EP0457898A1

EP0457898A1 - Proteinase inhibitory agents and methods for their use

Info

Publication number: EP0457898A1
Application number: EP91902042A
Authority: EP
Inventors: Roxanne M. Broadway
Original assignee: Cornell Research Foundation Inc
Current assignee: Cornell Research Foundation Inc
Priority date: 1989-12-11
Filing date: 1990-12-11
Publication date: 1991-11-27
Also published as: JPH04504730A; EP0457898A4; WO1991009060A1

Abstract

Un produit phytochimique à spécificité végétale, préparé à partir du chou et possédant une réponse spécifique d'inhibition dirigé contre les protéinases présentes dans les larves de Trichoplusia ni et Pieris rapae. On décrit également l'utilisation du produit phytochimique comme un moyen de protection de cette plante contre ces larves.A plant specific phytochemical, prepared from cabbage and having a specific inhibition response directed against the proteinases present in the larvae of Trichoplusia ni and Pieris rapae. The use of the phytochemical is also described as a means of protecting this plant against these larvae.

Description

PROTEINASE INHIBITORY AGENTS AND METHODS FOR THEIR USE

The herbivorous insects which are major pests for New York

State cabbage growers are the flea beetles (Phyllotreta cruciferae) , diamondback moth (Plutella xylostella), imported cabbage worm (Pieris rapae), and the cabbage looper (Trichoplusia ni). Of these pests, the imported cabbage worm and cabbage looper attack the plant during middle maturation, that is when the plants reach the 6 to 8 leaf stage of growth, at a time when the grower is most susceptible to a total loss of the cabbage crop. If the plants are destroyed at this stage of maturation, there is little chance of a second replanting during the growing season. It is important, therefore, that means be found to combat these pests which are agriculturally and environmentally acceptable.

Current literature supports the hypothesis that plant proteinase inhibitors - protein or polypeptides which occur in a wide variety of plants - provide the plant with significant protection against herbivorous insects. The proteinase inhibitors which have been most extensively studied are those that inhibit serine proteinases (e.g. trypsin, chymotrypsin) that are common digestive enzymes in animals but are not present in plants. Since plants do not contain serine proteinases,' the presence of serine proteinase inhibitors in plants suggests that they are acting as a defense against herbivorous insects.

The studies suggesting the protective nature of serine proteinase inhibitors in plants originate from experiments based upon (1) the incorporation of plant proteinase inhibitors into artificial diets for laboratory-grown pests, (2) feeding studies using plant tissue with or without- proteinase inhibitory activity, and (3) the use of plants that have been transformed with a gene for proteinase inhibitor. The potency of specific proteinase inhibitors is dependent on the presence of (1 ) susceptible proteinases in the target organism, and (2) other dietary factors (i.e. protein quality, polyphenyloxidase activity). The susceptibility of a particular proteinase protein is dependent upon (1) its type of proteinase activity (e.g. trypsin, chymotrypsin, carboxypeptidase), and (2); the structural configuration of its active site (the site of interaction between the enzyme and its inhibitor); the stronger the interaction between the inhibitor and enzyme, the more effective the inhibitor.

Thus, although proteinase inhibitors in general may contribute to the defense of plants against attack by invading pests, the efficacy of specific inhibitors from individual species of plants is dependent upon (1 ) the unique structure of the plant proteinase inhibitor, and (2) the susceptibility of the proteinase in the target organism. It appears that each plant species produces a proteinase inhibitor having a unique structure, and that while the proteinase inhibitor produced by one plant type may have a protective effect upon the pests which feed upon that type, the inhibitor may have little or no protective effect against the herbivorous pests feeding upon a second plant type.

