Partial Characterization of a Protease Inhibitor Which Inhibits the Major Endopeptidase Present in the Cotyledons of Mung Beans

Abstract

Germination of mung beans (Phaseolus aureus, Roxb.) is accompanied by an increase in the activity of the endopeptidase involved in storage protein metabolism. Enzyme activity in the cotyledons increases 25-fold during the first 5 days of germination. The cotyledons also contain inhibitory activity against the endopeptidase, and this inhibitory activity declines during germination, suggesting that inhibitors may play a role in regulating the activity of the endopeptidase. The inhibitory activity against the mung bean endopeptidase is due to the presence of two inhibitors which can be separated by chromatography on Sephadex G-100. The two inhibitors have approximate molecular weights of 12,000 and smaller than 2,000 daltons. The large inhibitor co-elutes with trypsin inhibitor on Sephadex G-100, but these two inhibi-tory activities can be separated by means of a trypsin affinity column. The inhibitory activity disappears slowly from crude extracts incu-bated at 6 C and more rapidly when the extracts are incubated at 25 C or 37 C. The disappearance of inhibitory activity is accompanied by a rise of the endopeptidase activity, but an examination of the kinetics of these two phenomena suggests that they are not causally related. Fractionation of the ceDular organelles on sucrose gradients shows that the inhibitory activity is not associated with the protein bodies, but rather with the cytosol. Our results suggest that the endopeptidase inhibitor(s) does not regulate the increase in endopeptidase activity which accompanies germination or the metabolism of storage protein. We, therefore, postulate that the inhibitor(s) may function in protecting the cytoplasm from accidental rupturing of the protease-containing protein bodies. geneity and found that more than 95% of the endopeptidase activity in the cotyledons of 5-day-old plants can be accounted for by a single enzyme with an apparent mol wt of 23,000 (Baumgartner and Chrispeels, in preparation). Cotyledons of leguminous seeds are a rich source of protease inhibitors (10). The inhibitors which inhibit proteases of animal or microbial origin (e.g. trypsin inhibitors and chymotrypsin inhibitors) have been studied rather extensively, while relatively little is known about the inhibitors which inhibit endogenous plant proteases (18). Germination is accompanied by a gradual decline in the level of trypsin inhibitors in the cotyledons (7, 16). There is no evidence, however, that the increase in proteolytic activity which also accompanies germination is causally related to the decrease in trypsin-inhibitor activity, and the physiological function of these inhibitors remains unknown (18). Royer et al. (17) have shown recently that the removal of trypsin inhibitors from extracts of cowpea cotyledons caused an increase in the caseolytic activity of these extracts. This led them to suggest that protease inhibitors play a role in the activation of pre-existing proteases and in the control of reserve protein metabolism. A similar mechanism has been proposed for the control of reserve protein metabolism in germinating lettuce seeds (18, 19). As part of our study on the control mechanism involved in storage protein metabolism in germinating legume seeds (3, 4, 6), we have analyzed extracts of cotyledons for the presence of inhibitors of the endopeptidase activity. The evidence presented here indicates that the cotyledons contain such inhibitors but suggests that these inhibitors are not involved in the regulation of the metabolism of storage proteins. The germination of leguminous seeds is accompanied by the metabolism of the reserve proteins stored in the cotyledons. These reserve proteins are located in special organelles called protein bodies or aleurone grains (5, 20), and these organelles appear to be undergoing autolysis during germination (2, 15). Protein bodies isolated from dry legume seeds contain a variety of hydrolytic enzymes including proteases (6, 12, 13, 21), and it has been suggested that they are capable of autolysis (13) although this process has never been demonstrated in vitro (6). Germination is accompanied by a dramatic increase in proteo-lytic activity in the cotyledons, and we have shown that this is due to the appearance of an endopeptidase (3). The metabolism of the storage proteins is dependent on the appearance of this enzymatic activity in the cotyledons. We have demonstrated both a temporal and a spatial relationship between the appearance of the enzyme activity and the metabolism of the reserve proteins. We have recently purified the endopeptidase to homo-I This work was supported by Grant GB 30235 from the National Science Foundation and a fellowship to B. B. from the Swiss National Science Foundation. MATERIALS AND METHODS Plant Material, Germination, and Extraction of Cotyledons. Seeds of mung beans (Phaseolus aureus, Roxb.) were obtained from a local dealer and grown as described in our earlier work (4). The cotyledons were homogenized with a mortar and pestle in 25 mm citrate-phosphate (pH 5.7) containing 10 mm 3-mercaptoethanol and 0.02% sodium azide. The homogenate was centrifuged at 20,000g for 20 min and the supernatant used as a source of endopeptidase. The enzyme could be stored at 3 to 5 C for up to 2 weeks, but was inactivated by freezing. For studies of endopeptidase inhibitor, the homogenate was incu-bated for 15 min at 70 C and then centrifuged at 20,000g for 20 min. The supernatant was adjusted to pH 4.8 with 1 M citric acid, again clarified by centrifugation, and this supernatant used as a source of inhibitors. Enzyme Assays. Endopeptidase activity was determined by viscometry as described before (3). Ten ml of 5% gelatin in 25 mm citrate-phosphate buffer (pH 5.7) and 0.02% NaN3 were mixed with 2 ml of 25 mm citrate-phosphate (pH 5.7) with 10 mM 2-mercaptoethanol containing an appropriate amount enzyme. The reaction was allowed to proceed at 40 C, and the viscosity of the gelatin solution was measured every 2 min for up www.plantphysiol.org on May 5, 2019-Published by Downloaded from