Effect of gallic acid on alkaline phosphatase and peptidase activities in rat intestine

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1 Indian Journal of Biochemistry & Biophysics Vol. 46, October 2009, pp Effect of gallic acid on alkaline phosphatase and peptidase activities in rat intestine Nidhi Mahajan and Akhtar Mahmood* Department of Biochemistry, Panjab University, Chandigarh, , India Received 08 May 2009; revised 17 August 2009 Gallic acid is a normal constituent of many edible foods, thus directly interacts with epithelial tissue in intestine. In the present study, the effect of gallic acid on intestinal alkaline phosphatase (IAP) and peptidase activities in rat intestine was evaluated. Gallic acid ( mm) inhibited activities of leucine aminopeptidase (LAP) and γ-glutamyl transpeptidase (γ- GTP) by over 90%, compared to controls in rat intestine. In contrast, mm gallic acid either had no effect or stimulated the activity of IAP in rat intestine. The observed inhibition of peptidases by gallic acid was reversible in nature. Kinetic analysis revealed no change in V max of LAP ( units/mg protein) and γ-gtp ( units/mg protein), while the values of apparent K m were increased 6-7 fold, exhibiting competitive-type of enzyme inhibition by gallic acid. The values of K i for LAP and γ-gtp were mm and mm, respectively. These observations indicate that gallic acid is a potent inhibitor of brush border peptidases, and thus may interfere in the digestion and absorption of proteins in the intestine. Keywords: Leucine aminopeptidase, γ-glutamyl transpeptidase, Intestinal alkaline phosphatase, Enzyme inhibition, Gallic acid, Rat intestine, Brush border membrane Gallic acid (3,4,5-trihydroxybenzoic acid), a naturally occurring polyphenol is abundantly present in fruits and nuts 1,2. Tea is an important source of gallic acid and contains about 4.5 g/kg fresh weight gallic acid 3, while red fruit, black radish and onions have relatively less concentrations of gallic acid 4. Inhibitory effects of gallic acid are well documented for a diverse array of enzymes. Gallic acid is reported to reduce tyrosine kinase activity in a dose-dependent manner 5, thus acting as anti-melanogenic agent. It also inhibits the activities of lysozyme, amylase and chymotrypsin over a broad ph range 6. The oxidized form of gallic acid produces 40% inhibition of brush border sucrase in rat intestine, while unoxidized molecule is essentially inert 7. Recently, the inhibitory effect of gallic acid on brush border disaccharidases has also been reported 8. However, whether the observed inhibitory effect of gallic acid is specific to disaccharidases or it also inhibits other intestinal enzymes, such as intestinal alkaline phosphatase (IAP) and peptidase activities is not known. Leucine aminopeptidase (LAP) and γ-glutamyl transpeptidase (γ-gtp) are not only * Author for Correspondence akhtarmah@yahoo.com Tel: Fax: involved in the digestion of proteins, but also play an important role in the transport of amino acids from intestine 9,10. Thus, in the present study, we have investigated the effect of gallic acid on IAP, LAP and γ-gtp activities in rat intestine. Materials and Methods All the chemicals used were of analytical grade and obtained from E. Merck, Mumbai, India or Glindia Ltd., Mumbai. p-nitrophenyl phosphate was purchased from SRL Pvt. Ltd., and gallic acid, bovine serum albumin, Tris (trihydroxymethyl amino methane), L-leucyl p-nitroanilide, γ-glutamyl-pnitroanilide, glycyl glycine were obtained from Sigma Chemical Co., Saint Louis, USA. Preparation of microvillus membranes Male albino rats (Wistar strain), weighing g were used to isolate and purify microvillus membranes, following the method described previously 11. Briefly, tissues were homogenized (10% w/v) in 2 mmol/l Tris buffer and 50 mmol/l mannitol, ph 7.2. The homogenate was filtered through cheese cloth and calcium chloride was added to the homogenate to a final concentration of 10 mmol/l. After 10 min, the contents were centrifuged at 2000 g for 10 min and the supernatant obtained was re-centrifuged at g for 35 min. The pellet was washed twice with 50 mmol/l sodium

2 MAHAJAN & MAHMOOD: EFFECT OF GALLIC ACID ON INTESTINAL ENZYMES 379 maleate (ph 6.8) by centrifugation (43000 g for 35 min) and re-suspension of the pellet. The final membrane preparation was suspended in 50 mmol/l sodium maleate (ph 6.8) and exhibited 9-11 fold enrichment of IAP activity over the crude homogenate. Preparation of soluble enzymes The homogenate (10%) of rat intestinal mucosa was prepared in 50 mmol/l sodium maleate (ph 6.8) and centrifuged at g for 35 min at 4ºC. The supernatant obtained was used as the source of soluble LAP and γ-gtp activities. Animals The experimental protocol for the use of animals was approved by the Ethical Committee of Panjab University, Chandigarh, India. Experiments on animals were performed in accordance with the guidelines for the use of laboratory animals approved by the Indian Council of Medical Research, New Delhi, India. Enzyme assays The activity of LAP (EC: ) was assayed using L-leucine p-nitroanilide as the substrate 12. γ-gtp (EC: ) activity was assayed as described earlier 13. IAP activity was determined using p-nitrophenyl phosphate as the substrate 14. Enzyme activities were expressed as units per mg protein. One unit of enzyme activity was defined as the amount of the enzyme which transformed 1 µmole of the substrate to product per min under the assay conditions. Protein was estimated by the method of Lowry et al 15 using bovine serum albumin as the standard. Effect of gallic acid on enzyme activities The effect of gallic acid on IAP, LAP and γ-gtp activities was determined by assaying the enzyme activities in the absence and presence of mm gallic acid. Kinetic studies The effect of gallic acid on kinetic parameters of LAP was studied by assaying the enzyme activity at different substrate concentrations ( mm) in absence and presence of 0.20 mm gallic acid. γ-gtp activity was determined using mm γ-glutamyl-p-nitroanilide hydrochloride as the substrate in the absence and presence of 0.13 mm gallic acid. The data were plotted according to Dowd & Riggs 16. From the slope and intercept of the lines, kinetic parameters K m, V max and K i were calculated. Reversibility of enzyme inhibition The enzyme preparation containing 0.20 or 0.13 mm gallic acid was dialyzed exhaustively against 20 mmol/l sodium maleate (ph 6.8) for 16 h with constant stirring at 4 C. A mock preparation without inhibitor was run simultaneously. LAP and γ-gtp activities were determined as described above. Effect of -SH group binding reagents on gallic acid-enzyme interactions Effect of hydroxylamine and sodium arsenite was studied on membrane bound and soluble LAP and γ-gtp activities in the absence and presence of 0.20 mm or 0.13 mm gallic acid, respectively. To the enzyme assay system, the reagents were added together with gallic acid and enzyme activity was determined. Statistical analysis Statistical analysis of the data was done using student t test. p<0.05 was considered significant compared to controls. All the results were expressed as mean ± SD. Results Increasing gallic acid concentration from 0.07 to 0.5 mm in the enzyme assay system reduced brush border LAP and γ-gtp activities in rat intestine. In contrast, the activity of IAP was not inhibited or stimulated by gallic acid under these conditions (Fig. 1a). Gallic acid (0.07 mm) produced 14% and 25% decrease in LAP and γ-gtp activities, respectively. Increasing the concentration of gallic acid to 0.5 mm produced 92-95% inhibition of LAP and γ-gtp activities under these conditions. Kinetic analysis of the inhibition of peptidase activities by gallic acid revealed that at all substrate concentrations used, there was nearly 50% inhibition of LAP and γ-gtp activities in the presence of 0.2 mm or 0.13 mm gallic acid. Kinetic parameters showed no change in V max of LAP ( units/mg protein) and γ-gtp ( units/mg protein), but the value of apparent K m enhanced 6-fold in case of LAP (0.25 mm in control vs 1.6 mm in presence of gallic acid) and 7-fold in case of γ-gtp activity (0.8 mm in control vs 6.9 mm in presence of gallic acid) (Table 1).

