Partial characterization of digestive tract proteinases from western corn rootworm larvae Diabrotica virgifera.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 19:285-298 (1992) Partial Characterization of Digestive Tract Proteinases From Western Corn Rootworm Larvae, Diabrotica virgiferd Jeffrey W. Gillikin, Steven Bevilacqua, a n d John S . Graham Department of Biological Sciences, Bowling Green State university, Bowling Green, Ohio We have partially characterized the proteolytic activity within luminal contents of the digestive tracts of larvae of the western corn rootworm, Diabrotica virgifera LeConte. At least 15 proteinaseswere detected based on chromatographic behavior on ion exchange high-performance liquid chromatography and their mobility o n sodium dodecyl sulfate gels containing gelatin. Inhibitors of proteolytic activity indicated that these enzymes are primarily sulfhydryl proteinases. Native polyacrylamide gel electrophoresis revealed that a single proteinaceous inhibitor, egg white cystatin, was capable of abolishing a substantial part of the proteolytic activity. O u r data suggest, accordingly, that gene transfer experiments utilizing the cystatin gene may generate lines of maize which have increased resistance to western corn rootworm larvae, o 1992 wiiey-Liss, Inc. Key words: sulfhydryl proteinases, proteinase inhibitors, zymograms INTRODUCTION Plants have developed a multitude of defensive mechanisms to minimize predation by insect pests [l]. One such mechanism is to make themselves unacceptable as a food source. The list of chemical compounds, referred to as allomones, produced by plants to discourage insect feeding is quite diverse and includes alkaloids, glycosides, terpenes, tannins, and saponins . These allomones often represent complex organic molecules whose biosynthetic pathways do not readily lend themselves to genetic manipulation, Plant compounds that can be genetically manipulated and may be synthesized for defensive purposes are the proteinase inhibitors. These proteins are Acknowledgments: We thank Philip Ganz for technical assistance and Lee French for helpful discussions on the handlin of rootworm larvae. We also thank Drs. G.S. Bullerjahn and S.L. Smith for critical review o f g i s manuscript. This work was supported by a grant to J.S.G. from the Agricultural Biotechnology Research Unit, CIBA-GEICY Corporation, Research Triangle Park, N.C. Received September 18,1991; accepted December 23,1991. Address reprint requests to John S. Graham, Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403. 0 1992 Wiley-Liss, Inc. 286 Cillikinet al. prevalent in storage and foliage tissues [3,4] and bind tightly (w0.lto 10 nM) to digestive proteinases of vertebrate, microbial, and insect origin . Recent reports have shown that proteinase inhibitors, when present in the diet of insects, can result in an increased mortality rate, decreased weight gain, and/or reduced fecundity [6,7]. Hence, the use of proteinase inhibitors to control insect populations is gaining favor as an alternative to use of applied chemical pesticides .The technology is available to introduce proteinase inhibitors into the plant genome through gene transfer techniques. Larvae of Diabroticu sp., important agronomic pests, cause considerable damage to the root system of maize . For the past 30 years research has focused on development of lines of maize with increased resistance to these pests. Some success has been attained by using conventional breeding programs [lo]. We have undertaken a project aimed at controlling field populations of the western corn rootworm, D. virgikra, by introducing genes coding for proteinase inhibitors specific for digestive tract proteinases into the maize genome. In this report we describe our initial efforts toward attainment of this goal. We present data describing biochemical characteristics of the proteolytic activity within the luminal contents of the western corn rootworm. We have identified at least fifteen distinct proteolytic activities and found that the majority are sulfhydryl proteinases. Moreover, we show that most digestive tract proteolytic activity can be abolished, in vitro, by a single proteinaceous proteinase inhibitor. MATERIALS AND METHODS Insects Second instar larvae of nondiapausing western corn rootworm, D. virgifera, were purchased from French Agricultural Research, Inc. (Lamberton, MN). Approximately 150 larvae were received on a mat of 40 to 50 three-week-old maize seedlings (Zea mays var. Pioneer B37 x H84). Isolation of Larvae from Corn Roots Larvae were removed from corn seedlings by placing