Purification and properties of an α-amylase inhibitor specific for human pancreatic amylase from proso (Panicium miliaceum) seeds

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1 J. Biosci., Vol. 7, Numbers 3 & 4, June 1985, pp Printed in India. Purification and properties of an α-amylase inhibitor specific for human pancreatic amylase from proso (Panicium miliaceum) seeds R. H. NAGARAJ and T. N. PATTABIRAMAN Department of Biochemistry, Kasturba Medical College, Manipal , India MS received 24 May 1984; revised 15 January 1985 Abstract. An α-amylase inhibitor was purified to homogeneity by acid extraction, ammonium sulphate fractionation, chromatography on carboxymethyl-cellulose, diethylaminoethylcellulose and Sephadex G-100 from proso grains (Panicium miliaceum). The calculated molecular weight was The inhibitor was fairly heat stable and stable under acidic and neutral conditions. The factor was more effective by two orders of magnitude in its action on human pancreatic amylase than on human salivary amylase. It did not inhibit on A. oryzae, B. subtilis and porcine pancreatic amylases. Pepsin rapidly inactivated the inhibitor. Chemical modification studies revealed that amino and guanido groups are essential for the action of the inhibitor. The inhibitor was found to protect both human salivary and pancreatic amylases against inactivation by acid. The mode of inhibition was found to be uncompetitive. Keywords. Panicium miliaceum; amylase inhibitor; specificity; pancreatic amylase; physicochemical properties. Introduction Even though several proteinaceous inhibitors of α-amylase have been isolated and characterized from wheat (Buonocore et al, 1977), a minor inhibitor with high degree of specificity towards human salivary amylase studied by O'Donnell and McGeeney (1976) is of special interest because of its application in clinical biochemistry. This inhibitor has been employed for the study of isoenzyme patterns in serum for diagnostic purposes (O'Donnell et al., 1977; Huang and Tietz, 1982). Preliminary studies in our laboratory (Chandrasekher et al., 1981) showed that the millet proso (Panicium miliaceum) had no action on human salivary amylase. Further studies indicated that it could inhibit human pancreatic amylase. In view of this, detailed study of the proso inhibitor was considered worthwhile both from fundamental and applied angles. In this communication, we report the isolation and characterization of this specific inhibitor. Abbreviations used: TNBS, Trinitrobenzene sulphonate; CHD, Cyclohexane dione; OMIU, O-methyl isourea; DTNB, 5,5'-dithiobis 2-nitrobenzoic acid; CM, carboxymethyl; DEAE, diethylaminoethyl; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulphate. 257