I have recently reported that cabbage has significantly higher levels of trypsin inhibitory activity than many other crucifers [Tryptic inhibitory activity in wild and cultivated crucifers. Phytochemistry , 28:755 (1989)], and that larval Trichoplusia ni and Pieris rapae both rely upon trypsin and chymotrypsin for digestion of protein [Characterization and ecological implications of midgut proteolytic activity in larval Pieris rapae and Trichoplusia ni. Journal of Chemical Ecology 15:2101 (1989)]. Based upon these findings, the initial scientific direction of the present invention was to isolate, purify, characterize, and determine the effect the proteinase inhibitory agents of cabbage would have have on T. ni and P. rapae larvae.

The following disclosure and examples are provided to allow one to receive a more complete understanding of the present invention. These examples are not intended nor provided to limit the scope of the present invention in any manner, and it would be improper for one to interpret them as doing so. EXAMPLE I Seeds for the cabbage cultivar Superpack™ were germinated in Cornell Mix™ in 2 gallon plastic pots, The seedlings were thinned to one plant per pot, and maintained under greenhouse conditions under 1 kw metal halide lamps at 28°C. The plants were watered daily and fertilized once a week with a water-soluble nutrient (16-32-16) mix.

Two differing whole plant bioassays were conducted: (1 ) each plant was placed in a large cage and larvae of Trichoplusia ni and Pieris rapae were allowed to move about freely on the entire plant; and (2) larvae were confined to a specific leaf in a 2-inch diameter cage which was moved when the foliage within the cage was consumed. For each type of bioassay, both young (10 to 12 true leaves) and mature (at least 3-inch diameter plants with 9 to 11 frame leaves) plants were used. In addition, a control plant without larvae infestation was placed in the same conditions as the treated plants and chemically analyzed at the end_. of the experiment.

For the first bioassay, 40 Trichoplusia ni eggs or 100 Pieris rapae eggs were applied to each of two mature plants per insect species. The larger number of P. rapae eggs placed on each plant was to overcome the greater loss of larval P. rapae during the experiments, possibly because of the influence of crowding. Each of the plants was individually housed in an insect proof cage to prevent the migration of insects to neighboring .plants. The entire experiment was terminated when a single type of foliage (e.g. the young leaf tissue on young plants) was substantially consumed. All the larvae were weighed, and the plants were chemically analyzed for tryptic inhibitory activity, chymotryptic inhibitory activity, total protein, and total glucosinolates.

For the experiments in which the larvae were confined to a specific leaf on a plant, 50 P. rapae eggs were placed inside the small cage (with a recovery of 10 to 20 larvae) and, as they grew, the larvae were separated into 3 to 5 larvae per cage. Thirty eggs for T. ni were placed inside a small cage and separated into 3 to 5 larvae per cage as the larvae grew. The cages were placed on an expanding leaf (leaf numbers 3 to 5) based on the designation of leaf number 1 as the youngest expanding leaf and each successive number indicating the next oldest leaf on the plant) on each young plant. For each mature plant, one cage was placed on a young expanding leaf (leaf numbers 2 to 4) and one cage was placed on a mature leaf (leaf numbers 8 to 10). The entire experiment was terminated when a single type of foliage (e. g. the young leaf tissue on a young plant) was substantially consumed. All the larvae were weighed, and the plants were chemically analyzed for tryptic inhibitory activity, chymotryptic inhibitory activity, total protein, and total glucosinolates.

