Biosci., Vol. 5, Number 3, September 1983, pp. 219 224. Printed in India. Proteases in germinating finger millet (Eleusine coracana) seeds Introduction U. VIDYAVATHI, B. SHIVARAJ and T. N. PATTABIRAMAN Department of Biochemistry, Kasturba Medical College, Manipal 576 119 MS received 12 February 1983; revised 24 May 1983 Abstract. Proteolytic activity was estimated in germinated finger millet seedlings using the endogenous trypsin/amylase inhibitor as substrate and also with haemoglobin and albumin as substrates. The maximal proteolytic activity was observed on the third day of germination. With the inhibitor as substrate, the proteolytic activity was maximal at ph 2.5. The protease that acted on the inhibitor required sulphydryl groups for maximal activity and was suppressed by diazoacetyl norleucine methyl ester and Pepstatin. The protease that acted on haemoglobin with optimum ph of 5.0, was more stable on storage, did not depend on sulphydryl groups for activity and was unaffected by reagents that react with carboxyl groups. Keywords. Proteases; finger millet; Eleusine coracana; trypsin/amylase inhibitor. Plant protease inhibitors and amylase inhibitors which are proteinaceous in nature are known to disappear during the process of germination (Ryan, 1973; Chrispeels and Baumgartner, 1978; Shivaraj and Pattabiraman, 1980). Veerabhadrappa et al. (1978) first showed that antitryptic activity and antichymotryptic activity were markedly reduced in the endosperm of finger millet on germination. This can be attributed to the proteolytic cleavage of the inhibitory proteins during germination. Characterization of many proteases from germinated cereals and legume seeds has been accomplished using exogenous as well as endogenous substrates (Ashton, 1976; Baumgartner and Chrispeels, 1977; Fujimaki et al., 1977). However, little information is available on the action of seed proteases on the endogenous protease inhibitors. An earlier communication from this laboratory reported the isolation and characterization of an inhibitor from finger millet acting on both trypsin and α-amylase (Shivaraj and Pattabiraman, 1981). Using this factor as a substrate, the proteolytic activity of finger millet during germination was studied and the results were compared with the proteolytic activities observed with haemoglobin and albumin as substrates. Materials and methods Chemicals Hog pancreatic amylase (EC 3.2.1.1, type IIIΑ, twice crystallised), Pepstatin, bovine haemoglobin, α-n-benzoyl DL-arginine p-nitroanilide (ΒΑΡΝΑ) and dia- Abbreviations used: ΒΑΡΝΑ, α-n-benzoyl-dl-arginine p-nitroanilide; DON, diazoacetyl DLnorleucine methyl ester; DTNB, 5, 5 -dithiobis nitrobenzoic acid; TAI, trypsin/amylase inhibitor. 219
220 Vidyavati et al. zoacetyl DL-norleucine methyl ester (DON) were purchased from Sigma Chemical Company, St. Louis, Missouri, USA. Bovine trypsin (EC 3.4.21.4, salt free and twice crystallized) was the product of Worthington Biochemical Corporation, Freehold, New Jersey, USA. -Dithiobis 2-nitro-benzoic acid (DTNB) was procured from Pierce Chemicals, Rockford, Illinois, USA. All other reagents were of analytical grade. Germination of finger millet This was carried out in batches. Two g of the grain was soaked in a wet cloth for 16 h and then allowed to germinate in a petri dish under diffused light at room temperature (25 28 C). At the end of 2, 3, 4, 5 and 6 days of germination, the seedlings were extracted with 20 ml of water at 4 C. The extract was centrifuged at 5000 g for 10 min in a Remi C-24 centrifuge at 4 C. The supernatant was used as the source of proteases. Isolation of trypsin/amylase inhibitor (TAI) This was done by the conventional purification method (Shivaraj and Pattabiraman, 1980) and also by affinity chromatography (Shivaraj et al., 1982). TAI prepared by both the methods behaved similarly when used as substrates for finger millet proteases. Hence, in all subsequent studies the inhibitor isolated by the affinity chromatographic method was employed. Assay of proteolytic activity of germinated finger millet extract using TAI as substrate An aliquot (20 100 µl) of the extract was incubated with 5.6 µg protein of TAI at 37 C in presence of 50 µl of 0.5% 2-mercapto-ethanol and 50 µl of 0.05 Μ buffers of varying ph (HCl-KCl, ph 1.5 2.0; glycine-hcl, ph 2.5 3.5; acetate, ph 4.0 5.0; sodium phosphate, ph 6.0 and Tris-HCl, ph 8.0) in a total volume of 0.2 ml for 3 h. To assay the residual antitryptic activity of TAI, trypsin solution (10 12 µg) was added to the incubation mixture followed by 2.0 µmol of ΒΑΡΝΑ and 100 µmol of phosphate buffer, ph 7.6 to a final volume of 3.0 ml. After 30 min incubation at 37 C, the tryptic activity was terminated by the addition of 2.0 ml of 30% acetic acid (v/v).p-nitro-aniline liberated was measured at 410 nm. Controls with heat treated (100 C, 2 min) extract of finger millet were run simultaneously. To measure the residual antiamylase activity of TAI after the action of the proteases, 0.1 ml of the incubation mixture (see above) was treated with hog pancreatic amylase (0.3 µg protein) at 37 C for 20 min in presence of 60 µmol of sodium phosphate buffer, ph 6.9 containing 10.5 µmol of NaCl (total volume 1.5 ml). The amylase action was initiated by the addition of 0.5 ml of 1% starch solution. After 5 min incubation at 37 C, the reaction was arrested with 1 ml of dinitrosalicylate reagent and processed further as described earlier (Shivaraj and Pattabiraman, 1980). Controls were performed during the assay with heat treated finger millet extract. One unit of tryptic activity was the amount that liberated 1 µmol of p-nitroaniline under the assay conditions. One unit of amylase activity was equivalent to the amount of amylase that liberated 1 µmol of reducing sugar (maltose equivalent) under the assay conditions. One unit of inhibitor, is the amount that decreased the tryptic (or amylase) activity by one unit.
Finger millet proteases 221 Protease activity of germinated finger millet extract with haemoglobin or albumin as substrate The extract (0.2 ml) was incubated with 20 mg of denatured haemoglobin (or albumin) in a volume of 2.5 ml in presence of 50 µmol of buffers of varying ph values (ph 2.5-8.0, see above). After 4 h incubation at 37 C, the reaction was terminated by the addition of 1.5 ml of 10% trichloroacetic acid (w/v). After 30 min, the precipitate formed was sedimented by centrifugation at 2500 g for 10 min and 1 ml of the clear supernatant was analyzed for the soluble peptides by the method of Lowry et al. (1951). One unit of proteolytic activity is equal to the amount that formed 1 mg of trichloroacetic acid soluble fragments under the assay conditions. Protein estimation Protein content of the finger millet extracts was measured by the dye binding method using Coomassie Brilliant Blue-G (Read and Northcote, 1981). Results The effect of the trypsin/amylase inhibitor isolated from dormant finger millet was tested for its action on the protease activity of germinated finger millet extracts at different ph values in the range 2.5 7.0. Haemoglobin was used as substrate in these studies. It was found that the inhibitor did not suppress the endogenous protease activity. Similarly TAI had no effect on the endogenous amylase activity of the millet extract when assayed at ph 6.9. These data indicate the possibility of the proteolytic digestion of TAI by the endogenous proteases in the germinated millet extract. When TAI was incubated with the seed extract and then tested for antitryptic and antiamylase activities, against bovine, trypsin, and hog pancreatic amylase respectively, there was a decrease of inhibitory potency. It was found that there was proportional loss of trypsin inhibitory activity and amylase inhibitory activity when TAI was treated with germinated millet extract. The data are shown in table 1. These results provide additional evidence for the earlier observation, that antiamylase and antitryptic activities reside in the same protein (Shivaraj and Pattabiraman, 1981). The proteolytic activity was found to be maximum in 3 days germinated finger millet extracts when estimated with either TAI or haemoglobin as substrates. The activity profiles at different stages of germination are depicted in figure 1. The results are based on the calculation of proteolytic activity at ph 2.5 for TAI substrate and at ph 5.0 with haemoglobin as substrate, since these were the optimum ph values required for the corresponding substrates (figure 2). The protease activity responsible for the inactivation of TAI, required SHgroups for maximal activity. Omission of 2-mercaptoethanol in the assay system at ph 2.5 led to a 65% reduction in enzyme activity. Inclusion of DTNB (500 µg) in the assay system completely abolished the enzyme activity. On the other hand, at ph 5.0 with haemoglobin as substrate, the proteolytic activity was not dependent on SH-groups. Addition of 2-mercaptoethanol did not increase the proteolytic activity at ph 5.0. Further, treatment of finger millet extract with DTNB (500 µg) both at ph 5.0 and at ph 7.5 did not cause any change in haemoglobin cleaving activity.