the mats of infested seedlings in Nalgene polypropylene funnels (150 mrn top diameter). Funnels were placed in 2 L beakers 30 cm below a 300 W reflector flood lamp and the larvae collected onto moistened filter paper. Most larvae (70%)were recovered after 24 h of constant light exposure. Isolation of Proteolytic Activity Digestive tracts were dissected from second instar larvae within 24 h after removal from seedlings. Isolated digestive tracts were placed on ice in PBS *Abbreviations used: BSA = bovine serum albumin; DANM = diazoacetylnorleucine methyl ester; D l 7 = dithiothreitol; E-64 = trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; & = dissociation constant; LBTl = limabean trypsin inhibitor; PME = p-mercaptoethanol; MES = 2-(N-morpholino) ethane sulfonic acid; NEM = N-ethyimaleimide; PBS = phosphate buffered saline; pCMB = p-chloromercuribenzoicacid; PMSF = phenylmethylsulfonylfluoride; SBBl = soybean Bowman-Birk inhibitor; SDS = sodium dodecyl sulfate; TCA = trichloroacetic acid; TLCK = Na-p-tosyl-L-lysine chloromethyl ketone; TPCK = Na-p-tosyl-L-phenylalanine chloromethyl ketone. Corn Rootworm Digestive Tract Proteinases 287 (140 mM NaCl and 6 mM sodium phosphate, pH 7.2), at a ratio of one digestive tract per 5 pl buffer. Fifty digestive tracts were typically collected per 0.5 ml Eppendorf tube. Intact digestive tracts were centrifuged for 5 min at 10,OOOg at 4°C. Supernatant, termed luminal content, was removed and stored at - 20°C. The pellet was washed twice with 250 p1 PBS. After centrifugation the supernatant fluid was discarded and the pellet, representing gut walls (termed digestive tract walls), was stored at - 20°C. Extraction of total soluble protein was accomplished by homogenization of intact larvae, carcasses (after removal of digestive tracts), or digestive tract walls in PBS by three 15 s pulses using a Tekmar (Cincinnati, OH) Tissuemizer with a microprobe. The homogenate was centrifuged for 5 min at l0,OOOg at 4°C. The supernatant fluid was removed and stored at - 20°C. Protein Determination Protein concentration was determined by the Bio-Rad assay system [ll] according to the manufacturer's procedures. BSA was used as standard protein. Proteinase Assay Proteinase assays were performed by mixing equal volumes of BSA assay buffer (40 mM sodium citrate (pH 4) 40 mh4 NaCI, 75 Fg/ml BSA, and 75 nCi/ml ['4C]methyl-BSA,5-50 pCi/mg; Sigma Chemical Co., St. Louis, MO) and either luminal contents or total soluble protein extract. The final assay volume was 50 pl. Reaction mixtures were incubated at 37°C for 30 min and then terminated by the addition of 20% TCA to a final volume of 100 pl. Samples were placed on ice for 15 min followed by centrifugation at 10,OOOg for 5 min at 4°C and 75 pl of supernatant fluid containing TCA-soluble fragments was pipetted into 2 ml of scintillation fluid (Packard, Downers Grove, IL). This mixture was vortexed for 30 s and counted in a Beckman (Fullerton, CA) LS 3801 scintillation counter. Proteolytic activity was also assayed by mixing equal volumes of casein assay buffer (40 mM MES (pH 6) 40 mM NaCl, 75 pg/ml casein, and 75 nCi/ml [14C]methyl-casein,5-50 pCi/mg; Sigma Chemical Co.) and total soluble protein extract. Reaction mixtures were TCA precipitated and counted as described above. The amount of extract added to reaction mixtures was adjusted so that rates of hydrolysis were linear with time. Proteolytic activity is defined as the increase in TCA-soluble counts/h compared with reactions containing no enzyme. Specific activity is defined as the increase in TCA-soluble counts/ h/mg protein. All proteinase assays were performed in triplicate. Determination of pH Optimum Proteinase assays were performed as described above except that the following buffers were substituted: pH 3-5, 20 mM citrate; pH 6, 20 mM MES; pH 7-9, 20 mM Trizma-Base; pH 10, 20 mM NaHC03. Heat denatured BSA was prepared by boiling the assay buffers for 5 min followed by placing samples on ice before addition of the enzyme. All reactions were performed in triplicate and values presented represent the mean. Inhibitor Studies Luminal contents were preincubated with inhibitors or activators of proteinases for 10 min at 21°C prior to the addition of BSA assay buffer. These 288 Cillikin et al. inhibitors and activators include the following: EDTA, E-64, NEM, iodoacetamide, iodoacetic acid, pCMB, cystatin, CuSO4, leupeptin, chymostatin, antipain, bromelain inhibitor, SBBI, LBTI, PMSF, aprotinin, benzamidine, TPCK, TLCK, pepstatin, cysteine, DTT, and PME. All inhibitors and activators were purchased from Sigma Chemical