2 258 Nagaraj and Pattabiraman Materials and methods Materials Proso grains (Cultivar No. 1595) was procured from Tamil Nadu Agricultural University, Coimbatore. Human salivary amylase was partially purified as described earlier (Bernfeld, 1955). An acetone powder preparation of human pancreas served as the source of human pancreatic amylase. Porcine pancreatic amylase (twice crystallized), Aspergillus oryzae α-amylase, Bacillus subtilis α -amylase, Dalton mark VI, pronase, pepsin, ninhydrin and trinitrobenzene sulphonate (TNBS) were purchased from Sigma Chemical Company, St. Louis, Missouri, USA. Bovine trypsin (twice crystallized) and bovine α-chymotrypsin (thrice crystallized) were obtained from Worthington Biochemical Corporation, Freehold, New Jersey USA. 1,2-Cyclohexane dione (CHD) and O-methyl isourea (OMIU) were purchased from Aldrich Chemicals, Milwaukee, Wisconsin, USA. Carboxymethyl (CM)-cellulose, diethylaminoethyl (DEAE)-cellulose and BioGel P-30 were from BioRad Laboratories, Richmond, California USA. 5,5'-Dithiobis 2-nitrobenzoic acid (DTNB) was purchased from Pierce Chemical Company, Rockford, Illinois, USA. Sephadex G-100 was from Pharmacia Fine Chemicals, Uppsala, Sweden. General methods Amylase activity was measured as described by Bernfeld (1955) with the following modifications. The assay mixture contained the enzyme, 30 µmol of sodium phosphate buffer ph 6 9 and 10 5 µmol of NaCl in volume of 1 5 ml. The reaction was initiated by the addition of 0 5 ml of 1% starch solution. After 5 min incubation at 37 C, the enzyme action was arrested by 1 0 ml of dinitrosalicylate reagent. The solution was kept in a boiling water bath for 10 min, cooled and diluted to 11 0 ml with water. The colour was measured at 540 nm. One unit of amylase is the amount that liberated 1 µmol of reducing equivalent (as maltose). Human salivary amylase (1 2 µg), porcine pancreatic amylase (0 2 µg), human pancreatic amylase (18 µg), A. oryzae amylase (15 4 µg) and B. subtilis amylase (8 2 µg) provided 4 units of activity under the assay conditions. To measure the inhibitory activity, suitable aliquots of the amylase inhibitor solution was incorporated into the assay medium. After 20 min incubation at 37 C, the substrate was added and the system was processed as described above. One unit of inhibitor is the amount that suppressed the amylase activity by one unit. Protein was estimated by the method of Lowry et al. (1951) using bovine serum albumin as standard. Isolation of proso amylase inhibitor All Operations were carried out at room temperature (28-30 C) unless stated otherwise. Proso seed powder (500 g) was homogenized with 1500 ml of 0 1 Μ HCl containing 0 15Μ NaCl. After stirring for 20 min, the suspension was centrifuged at 10,000 g for 30 min. The supernatant (designated as acid extract) was adjusted to a ph of 7 0 by the addition of 5 Ν NaOH. The protein precipitated was removed by centrifugation at 10,000 g for 10 min. The supernatant (neutral extract) was subjected to ammonium

3 Amylase inhibitor from proso seed 259 sulphate fractionation by the addition of salt (297 g) to 50 % saturation. After standing for 48 h at 4 C, the precipitate containing the activity was collected by centrifugation at 10,000 g for 20 min, dissolved in 80 ml of 0 02 Μ acetate buffer ph 5 0 and dialyzed against 20 volumes of the same buffer for 24 h. The dialysate was centrifuged at 10,000 g for 30 min. CM-Cellulose chromatography: The clear supernatant (designated as ammonium sulphate fraction) was passed through a column of CM-cellulose ( cm bed vol 30 ml) equilibrated with 0 02 Μ acetate buffer ph 5 0. The column was eluted with 500 ml of the equilibration buffer followed by elution with a linear gradient formed between 100 ml of 0 02 Μ acetate buffer ph 5 0 and 100 ml of the buffer containing 0 2 Μ NaCl. Ten ml fractions were collected at a flow rate of 25 ml/h. The 'active fractions' (tube numbers 26 34, figure 1) were pooled. The solution was concentrated to 36 ml by ultrafiltration and dialyzed against 40 volumes of 0 02 Μ Tris-HCl buffer ph 9 0 for 24 h. This was designated as CM-cellulose fraction. Figure 1. Chromatography of the ammonium sulphate fraction on CM-cellulose column Experimental details are described under materials and methods. DEAE-cellulose chromatography: The above solution was applied onto a column of DEAE-cellulose ( cm, bed vol 16 0 ml) equilibrated with 0 02 Μ Tris-HCl buffer ph 9 0 The column was washed with 200 ml of the buffer and the inhibitor was eluted with a linear gradient (45 ml of the buffer + 45 ml of the buffer containing 0 2 Μ NaCl) Fractions (8 5 ml) were collected at a flow rate of 30 ml/h. 'Active fractions (tube numbers 14 17, figure 2) were pooled and concentrated to 5 0 ml by ultrafiltration, (DEAE-cellulose fraction).