With regard to total plant protein bioassay, the total protein fractions were isolated from three different types of cabbage foliage: (1) young leaves (leaf numbers 1 to 4) from mature plants, (2) mature leaves from mature plants, or (3) leaves from young plants. The foliage (700 grams) was homogenized in 500 ml of 0.01 M sodium citrate, 1 M potassium chloride, pH 4.5 buffer. The supernatant was retained and the homogenate was pressed through two layers of cheesecloth, and the liquid was collected, on ice. The leaf tissue was homogenized a second time in 500 ml of buffer, pressed through the cheesecloth, and the collected liquid was pooled with the first supernatant. The liquid was centrifuged at 4200 x g for 15 min at 4°C. The supernatant was collected, and ammonium sulfate was added to 70% saturation. The solution was stored Overnight at 4°C, and centrifuged at 4200 x g for 15 min at 4°C. The pellet was resuspended in a small volume of distilled water, and subsequently dialyzed (12,000 to 14,000 MWCO) against distilled water. The dialysate was centrifuged at 4200 x g for 15 min at 4°C, and the supernatant was lyophilized. The lyophilized powder was chemically analyzed for tryptic inhibitory activity, chymotryptic inhibitory activity and total protein. The powder remaining after analysis was used to prepare 600 ml of wheat germ based diet for the determination of the effect of this foliar total protein on larval growth and development. The diet used in these tests was the same as that used for mass rearing of larval P. rapae except that the concentration of casein in the diet was reduced from 3.2% to 1.6 % (weight/volume). The bioassays for both insect species included 2 treatments, 3 cups per treatment, 30 eggs per cup. Each bioassay was replicated. The larvae were provided with diet, ad libitum from neonate until the controls reached the ultimate instar at which point tall the larvae were weighed. With regard to chemical analyses, a standard spectrophotometric assay was used to determine the presence of tryptic inhibitory activity in the cabbage foliage. Bovine trypsin (0,1 mg/ml, 1 mM HCI) was mixed (1 :1 volume/volume) with cabbage leaf juice acquired by grinding the foliage with a mortar and pestle, centrifuging the tissue, collecting the supernatant, and diluting the supernatant to 0.1 x with 1 mM HCI. The resulting material was then incubated at room temperature for 10 min. Then 100 μl of the mixture was added to 2.9 ml of buffer (0.05 M Tris, pH 8.0) containing 1.04 M p-toluene-sulphonyl-L-arginine methyl ester. Tryptic activity was monitored at 247 nm for 3 min utilizing a 50 μl aliquot of trypsin to determine uninhibited tryptic activity.

Chymotryptic inhibitory activity was determined by mixing (1 :1 volume/volume) the diluted leaf juice as prepared above with TLCK-treated bovine chymotrypsin (0,1 mg/ml 1 mM HCI) for 10 min at room temperature. 100 μl of the mixture was then added to 2.9 ml substrate (1 mM benzoyl-L-tyrosine ethyl ester in 50% MeOH, mixed 1 :1 with 0.05 M Tris buffer at pH 8.0), and monitored spectrophometrically at 256 nm for 3 min. A 50 μl aliquot of chymotrypsin was used to determine uninhibited chymotryptic activity.

To quantify glucosinolates in cabbage foliage, leaves were ground with a mortar and pestle, and the fluid was applied to a 1 ml 5 DEAE Sephadex A25 column, The column was washed with distilled water followed by duplicate washings with 0.5 ml 0.02 M pyridine/acetic acid buffer. Myrosinase (250 μl of 25 mg/ml pyridine buffer) was applied to the column and allowed to incubate at room temperature for 2 hours. The column was then eluted with 1.0 distilled water, collecting 1.25 ml of eluant. Three 250 μl aliquots of the eluant were assayed for glucose with sinigrin used as the standard.

With regard to total protein concentration, bicinchoninic acid reagent was used with purified protein from cabbage foliage being 1 5 used as the standard.

Following these experimental procedures, the larvae that hatched on plants in the large cages were allowed to select their feeding sites. Larval T. ni fed on the underside of the oldest leaves of both the young and mature plants, although they would move up the plant to the fully expanded mature leaves as the oldest leaf tissue was consumed. The preferred feeding site for larval P. rapae was the youngest tissue on the plant. They consumed the apical meristem of the plant first, then moved downward feeding on the non-vascular tissue at the base of the youngest leaves. They generally did not feed on the fully expanded or oldest leaves of either the young or mature plant.

