Vol. 92, No. February 4, 1980 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 27, 1980 SURVEY DF PLANTS Albert Department of FCR ENTEROKINASE Lau and 1243-1249 INHIBITORS Harry Ako Agricultural Biochemistry and Margaret Werner-Washburne' Department University Honolulu, Received January of Botany of Hawaii Hawaii 96822 3,198O SUMMARY A radioisotopic assay procedure was developed for the assay of enterokinase inhibitors. This assay was used to test a number of plant species for enterokinase inhibiting activity. Enterokinase inhibiting activity was found in 5 of the tissues surveyed. These included peanut seeds, leaves of two tomato varieties, and tubers of two ootato cultivars. Peanut seeds contained protein enterokinase inhibitors that were separated by gel filtration on Seohadex G-50 after quantitative recovery by affinity chromatography on anhydrotrypsinSeoharose. Enterokinase inhibitina activity was found to accumulate in tomato leaves as a result of wounding. It was concluded that some olants contain enterokinase inhibitors. Enterokinase activation when initiates of digestive enterokinase activates role exDected be to occurring inhibitors studied. Their their oossible properties to about 1 have study as the enterokinase Submitted M.S. in Journal of of other orotein the in all been plant oroteolytic enzymes form (1,2). Because proteolysis. diaestive their protection have However, with their and of the be been proteases, anti-nutritive enterokinase proteinases turn, Naturally proteinases and in would interactions the begins which, of other in trypsin inhibitors diqestive results reaction enterokinase examined. digestive The to chemistrv, roles which animals. tryosinoqen inhibitors of reaction in enterokinase, potent other cascade proteinases activates zvmogens initiatinq the little is is not as easy known inhibitors. b.y Marqaret Werner-Washburne Botanical Sciences. Hawaii Series No. 2463. in oartial Agricultural fulfillment Exneriment of the Station, 0006-291X/80/041243-07$01.00/0 1243 All Copyright i 1980 rr,vhir of reproductron b-v Academic in any.forrn Pms, Inc.. merwd. Vol. 92, No. 4, 1980 The BIOCHEMICAL purpose investiaation into kinase inhibitors. several plant followinq assay present a new class As will of a first for can step, used to was entero- tmade inhibitors. that in some In plants of amonq the contain a [%]benzo,yl studies COMMUNICATIONS initiate inhibitors, a search that and be RESEARCH is nroteinase enterokinase activities procedure BIOPHYSICAL communication be demonstrated inhibiting ester the soecies it kinase of AND arginine enteroethyl enterokinase inhibitors. MATERIALS Radiochemicals and supolies such as butyl-PBD and tritiated ethanol were obtained from New England Nuclear. Tritiated BzArqOEt* was prepared according to Roffman (3) as modified by Anderson --et al. (4) except that the r3H]BzArgOEt had to be purified on a 1 x 60 --et al. column of Sephadex LH-20 run in methanol prior to use. Enterokinase was prepared from bovine duodenal mucosa according to (L. Fang, F. Sumida and H. Ako, in preparation). Fong -et -*. al Enterokinase purified in this manner gave one major band after electrophoresis in sodium dodecylsulfate in the absence of reducing agent. Anhydrotrypsin Sepharose was prepared by coupling crude anhydrotrypsin (5) to Sepharose 6B using the CPlBr procedure. Plant materials, except where noted, were collected at maturity in Hawaii. Solanum tuberosum cultivars were flown in bulk from ti!ashington state and stored frozen. Fresh or thawed tissues were homogenized in Extraction techniques. a Warinq Blender with 1 to 4 ml of 5DmM HEPES, pH 8, per gram fresh weight of tissue. Extracts were centrifuged for 30 min at 16,000 x g. Extracts of cultivars of S. tuberosum were prepared as described above except that tissues were extracted with 0.4 M NaCl at pH 8 (1.5 ml/g Tissues which contained low but untitratable levels of tissue). inhibitory activity were concentrated 10 fold by lyophilization. Arachis hypopaea extracts were prepared by Tur-Sinai (6). -et --al Bonnie Best plants were qrown from seed Lycopersicon esculentum cv. according to Green and Ryan (7,8) in a growth room at 24°C with approxiPlants mately 175~ Einsteins/m2/second