Abstract: Studies were carried out to ascertain some kinetic properties of alkaline phosphatase (ALP) extracted from Lepus townsendii liver. Incubation of ALP extract with 4-nitrophenylphosphate (4-NPP) in glycine-NaOH buffer mixture at 37°C for 30 min formed the basis for determination of enzyme activity. Spectrophotometric method was used to assay for the enzyme activity for 30 min and the kinetic constants-maximum enzyme velocity (Vmax) and Michealis-Menten constant (Km) were evaluated. The Km and Vmax values were 0.5x10-3 M and 20x10-6 M min-1, respectively. Inhibition studies showed that ALP activity was competitively inhibited by 0.67 mM sodium hydrogen orthophosphate (NaH2PO4) and the inhibition constant (Ki) was 0.9x10-3 M. The optimum pH value for ALP activity was about 9.2 and optimum temperature registered 45°C. ALP activity exhibited linear Arrhenius relationship at temperature greater than 44.95°C with corresponding catalytic energy of activation (Ea) = 15.23 KJ mole-1. The present study gave insights into characteristic catalytic properties of ALP extracted from L. townsendii liver.
INTRODUCTION
Orthophosphoric monoester phosphohydrolase (E.C: 3.1.3.1), also referred to as Alkaline phosphatase (ALP) is a generic term that describes a group of catalytic proteins sharing the capacity to hydrolyse phosphate esters in alkaline medium (Zhang et al., 2004; Saini et al., 2005). The enzyme contains a zinc atom (Zn2+) near the active site that is viewed to be responsible for its catalytic activity (Du et al., 2001; Dean, 2002). Also, activation of ALP activity is facilitated by divalent cations such as magnesium (Mg2+) (Kim and Wyckoff, 1990; Du et al., 2001; Dean, 2002) and colbalt (Co2+) (Arise et al., 2008). Conversely, ALPs are inactivated/inhibited by wide range of compounds, depending on the source or/and isoenzymic form of the enzyme (Hummer and Millan, 1991; Hoylaerts et al., 1992). For instance, whereas bone and kidney ALPs are inhibited by urea, those obtained from planceta and gastrointestinal are unaffected but inhibited by phenylalanine (Dean, 2002). Also, ALP extracted from Escherichia coli is not inhibited by phenylalanine (Hoylaerts et al., 1992). The wide distribution of ALP in biologic systems coupled with its broad specificity makes the enzyme a readily available parameter for diagnostic and research studies (Garba and Gregory, 2005).
Reports showed that ALP extracts from different tissue/organ and various biological systems exhibit variable but characteristic kinetic properties that serve as basis for distinguishing ALP isoforms (Mayer-Sabellek et al., 1988; Dean, 2002). Therefore, the focus of the present study is to ascertain some kinetic parameters of ALP in liver extract of L. townsendii. The findings will contribute to the characterization of the enzyme.
MATERIALS AND METHODS
Animal handling: Five male rabbits between 8-9 weeks old were obtained between August and September, 2002, from the Experimental Animal House, Department of Biological Science, Usman Danfodiyo University, Sokoto, Nigeria. The animals were healthy and showed no sign of stress. They were fed ad libitium with hay and water for a period of 1 week for acclimatization before the animals were sacrificed. The institutional review board of the Department of Biochemistry, University of Port Harcourt, Port Harcourt, Nigeria, granted approval for this study in accordance with the guidelines of the National Institute for Animal Research, Nigeria.
Preparation of liver extract of alkaline phosphatase: The extraction and purification of the liver enzyme was by the methods described by Malomo et al. (2003) and Arise et al. (2008), but with minor modifications. The rabbit was dissected and the liver carefully removed and washed in physiological saline solution. Two grams of the liver was homogenized in 40 mL of 0.1 M glycine-NaOH buffer (pH = 9.9) solution. The homogenate was centrifuged in a Shermond bench centrifuge at 5000x g for 40 min. The supernatant constituted the crude enzyme extract and was carefully harvested using a Pasteur pipette. To the supernatant, 4.17 M (NH4)2 SO4 was gradually added while stirring until 30% saturation was achieved. The solution was stored at 4°C for 1 h to precipitate the enzyme (Hulett et al., 1990). The precipitate was collected by centrifugation at 5,000x g for 20 min and re-dissolved in 5 mL of 0.1 M glycine-NaOH buffer (pH = 9.9) solution. The solubilized enzyme was subjected to dialysis at 2°C for 24 h to remove contaminating salt constituents (Ali-ul-Qader et al., 2009) and further purified on a sephadex G-100 column to obtain a highly active ALP fraction. In total, ALP extracts from 5 rabbits were used. Protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as the standard. One unit of ALP activity (U) was expressed as 1 μM of 4-NP produced per min. The procedure for ALP purification is summarized in Table 1. The procedure for ALP purification is summarized in Table 1.