When larval 7. ni were free to select their feeding sites (thus feeding on the oldest leaves on the plant), there was no significant difference in growth between larval 7. ni feeding on the young or mature plants (p = 0.460, n = 220). The chemical analyses of the leaf tissue indicated that there was no significant difference in the tryptic inhibitory activity (p = 0.383, n = 35), chymotryptic inhibitory activity (p = 0.389, n = 35), total protein (p = 0.563, n = 35), or total glucosinolate p > 0.05, n = 35) in the oldest foliage from the young and mature plants. Larval P. rapae that fed on the youngest foliage on mature plants were significantly smaller than larvae that fed on the youngest foliage on young plants (p < 0.001 , n = 87). A comparison of the chemical composition of plants indicated that the tryptic and chymotryptic inhibitory activity in the young foliage on the mature plant was significantly higher than that on the young plant (p < 0.001 , n = 35 for both trypsin and chymotrypsin).

Larval 7. ni that were confined to feeding on the oldest leaves on the mature plant were significantly larger than the larvae feeding on the young leaves on the young plant (p < 0.001 , n ^■= 45). The larvae restricted to feeding on the young leaves on the mature plant attempted to feed, but there was 100% mortality of the larvae in the second instar. Examination of the chemical analyses of the plant foliage indicated that there was a significant inverse correlation between larval growth and the level of tryptic or chymotryptic inhibitory activity. The tryptic and chymotryptic inhibitory activity was significantly highest in the young leaves on the mature plant (p < 0.001 , n = 35), at an intermediate level in the young leaves on the young plant (p < 0.001 , n = 35), and at the significantly lowest level in the old leaves on the mature plant (p < 0.001 , n = 35).

With the findings of Example I, the effect of cabbage proteinase inhibitor on the growth and development of larval 7. ni and P. rapae were examined in greater detail according to the following example. EXAMPLE II

Proteinase inhibitors were extracted from cabbage by homogenizing the foliage in 0.01 M sodium citrate I M potassium chloride buffer at pH 4.5, centrifuging the homogenate at 4200 x g for 10 min at 4°C, and collecting the supernatant. The supernatant was incubated for 10 min at 70°C, cooled on ice, and centrifuged at 6000 x g for 60 min. The supernatant was adjusted to pH 8.0, ammonium sulfate was added to 70% saturation, and the resulting solution was stored overnight at 4°C. The solution was then centrifuged at 6000 x g, and the pellet was resuspended in distilled water, dialyzed (MWCO 12,000 to 14,000) against water to remove the salt. The dialysate was centrifuged at 6000 x g for 20 min, and the supernatant lyophilized and labeled as "semi-pure proteinase inhibitor".

The semi-pure proteinase inhibitor was purified in a two-step process. First, 100 mg of semi-pure material was applied to a Sephadex-G75 column (2.2 x 50 cm) previously equilibrated and eluted with 0.05 M Tris pH 9.0 buffer, and monitored at 280 nm. Protein fractions with tryptic inhibitory activity were pooled, dialyzed against distilled water, lyophilized, and labeled "Sephadex- purified proteinase inhibitor". The second step in the purification process involved trypsin-bound cyanogen bromide activated Sepharose 4B affinity chromatography. The proteinase inhibitor was applied to the affinity column (1.5 x 33 cm) in 0.01 M Tris 0.1 M calcium chloride buffer at pH 8.1. The column was washed with the buffer until the optical density (at 280 nm) approached zero. The trypsin inhibitor was then eluted with 8 M urea at pH 3.0, dialyzed against distilled water, lyophilized, and labeled "affinity-purified proteinase inhibitor".

To determine the purity of the proteinase inhibitors prepared as above, a sample (4 mg/ml) of each preparation was applied to a vertical, discontinuous, non-denaturing 12.5% polyacrylamide gel with a 4% stack. Following electrophoresis, the gel was stained with 0.1% Coomassie brilliant blue R in 20% MeOH, 30% acetic acid, and destained with 30% MeOH, 10% acetic acid.

The presence of tryptic and chymotryptic inhibitory activities in the sample preparations was determined using procedures modified slightly from those described in Example 1.