4 260 Nagaraj and Pattabiraman Figure 2. Chromatography of the CM-cellulose fraction on DEAE-cellulose. Experimental details are described under materials and methods. Gel chromatography: The above solution was allowed to flow through a column of Sephadex G-100 ( cm, bed vol 130 ml) equilibrated with 0 02 Μ Tris-HCl buffer ph 9 0 containing 0 1 Μ NaCl. Gel filtration was done at a flow rate of 10 ml/h, with the same buffer and 5 0 ml fractions were collected. 'Active fractions' (tube numbers 13 17, figure 3) were pooled and stored at 5 C (Sephadex fraction). Figure 3. Chromatography of DEAE-cellulose fraction on Sephadex G-100 column. Experimental details are described under materials and methods

5 Electrophoretic studies Amylase inhibitor from proso seed 261 The purified inhibitor was subjected to polyacrylamide gel electrophoresis (PAGE) in Tris-glycine buffer ph 8 6 according to the method of Weber et al. (1972) using 5 % gel. The run was for 4 hat a current of 1 5 ma per tube. Sodium dodecyl sulphate (SDS)- PAGE with and without mercaptoethanol treatment was also done under similar conditions with 10 % gel. Electrophoresis was also performed on Cellogram (Shandon) strips in 0 02 Μ citrate buffer ph 5 0 and in 0 02 Μ Tris-glycine buffer ph 8 6. The run was for 2 h at a current of 1 5 ma per cm of the strip. The protein bands were stained with Coomassie brilliant blue R-250 (0 25 % in methanol: acetic acid: water, 5:1:5). Destaining was done by washing with 7 % acetic acid. Molecular weight determination This was done by SDS-PAGE as described above. Dalton mark VI containing bovine serum albumin, ovalbumin, pepsin, trypsinogen, β-lactalbumin and lysozyme was used for standardization. Molecular weight was also estimated by gel chromatography on BioGel P-30 ( cm, bed vol 36 ml) using 0 02 Μ Tris-HCl buffer ph 9 0 containing 0 1 Μ NaCl as eluant. One ml fractions were collected at a flow rate of 6 0 ml/h and assayed for protein and inhibitor. Myoglobin, cytochrome-c and chymotrypsin were used as standards for calculating the molecular weight. Studies on the stability of the inhibitor The inhibitor (40 µg) dissolved in 1 ml of water was heated at 100 C for different intervals of time. Aliquots were withdrawn, cooled and assayed for residual inhibitory activity. The inhibitor (5 µg) in 0 05 ml was mixed with 0 05 ml of systems of varying ph (0 1 Ν HCl ph 1 0,0 1 Ν citrate-phosphate ph 3 0,0 1 Ν acetate ph 4 0, ph 5 0,0 1 Ν citrate-phosphate ph 6 1,0 1 Ν phosphate ph 6 9,0 1 Ν Tris-HCl ph 8 0, ph 9 0, 10 0, 0 1Ν NaOH ph 12 0) and incubated for 18 h at 4 C. The solution was added to the assay medium and tested for inhibitory activity. Effect of ph on the interaction of amylases with inhibitor The inhibitor (4 µg) was preincubated with human pancreatic or salivary amylase in the presence of 5 µmol of the following buffers for 20 min at 37 C in a volume of 0 3 ml HCl-KCl ph 2 0, acetate ph 3 0, ph 4 0, ph 4 5, 5 0, phosphate ph 6 0, 6 9, Tris-HCl ph 8 0, 8 5. The amylase activity was measured by the addition of 30 µmol of sodium phosphate buffer ph 6 9,10 5 µmol of NaCl and 0 5 ml of starch solution as described above. Controls without inhibitor were run simultaneously. Action of proteases on the inhibitor To study the effect of pepsin, the inhibitor (40 µg) was incubated with pepsin (10 µg) in the presence 100 µmol of 0 2 Ν HC1-KC1 (ph 2 0) in 1 5 ml at 37 C. Aliquots were withdrawn at different intervals of time and assayed for residual inhibitory activity. To study the effect of neutral proteases the inhibitor (40 µg) was treated with trypsin (10 µg), chymotrypsin (10 µg) or pronase (40 µg) in the presence of 100 µmol of sodium phosphate buffer ph 7 6 in 3 0 ml at 37 C. At definite time intervals aliquots were

