Immunological Comparisons of Chymotrypsin Inhibitor I among Several

Microcomplement fixation was employed to compare the immunological differences that occur between purified inhibitor I from potato tubers, the four purified protomers that comprise it, and inhibitor I from tuber and leaf extracts. Total inhibitors of chymotrypsin and trypsin in leaves of seven genera of the Solanaceae were identified by enzymatic assay. In leaves of three genera, Solanum, Lycopersicum, and Datura, chymotrypsin inhibitor I was identified immunologically. In petals of all seven genera inhibitor I was also identified immunologically. With the microcomplement fixation technique inhibitor I from leaf or petal extracts of eight Solanaceae genera were compared. An immunological relationship of inhibitor I among seven of these genera was established. Chymotrypsin inhibitor I, a tetrameric protein of M.W. 39,000 (6) isolated from potato tubers was identified, with immunological techniques, in tissues of certain other members of the Solanaceae family besides Solanum tuberosum (13). The inhibitor accumulated in detached green leaves of Solanum tuberosum and Lycopersicum esculentum in large quantities (9), but not in green leaves of Nicotiana tabacum (12). However, in etiolated or white mutant tobacco leaf tissue the inhibitor did accumulate (12). Recently, we found that insect damage or mechanical wounding of potato and tomato leaves caused a rapid accumulation of inhibitor I throughout the attacked plants (2). We hypothesized that the inhibitor was produced as a defense mechanism to ward off the marauding insects by inhibiting their digestive proteinases. In the present communication we present the results of a search for inhibitor I in other members of the Solanaceae family. With immunological techniques we have identified inhibitor I in tissues from five additional species of solanaceous plants and have compared their serologic reactivities by the microcomplement fixation technique. 'This research was supported in part by United States Public Health Grants G.M. 12505 and 2-K3-GM 17059, and by United States Department of Agriculture, Cooperative State Research Service Grant 915-15-29. College of Agriculture Research Center Scientific Paper No. 3823, Project 1791. 'Present address: Department of Chemistry, Washington State University, Pullman, Wash. 3 Career Development Awardee of the United States Public Health Service. To whom reprint requests should be made. MATERIALS AND METHODS Bovine alpha chymotrypsin and trypsin were purchased from Worthington. Benzoyl-L-arginine methyl ester was purchased from the British Drug House, Ltd. and TAME' from Nutritional Biochemical Corp. Inhibitor I employed in complement fixation assays was "dissociation-purified inhibitor I" (6) prepared from the juice of white potatoes. Inhibitor I subunits (called A, B, C, and D) were purified as described previously (6). Inhibitor I used for preparing anti-inhibitor I serum was four times crystallized by the method of Balls and Ryan (1). This method of preparation preferentially crystallizes a protomer species of inhibitor I that resembles protomer type C. Microcomplement fixation was performed by the method of Levine (4). Rabbit anti-inhibitor I serum was obtained from 4to 5-lb rabbits by biweekly subcutaneous injections of 1 mg of four times crystallized inhibitor I emulsified in 1.0 ml of complete Freund's adjuvant. After six injections the rabbits were allowed to rest at least 3 months before resuming injections for 2 more weeks. Ten days after the final injection, blood was collected from an ear vein. The serum was recovered by centrifugation and stored at -20 C. Serum used for complement fixation was heated to 60 C for 20 min to remove complement. The antiserum produced a single sharp precipitin band in both Ouchterloney and immunoelectrophoretic assays with pure inhibitor, potato tuber juice, and potato leaf juice (10). Plants were grown from seed in a greenhouse under natural light during the summer months. Flower petals were removed from young plants for analysis during the first flowering. Only freshly blossomed fiowers were used. Expanding young leaves were utilized from plants 3 to 6 weeks old and were usually removed from the third or fourth petiole down from the apex. Extracts of leaves or flower petals were made for complement fixation by macerating the tissue with a ceramic mortar and pestle at 50 and expressing the juice with a hand garlic press followed by centrifugation at 144,000g at 0 C for 1 hr in a Spinco Model L ultracentrifuge. The clear supernatant was used for assay. For enzymic analysis of inhibitors, 0.1 ml of 1 M ascorbate was added per gram of leaves before maceration. After high speed centrifugation the pH was adjusted to 7.9 with 1 N NaOH and centrifuged at 2000g to clarify. The following plants were included in the study: Solanum tuberosum, Solanum nigruin, Lycopersicum esculentum, Nicotiana tabacum, Datura stramonium, Petunia hybrida, Atropa belladonna. Physalis floridiana, and Capsicum annuum. Lighting for constant light studies consisted of Gro-Lux fluorescent lamps supplemented with 60-w incandescent bulbs. A I Abbreviations: BTEE: N-benzoyl-L-tyrosine ethyl ester; TAME: p-toluenesulfonyl-L-arginine methyl ester.