Determination of alkaline phosphatase activity: The enzyme activity represents the rate of hydrolysis of 4-Nitrophenylphosphate to 4-Nitrophenol described as follows:
Table 1: | Purification protocol for liver ALP extract |
The extract specific enzyme activity = 9.43±1.09 Unit mg-1 protein |
The enzyme assay was carried out by method described by Glogowski et al. (2002), but with minor modifications. Five test tubes containing 0.45 mL dilutions of 4, 3.2, 2.4, 1.6 and 0.8 mM of 4-NPP solutions were introduced into five corresponding test tubes containing 0.45 mL of 0.1 M glycine-NaOH buffer (pH = 9.9). The 4-NPP/glycine-NaOH buffer mixture was incubated at 37°C for 30 min. The enzyme reaction was started by adding 0.1 mL of enzyme extract into the various test tubes. At a regular time interval of 5 min for 30 min, 5.0 mL of 0.2 M NaOH was added to the assay mixture to terminate ALP activity. The mixture was transferred into a cuvette and absorbance was measured with a spectrophotometer (SPECTRONIC 20, Labtech-Digital Blood Analyzer®) at maximiun wavelength (λmax) of 405 nm. The concentrations of 4-NP produced in the various tubes were interpolated from standard curves (not shown).
Inhibition studies using NaH2PO4 as inhibitor: The enzyme assay was carried out as described above. The glycin-NaOH buffer was substituted with 0.1 M glycine-NaOH buffer/2 mM NaH2PO4 mixture {ratio 2:1 (v/v)}. Final concentration of inhibitor = 0.67 mM.
Effect of pH on Enzyme Activity: The activity of ALP was investigated under varying pH values of 8.4, 8.8, 9.2, 9.6, 9.9, 10.0 and 10.4 of 0.1 M glycine-NaOH buffer. A substrate concentration of 4 mM 4-NPP was used for the enzyme assay.
Effect of temperature on enzyme activity: The enzyme assay was carried out by incubating the enzyme mixture containing 4 mM 4-NPP in varying temperature values of 5, 15, 25, 35, 45 and 55°C.
Evaluation of kinetic parameters: The kinetic parameters were evaluated with the plot of the reciprocal of initial velocities (VI) against corresponding reciprocal of substrate concentrations [4-NPP], i.e., 1/Vi vs 1/[S] (Lineweaver and Burk, 1934).
Statistical analyses: The experiments were designed in a completely randomized method and data collected were analyzed by the analysis of variance procedure while treatment means were separated by the Least Significance Difference (LSD) incorporated in the Statistical Analysis System (SAS) package of 9.1 version.
RESULTS
The activity (Vi) of ALP measured in the absence and presence of inhibitor ([I] = 0.67 mM NaH2PO4) is represented by the Lineweaver-Burk plot (Fig. 1).
By inspection, the Lineweaver-Burk plot (Fig. 1) showed that ALP activity was competitively inhibited by 0.67 mM NaH2PO4. Also, the plots representing the presence and absence (control) of the inhibitor intersected the x-axis at -1.1 and -2, respectively. Furthermore, the two plots converged at a common point of intersection on the y-axis: 5x104 μM-1/min-1x104. The kinetic properties of ALP deduced from the Lineweaver-Burk plot above are summarized in Table 2.
The activity of ALP investigated in varied alkaline pH environment is presented in Table 3.
A cursory look at Table 3 shows that the activity of ALP exhibited two phase profile within the range of values of pH = 8.4 to 10.4. The first phase showed increasing ALP activity as pH values were adjusted from 8.4 to 9.2.
