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 coelutes with trypsin inhibitor on Sephadex G-100, but these two inhibitory activities can be separated by means of a trypsin affinity column.The inhibitory activity disappears slowly from crude extracts incubated 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 re...

Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis)

“…Later during germination the loss of protein from the cotyledons was probably associated with the rapid increase in -SH EPA at pH 4 and 5 and later at pH 6. Mixing experiments indicate that the rise in EPA at pH 6 and in CPA later in germination may be the result of lower levels of the substances which inhibited these activities early in germination. A similar situation occurs in wheat (23) and an endogenous inhibitor of EPA at pH 6 has been reported in mung beans (3). EPA at pH 7 and 8 did not appear to play a major role in reserve protein catabolism in jojoba seeds.…”

“…An expected value of additive activity was calculated from control assays of the 3-and 37-day extracts alone. A measured activity of the mixed extracts that was lower than the expected additive activity was taken to indicate the presence of an endogenous inhibitor (3,29).…”

“…The possible presence of endogenous inhibitors of EPA, APA, and CPA in the germinating jojoba cotyledons was assayed by standard mixing experiments (3,29). Equal volumes of gelfiltered extract from 3-and 37-day postimbibition cotyledons were mixed and then preincubated at room temperature for 15 min.…”

Naturally Occurring Protein Crystals in the Potato

“…The work has been repeatedly cited (26,29) although the data have not been previously published. A sulfhydryl proteinase inhibitor from mung bean cotyledons has been partially characterized (5) and a family of low molecular polypeptides inhibitor to bromelain has been isolated from pineapple stem (24). Rele et al (25) (10) prior to hydrolysis for determination of the sulfur amino acids.…”

Physiological Control of Exo- and Endoproteolytic Activities in Germinating Wheat and Their Relationship to Storage Protein Hydrolysis

“…These results, in addition to the insensitivity of these increases to GA, ABA, and inhibitors of protein and RNA synthesis indicated that early increases in endoproteolytic activity were not due to de novo synthesis but rather due to the activation of enzymes which have previously been shown to be present in the aleurone of ungerminated wheat (20). Previous studies have shown that the disappearance of inhibitors present in the ungerminated seed of lettuce (24), cow pea (23), barley (13), and mung bean (1) may be partially responsible for increases in proteolytic activity during germination. Warchalewski and Skupin (29) have also shown that the antiproteolytic activity of inhibitors present in ungerminated barley against native proteases is lost during seed storage.…”