6 262 Nagaraj and Pattabiraman withdrawn and assayed for amylase inhibitory activity. Under the assay conditions the proteases had no effect on the amylase. Chemical modification of the inhibitor Lysine residues were modified with TNBS (Haynes et al., 1967). The inhibitor (150 µg) was treated with 500 µg of TNBS in the presence of 200 µmol of sodium phosphate buffer ph 7 6 in 2 0 ml. After definite intervals of time, the solution was dialyzed against 500 ml of water for 8 h. Lysine residues were also modified with O-methyl isourea (OMIU) (Kimmel, 1967). The inhibitor (100 µg) was treated with 500 µg of OMIU in the presence of 200 µmol of Tris-HCl buffer ph 9 5 in 5 0 ml. Aliquots at intervals of time were withdrawn and dialyzed against 500 ml of water for 8 h. Arginyl residues were modified by treatment with cyclohexane dione (CHD) (Abe et al., 1978). The inhibitor (100µg protein) was incubated with 550 µg of CHD in the presence of 250 µmol of Tris-HCl buffer ph 9 0 in 2 0 ml. After definite periods of reaction the solution was dialyzed against 500 ml of water. Arginyl groups were also modified with ninhydrin (Chaplin, 1976). The inhibitor (50 µg) was reacted with 200 µg of ninhydrin in the presence of 200 µmol of Na 2 HPO 4 (ph 9 2) in 2 0 ml. The samples after varying periods of reaction were dialyzed against 500 ml of 0 02 Μ sodium acetate buffer ph 4 0 for 8 h. Sulphydryl groups were modified with DTNB (Ellman, 1959). The inhibitor (150 µg) was treated with 500 µg of DTNB in the presence of 50 µmol of sodium phosphate buffer ph 8 0 in 1 0 ml. After fixed periods of reaction the solution was dialyzed against 500 ml of water for 8 h. Disulphide bridges in the inhibitor (50 µg) were reduced by treatment with 0 1 ml of 1 % 2-mercaptoethanol in the presence of 50 µmol of sodium phosphate buffer ph 7 3 for 5 h in a volume of 1 0 ml. The residual inhibitory activity in all these cases of the modified protein was measured. Controls without the chemical modifiers were run simultaneously. Results The purification of proso α-amylase inhibitor is summarized in table 1. A 30-fold purification with an yield of 20 % was accomplished. The inhibitor on SDS-PAGE in Table 1. Purification of the proso amylase inhibitor.

7 Amylase inhibitor from proso seed 263 Figure 4. A. SDS-PAGE of the purified inhibitor in presence of mercaptoethanol. Experimental details are given under electrophoretic studies. B. SDS-PAGE of the purified inhibitor without mercaptoethanol. Experimental details are described under electrophoretic studies. C. PAGE of the purified inhibitor. Details are given under electrophoretic studies. the presence of 2-mercaptoethanol and in its absence showed single protein bands (figure 4A, B). During gel chromatography on BioGel P-30, it was eluted as a sharp single peak with a Ve/Vo value of 1 46 and with constant specific activity across the peak. On cellulose acetate electrophoresis at ph 5 0, the purified inhibitor moved as a single protein band 15 mm towards the anode. However on cellulose acetate electrophoresis at ph 8 6, the inhibitor separated into 3 closely moving fractions of roughly equal proportion. Similarly PAGE at ph 8 6 also revealed three protein bands (figure 4C). These data suggest that the purified inhibitor could represent three charge isomers having similar size. However attempts to elute the three bands and demonstrate the inhibitory activity in them were not successful. The molecular weight calculated based on the mobility during SDS-PAGE was 14,000 which agreed well with the value of 13,000 obtained based on the data on gel chromatography on BioGel P-30. The purified inhibitor had no effect on B. subtilis, A. oryzae and porcine pancreatic amylases even at a concentration of 100 µg. At this level it showed a weak inhibition on human salivary amylase. The relative ratio of action on human pancreatic and salivary amylases was calculated to be 87. The inhibitor exhibited linear pattern of action upto