The presence of tryptic inhibitory activity in the cabbage extracts and purification fractions was determined by mixing bovine trypsin (0.1 mg/ml, 1 mM HCI) 1 :1 (volume/volume) with the plant extract (2 mg/ml, 1 mM HCI), incubating the resulting mixture for 10 min at room temperature, and then adding 2.9 ml of buffer (0.05 M Tris at pH 8.0 and containing 1.04 M p-toluene-sulphonyl-L-arginine) to 100 μl of the mixture. Tryptic activity was monitored at 247 nm for 3 min. A 50 μl aliquot of trypsin was used to determine uninhibited tryptic activity. Chymotryptic inhibitory activity was determined by mixing (1 :1 volume/volume) the test solution (2 mg/ml, 1 mM HCI) for 10 min at room temperature. 100 μl of the mixture was then added to 2.9 ml of substrate (1 mM benzoyl-L-tyrosine ethyl ester in 50% MeOH, mixed 1 :1 with 0.05 M Tris pH 8.0 buffer), and monitored spectrophotometrically at 256 nm for 3 min. A 50 μl aliquot of chymotrypsin was used to determine uninhibited activity.

To determine the effect of cabbage proteinase inhibitors on larval growth and development, larvae were reared on a wheat germ- based diet that was supplemented with plant proteinase inhibitors. The basic diet was the same as that used in Example I. Initial experiments were performed using semi-purified proteinase inhibitor. Soybean trypsin inhibitor was used as a standard for comparison. Each bioassay included 5 treatments, 3 cups/treatment, 30 eggs/ cup. To ensure the results for the semi-purified proteinase inhibitor were due to the inhibitor ans not a contaminant in the preparation, the bioassay was repeated using the Sephadex-purified proteinase inhibitor. As a final confirmation of the toxicity of cabbage trypsin inhibitor to larval 7. ni and P. rapae, a bioassay was also performed using affinity-purified trypsin inhibitor. For all tests, the larvae were started on the diets as neonates, and were maintained on the diets until the controls reached the ultimate instar. All larvae were then weighed. The percent pupation and adult emergence was based on the total number of larvae weighed for each diet.

Following the incorporation of the different proteinase inhibitor preparations into the artificial diet, the soybean trypsin inhibitor had no significant effect on the growth of larval 7. ni (p = 0.225, n = 825) or P. rapae (p = 0.206, n = 991) even at a concentration of 0.5% (weight/volume) in the diet. This, evidences an adaption of each plant species to produce its own proteinase inhibitor specific for the trypsin and chymotrypsin inhibition required to protect itself against the species of larvae which utilizes the host plant as a source of food. Growth of larval 7. ni and P. rapae, however, was significantly reduced by dietary supplementation with semi-purified (p < 0.001 , n = 1252 for 7. ni ; p < 0.001 , n •= 1557 for P. rapae); Sephadex-purified (p < 0.001 , n ^■= 264 for 7. ni ; p <0.001 , n = 270 for P. rapae); and affinity-purified proteinase inhibitory factors. In addition, the dietary concentration of the proteinase inhibitor acted as a predictor of larval growth.

The relative proportion of larvae that pupated was also found to be significantly influenced by the presence of dietary cabbage inhibitor. In addition, pupal deformities were also seen as being common for larvae feeding on diets containing proteinase inhibitor. The dietary concentration of proteinase inhibitor also acted as a predictor of percent pupation of larval 7. ni and P. rapae.

Although many plant proteinase inhibitors have been shown to reduce the growth and development of insects, the remarkable feature about the results from Examples I and II is the level of dietary cabbage proteinase inhibitor required to significantly reduce larval growth and development. With cabbage proteinase inhibitor, a dietary concentration of 0.1% (1 mg/ml) will reduce larval growth as much as 66%, reduce pupation by 93%, and reduce adult emergence by 60%. Thus, these proteinase inhibitors have a sufficient detrimental effect on the growth and development of larval pests to make them potentially valuable as insecticides and larvacides for commercial use.

