. M and Knox W. E. 1972).  Studies have suggested that arginase may play a role in modulating the immune response, since an inhibitor of the mixed lymphocyte reaction purified from rat liver has high N-terminal sequence homology with rat liver arginase and also displays arginase activity (Shinomiya T. et al 1990).

Arginase activity is not restricted to ureotelic organisms; the enzyme has been detected in the liver of a variety of uricotelic organisms such as reptiles and amphibians (Brown G. N.jnr and Cohen P. P. 1960) as well as in plant (Hellerman L. 1935, Anderson A. B. 1945, Kang J. H. and Cho Y. D. 1990), fungi (Davies R. H 1986) and certain strain of bacteria (Cunin R. et al 1986).

A common feature of all arginase is thus far studied, whether of eukaryotic or prokaryotic origin is the requirement for divalent cation activators. Metal activation appears to be species dependent and although Co2+, Ni2+ and Cd2+activate the enzymes from some sources (Hellerman and Perkins M.E> 1935, Harrel D. and Sokolovsky M. 1972). Mn2+ provides the most potent stimulation of activity and it’s thought to be the physiological activator.


1.4.1    ARGINASE  

Arginase (EC, arginine transaminase, arginine amidinase, L-arginase) is a manganese-containing enzyme. Arginasecatalyses the hydrolysis of L-arginine to produce L-ornithine and urea. It is the final enzyme of the urea cycle (Di Constanzo et al, 2007). Arginase belongs to the ureohydrolase family of enzymes. Arginasecatalyses the fifth and last step of the urea cycle; a series of biochemical reactions during which the body disposes of harmful ammonia. Specifically, arginase converts arginine into ornithine and urea.

Arginine + H2O = Ornithine + Urea

Arginase also exists in a number of non-hepatic tissues that lack a complete urea cycle; the reaction is thought to provide ornithine, a major biosynthetic precursor of proline and polyamines. For example, in lactating mammary gland, a major conversion of arginine to proline occurs without the formation of citrulline (Mepham and Linzell, 1996, 1997). This suggests that the urea cycle is inoperative and that arginine can be diverted through the reaction of arginase and the transamination of ornithine to proline. Arginase activity rises to about 25% that found in the liver in order to supply the required for milk protein biosynthesis (Yip et al, 1972).


Arginase as the fifth and last enzyme of the urea cycle occurs in two forms, arginase 1 and arginase 11. Both isoforms catalyse the same reaction but differ in tissue distribution, subcellular location and regulation of expression. The isoform arginase 1, is expressed in the liver as one of the enzymes in the urea cycle which detoxifies ammonia in mammals. Arginase 1 is highly distributed in the cytoplasm of the liver. Human liver arginase 1 is a 322-amino acid protein and demonstrates 58% sequence identity to human arginase 11. It was cloned more than 20 years ago (Dizikes et al, 1986; Haraguchi et al, 1987) and the gene for human arginase 1 was localized on chromosome 6q 23 (Sparkes et al,1986). Arginase 1 may also play a role in the regulation of polyamines by the production of macrophage in the conditions of wound healing (Shearer et al, 1997).

Arginase 11 functions in the regulation of arginine/ornithine concentrations in the cell. It is  located in the mitochondrial of several tissues in the body, with most abundance in the kidney and prostate. The second isoenzyme can be found in the absence of other urea cycle enzymes (Di Constanzo, 2007). Human arginase 11 was cloned in 1996 (Gotoh et al, 1996; Vockey et al, 1996) and the gene was mapped to chromosome 14q 24:1-24.3 (Gotoh et al, 1997a).



Different values of molecular weight of arginase have been reported for higher organisms, invertebrates, vertebrates, mammals, plant species and bacteria. The ureotelic arginases have molecular weight of approximately 130000 and the uricotelic have 280000 (Mora et al, 1956). Vertebrates hepatic arginases have molecular weights of 120000 and 240000, the enzyme have quartenary structure and each subunit has a molecular weight of 30800 (Hirshklob and Greenberg, 1968).