8 264 Nagaraj and Pattabiraman 60 % with respect to concentration (1 7 µg) on human pancreatic amylase. Beyond this range the inhibition was progressive but non-linear, culminating in complete inactivation of the amylase at 100 µg of inhibitor protein. The magnitude of inhibition was dependent on the preincubation of the inhibitor with the amylase. A minimum of min preincubation was necessary to elicit the maximal response. Pretreatment of the inhibitor with starch solution for 10 min before the assay resulted in complete disappearance of the inhibitory activity. The inhibitor was fairly heat stable. Exposure to 100 C for 10,20,30 and 60 min resulted in the loss of 77 % 63 %, 58 % and 50 % of the activity respectively. The effect of ph on the stability of the inhibitor is shown in figure 5. In the alkaline ph range there was a gradual loss of activity. The inhibitor was more stable under acidic condition than in neutral solution. Figure 5. Effect of ph on the stability of the inhibitor. Experimental details are described under materials and methods. The effect of H + concentration on the interaction of human pancreatic amylase with the proso inhibitor during the preincubation period was studied and the results are shown in figure 6A. In the ph range 6 7 the interaction was strong as indicated by maximal inhibition. In the alkaline range there was a decrease in inhibitory activity. In the acidic range the picture is complicated because of the inactivation of amylase itself in the control tubes which was maximal at ph 4 5. Below ph 4 0 instead of inhibition an activation was observed in the test systems. This suggests that the inhibitor by binding with the amylase protects the enzyme against inactivation by the acidic conditions. Interestingly, a similar phenomenon was observed with human salivary amylase (figure 6B), prominently in the ph range , even though the inhibitor had no enzyme-blocking action per se on the enzyme at the concentration used.

9 Amylase inhibitor from proso seed 265 Figure 6. Α. Effect of ph during pre-incubation of the inhibitor with human pancreatic amylase.( ) Amylase activity; ( ), activation/inhibition of amylase in presence of inhibitor; (O), inhibitory activity. Experimental details are described under materials and methods. B. Effect of ph during pre-incubation of the inhibitor with human salivary amylase. ( ), Amylase activity; ( ), activation/inhibition of amylase with inhibitor; (O), inhibitor activity. Experimental details are described under materials and methods. Figure 7. Effect of digestion of the inhibitor with ( ), pepsin; (O) pronase (Δ), chymotrypsin ( ) trypsin Details are given under action of protease on the inhibitor.

10 266 Nagaraj and Pattabiraman The action of proteolytic enzymes on proso inhibitor is shown in figure 7. Pepsin was found to rapidly inactivate the inhibitor and complete abolition of amylase inhibitory activity was observed in min. Trypsin and chymotrypsin were relatively slow in this respect. Only after 8 h interaction with these enzymes the proso inhibitor lost its activity completely. Pronase was found to inactivate the inhibitor in 2 h. The data on the effect of chemical modifiers on the biological activity of proso inhibitor are shown in table 2. TNBS treatment resulted in a rapid loss of the activity of the inhibitor implicating the amino groups in the process of interaction of the inhibitor with human pancreatic amylase. OMIU, another amino group modifier was however sluggish in bringing about the loss of activity. Ethyl acetamidate had no action at all under the conditions of the study. Ninhydrin also caused rapid loss of the action of the inhibitor indicating that arginyl groups of the inhibitor are also essential for the expression of the biological activity. This is supported by the data on CHD which however was slow in bringing about the change. Free sulphydryl groups do not appear to be essential for the action or stability of the inhibitor since DTNB had no effect. Similarly, the disulphide bridges if any, do not appear to play a role since mercaptoethanol did not alter the inhibitor potency. The mode of inhibition of human pancreatic amylase by the proso inhibitor was found to be uncompetitive (figure 8). The K i value was calculated to be 0 17 µm based on a molecular weight of 14,000 for the inhibitor. Table 2. inhibitor. Effect of chemical modifiers on the