provided by fluorescent lights. were growin in vermiculite and fertilized weekly with one-half pram per plant of Stern's 15-30-15 mixture supplemented with trace elements. After 4 weeks, plants were wounded once on the midvein of the first leaf above the cotyledons for two consecutive days. Wounds were made by pressing a leaflet between a rat-tail file and an 8 mm dowel. Both wounded and control plants (44 plants per croup) were kept under constant light after wounding and, after 48 hours, stem apices were harvested and extracted as described above. METHODS detect 2 A continuous, enterokinase Abbreviation: liquid scintillation inhibitors. Extracts BzArgOEt, benzoyl-L-arginine 1244 assay procedure tested for ethyl (4) was enterokinase ester used to Vol. 92, No. BIOCHEMICAL 4, 1980 AND BIOPHYSICAL RESEARCH COMMUNICATIONS inhibitors were pre-incubated with enterokinase in the presence of buffer for 1 hr prior to addition of substrate which initiates the assay. Assay Final concentrations of HEPES, pH 8, and volumes were kept at 100 111. The assay is run by placing substrate were 50 mM and 50 $4 respectively. the assay mixture on the bottom of a scintillation vial, layering scintillation fluid (7 g/l butyl-PBD) carefully over the mix and counting at intervals. As substrate is cleaved, [SH]ethanol diffuses out of the assay mixture into the scintillation fluid and is counted. Since sub-saturating concentrations of substrate are used (50 l!M as compared with a Km found to be 470 pM), substrate does not interfere with inhibitor binding. Since 1 x 10-9 M enterokinase is present in the assay, given tiqht inhibitor binding (Ki = 10-9 II) it is possible to detect 1 x 10-9-M inhibitor. These limitations do not appear to be unduly restrictive since many inhibitors have Ki values lower than 10-9 and are present in tissues in concentrations higher than 10-9 M (9). RESULTS The 10 survey plant Ricinus and Psilotum It Solanaceae inhibitors. variation was either Table nudum. found activity was detected that a wide I. 5 of 5. range Cornnon name Organ assayed potato tuber by titration for Allamanda _____. inhibitory peanut as reviewed seed (8). 1245 the 20 first -cathartica, activity tissues tested activities (Table from the was (Table showed 1). equivalents may Equivalents Tissues Nanomoles enterokinase inhibited per gram of tissuea (unwounded) (wounded) .0242 .0506 not titrated 4.36 1). a These tomato hybrid aDetermined 15 of .062 .0009 leaf leaf leaf hypogaea the tuberosum ---_~ of in species inhibiting inhibitinq Enterokinase Inhibitory in Various Plant escu- Leguminosae Arachis qiqantea, - - only of 15 was Enterokinase in cultivars Solanaceae Solanum tuberosum cv. Russet Burbank cv. Russet Norgold N-52 this representing activity Enterokinase Plant Lycopersicon lentum cv. Bonnie tissues Mucuna - present between suqgest which charantia, widespread. values in Yomordica 20 inhibiting Species communis 60-fold included Trypsin tested. included less plants families. tissues time of be Vol. 92, No. found in that BIOCHEMICAL 4, 1980 different there may parameters be such In as potato order to a result assayed of for greater inhibiting controls (Fig increased by here Green activity in the Table I). in these leaves. Ryan may be of be tubers a column of (Table 0.5 NH&OH. (10) on o,ther former higher latter stress in to proteinase peanut Subsequent than separation into plants used results (8,ll). activity tomato 0.05M M formic two plants which activity with 0.5 in from inhibiting inhibiting seeds with the wounding inhibiting eluted the inhibitors washed and as fractions leaves was Titration unwounded leaves. of enterokinase (closed circles) with increasing and wounded 1246 amount of (open circles) or loaded Tris buffer, acid adjusted on extract tomato on pH Sephadex &I of Extract 1. Z-fold also enterokinase Enterokinase in was than that way that there activity same response Enterokinase M NaCl the diisopropyl plants suggests suggests of of that wounded increased 01234567 Fig or subsequently inhibiting The inhibitors. I). showed from anhydrotrypsin-Sepharose, containing with extracts and Titration extracts a battery somewhat others activity wounded activity. a systematic of COMMUNICATIONS