native Kunitz soybean trypsin inhibitor of Amsoy 71, Ti'. Protease Kl cleaves Ti5 to Ti',, the inhibitor form lacking the five carboxyl-terminal amino acid residues ...
Plant Physiol. (1988) 88, 355-360 0032-0889/88/88/0355/06/$01.00/0
Survey of the Proteolytic Activities Degrading the Kunitz Trypsin Inhibitor and Glycinin in Germinating Soybeans (Glycine max)1 Received for publication March 4, 1988 and in revised form April 26, 1988
KARL A. WILSON*, GREGORY PAPASTOITSIS, PHILIPPE HARTL, AND ANNA L. TAN-WILSON Department of Biological Sciences, State University of New York at Binghamton, Binghamton, New York 13901 ABSTRACT
The cotyledons of the soybean (Glycine max [L.] Merrill cv Amsoy 71) were examined for proteolytic activities capable of degrading soybean seed proteins. Three distinct activities were identified that attack the native Kunitz soybean trypsin inhibitor of Amsoy 71, Ti'. Protease Kl cleaves Ti5 to Ti',, the inhibitor form lacking the five carboxyl-terminal amino acid residues relative to Ti'. Protease Kl is a cysteine protease that peaks in activity on day 4 after the beginning of imbibition, with maximal activity toward Ti5 at pH 4. The characteristics of protease Kl are consistent with the involvement of this protease in the initial proteolysis of the Kunitz inhibitor during germination. Protease K2 also degrades Ti5 at pH 4 but produces no electrophoretically recognizable products. It peaks later in seedling growth, at day 8. Protease K3 degrades Ti' to products other than Ti5.. However, it is active at pH 8. Two proteolytic activities were identified that attack the major storage protein, glycinin. Protease Gl (which appears by 4 days after imbibition) specifically cleaves the acidic polypeptides of glycinin at pH 4, yielding a product approximately 1.5 kilodaltons smaller. Protease Gl is inhibited by metal chelators as well as by reagents reactive toward thiols. Protease G2 also degrades the glycinin acidic chains at pH 4, but without the appearance of electrophoretically recognizable products. Protease G2, while present at low levels in the dry seed, is found primarily in the cotyledons after 8 days of growth.
cinin exhibit similar, initially limited, proteolysis, with one or more specific intermediates accumulating, at least transiently, during the degradation of these proteins (24). We have now identified at least three proteolytic activities that are capable of degrading KSTI in vitro. One enzyme converts KSTI-Tia to KSTI-Tiam, suggesting that this protease is responsible for this reaction in vivo. Another protease has been identified that catalyzes a limited specific proteolysis of the acidic chains of glycinin.
MATERIALS AND METHODS Plant Materials and Extracts. Soybeans, Glycine max (L.) Merrill cv Amsoy 71, were purchased from May Seed and Nursery Co., Shenandoah, IA. Germination and growth were carried out as previously described (24) for periods of up to 14 d. All ages quoted are reckoned relative to the beginning of seed imbibition. Cotyledons derived from these plants were harvested and stored at -20° C until needed. Extracts for enzyme assays were prepared by homogenizing cotyledons with ice-cold 50 mM Tris-Cl + 1 mm 2-mercaptoethanol, pH 8.0, at the rate of 5 mL of buffer per g of tissue. Extracts for the quantitation of storage protein and inhibitor species were prepared using 50 mM Na phosphate + 250 mM NaCl + 0.5 mm Na iodoacetate + 0.3 mM PMSF, pH 7.0, at the same ratio of buffer to tissue. In both cases the homogenate was filtered through cheesecloth and centrifuged at 34,800g for 1 h. The resulting clarified extracts were frozen as aliquots at -20° C. Reagents and Substrates. The native form of KSTI, Tia, was purified from Amsoy 71 beans by the method of Hartl et al. (6) Dicot, and particularly legume, seeds contain reserve proteins or was purchased from Worthington Biochemical Corp. The that are degraded during the germination and the subsequent modified form of KSTI, Tiam, was purified from germinated early growth of the seedling. The proteolysis of these storage seeds in a similar manner. Concentrations of KSTI solutions molecules supplies amino acids and their derivatives for the were determined spectrophotometrically, assuming a 1 mg/mL biosynthetic and energy needs of the developing plant (1). The solution to have an A280 