Isolation and primary structures of seven serine proteinase inhibitors from Cyclanthera pedata seeds Jolanta Kowalska ⁎ , Agnieszka Zabłocka, Tadeusz Wilusz Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland Received 3 January 2006; received in revised form 13 March 2006; accepted 13 March 2006 Available online 5 April 2006 Abstract Seven new trypsin inhibitors, CyPTI I–VII, were purified from ripe seeds of Cyclanthera pedata by affinity chromatography on immobilized chymotrypsin in the presence of 5 M NaCl followed by preparative native PAGE at pH 8.9. The CyPTIs (Cyclanthera pedata trypsin inhibitors) belong to a well-known squash inhibitor family. They contain 28–30 amino acids and have molecular weights from 3031 to 3367 Da. All the isolated inhibitors strongly inhibit bovine β-trypsin (K a N 10 11 M - 1 ) and, more weakly, bovine α-chymotrypsin (K a ≈ 10 4 –10 6 M - 1 ). In the presence of 3 M NaCl the association constants of CyPTIs with α-chymotrypsin increased a few hundred fold. Taking advantage of this phenomenon, a high concentration of NaCl was used to isolate the inhibitors by affinity chromatography on immobilized chymotrypsin. It was found that although one of them, CyPTI IV, had split the Asn25–Gly26 peptide bond, its inhibitory activity remained unchanged. The hydrolyzed bond is located downstream of the reactive site. Presumably, the inhibitor is a naturally occurring, double-chain protein arising during posttranslational modifications. © 2006 Elsevier B.V. All rights reserved. Keywords: Cyclanthera pedata; Double-chain inhibitor; Squash inhibitors 1. Introduction The inhibitors isolated from the seeds of Cucurbitaceae plants are one of several inhibitor families and include over 50 peptides with molecular weights of about 3300 Da. With the exception of a cyclic, 14 amino-acid-residue inhibitor from sunflower seeds, SFTI-1 [1], the squash family inhibitors are the smallest known natural serine protease inhibitors, consisting of 27–33 amino-acid residues cross-linked with three disulfide bonds. Some of them have pyroglutamic acid at the N-terminal end (see Fig. 8), and two inhibitors, MCoTI I and II, have a cyclic structure [2]. All known squash inhibitors, with the exception of MCEI, have a Lys or Arg residue at the P1 position. MCEI has Leu at the P1 position and therefore exhibits antielastase activity [3]. The physiological function of the inhibitors of the family of Cucurbitaceae remains unknown. A potential role of squash inhibitors in the plant cell cycle was reported by Wiśniowska et al. [4]: CPPTI showed a similar effect to gibberellic acid. In addition CMTI I and CMTI III (both in their virgin and modified forms, with a cleaved reactive-site peptide bond) stimulated the proliferation of mouse fibroblasts [5]. It was also found that CPPTI activated RNA polymerase I and II [4]. In vitro, the inhibitors of this family strongly inhibit bovine β-trypsin, with K a ≈ 10 10 –10 12 M - 1 [6,7], to a smaller degree human plasmin [8], cathepsin G [8,9], plasma kallikrein [8], and β-factor XIIa [10], and considerably weakly trombin, the blood clotting factors VIIa, IXa, Xa, and XIa, and the microbiological enzymes subtilisin BPN′ and proteinase from Streptomyces griseus (SGPB) [8,11]. Bovine α-chymotrypsin is inactivated by squash inhibitors only in the presence of a high NaCl concentration [12,13]. Endogenous proteinases inhibited by squash family inhibi- tors are still unknown, but proteinases capable of degrading inhibitors were isolated from Cucurbitaceae family seeds. Pepsin-like proteinase from Cucurbita ficifolia seeds hydro- lyzes the Leu7–Met8 peptide bond in CMTI I, causing loss of its inhibitory activity [14], whereas a serine protease from the same source only splits off an N-terminal Arg residue without Biochimica et Biophysica Acta 1760 (2006) 1054 – 1063 http://www.elsevier.com/locate/bba ⁎ Corresponding author. Tel.: +48 71 3752 936; fax: +48 71 3752 608. E-mail address: jolanta.kowalska@bf.uni.wroc.pl (J. Kowalska). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.03.011