based inhibiting Chymotrypsin wound The part hypogaea to potato pH 3.0 (8). accumulation appears in to by content were dialyzed 1 and and Arachis 8.0, (7), responded found inhibitor plants inhibiting activity Z-fold been enterokinase tomato treated RESEARCH status. whether enterokinase has in physioloqical test BIOPHYSICAL as variations wounding, flurophosphate tested cultivars large as AND from to Vol. 92, No. 4, 1980 BIOCHEMICAL G-50 is shown also be separated both fractions studied may be inhibit trypsin related to in Table RESEARCH II. and peanut the can Since cellulose. chymotrynsin, protease COMMUNICATIONS The two fractions on carboxymethyl inhibitors inhibitors studied by (6,lZ). FRACTION Fig BIOPHYSICAL 2 and summarized by chromatography also here others in Fig AND 2. NUMBER Separation of enterokinase inhibiting activities from peanuts. A 1.5 x 98 cm column of Sephadex G-50 (superfine) was used. It was equilibrated and eluted with O.lpl ammonium bicarbonate adjusted to pH 7 with acetic acid. Tube numbers 36-39 and 41-46 were pooled and referred to as the 1st and 2nd fractions, respectively, in Table II. Table II. fraction Purification of b atotal protein extract Peanut (mg) total activity 532 Enterokinase Inhibitors yield (%I purification .333 anhydrotrypsinSepharose 8.32 .318 95 61 G-50 1st peak 1.01 .147 44 231 .102 31 179 G-50 2nd peak a as .911 determined b umoles bythe enterokinase Lowry inhibited method as determined 1137 by titration (8) Vol. 92, No. 4, 1980 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS DISCUSSION One objective studying the be suitably with of inhibitor at have data and is thus should studying in be protease attack and as With present work proteinase adds contain proteins to function B (15,16,17) latter, the physiologically kinase inhibition times pancreatic a potent more potent than hypertrophy inhibitor in of why other rats no considered when herbivore as well that note that the the list of Potato as trypsin With inhibitors is trypsin raised. inhibitor in Pancreatic for chymotrypsin, inhibitors inhibitors tubers, enterokinase. some pancreatic enterokinase be to trypsin, (19). are enterokinase to inhibit trypsin Thus, there to Solanaceae. enterokinase explain now activity possibility as may can can that that interesting the which A and the in it nutrition. inhibiting proteins proved interfere system deterrents is achieved be (2,13). that must human it not for enterokinase others suqgests possible former, why has can results) notion and in enterokinase carboxypeptidases respect the inhibiting instance, studied toxicants to by It as do assay reason the It and that the was system. unpublished nature. inhibitors regard Ako, a methodology objective enterokinase undetected further possible This ["H]BzArgOEt challenges inhibitors inhibition H. develop assay with the gone here plants. orobably far presented enterokinase 10 Lau to concentrations With interference inhibitors use substrate (P. was [3H]BzArgOEt for binding. be demonstrated work in the sensitive run substrate present inhibitors development conveniently the the enterokinase with to of Enterois nearly inducing trypsin inhibitor is useful comments made (14). ACKNOWLEDGEMENTS The during authors preparation wish of to this thank Dr. C. manuscript. 1248 A. Ryan for Vol. 92, No. BIOCHEMICAL 4. 1980 AND BIOPHYSICAL RESEARCH COMMUNICATIONS REFERENCES 1. 2. 3. 4. Neurath, Maroux, 5031-5039. Roffman, 11-17. Anderson, H. S., and Walsh, K.A. Baratti, J. and S., L.W., Sanocka, Walsh, U. and K.A. (1976) Proc. Nat. Acad. Desnuelle, P. (1971) J. Troll, and W. Neurath, (1970) H. Anal. (1977) Sci. Biol. 73, 3825-3832. Chem. 246, Biochem. Biochem. 36, 16, 3354- 3360. 5. 6. 7. 8. 9. 10. Ako, H., Foster, R.J. and Ryan, C.A. (1972) Biochem. Biophys. Res. Commun. 47, 1402-1407. Tur-Sinai, A., Birk, Y., Gertler, A., and Rigbi, M. (1972) Biochim. Biophys. Acta 263, 666-672. Walker-Simmons, M. and Ryan, C.A. (1977) Biochem. Biophys. Res. Commun. 74, 411-416. Green, T.R. and Ryan, C.A. (1972) Science 175, 776-777. Laskowski, M. Jr. and Sealock, R.W. (1971) Enzymes III, 376-457. Ryan, C.A., Kuo, T., Pearce, G. and Kinkel, R. (1976) Am. 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