3 380 INDIAN J. BIOCHEM. BIOPHYS., VOL. 46, OCTOBER 2009 Fig. 1 (a): Effect of gallic acid on alkaline phosphatase (IAP), leucine aminopeptidase (LAP) and γ-glutamyl transpeptidase (γ-gtp) activities in rat intestine. Gallic acid ( mm) was added to the reaction mixture and enzyme activities were determined as described under methods and materials. [Bars indicate S.D of the mean, n = 4]; (b): Effect of gallic acid on soluble and membrane bound LAP activities. [Bars indicate S.D of the mean, n = 4]; and (c): Effect of gallic acid on soluble and membrane bound γ-gtp activities. [Bars indicate S.D of the mean, n = 4] Table 1 Effect of gallic acid on kinetic parameters of LAP and γ-gtp and IAP activities in rat intestine [Values are mean of preparations] Enzyme Gallic acid K m V max (mm) (mm) (mm) K i (µmolmin -1 mg -1 protein LAP γ- GTP IAP Reversible nature of the enzyme inhibition by gallic acid was elucidated by performing exhaustive dialysis of enzyme preparation containing 0.20 mm gallic acid. LAP activity was reduced by 57% in presence of the inhibitor. However, dialysis of the preparation restored the enzyme activity to nearly control levels (98%). Similar experiments with γ-gtp activity revealed that addition of 0.13 mm gallic acid to the enzyme assay system produced 88% inhibition of the enzyme activity, but removal of the inhibitor restored approximately 89% of the enzyme activity (Table 2). Since LAP and γ-gtp both exist in the soluble and membrane bound forms in rat intestine 17, the effect of gallic acid on soluble enzymes was also studied. As shown in Fig. 1b, increasing gallic acid concentration from 0.06 mm to 0.27 mm inhibited membrane bound LAP activity by 14 to 64%. Similar inhibition was observed for soluble LAP activity (17 to 68%) under these conditions. Experiments with γ-gtp revealed that membrane bound enzyme activity was inhibited by 25% at mm gallic acid concentration, compared to 23% inhibition of soluble γ-gtp preparation. Increasing gallic acid Table 2 Reversibility of γ- GTP and LAP inhibition by gallic acid [Values represent mean ± S.D., n = 4] Assay system γ- GTP Activity LAP Activity Control ± ± ± 0.026* ± 0.011* (88) (57) dialysed ± (11) Values in parenthesis indicate % inhibition ± (2) concentration from mm to 0.27 mm induced 75% to 93% inhibition of soluble γ-gtp activity (Fig. 1c). To examine the mode of action of gallic acid on peptidase activity, the effect of SH binding reagents on gallic acid interactions with LAP and γ-gtp was also studied. Presence of hydroxylamine or sodium arsenite in the enzyme assay tube, together with 0.2 mm gallic acid produced 59% inhibition of LAP activity, in contrast to 46% inhibition observed in presence of 0.2 mm gallic acid alone. In presence of hydroxylamine alone, 11% enzyme inhibition was observed. Sodium arsenite was essentially inert, but together with gallic acid gave 25% enzyme inhibition (Table 3). γ-gtp activity was inhibited by 61% by hydroxylamine and gallic acid (0.13 mm), while 43% inhibition was observed in the presence of 0.13 mm gallic acid or 30% in the presence of 2 mm hydroxylamine alone (Table 4). Sodium arsenite was essentially inert and produced 3% enzyme inhibition, which was augmented to 43% when gallic acid was added to the enzyme assay system.