Fig. 1: | Double reciprocal plot of ALP assay in the presence of inhibitor NaH2PO4 ( ) and the control ( ) analysis. [I]: Concentration of inhibitor = 0.67 mM |
Table 2: | Kinetic parameters of ALP |
Values are Mean±S.D of five (n = 5) determinations |
Table 3: | Levels of ALP activity with corresponding pH values |
Values are Mean±S.D of five (n = 5) determinations. Means in the rows with the same letter are not significantly different at p<0.05 according to LSD |
Table 4: | Levels of ALP activity with corresponding temperatures (T°C) |
Values are Mean±S.D of five (n = 5) determinations. Means in the rows with the same letter are not significantly different at p<0.05 according to LSD |
Noteworthy, the pH value of about 9.2 corresponded to the maximum enzyme activity attained within the experimental pH range. Therefore, about pH = 9.2 represented the optimum pH value of ALP extracted from L. townsendii liver. However, ALP activity at pH 10.0 was not significantly different (p<0.05) from the activity at optimum pH. Further increases in pH values beyond the optimum engendered decreasing level of ALP activity. The relationship between ALP activity and experimental incubation temperature of the enzyme assay is presented in Table 4.
The pattern ALP activity in relation to changes in experimental temperature showed two phase profile. The results in Table 4 showed the optimum temperature of ALP activity was about 45°C. The maximum enzyme activity attained at optimum temperature was 5.01±0.56 μM min-1. ALP activity at 35°C (4.67±0.79 μM min-1) was not significantly different (p<0.05) compared to activity at 55°C (4.53±0.83 μM min-1). Using Arrhenius plot, log V versus 1/T, the activation energy (Ea) of catalysis was evaluated thus:
Evaluating for Ea: Log V = Ea/2.3RT where, R = 8.31447 J K-1 mol-1: molar gas constant But gradient of plot: -800 = Ea/2.3R, Therefore, Ea= 15.23 KJ mole-.
DISCUSSION
This study demonstrated the activity of ALP in liver extract of L. townsendii. The occurance and derversity of ALP in biological systems has been reported by several authors (Coburn et al., 1998; Zhang et al., 2004). For instance, Weissig et al. (1993), reported on sequence comparisons between different ALP indicated that about 25-30% homology exist between mammalian and Escherichia coli ALP. Despite differences in amino acid sequences and three-dimensional structures, all ALP catalyze the hydrolysis of almost any phosphomonoester with release of inorganic phosphate and alcohol. In addition, although ALP exhibits broad specificity for wide range of phosphorylated substrates, the enzyme displays variable affinity for these substrates depending on the molecular nature of the substrate, isoform and source of the enzyme. Szalewicz et al. (2003) noted that the equivalent enzyme, acid phosphatase, of frog Rana esculenta liver displayed higher affinity for peptidic substrates than for small phosphate esters. Also, Coburn et al. (1998), from their findings stated that in the presence pyridoxal phosphate as substrate, bovin kidney ALP Km was 0.42±0.04 μmol L-1, whereas enzyme extracted from the intestine gave 1.6±0.2 μmoL-1 and placenta gave 0.42±0.08 μmoL-1. Comparatively, the present investigation showed that ALP liver extract of L. townsendii exhibited higher affinity for 4-NPP (Km = 0.5±0.25 mM) (Table 2) than enzyme extracts from rat Rattus novergicus kidney (Km = 6.41 mM; Arise et al., 2008) and human placental isoenzyme (Km = 5.55 mM; Saini et al., 2005). Comparatively, the present investigation showed that ALP liver extract of L. townsendii exhibited higher affinity for 4-NPP (Km = 0.5±0.25 mM) (Table 2) than enzyme extracts from rat Rattus novergicus kidney (Km = 6.41 mM; Arise et al., 2008) and human placental isoenzyme (Km = 5.55 mM; Saini et al., 2005). These findings serve to highlight and confirm the occurance of isoforms of ALP amongst the mammalian genera.