Although proteinase inhibitors are believed to have a role in the defense of plants against attack by herbivorous insects, detailed characterization of specific proteinase inhibitors is necessary to determine there effectiveness against specific attacking organisms. The effectiveness of a proteinase inhibitor as a defensive agent is directly related to its stability (both pH and temperature), specificity, binding constant, and concentration. Such information has been reported for proteinase inhibitors from legumes, solanum, and grains. However, before the making of the present invention, nothing was known about the chemical structure and biological activity of the proteinases from Cabbage. Accordingly, the following example was conducted with the specific purpose of determining the chemical structure and characteristics of the protein inhibitors isolated and tested in Examples I and II. EXAMPLE III

Proteinase inhibitor was extracted from cabbage by homogenizing 500 grams of fresh foliage in 900 ml of 0.01 M sodium citrate, 1 M KCI, pH 4.5 buffer. The homogenate was squeezed through a double layer of cheesecloth, and the liquid was centrifuged at 4200 x g for 10 min at 4°C. The supernatant was incubated for 10 min at 70°C, then cooled on ice for 20 min and centrifuged at 6000 x g for 60 min. The supernatant was adjusted to pH 8.0 with NaOH, and the protein was precipitated with ammonium sulfate (70% saturation) at 4°C overnight. The sample was centrifuged at 6000 x g for 20 min, and the pellet was resuspended in dwater, and dialyzed (MWCO 12,000 to 14,000) against dwater to remove the salt. The dialysate was then lyophilized and labeled as "semi-purified proteinase inhibitor".

The semi-purified proteinase inhibitor was purified by column chromatography. A 200 mg sample of the inhibitor was applied to an anion exchange 4.5 x 9 cm column with a DEAE-25A Sephadex bed, and the column was washed with 10 column volumes of 0.05 M Tris pH 9.0 buffer (until the optical density at 280 nm returned to zero). This removed most of the chlorophilic material from the sample. Then, collecting 9 ml fractions, the proteinase inhibitor material was eluted with 0.2 M Tris pH 8.5 buffer (an additional protein beak was eluted with 0.5 M Tris, pH 7.8 buffer but showed no proteinase inhibitory activity). The proteinase inhibitory active fractions from the column were applied to an affinity column of trypsin bound to cyanogen bromide activated Sepharose 4B at 6°C. The column was washed with 10 column volumes of 0.01 M Tris 0.1 calcium chloride pH 8.1 buffer (until O.D. drops equaled 300 to 500 ml). The proteinase inhibitor was eluted with 8 M urea at pH 3.0 (until O.D. dropped <100 ml), and the fractions from the entire protein peak (280 nm) were pooled. The protein peak (50 to 60 ml) was dialyzed against dwater (16 to 20 liters) overnight, concentrated to 2 ml, and analyzed for tryptic and chymotryptic inhibitory activity.

The thermal stability of the proteinase inhibitory activity was tested by incubating aliquots of a solution of semi-purified proteinase inhibitor (2 mg/ml, 1 mM HCI) at designated temperatures for specific lengths of time. Each solution was then mixed (1 :1 volume/volume) with bovine trypsin or alpha- chymotrypsin (0.1 mg/ml, 1 mM HCI), incubated for 10 min at room temperature, and tested for enzyme activity.