4 MAHAJAN & MAHMOOD: EFFECT OF GALLIC ACID ON INTESTINAL ENZYMES 381 Table 3 Effect of 0.2 mm gallic acid in absence and presence of 2 mm hydroxylamine and 2 mm sodium arsenite on brush border LAP activity in rat intestine. [Values represent mean ± S.D., n=4] Assay system Activity % Decrease Control ± (0.20 mm) ± * ±.0060* 11 and Gallic ±.0044* 59 acid (0.20 mm) ±.0028* 2 and Gallic acid (0.20 mm) ± 0.023* 25 Discussion The data presented herein indicate that gallic acid is a potent inhibitor of LAP and γ-gtp activities, but either does not inhibit or stimulate IAP activity in rat intestine. Although all these enzymes have same location in brush border membrane (BBM), but they are differently affected by gallic acid, suggesting differences in the mode of their interations. The observed inhibition of LAP and γ-gtp activities, in general, is similar to that reported for intestinal disaccharidases in various animal species 8,18,19. However, K i values of LAP (0.037 mm) and γ-gtp (0.017 mm) inhibition are much lower, compared to those reported for brush border sucrase (0.9 mm) 8 and yeast invertase (0.06 mm) 20. Experiments with the membrane bound and soluble enzyme iso-forms reveal that the enzymes are equally sensitive to inhibition by gallic acid, except that in case of γ-gtp, increasing inhibitor concentration from mm to 0.27 mm induces 75% to 93% enzyme inhibition of membrane bound activity, in contrast to 59% to 73% inhibition of soluble γ-gtp activity. These findings indicate that soluble and membrane bound LAP and γ-gtp activities are inhibited essentially to the same degree by gallic acid in rat intestine. Thus, lipid microenvironment in brush borders is not involved in enzyme-gallic acid interactions, although gallic acid is a partially hydrophobic molecule. The kinetic studies reveal that observed inhibition of LAP and γ-gtp activities by gallic acid is of competitive type, since enzyme K m increases 6-7 fold Table 4 Effect of 0.13 mm gallic acid in absence and presence of 2 mm hydroxylamine and 2 mm sodium arsenite on brush border γ-gtp activity in rat intestine [Values represent mean ± S.D., n=4] Assay system Activity % Decrease Control ± (0.13 mm) ± 0.037* ±.022* 30 and gallic ± 0.016* 61 acid (0.13 mm) ± 0.014* 3 and gallic acid (0.13 mm) ± * 43 in presence of gallic acid, compared to controls. These findings are apparently similar to those described for brush border sucrase 8 and invertase 20, where gallic acid also acts as competitive inhibitor of the enzymes. Due to the presence of polyhydroxy constellation in gallic acid, it is structurally similar to sugars, thus competes with enzyme substrate for the active site 8. However, this kind of assertion can not explain the observed competitive nature of inhibition of LAP and γ-gtp by gallic acid, since there is no structural similarity with the enzyme substrates. Hence, it is likely that gallic acid may bind to the enzyme protein at a site away from the active centre, but because of bulky nature may block the active site for accessibility of the substrate and thus it behaves as a fully competitive inhibitor of LAP and γ-gtp activities. Such a mechanism of action has earlier been proposed for sucrase inhibition by harmaline 21. Recently, it has been shown that polyphenols bind to various amino acid residues in proteins, which may be responsible for the observed enzyme inhibition 22. Dual effects of polyphenols on human salivary amylase activity have been reported 23. Fluorescence quenching studies reveal the structural changes in the enzyme protein, leading to the exposure of tryptophan residues upon polyphenol binding to proteins. Similar conclusions have been reported using circular dichroism and fluorescence measurements of gallic acid interactions with purified brush border sucrase in mice intestine 24.