of 1.00 for both Tia and Tiam. Glycinin soybean, Glycine max (L.) Merrill contains two major reserve was purified from Amsoy 71 soybeans as described by Wilson et globulins, glycinin and ,3-conglycinin (12, 17). In addition, the al. (24). Concentrations were determined assuming an A280 of soybean typically contains large amounts of protein proteinase 0.81 for a 1 mg/mL solution of glycinin. inhibitors, in particular KSTI.2 a-N-Carbobenyloxy-L-phenylalanyl-L-alanine, L-alanylglycine, We have recently examined the early stages of degradation of BAPA, azocasein, leupeptin, antipain, chymostatin, and pepstathese proteins in the Amsoy 71 cultivar of soybean. KSTI is tin A were from Sigma Chemical Company (St. Louis, MO). Einitially cleaved at the carboxyl-terminus, removing five amino 64, N-[N-(L-3-transcarboxyl-oxiran)-2-carbonyl)-L-leucyl]-agaacid residues from the native inhibitor, KSTI-Tia, to produce the matine, was from Boehringer Mannhein (Indianapolis, IN). All modified form, KSTI-Tiam (6, 21). Both glycinin and ,B-congly- other chemicals were reagent grade or better. All pH adjustments were performed at room temperature (21 ± 1°C) and twice 'Supported by National Science Foundation grant PCM 8301202. distilled water was used throughout. 2Abbreviations: BAPA, a-N-benzoyl-D,L-arginine p-nitroanilide; Quantitation of Glycinin and KSTI Species in Soybean ExKSTI, Kunitz soybean trypsin inhibitor; pCMPS, p-chloromercuriphen- tracts. The determination of glycinin chains and their degradaylsulfonate; PMSF, phenylmethylsulfonyl fluoride. tion products was carried out as detailed by Wilson et al. (24). 355
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PAGE (5) was utilized for the quantitation of KSTI in both its native form, Tia, and initial degraded form, Tiam (6). Electrophoresis was in 1.5 mm thick 10% (w/v) gels. Gels were stained with Coomassie blue G (Serva Chemical Co.). For quantitation, gels were scanned using a Hoefer model GS300 scanning densitometer. Samples of purified KSTI-Tia and KSTI-Tiam were used to establish the range in which sample size was directly proportional to staining intensity. Tia and Tiam were found to stain identically on a weight basis within this range. Total KSTI cross-reactive material in the extracts was quantified by radial immunodiffusion using KSTI-Tia as standard (21). Assay of KSTI-Degrading Activities. The assay reaction for KSTI-degrading activity consisted of 15 ,tL of substrate mix, 25 ,uL of buffer, and 20,uL of the desired sample. The substrate mix consisted of 500 ,ug/mL of Tia + 9.3 mm 2-mercaptoethanol + 20 ,ug/mL of amphotericin B + 400 ,ug/mL of kanamycin in water. Citrate/phosphate buffers (14) were used over the pH range 3.0 to 7.0, while 0.1 M Tris-Cl was used at pH 8.0. For each sample, two identical reaction mixtures were used. One was immediately frozen to serve as a zero time reaction. The second was incubated for 24 h at 30°C. Sample buffer, 380 mM glycine + 50 mM Tris + 20% (w/v) sucrose, pH 8.3, 40 ,uL, was added to both the zero time and 24-h reaction mixtures, and aliquots were subjected to electrophoresis in the system described by Davis (5). The stacking gel was omitted for routine assays, and a 10% (w/v) separating gel was used. The gels were stained for protein with Coomassie blue G (Serva) (0.1 % [w/v] in 50% [v/ v] methanol + 10% [v/v] acetic acid) and were destained in 12% (v/v) 2-propanol + 10% (v/v) acetic acid. For quantitation the stained gels were scanned using a Hoefer model GS300 scanning densitometer. Results were reported as the percentage of the Tia present at zero time converted to Tiam or as the percentage of that initially present converted to forms other than Tiam after 24 h. Assays for Tiam degrading activity were carried out in an analogous manner. Assay of Glycinin-degrading Activity. Reaction mixtures contained 5 ,uL of sample, 17 ,L of glycinin substrate mixture, and 18,uL of the desired citrate/phosphate buffer. The final reaction mixture (40 ,uL) contained 40 ,ug of glycinin and, if added, 0.5 mM DTT. The mixture was incubated at 37°C for 24 h. After this time, 40,gL of 2x Laemmli treatment buffer (13) was added, and the mixture was heated at 90°C for 15 min. Zero time reaction mixtures were made in an identical manner, except that the treatment buffer was added immediately to