Co. The reaction mixtures were then incubated at 37"C, TCA precipitated, and counted as described above. Control samples containing only inhibitor buffers were assayed simultaneously and values obtained were used to calculate percentage of residual activity. All reactions were performed in triplicate. Gel Electrophoresis Electrophoretic mobility of proteolytic activity on 10% SDS-PAGE gels was determined by the method of Heussen and Dowdle  with the following modification: plasminogen was replaced with 0.25% gelatin. Ten microliters of luminal contents (the equivalent of two larval guts) were diluted 4:l with SDS-PAGE sample buffer (2%SDS, 20% glycerol, 200 mM Tris-HC1 (pH 7) and 0.1% bromphenol blue). Molecular weight standards containing rabbit muscle phosphorylase b (M, 97,400), bovine serum albumin (M, 66,200), hen egg white ovalbumin (M, 45,000), bovine carbonic anhydrase (M, 31,000), soybean trypsin inhibitor (M, 21,500), and hen egg white lysozyme (M, 14,400) (BioRad, Richmond, CA)were loaded onto gels at 50 times the manufacturer's recommended amount. This amount of molecular weight standards was required for visualization above the gelatin background. Samples were loaded directly onto the gel and electrophoresed at 150 V/gel(l5 x 17 cm x 1.5 mm) at 4°C. After electrophoresis was complete, gels were placed in 2.5% Triton X-100 in H20 for 30 min with shaking to remove the SDS. Gels were rinsed twice with distilled water and transferred to 100 mM MES, 10 mM cysteine-HC1 (pH 6) for 3 h at 37°C with gentle shaking. SDS gels were stained for 1h in 25% methanol and 7.5% acetic acid containing 0.5% brilliant blue R-250. Proteolytic activity and molecular weight markers were visualized by destaining gels in 25% methanol and 7.5% acetic acid. Electrophoretic mobility of proteolytic activity on PAGE gels was determined as described above except SDS was omitted from the gel. Two luminal content equivalents were diluted 1:l in native sample buffer (10% sucrose, 100 mM Tris-HC1 (pH 7) and 0.1% bromphenol blue). Samples were loaded directly onto the gel and electrophoresed at 150 Vigel(l5 x 17 cm x 1.5 mm) at 4°C. After electrophoresis was complete, gels were placed in 100 mM MES, 10 mM cysteine-HCl (pH 6) for 2 h at 37°C with gentle shaking. Native gels were stained for 1h in 25% methanol and 7.5% acetic acid containing 0.5% brilliant blue R-250. Proteolytic activity was visualized by destaining gels in 25% methanol and 7.5% acetic acid. Fractionation on DEAE-HPLC Luminal contents (from 100 larvae) were chromatographed on a Waters (Milford, MA) HPLC DEAE 8HR anion exchange column (1 x 10 cm) equilibrated in 20 mM potassium acetate, 2 mM cysteine-HC1 (pH 5) at a flow rate of 1 mumin. The column was washed for 10 min before beginning a linear gradient of 0 to 500 mM potassium chloride over 80 min at a flow rate of 1mumin. Corn Rootworm Digestive Tract Proteinases 289 One milliliter fractionswere collected and assayed for proteolytic activity using [14C]methyl-BSA.Proteolytic activity was also visualized by SDS gels using 25 p1of the HPLC fractions. RESULTS Determination of pH Optimum The effect of pH on proteolytic activity from larval extracts was examined by using ['*C]methyl-BSA, heat denatured [14C]methyl-BSA,and [14C]methylcasein over a ran e from pH 3 to 10 (Fig. 1). The highest level of proteolytic activity against [ Clmethyl-BSA was observed at pH 4. The optimal pH for proteolytic activity against ['4C]methyl-casein was found to be between pH 5 and 6; however, significant proteolytic activity was observed over the range of pH 4 to 9 (Fig. 1). Proteolytic activity was also examined using Azocoll (Calbiochem, La Jolla, CA) as a substrate and the pH profile was similar to that observed with [14C]methyl-casein(data not shown). This broad pH optimum is indicative of multiple proteolytic activities. The difference in pH optima between BSA and casein may also be explained by differences in tertiary structure of the two substrates. Indeed, heat denatured BSA shows a pH profile similar to that of casein and a broader pH optima than native BSA (Fig. 1). 4 Qualitative Analysis of Larval Proteolytic Activities Prior to characterization of digestive tract proteolytic activities, we analyzed specific activity of second instar larval extracts. Specificactivity values of intact larvae and larval carcasses are 10.3 X lo3 (SE 4 0.7) and 11.6 X lo3 (SE f 0.8) TCA-solubilized counts/h/mg protein, respectively. Compared to intact larvae, digestive tract walls and luminal contents show specific activities 32-fold and 165-fold higher, respectively (data not shown). The high specific activity observed in the luminal contents, relative to intact larval extracts, is expected 1200 , 800 400 0 Fig. 1. Effect of pH on proteolytic activity from soluble protein extracts of intact larvae. Proheat , denatured ['4C]methyl-BSA teolytic activity was determined using ['4Clmethyl-BSA (B) c.),and ['4Clmethyl-casein@jand represents the increase in TCA-soluble counts/h compared with reactions containing no enzyme. 