222 Vidyavati et al. Table 1. Action of proteases of 3-day germinated finger millet extract on the antitryptic and antiamylase activities of TAI. TAI was incubated with finger millet extract for 3 h at 37 C at different ph values and residual inhibitory activities were determined. Other details are given under materials and methods. Figure 1. Proteolytic activity of germinated finger millet extract at different days of germination. Using TAI as substrate at ph 2.5, (O). The protease activity was measured on the basis of disappearance of antitryptic activity of the inhibitor. Using haemoglobin as substrate at ph 5.0, ( ). Addition of DON (250 µg) or Pepstatin (250µg) to the germinated finger millet extract abolished the protease action on TAI. On the other hand, these modifiers did not affect the haemoglobin splitting activity at ph 5.0. The results are shown in table 2. These data suggest that the protease that acts on TAI is a carboxyl proteinase. The protease that inactivates TAI was found to be highly labile on storage. The activity disappeared completely on standing of the millet extract at 4 C for 72 h. In contrast, 65% of the original haemoglobin splitting activity at ph 5.0 remained on storage of the extract at 4 C for 6 days. The relative stabilities of the two activities
Finger millet proteases 223 Figure 2. Prpteolytic activity of 3 day germinated ragi extract at different ph values. Using TAI as substrate, (O). The protease activity was measured on the basis of disappearance of antitryptic activity of the inhibitor. Using haemoglobin as substrate, ( ) Table 2. Action of carboxyl group modifiers on the finger millet proteases. *The protease activity was measured in terms of disappearance of antitryptic activity of TAI. are shown in figure 3. When the proteolytic activity in germinated finger millet extracts was measured with albumin as substrate, the ph optimum was 3.5. However, the activity with albumin as substrate was only 25% compared to the activity with haemoglobin as substrate. Discussion The present studies show that there are at least two different proteases in germinating finger millet, one that has an optimum ph 2.5 and the other with an optimum ph of 5.0. The latter enzyme was relatively more stable and was not dependent on SH-groups for activity. The former enzyme was relatively labile and required SH-groups for maximal activity. Sulphydryl dependent acid proteases have been isolated and characterized during germination from barley (Burger, 1973), mung bean (Baumgartner and Chrispeels, 1977), kidney bean (Vavreinova and Turkova, 1975) and corn (Abe et al., 1978). The corn enzyme is reported to have the lowest ph optimum of 3.0 for all the sulphydryl proteases known. The
224 Vidyavati et al. Figure 3. Effect of storage of 3 day germinated ragi extract at 4 C on the endogenous protease activity. Using TAI as substrate at ph 2.5, (O). The protease activity was measured on the basis of disappearance of antitryptic activity of the inhibitor. Using haemoglobin as substrate at ph 5.0, ( ). acid protease in finger millet reported here has a ph optimum lower than that. The active site of the enzyme probably has a carboxyl group since Pepstatin and DON inhibited its action. The SH-groups may be essential for the conformational stability of the enzyme. Further studies with purified enzymes are needed to confirm this suggestion. To the best of our knowledge, this report is the first that demonstrates the presence of a protease that needs a carboxyl group for activity in germinating plant seeds. It is probable that many of the sulphydryl dependent acid proteases reported in the plant systems could in fact be carboxyl proteases requiring SH-groups for stability. Acknowledgements This work was supported by a grant-in-aid from the Indian Council of Agricultural Research, New Delhi. The authors are thankful to Dr. A. Krishna Rao, Dean of this college for the keen interest and encouragement. References Abe, M., Arai, S. and Fujimaki, M. (1978) Agric. Biol. Chem., 42, 1813. Ashton, E. M. (1976) Ann. Rev. Plant Physiol., 27, 95. Baumgartner, B. and Chrispeels, M. J. (1977) Eur. Biochem., 77, 223. Burger, W. C. (1973) Plant Physiol., 51, 1015. Chrispeels, M. J. and Baumgartner, B. (1978) Plant Physiol., 61, 617. Fujimaki, M., Abe, M. and Arai, S. (1977) Agric. Biol. Chem., 41, 887. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Biol. Chem., 193, 265. Read, S. M. and Northocote, D. H. (1981) Anal. Biochem., 116, 53. Ryan, C. A: (1973) Ann. Rev. Plant Physiol., 24, 173. Shivaraj, B. and Pattabiraman, T. N. (1980) Indian Biochem. Biophys., 17, 181. Shivaraj, B. and Pattabiraman, Τ. Ν. (1981) Biochem., 193, 29. Shivaraj, B., Nayana Rao, Η. and Pattabiraman, T. N. (1982) Sci. Food Agric., 33, 1080. Vavreinova, S. and Turkova, J. (1975) Biochim. Biophys. Acta, 403, 506. Veerabhadrappa, P. S., Manjunath, N. H. and. Virupaksha, T. K. (1978) Sci. Food Agric., 29, 353.