the reaction mixture and heated at 90°C without the normal 24-h incubation at 37°C. The treated reaction mixtures were subjected to SDS PAGE as described by Laemmli (13) using 1.5 mm thick 12.5% (w/v) gels. The gels were stained, destained, and scanned as described above. Some assay incubations were made containing amphotericin and kanamycin as described above. Identical results were found with or without the addition of the antibiotics. Inhibition Studies. To determine the effect of various reagents on the KSTI-degrading activities, 125,uL of the extract of interest, 15 1,L of 1 M citrate/phosphate buffer (pH 4.0) (14), and the reagent were incubated together in a total volume of 155 ,uL. After 2 h at 0°C, aliquots of the reaction mixtures were assayed for remaining activity as described above. A control reaction with no added reagent was treated in an identical manner. Analogous studies were also carried out with the glycinindegrading activity. In this case, 50 ,L of extract, 15 ,uL of 500 mM Tris-Cl (pH 8.0), and the reagent of interest were incubated at 0°C for 3 h in a final volume of 100 uL. Assay of Other Proteolytic Activities. Carboxypeptidase (25) and dipeptidase (16) activities were determined using a-N-carbobenzyloxy-L-phenylalanyl-L-alanine and L-alanyl-glycine, respectively, as substrates, while leucine aminopeptidase activity was determined using L-leucine p-nitroanilide (1 1). Hydrolytic
Plant Physiol. Vol. 88, 1988
activity toward BAPA was assayed by the method of Catsimpoolas et al. (4). One unit of activity was defined as hydrolyzing 1 ,umol of substrate per min, with the exception of BAPA hydrolysis, where 1 unit was defined as producing an absorbance
increase of 1.0 in the assay system. General proteolytic activity
was determined with azocasein as substrate. The assay mixture consisted of 1 mL of 1% (w/v) azocasein in 1 mm DTT, 0.5 mL of sample, and 1 mL of buffer (0.1 M Na acetate + 1 mM DTT [pH 5.0] or 0.1 M K phosphate + 1 mm DTT [pH 7.0]. After 2 h at 30C, 0.5 mL of 50% (w/v) trichloroacetic acid was added.
The mixture was centrifuged, 2 mL of the clarified supernatant was mixed with 2 mL of 2 M NaOH, and the absorbance at 430 nm was determined. Blank reactions were carried out in an identical manner except that the sample was added after the addition of the trichloroacetic acid. An increase in absorbance of 1.0 over the blank was defined as 1 unit of activity in this assay.
RESULTS Time Course of KSTI Degradation. The electrophoretic pattern of extracts from cotyledons of seedlings varying in age from 0 to 14 d is shown in Figure 1. In the Davis PAGE system (5) used, both Tia and Tiam were well separated from the much less mobile glycinin and (3-conglycinin. In the first 2 d of growth, only the native inhibitor form Tia was present. However, by day 4 the proteolytically modified form Tiam began to appear. By day 6, Tiam accounted for approximately 30% of the total detected KSTI (Fig. 2A). The appearance of Tiam was accompanied by the disappearance of Tia. By day 14, both Tia and Tiam had essentially
disappeared. During the first 8 d of growth, the sum of Tia and Tiam was essentially constant and equal to the amount of Tia in the dry seed. This suggests that no additional partially degraded KSTI species were formed in significant amounts over this time period. However, after day 8 the total amount of KSTI declined precipitously in the cotyledons, either measured as the sum of Tia + Tiam or measured by radial immunodiffusion (Fig. 2A, B). The degradation of the major soybean storage proteins in these extracts was also examined. Essentially the same kinetics of degradation of both glycinin (Fig. 2C) and ,B-conglycinin (not shown) were found as we have previously described (24). In terms of these kinetics, the degradation of the glycinin acidic chains Al, A2, and A4 appears to have been quite similar to that of KSTI. Degradation of the native protein began approximately
IIrnIIl
a
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mT a FIG. 1. PAGE of extracts of cotyledons at various stages of germination and seedling growth, S. Tia, and Tiam standards; A to H, extracts from days 0, 2, 4, 6, 8, 10, 12, and 14, respectively. The amount of extract in each case is equivalent to 0.05 cotyledons.
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