Jenkinson et al 1996, reported that subunit molecular mass is estimated as 31,000-34,000Da in several bacteria. The bacterium, Rhodobactuscapsulatus contain an apparent mass of 120000Da (Morano Vivian et al, 1992). Porembska 1973, reported that arginase from the liver of adult frog has a molecular mass of 160000Da, that of tadpole is 80,000Da. The molecular mass of arginase from liver and kidney of frog Ranatigerina varies with the protein concentration of extracts increasing from 26,000-94,000Da due to protein aggregation (VenkataKrisshnan and Roddy,1993).

Mammalian arginase has been reported by Roddy and Campbell 1970, to have a molecular mass of 120,000Da, whereas the estimated molecular native mammalian arginase varies from 100,000-140,000Da with an average value of about 120,000Da (Jenkinson et al, 1996).


There are few reports on the amino acid composition of arginase. Rat liver arginase is reported to contain high values of lysine, threonine, proline and lower values of arginine, aspartic acid, glutamic acid, alanine and leucine.

Chicken liver arginase amino acid was analysed by the hydrolysis of protein in 6M HCl for 24hours at 110°C in evacuated and sealed tubes. The hydrosylate was evaporated to dryness five times and finally dissolved in citrate buffer, pH 2.2. Cysteine was determined as cysteic acid after performic acid oxidation, tryptophan by the bromosucinimide method and amino acid analysis was performed in a Beckman model 120B analyser (Grazi and Magri, 1971).



A common feature of all arginase studied so far, either of eukaryotic or prokaryotic origin, is the requirement of divalent cations for activity. Mn2+ is the physiological activator, although the divalent cation requirement for certain arginases has been reported to be satisfied by Co2+ and Ni2+ (Hellerman& Perkins, 1935, Mora et al, 1965, Brown, 1966) and in some cases by Fe2+, Vo2+, Cd2+ (Anderson, 1995, Edblacher and Baur,1958). Most arginases have an alkaline pH optimum, with maximum velocities observed in the range of pH 9.0-9.5 (Robolt and Greenberg, 1956). Substrate specificity depends on

·         The presence of a guanidinium group

·         The proper length and hydrophobicity of the side chain

●     The stereochemistry and nature of substituents at Cα

1.6.2  EFFECT OF pH

The effect of pH on arginase has been determined in different organisms. Bach and Kilip, 1961, reported an optimum p H of 9.3 for arginase from sheep and horse liver. Arginase from rat liver, kidney, brain and mammary gland has been reported to be between 9.4 and 10.2 (Gasiorowska et al, 1970, Baranezyk-Kuzma et al, 1976, Herzfeld and Raper, 1976). The activity of cotyledon determined in the buffer pH range from 6.0-11.0. The optimum p H of cotyledon arginase was found to be 10.0 (Mastsubara S, Suzuki Y, 1984). Generally, mammalian arginases appear to have basic pH optima of 9.5-10.5, although some exceptions have been noted (Jenkinsonet al, 1996). Patilet al, 1990 noted optimum p H 11.5 for ox-erythrocyte arginase.


Few temperature optima have been reported for arginase. The optimum temperature of Pistapacifica is about 60°C (O’Malley and Terwilliger, 1974). Helixpomatia and Helixaspersa enzymes have optima temperature of 60°C and 65°C respectively and horse liver arginase has a temperature optimum of 45-50°C (O’Malley and Terwilliger, 1974).


Arginase is a metalloenzyme in which manganese acts as a cofactor as well as an activator in almost all reported arginases (Mora J et al, 1965, Weeks et al, 2000). In addition to manganese, Co2+,Ni2+ and Fe2+ also act as cofactors for arginase.

The amount required for optimum activity for the enzyme varies. Arginaseapoenzyme forms stable complexes with the activatingcations and cations protect the enzyme against heat denaturation (Midlehoven, 1969). Replacement of Mn2+ ion with other metal ions such as Mg2+ restores almost the original arginase activity whereas Ca2+, Ni2+, Co2+ and Cd2+ restores about 40-50% and Fe2+ and Zn2+ completely inactivates arginase activity.