To determine the purity of the affinity purified proteinase inhibitor, 75 μl of concentrate was applied to a vertical, discontinuous, non-denaturing, 12.5% polyacrylamide gel with a 4% stack (using 0.025 M Tris 0.192 M glycine pH 8.3 buffer as the reservoir buffer). Following electrophoresis, the gel was stained with Coomassie brilliant blue R (20% MeOH, 30% acetic acid, ).1% Coomassie) and de-stained with 30% MeOH, 10% acetic acid to detect all protein bands. To determine those protein bands with tryptic inhibitory activity, a modification of the method from Filho and Moreira (1978) was used. The native gel was washed 3 times with 30% MeOH, 20% acetic acid to fix the protein, and then rinsed with distilled water 2 times. The gel was equilabrated, overnight, in 0.1 M sodium phosphate buffer at pH 7.8, and then incubated in a trypsin solution (0.1 ml trypsin/ml 0.1 M phosphate buffer at pH 7.8) for 30 min at 37°C. The gel was rinsed twice with distilled water, then covered with a freshly prepared solution of 2.5 mg acetyl- phenylanine-β-naphthyl-ester in 1 ml dimethylformamide plus 9 ml of 0.55 mg/ml tetrazotized O-dianisidine in 0.1 M phosphate buffer at pH 7.8. The gel was incubated in the acetyl-phenylalanine-β- naphthyl-ester solution for 30 min at 37°C, then rinsed with distilled water. Clear bands in the final gel indicated the presence of trypsin inhibitor.

The molecular weights of the proteins with proteinase inhibitory activity were determined on PAGE-SDS. A 1.5 mm discontinuous polyacrylamide gel consisting of a 15% acrylamide- 0.34% bis-acrylamide separating gel and a 4% acrylamide-0.1% bis- acrylamide stacking gel were used. A 75 μl sample plus molecular weight markers with molecular weight range of 14,000 to 70,000 were employed in the PAGE-SDS method.

The isoelectric point for each trypsin inhibitor was also determined using conventional methods known in the art. A standard spectrophotometric assay was used to determine the presence of tryptic inhibitory activity in the cabbage extracts and purification fractions. Bovine trypsin (0.1 mg/ml 1 mM HCI) was mixed (1 :1 volume/volume) with the plant extract, and then incubated at room temperature for 10 min. 100 μl of the mixture was then added to 2.9 ml of buffer (0.05 M Tris at pH 8.0) containing 1.04 M p-toluene-sulphonyl-L-arginiπe methyl ester. Tryptic activity was monitored at 247 ήm for 3 min.

Chymotryptic inhibitory activity was determined by mixing the test solution (1 :1 volume/volume) with TLCK-treated bovine chymotrypsin (0.1 mg/ml 1 mM HCI) for 10 min at room temperature. 100 μl of the mixture was then added to 2.9 ml substrate (1 mM benzoyl-L-tyrosine ethyl ester in 50% MeOH, mixed 1 :1 with 0.05 M Tris buffer at pH 8.0), and monitored at 256 nm for 3 min.

Following the procedures outlined in example III, both tryptic and chymotryptic inhibitory activity were detected in the crude extract of homogenized foliage from mature plants. However, with each purification step, the ration of tryptic to chymotryptic inhibitory activity increased (i.e., the level of tryptic activity increased) as depicted in the following table:

TABLE I Trea ment Trvptic:Chvmotryptic

Inhibitory ratio

Crude leaf juice 2:1

Semi-purified proteinase inhibitor 2:1 DEAE-purified proteinase inhibitor 10:1

Affinity-purified proteinase inhibitor 70:1

In addition, both types of inhibitory activity were very stable at high temperatures, in an acidic environment, and were found to be significantly reduced only when lyophilized.

After the initial isolation and removal of thermally unstable proteins, DEAE chromatography removed the majority of the chlorophyll and a significant proportion of contaminating protein. Affinity chromatography was used to purify the trypsin inhibitors. Approximately 5 to 6 hrs were required to apply the 300 to 400 of DEAE-purified trypsin inhibitor samples to the affinity column. The column was then washed with buffer until the column eluates no longer contained material absorbing at 280 nm. Subsequent elution with <100 ml of 8 M urea, pH 3.0, eluted a single peak of 280 nm absorbing material which contained tryptic inhibitory activity. Washing the column with 1 mM HCI, 0.1 M CaCl2 eluted a second protein peak that had no tryptic inhibitory activity.