290 Cillikin et al. Fig. 2. Proteolytic activity in second instar larvae of D. virgifera. lane 1: SDS gels of soluble protein extracts from two second instar larvae. lane 2: Two second instar larvae after removal of the digestive tracts. lane 3: Two digestive tract wall equivalents. lane 4: Two luminal content equivalents. since gut extracts should contain the proteinases which function in hydrolysis of ingested plant material. We utilized SDS gels containing a proteinaceous substrate  to analyze the complexity of proteolytic enzymes within the various extracts. Figure 2 shows the distribution of proteolytic activities in intact larva, carcass, digestive tract wall, and luminal contents. Three groups of proteolytic activities, which appear as cleared zones against a dark background, are apparent in extracts of whole larvae (Fig. 2, lane 1). We have categorized the proteolytic activities of Figure 2 into three groups based solely on mobility in SDS gels; these are referred to as Group I, Group 11, and Group I11 proteinases. Lane 1 also shows a zone of clearing at the top of the gel which is due to the entrapment of proteinases resulting from the high level of protein and contaminating fats loaded onto the gel. When carcass extracts are .analyzed by this technique, the presence of the three groups of proteolytic activity is not evident (Fig. 2, lane 2). This suggests that the proteinases observed in whole larvae originate from the digestive tract. Separation of the digestive tract into its two components, digestive tract walls and IuminaI contents (Fig. 2, lanes 3 and 4), shows that all three groups are present in the luminal contents, but only Group I1 activity is detectable in digestive tract walls. Proteolytic activity present within the lumen of the digestive tract must originate from cells within the digestive tract wall. Group I and I11 proteolytic activities observed in the rootworm digestive tract lumen may have been synthesized as inactive zymogens and required further processing in the lumen for activation . We have eliminated the possibility that the observed proteolytic activity seen in Figure 2 is of plant origin since corn kernel and root tissue do not display similar proteolytic patterns (data not shown). Visual inspection suggests that Group I consists of at least four proteolytic Corn Rootworm Digestive Tract Proteinases 291 activities with M,s between approximately 20,000 and 25,000; Group I1 consists of at least two proteolytic activities with M,s between 33,000 and 36,000; Group 111 consists of at least two proteolytic activities with M,s above 45,000. The assignment of two proteolytic activities to Group I11 is tentative due to low resolutiodactivity in this region. In addition to those proteolytic activities which fall into the three distinct groups, there is another proteinase which migrates between Groups I and 11. Therefore, we are able to detect at least nine distinct proteolytic activities based solely on differences in mobility on SDS gels. It should be noted that proteolytic activities detected with this technique are those that are capable of surviving the denaturatiodrenaturation step. Moreover, proteolytic activities dependent on quaternary structure would not be observed with this technique. SDS gels containing casein or BSA as substrates showed similar banding patterns to that observed with gelatin; however, these required 24 to 48 h incubation times for visualization (data not shown). Fractionation of Proteolytic Activity Within Luminal Contents Luminal contents from 100 second instar larvae were fractionated on a Waters HPLC DEAE 8HR anion exchange column(1 x 10 cm). The elution profile of the protein and proteolytic activity is shown in Figure 3. Six peaks of proteolytic activity are observed eluting from the anion exchange column. Individual fractions were analyzed by SDS gels to determine the composition of proteinase activities present within each peak (Fig. 4). Members of Group I activities appear in fractions 49-69 and have the highest affinity for the anion exchange column. Based on electrophoretic mobility on the SDS gels of Figure 4,there are 5 distinct activities comprising