The active site holds L-arginine in place via hydrogen bonding between the guanidinium group with Glu227. The bonding orients L-arginine for nucleophilic attack by the metal-associated hydroxide ion at the guanidinium group which results in a tetrahedral intermediate.The manganese ions act to stabilize both the hydroxyl group in the tetrahedral intermediate, as well as the developing sp3 lone electron pair on the NH2 group as the tetrahedral intermediate is formed.

Arginase’s active site is extraordinarily specific. Modifying the substrate structure and/or stereochemistry severally lowers the kinetic activity of the enzyme. This specificity occurs due to the number of hydrogen bonds between the substrate and enzyme; direct or water-facilitated hydrogen bonds exist, saturating bvoth the four acceptor positions on the alpha carboxylate group and all three positions on the alpha amino group. N-hydroxy-L-arginine (NOHA), an intermediate of NO biosynthesis, is a moderate inhibitor of arginase. Crystal structure of its complex with the enzyme reveals that it displaces the metal-bridging hydroxide ion and bridges the bionuclear manganese cluster (Reczkowski RS, Ash DE, 1994).

Additionally, 2(S)-amino-6-boronohexonic acid(ABH) is an L-arginine analogue that also creates tetrahedral intermediate similar to that formed in the catalysis of the natural substrate, and is a potent inhibitor of human arginase (Cox et al, 1999).


Arginase deficiency (argininemia, hyperargininemia) is a type of amino acid disorder. It occurs when the enzyme “arginase” is either missing or not performing its duty effectively. The enzyme’s job is to break down its substrate ‘arginine’ and help remove ammonia from the system. When arginase is not functioning well, both arginine and ammonia can be built up in the blood resulting in serious health problems. Symptons of argininemia are poor growth, poor co-ordination, balance problem, learning delays, vomiting and excessive sleeping. When argininemia is left untreated, other symptons such as muscle weakness, seizures, breathing problem, swelling of brain, coma and even death may follow.

Argininemia is a hereditary disorder. Treatment of argininemia requires the intervention of a metabolic doctor and a dietician. A victim of argininemia should take low-protein diet e.g fruits and green vegetables and should avoid highly-rich protein containing foods.


Arginase is a widely distributed enzyme that occurs in all living organism. It catalyses the last step of urea cycle where it hydrolyses arginine into ornithine and urea. It is involved in a variety of metabolic roles: the biological significance is therefore related to its role in the control of cellular level of arginine and ornithine since these amino acids are required for various critical metabolic processes which include the urea cycle in the liver of ureotelic animals, proline biosynthesis in mammary gland (Mepham and Linzell, 1967; Yip and Knox, 1972), polyamine biosynthesis in male genital tract (Della Pietra et al, 1973), in mammary gland (Oka and Perery, 1974).

Arginase type 1 and 11 are believed to participate in the regulation of nitric acid production, although arginase type 11 probably plays a more important role in this regard (Kanyo et al, 1996; Scolnick et al, 1997; Cox et al, 2001). Nitric oxide inhibits proliferation of many cell lines (Singh et al, 2000). Arginase 1 may also play a role in the regulation of polyamines by the production of macrophages in condition of wound healing (Shearer et al, 1997). Arginase also plays a role in sexual arousal.

Nitric acid promotes smooth muscle relaxation, enhancing the flow of blood into the genital tissue, therefore type 11 is a potent target for the treatment of male and female sexual arousal disorder (Wikipedia).

Arginase may also be used as a marker of cancer. Studies show higher arginase activity in cancerous tissue than their normal counterparts. Jamhidzadeh et al, 2001 reported higher arginase activity in oesophagus, stomach and lung carcinomas then their normal counterparts. High level of arginase has also been detected in gastric cancer (Wu et al., 1996). It has also been reported that arginase specific activity is significantly greater in

prostatic carcinoma than in prostatic hyperplasia (Prestlow et al., 1985).






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