Three protein bands for the affinity purified trypsin inhibitory agents according to the present invention were detectable when examined in a non-denaturing, discontinuous 12.5% polyacrylamide gel. The two major bands represented approximately 45% each of the total protein, while the minor band represented the remainder. All three bands were found to have tryptic inhibitory activity. The molecular weights of these three proteins was found to be 23,300, 22,250, and 18,370 daltons; the isoelectric points for the three proteins were 5.05, 5.13, and 5.99. The proteinase inhibitors of the present invention have a number of potential uses as insecticides for herbivorous pests which attack cabbage plants. The proteinases inhibitors having the molecular weights and isoelectric points isolated from cabbage tissues may be utilized in sprays or powders (in combination with conventional insecticides, surfactants, buffers, fillers, binding agents and other materials commonly compounded with insecticidal sprays and powders, including materials to inhibit the degrading of the proteinase inhibitor by environmental means such as sunlight or bacteria after application) which are meant to be applied to the growing cabbage plant as a means to control and prevent 7. ni and P. rapae larval infestation of the plant. Of course, rather than physical application of proteinase inhibitors to growing cabbage plants, it is also within the intention of the present invention to encompass the use of such inhibitory agents through biotechnical means. For example, using conventional technology, the amino acid sequence of the inhibitory agents may be determined; once known, the genetic sequence necessary to produce the inhibitory agent by the plant may be determined; and this sequence may then be incorporated, along with an appropriate genetic promoter to initiate expression of the sequence if necessary, within the genome of the plant, thus giving the plant the innate ability to produce sufficient amounts of the inhibitory agent to protect all its plant tissues from attack by susceptible pests, Thus, while I have illustrated and described the preferred embodiment of my invention, it is to be understood that this invention is capable of variation and modification, and I therefore do not wish or intend to be limited to the precise terms set forth, but desire and intend to avail myself of such changes and alterations which may be made for adapting the invention of the present invention to various usages and conditions. Accordingly, such changes and alterations are properly intended to be within the full purview of the the following claims. The terms and expressions which have been employed in the foregoing specification are used therein as terms of description and not as terms of limitation, and thus there is no intention, in the use of such terms and expressions, of excluding equivalents of the features shown and described or portions thereof, it being recognized that the scope of the invention is defined and limited only by the claims which follow.

. Having thus described my invention and the manner and process for making and using it in such full, clear, concise, and exact terms so as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected to make and use the same,

Claims

I CLAIM:

1. A proteinase inhibitory agent which is a peptide having a molecular weight of from about 18,370 to about 23,300 daltons, an isoelectric point of from about 5.05 to about 5.99, and which is inhibitory to trypsin and chymotrypsin produced in Trichoplusia ni and Pieris rapae larvae.

2. A proteinase inhibitory agent according to claim 1 wherein the molecular weight is about 18,370 daltons and the isoelectric point is about 5.99.

3. A proteinase inhibitory agent according to claim 1 wherein the molecular weight is about 23,300 daltons and the isoelectric point is about 5.05.

4. A proteinase inhibitory agent according to claim 1 wherein the molecular weight is about 22,250 daltons and the isoelectric point is about 5.13.

5. A method for inhibiting the growth and development of Trichoplusia ni and Pieris rapae larvae which comprises providing said larvae a proteinase inhibitory agent having a molecular weight of from about 18, 370 to about 23,300 daltons and an isoelectric point of from about 5.99 to about 5.05, and allowing said larvae to ingest the agent.

6. A method according to claim 5 wherein said agent is provided by applying said agent to the foliage of a cabbage plant.

7. A method according to claim 6 wherein said agent is in solution and applied as a spray.

8. A method according to claim 6 wherein said agent is a solid and applied as a powder.

9. A method for providing a cabbage plant with the ability to protect itself against damage caused by the infestation of larvae of Trichoplusia ni or Pieris rapae which comprises inserting the genetic sequence for the production of a proteinase inhibitory peptide specific for a proteinase found in the body of said larvae into the genome of said plant, providing means to allow said transformed genome to initiate expression of said sequence, and initiating the expression of said inhibitory peptide.