11 Amylase inhibitor from proso seed 267 Figure 8. Lineweaver-Burk plot for the mode of inhibition ( ).Human pancreatic amylase; (Δ) human pancreatic amylase with inhibitor (4 0 µg). The assay system is as described under materials and methods except for varying concentration of starch, expressed as mg in the assay mixture. V is described as µmol of maltose equivalent liberated under the assay conditions. Discussion The inhibitor from proso described here is unique in that its action is two orders of magnitude higher on human pancreatic than on salivary amylase. To the best of our knowledge only one such inhibitor with the reverse specificity has been described in wheat (O'Donnell and McGeeney, 1976). The exact mechanism of action of these two inhibitors on amylases is not known. Further studies are needed to understand the reason for high specificities of these inhibitors. The molecular weight of the inhibitor is relatively low compared to the wheat inhibitor (O'Donnell and McGeeney, 1976) and pearl millet inhibitor (Chandrasekhar and Pattabiraman, 1983) but is identical to the subunits of the 0 19 inhibitor of wheat (Silano et al., 1973) and the pearl millet inhibitor (Chandrasekhar and Pattabiraman, 1983). In other physicochemical properties the proso inhibitor resembles the other cereal inhibitors. The inactivation by starch, essentiality of pretreatment with the target enzyme for maximal activity are reminiscent of the cereal inhibitors in general. Like the amylase inhibitors-1 and -2 of wheat (Shainkin and Birk, 1970) and inhibitor-1 of ragi (Shivaraj and Pattabiraman, 1981) both amino and guanido groups appear to be essential for the action of the proso inhibitor. The proso inhibitor may not be of much nutritional relevance since pepsin was found to rapidly destroy its biological action. However the inhibitor will find application in differentiating salivary and pancreatic amylases in the serum for diagnostic purposes.

12 268 Nagaraj and Pattabiraman Acknowledgements This work was supported by a grant from the Department of Science and Technology, New Delhi. The authors thank Dr. A. Krishna Rao, Dean of this college for his encouragement. References Abe, O., Ohata, J., Utsami, Y. and Kuromizu, K. (1978) J. Biochem., 83, Bernfeld, P. (1955) Methods Enzymol., 1, 149. Buonocore, V., Petrucci, T. and Silano, V. (1977) Phytochemistry, 16, 811. Chandrasekhar, G., Prasad Raju, D. and Pattabiraman, T. N. (1981) J. Sci. Food Agric., 32, 9. Chandrasekhar, G. and Pattabiraman, Τ. Ν. (1983) Ind. J. Biochem. Biophys., 20, 241. Chaplin, Μ. F. (1976) Biochem. J., 155, 457. O'Donnell, Μ. D. and McGeeney, Κ. F. (1976) Biochim. Biophys. Acta, 422, 159. O'Donnell, M. D., Fitzgerald, Ο. and McGeeney, K. F. (1977) Clin. Chem., 23, 560. Ellman, G. L. (1959) Arch. Biochem. Biophys., 82, 70. Haynes, R., Osuga, D. T. and Feeney, R. F. (1967) Biochemistry, 6, 541. Huang, W. Υ. and Tietz, Ν. W. (1982) Clin. Chem., 28, Kimmel, J. R. (1967) Methods Enzymol., 9, 481. Lowry, Ο. Η., Rosebrough, Ν. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem., 193, 265. Shainkin, R. and Birk, Y. (1970) Biochim. Biophys. Acta, 221, 502. Shivaraj, Β. and Pattabiraman, Τ. Ν. (1981) Biochem. J., 193, 29. Silano, V., Pocchairi, F. and Kasarda, D. D. (1973) Biochim. Biophys. Acta, 317, 139 Weber, K., Pringle, J. R. and Osborn, Μ. (1972) Methods Enzymol., 26, 3.