this group. However, based on A 2J 0 s0 5 800 5' 0.8 0.4 100 0.0 0 60 30 90 Fraction No. Fig. 3. Fractionation of luminal contents on a Waters DEAE 8HR HPLC column. One hundred luminal content equivalentswere applied to a Waters DEAE 8HR column equilibrated in 20 m M potassium acetate, 2 mM cysteine-HCI, pH 5 (flow rate 1 m h i n ) . The column was washed for 10 min; then proteins were eluted with a linear gradient of 0-500 rnM potassium chloride in 20 m M potassium acetate, 2 m M cysteine-HCI (pH 5) over 80 min. One milliliter fractions were collected. Proteolytic activitywas determined using ['4Clmethyl-BSA and represents the increase in TCA-soluble countslh compared with reactions containing no enzyme. Absorbance at 280 nm (-); proteolytic activity (-----). 292 Gillikin et al. Fig. 4. Analysis of DEAE-HPLC fractionated luminal contents on SDS gels. Every other fraction (1-69) of Figure 3 was analyzed for proteolytic activity using SDS gels as described in the Materials and Methods. CC, unfractionated luminal contents. elution from the HPLC column, several components of similar electrophoretic mobility differ in their retention times. Taken together, the data of Figure 4 suggest the presence of nine proteolytic species within the Group I proteinases. Group I1 activities are found in fractions 7,21,31-33, and 65. The differences in chromatographic behavior of Group I1 activities has provided for the detection of at least 5 distinct activities. Group I11 activities have not been observed on SDS gels after fractionation by HPLC, suggesting these activities are unstable or are tightly bound to the column. Fraction 51 contains the activity with intermediate electrophoreticmobility between Groups I and 11. Therefore, DEAE anion exchange chromatography coupled with the SDS gels reveal the presence of 15 proteolytic activities. Corn Rootworm Digestive Tract Proteinases 293 TABLE 1. Effect of Inhibitors and Reducing Agents on the Proteolytic Activity* With Luminal Contents Inhibitor EDTA E-64 NEM Iodoacetamide Iodoacetic Acid pCMB Cystatin cu+ + Leupeptin Chymostatin Antipain Bromelain Inhibitor SBBI LBTI PMSF Aprotinin Benzamidine TPCK TLCK Pepstatin Cysteine DTT PME Concentration (mM) 10.0 0.02 1.0 1.0 1.0 1.0 0.01 1.0 0.5 1.0" 1.0" 1.0" l.oa 1.0" 10.0 l.Ob 1.0 0.01 0.01 5.0" 5.0 5.0 5.0 Activity remaining (%) 79 70 99 91 57 61 31 21 35 41 43 95 100 100 89 90 102 103 67 38 221 172 211 (6.5)E (6.0) (1.3) (5.5) (2.2) (3.1) (1.5) (1.6) (1.3) (1.8) (2.5) (3.4) (3.7) (3.5) (4.6) (4.0) (3.6) (2.3) (1.5) (2.8) (1.7) (2.4) (1.5) *Pmteolyticactivity determined using ['4C]rnethyl-BSA and represents the increase in TCA-soluble countslh compared with reactions containing no enzyme. aConcentrations in kg/mL. bTrypsin inhibitor units. CNumbersin parentheses are the SE values (n = 3). Inhibitor Studies Inhibitors diagnostic for the 4 mechanistic classes of proteinases were employed to determine those classes present in luminal contents of second instar larvae. Effects of these proteinase inhibitors are presented in Table 1. The study demonstrates that no single proteinase inhibitor completely abolishes the proteolytic activity isolated from luminal contents. This result is not surprising given the complexity of activities observed in Figure 4. Inhibitors diagnostic of the sulfhydryl class of proteinases were most effective. E-64, pCMB, and cystatin, when utilized at concentrations which resulted in maximal inhibition, inhibited 30, 39, and 69% of digestive tract proteinase activity, respectively (Table 1). In addition to these compounds, the proteolytic activity was strongIy inhibited by antipain, leupeptin, and chymostatin which can affect both serine and sulfhydryl class proteinases , Further evidence to support the presence of sulfhydryl proteinases in the luminal contents is the 79% inhibition by C u + +  and the stimulation of activity by reducing agents such as cysteine, DTT, and g-mercaptoethanol (Table 1). We were interested in determining whether the stimulation by cysteine was a result of increased activity of a subset of the luminal proteinases or a gen- 294 Gillikin et al. Fig. 5. Effect of cysteine on proteolytic activity. Two luminal content equivalents were electrophoresed on SDS gels in the presence of 5 mM cysteine (lane 1) and in the absence of cysteine (lane 2). era1 stimulation of the entire proteolytic complement. The stimulatory effect of cysteine is shown in Figure 5. The presence of 5 mM cysteine in the incubation buffer stimulated all of the proteolytic activities (Fig. 5, lane 1).It should be noted that the activity profile of Figure 5, lane 