5 382 INDIAN J. BIOCHEM. BIOPHYS., VOL. 46, OCTOBER 2009 Compared to other known inhibitors of LAP, such as amastatin and bestatin, which have K i of 1 µm and 20 nm, respectively 25,26, Ki for gallic acid- LAP interaction is found to be low (0.037 mm), indicating low affinity for the enzyme. However, affinity of gallic acid for LAP and γ-gtp is much stronger, compared to the intestinal disaccharidases and invertase 8,20. The observed inhibition of LAP and γ-gtp activities is reversible in nature, since addition of the inhibitor to the enzyme preparation produces significant decrease in enzyme activity which is restored to control values upon removal of the inhibitor by exhaustive dialysis. The tannins, in general have been reported to bind to a variety of proteins by interacting with free NH 2 or SH groups 27. The present data indicate that sodium arsenite which is known to block the SH group in the enzyme molecule causes protection of LAP and γ-gtp activities against inhibition by gallic acid. Also, the effect of hydroxylamine and sodiun arsenite alone or in combination with gallic acid reveals that the binding sites for hydroxylamine and sodium arsenite on the enzyme molecules are distinct from that for gallic acid, as the enzyme inhibition is additive in nature under experimental conditions. LAP and γ-gtp activities have been implicated not only in the digestion of peptides in intestinal lumen, but also have been reported to play an important role in the absorption of peptides and free amino acids in the intestine 9,10. The present findings indicate that these peptidases are strongly inhibited by low concentrations of gallic acid, which is a common constituent of edible foods 1,2. Thus, the intake of such compounds as dietary constituents may interfere with the absorption of amino acids from intestine. In conclusion, the present data support the contention that gallic acid, a constituent of dietary polyphenols may interfere in the digestion and absorption of proteins in rat intestine. Although the site of gallic acid interaction with LAP and γ-gtp is unknown, but several amino acid residues have been suggested as a target site for polyphenol interactions with the proteins 22. References 1 Amakura Y, Okada M, Tsuji A & Tonozai Y (2000) J Chromatogr B 896, Clifford M N & Scalbert A (2000) J Sci Food Agric 80, Tomas-Barberan F A & Clifford M N (2000) J Sci Food Agric 80, Shahidi F & Naczk M (1995) Food Phenolics Sources, Chemistry and Apllications, Lancaster, PA: Technomic Publishing Co. Inc 5 Kim Y J (2007) Biol Pharm Bull 30, Kroll J J, Noack H, Rawel R & Kroeck J (1994) J Sci Food Agric 65, Welsch C A, Lachance P A & Wasseman B P (1989) J Nutr 119, Gupta N, Gupta S & Mahmood A (2007) Nutr Res 27, Meister A (1973) Science 180, Louvard D, Marous S & Baratti J (1973) Biochem Biophys Acta 321, Kessler K, Acuto O, Storelli C, Murer M & Semenza G A (1978) Biochem Biophys Acta 506, Goldberg J A & Rutenberg A N (1958) Cancer 11, Naftalin B, Sexton M, Whitaker J F & Tracey D (1969) Am J Physiol 272, L739-L Bergmeyer M V C (1963) Academic Press New York 40, Lowry O H, Rosenbrough N J, Farr A L & Randall R J (1951) J Biochem 193, Dowd D & Riggs D S (1965) J Biol Chem 240, Kaur J, Jaswal V M S & Mahmood A (1994) Indian J Exp Biol 28, Ahmed A E, Smithoard R & Ellis M (1991) Br J Nutr 65, Chauhan A, Gupta S & Mahmood A (2007) Indian J Exp Biol 45, Mittal S (2006) M.Sc. Thesis, Effect of tannic acid and gallic acid on invertase activity in Saccharomyces cerevisiae and intestinal uptake in rats. Dept. of Biochemistry, Panjab University, Chandigarh 21 Mahmood A & Alverdo F (1975) Arch Biochem Biophys 168, Soares S, Mateus N & Freitas V D (2007) J Agric Food Chem 55, Rawel H M, Frey S K, Meidtner K, Kroll J & Schweigert F J (2006) Mol Nutr Food Res 50, Gupta S, Mahmood S, Khan R H & Mahmood A (2009) Biosci Rep (In Press) 25 Rich D H, Moon B J & Harbeson S (1984) J Med Chem 24, Burkey S K, David P R, Taylor A & Lipscomb W N (1991) Proc Natl Acad Sci (USA) 87, Rawel H M, Meidtner K & Kroll J (2005) J Agric Food Chem 53,

Effect of tannic acid on brush border disaccharidases in mammalian intestine

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