More than five decades ago, Hilliard et al. (1965), demonstrated that endogenous phosphate interferes with the determination of ALP in urine and suggested that the wide variation in serum inorganic phosphate concentrations in diseases such as uremia or renal tubular disease might interfere with ALP measurements in serum. From inspection of the double reciprocal plot (Fig. 1), Vmax of the enzyme was not altered in the presence of inhibitor. (Therefore, the present kinetic studies showed that phosphate salt, NaH2PO4, is a competitive inhibitor of ALP activity. This finding is in concord with previous research reports by Fox (1978), Coburn et al. (1998) and So et al. (2007), in which they noted that ALP is competitively inhibited by physiological concentrations of inorganic phosphate. Therefore, the present kinetic studies showed that phosphate salt, NaH2PO4, is a competitive inhibitor of ALP activity. This finding is in concord with previous research reports by Fox (1978), Coburn et al. (1998) and So et al. (2007), in which they noted that ALP is competitively inhibited by physiological concentrations of inorganic phosphate. They further averred that inorganic phosphate is an important physiologic regulator of extracellular ALP activity. Incongrously, the possible effects of endogenous phosphate on phosphatase activity with natural substrates under physiological conditions have not been rigorously investigated (Coburn et al., 1998).
Although, the pH optimum of an enzyme often reflects the pH of its normal environment, the optimum value may not be precisely the same in every biological system. Noteworthy, as far back as five decades ago, Garen and Levinthal (1960), reported optimal pH for ALP activity of E. coli to be about 8.0 while, the bovine enzyme optimum pH was slightly higher at about 8.5 (Harada et al., 1986). Furthermore, study of ALP from human hydatidiform by Aberomand et al. (2008), gave pH optimum of 10.8. The current study reported optimum pH value of ALP liver extract of L. townsendii to be about 9.2. These findings are consistent with prevoius studies by Zappa et al. (2001), The current study reported optimum pH value of ALP liver extract of L. townsendii to be about 9.2. These findings are consistent with prevoius studies by Zappa et al. (2001), in which they observed that mammalian ALPs possess higher pH optima than procaryotes (E. coli) enzyme and proposed that substitution of two amino acid residues with corresponding histidine molecules in the mammalian enzymes are responsible for this increase in optimum pH (Holtz and Kantrowitz, 1999; Murphy et al., 1995). These variations in ALP activity which are consequence of adjustments in Km or Vmax values or both, suggest that pH/activity relationship of ALP may be a factor in the intracellular regulation of its activity.
Enzyme activity/temperature linked properties reported elsewhere (Suzuki et al., 2005; Lee et al., 2007; Copeland et al., 1985; Aberomand et al., 2008) defined the so-called enzyme "temperature optimum" of ALP activity. The present report suggests that at experimental temperature of about 45°C, ALP extract of L. townsendii exhibited maximum conformational flexibility to accommodate the substrate and enhanced conformational changes required for maximum catalysis. Contrary to the present suggestion, Daniel et al. (2001), proposed that temperature optimum is not an intrinsic property, being derived from a complex mixture of both activity and thermal stability effects.
The present report suggests that at experimental temperature of about 45°C, ALP extract of L. townsendii exhibited maximum conformational flexibility to accommodate the substrate and enhanced conformational changes required for maximum catalysis. Contrary to the present suggestion, Daniel et al. (2001), proposed that temperature optimum is not an intrinsic property, being derived from a complex mixture of both activity and thermal stability effects.
The Arrhenius plot (Fig. 2) conformed to the pattern previously described by Copeland et al. (1985). They averred that all enzymes of human origin exhibited similar linear Arrhenius relationships over the range of 20-37°C with corresponding Ea values between 30 and 36 KJmol-1. In the present study, ALP activity exhibited linear Arrhenius relationship at values of temperature greater than 44.95°C with corresponding Ea= 15.23 KJ mol-1 (Fig. 2). Although, ALP obtained from human and L. townsendii showed linear Arrhenius relation within variable temperature range, Ea values of catalysis were significantly different. Again, in concord with the present investigations, studies by Copeland et al. (1985) reported that porcine kidney enzyme obeyed an Arrhenius relationship that was slightly but significantly different from the isoenzymes of human origin.
Although, ALP obtained from human and L. townsendii showed linear Arrhenius relation within variable temperature range, Ea values of catalysis were significantly different.
Fig. 2: | Arrhenius Plot: Effect of Temperature on ALT Activity. Where 1/Topt: Reciprocal of Optimum Temperature = 3.144x10-3 K-1. ALP exhibited linear Arrhenius relationship within the temperature range: 35-55°C |
Again, in concord with the present investigations, studies by Copeland et al. (1985) reported that porcine kidney enzyme obeyed an Arrhenius relationship that was slightly but significantly different from the isoenzymes of human origin.