2, after a fivefold increase in the incubation period, looks identical to that of Figure 5, lane 1. The data of Table 1 in conjunction with the stimulation of proteolytic activity by cysteine (Fig. 5) provide strong evidence that sulfhydryl proteinases represent the dominant class of proteolytic activity within luminal contents of the western corn rootworm. EDTA inhibited the proteolytic activity by 21% indicating that metalloproteinases may be present in luminal contents. An alternative possibility is that one or more of the proteolytic activities requires divalent cations for optimal activity. Proteolytic activity was reduced by 33% with TLCK, which suggests the presence of a proteinase(s) with trypsin-like specificity. This inhibition may not be due to the inhibition of serine type proteinases because proteolytic activity was not significantly affected by aprotinin, SBBI, LBTI, benzamidine, or PMSE This inhibition could be explained by the fact that TLCK has been shown to affect certain sulfhydryl proteinases in addition to serine proteinases . Table 1also indicates that a significant portion of the proteolytic activity may be due to aspartic acid proteinases based on the 62%inhibition by pepstatin. This result is a constant feature of the luminal proteinases; however, we were unable to bind the activity to a pepstatin agarose column or inhibit the activity with DANM, another aspartic acid proteinase inhibitor. Also, we have been unable to detect inhibition by pepstatin on native gels. Therefore, the presence of this mechanistic class in the luminal contents is uncertain. Corn Rootworm DigestiveTract Proteinases 295 Gel Analysis of Proteinase Inhibitors The use of zymograms enables us to determine the reactivity of individual proteinases toward a given inhibitor. There are, however, some limitations that must be addressed. First, the inhibitodenzyme binary complex formed during the preincubation period must be of a covalent nature to remain associated during migration on SDS gels, If a reversible inhibitor (i.e., leupeptin) is to be examined, the inhibitor must be introduced during the incubation period. SDS gels were used to examine inhibition of proteinases by Cu' + and E-64. Results of the Cu+ * inhibition study (Fig. 6A) show that most proteolytic activities are sensitive to this metal ion (Fig. 6A, lane 2). Inhibitory effects of E-64 on proteolytic activity are shown in Figure 6B. Although all activities seem to be reduced slightly, inhibition by E-64 is limited to primarily two proteolytic activities (denoted in the figure with asterisks). The proteinase activity migrating between Group I and I1 and an activity within Group I11 are affected by this compound. This result is consistent with the data of Table 1 which show a relatively low level of inhibition. Native gels were used to analyze effects of cystatin, a proteinaceous inhibitor found in egg white , on the proteolytic activity of D. vivgiferu. The dissociation of the cystatidenzyme complex in the presence of SDS, coupled with the inability of cystatin to efficiently permeate gels during the incubation period, required the use of gelatin PAGE. The activity profiles of Figure 6C show that the presence of 10 p M cystatin inhibits a significantfraction of proteolytic activity present in the luminal contents of second instar larvae. This result is conSubstrate Fig. 6 . Effect of proteinase inhibitors on luminal content proteolyticactivity. A: SDS gel showing the effect of Cu + on proteolytic activity. Gel slices containing two luminal content equivalents were incubated in the absence (lane 1) and in the presence (lane 2) of 1 m M CuSO,. B: SDS gel showing the effect of E-64 on proteofytic activity. Two lurninal content equivalentswere preincubated, prior to application to the gel, in the absence (lane 1) and presence (lane 2) of 20 FM E-64. Asterisk denotes proteinase activity which is strongly inhibited by E-64. C: Native gel showing the effect of cystatin on the proteolytic activity. Two luminal content equivalents were preincubated in the absence (lane 1) and presence (lane 2) of 10 pM cystatin. + 296 Cillikin et al. sistent with the inhibition data of Table 1. Native gels do not achieve the degree of resolution observed on denaturing gels (compare Fig. 6B and C). The poor resolution obtained is due, in part, to the narrow range of isoelectric points displayed by luminal proteolytic activities, pH 3.5-4.5 (data not shown). DISCUSSION We have examined the proteolytic activity within the luminal contents of second instar larvae of the western corn rootworm. Evidence indicates that the corn rootworm possesses a complex mixture of proteinases. At least 15 distinct activities are detectable on SDS gels after an initial fractionation of luminal contents by DEAE HPLC. We recognize that substrate gels only allow for the detection of those activities capable of hydrolyzing gelatin. This fact, coupled with the requirements of renaturation and possibly higher order structure may result in an underestimation of the number of proteolytic activities. Also, it is doubtful that peptidases would be detected by this technique although we did not assay for activity using peptidase specific substrates. Since we examined the entire luminal contents, we do not know whether various proteinases are secretedAocalized in specific regions of the digestive tract (i.e., foregut, midgut, or hindgut) . Although our data suggest a large number of distinct proteolytic activities, the occurrence of post-translational modification cannot be ignored. It may be that the three groups of activities represent only a few gene products which may be modified (e.g., limited proteolysis) in the lumen of the digestive tract. If this mechanism is operative, then the complex pattern of proteolytic activities after fractionation of luminal contents on DEAE-HPLC and SDS gels would be observed. The resolution of our observation will require further characterization to include the isolation of individual activities and the determination of their primary structures. Our data with inhibitors and activators indicate that the digestive tract of the corn rootworm contains proteinases of the sulfhydryl mechanistic class. These findings are consistent with those of Murdock et al. [MI. However, our data do not show the large degree of inhibition (92%)by pCMB presented by Murdock et al. This may be due to the difference in pH of the assay and/or the fact that the entire midgut, as opposed to the luminal contents, was used. The use of inhibitors of insect digestive enzymes as a means of controlling insect populations was originally put forward by Green and Ryan .Since that time, numerous studies have focused on the characterization of insect proteinases [ 15,20-231 and the incorporation of proteinase inhibitors in diets of insects to assess their effects on mortality, fecundity, growth, and development [6,7,24,25]. Indeed, such studies have shown that the presence of exogenous inhibitors can result in a disruption of normal growth and development. Broadway and Duffey  suggested that the disruption is not necessarily due to an inhibition of protein digestion, but instead causes hyperproduction of digestive enzymes which disturb the pool of sulfur-containing amino acids. Effects of sulfhydryl proteinase inhibitors on the digestive physiology of insect pests is an important area of study. Recent characterization studies have shown that the sulfhydryl proteinases are well represented within the Coleoptera Corn Rootworm Digestive Tract Proteinases 297 [18,26] and other studies have shown that the incorporation of E-64, a specific sulfhydryl proteinase inhibitor from Aspergillis japotzicus , into the diets of the cowpea weevil (Cullosobruchus maculutus)  and larvae of the Colorado potato beetle (Leptinotursu decemZineutu)  does have adverse physiological effects on these organisms. Recently, the introduction of stable genes coding for proteinase inhibitors into the tobacco genome has resulted in the development of plants with enhanced resistance to insect pests [28,29], Advances in the transformation and regeneration of maize will allow for genetic manipulation of this economically important crop . Our data suggest that cystatin may serve as a useful candidate for gene transfer studies in maize. Indeed, the appropriate expression of this proteinase inhibitor in maize may result in the development of a line with increased resistance to the western corn rootworm larvae. LITERATURE CITED 1. Smith CM: Plant Resistance to Insects: A Fundamental Approach. John Wiley & Sons, Inc., New York, pp 8-86 (1986). 2. Rosenthal GA, Janzen DH, eds. Herbivores: Their Interactions with Plant Secondary Compounds. Academic Press, New York, 718 pp (1979). 3. Ryan CA:Proteolytic enzymes and their inhibitors in plants. Annu Rev Plant Physiol 24, 173 (1973). 4. Richardson M: Protein inhibitors of enzymes. J Food Chem 6,235 (1980). 5. Ryan CA:Wound-regulated synthesis and vacuolar compartmentation of proteinase inhibitors in plant leaves. In: Current Topics in Cellular Regulation. Horecker BL, Boulter D, eds. Academic Press, New York, vol17, pp 1-19 (1981). 6. 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