Pergamon PH: S0031-9422(96)00193-8 Phytochemistry, Vol. 43, No. 1, pp. 39-44, 1996 Copyright ~) 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/96 $15.00 + 0.00 MAJOR PROTEINASE HYDROLYSING GLIAD1N DURING WHEAT GERMINATION ANDREABOTrAR1,ANTONELLA CAPOCCHI, DEBORA FONTANINI and LUCIANO GALLESCHI* Department of Botanical Sciences, University of Pisa, Via L. Ghini 5, 56126-Pisa, Italy (Received in revisedform 30 January 1996) Key Word Index--Triticum durum; Gramineae; wheat; purification and characterization; pro- teinases; cysteine proteinase. Abstract--A proteinase, representing the bulk of the enzyme activity for the hydrolysis of gliadin, was extracted from endosperms isolated from germinated seeds (four days) and was purified by ion-exchange chromatography and preparative isoelectric focusing. The optimal pH for gliadin hydrolysis was 4.25. The M r, determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 30 000; the isoelectric point was 4.5. The enzyme activity was totally inhibited by E-64 and cystatin, while inhibitors of other classes of proteinases were barely effective or ineffective. The activity was stimulated by sulphhydryl compounds. The proteinase hydrolysed to small peptides the gliadins from durum and soft wheat seeds. Other protein substrates were weakly degraded or not degraded. The proteinase appears to belong to the cysteine class and to play a key role in the initial mobilization of the main reserve protein in the starchy endosperm. Copyright © 1996 Elsevier Science Ltd INTRODUCTION The mobilization of seed storage proteins represents one of the most important post-germinative events in growth and seedling development. In fact, hydrolysis of these reserves to soluble products supports seedling growth prior to the development of autotrophy. In a wheat grain most of the nitrogen is found in the storage proteins, known as gliadins, which are soluble in alcohol solution. They are deposited in the starchy endosperm where they constitute up to 50% of the total proteins [1]. It is known that cereal storage proteins are degraded during early seedling growth by endo- and exopeptidases, although the cooperative role of those enzymes in protein breakdown still remains to be elucidated in many of the plant materials studied [2]. Carboxypeptidases and aspartic proteinases are already present in dry wheat seeds [3-5]. In in vitro experi- ments they showed a weak hydrolytic action on the gliadin, which increased by about 25% when both enzymes were added simultaneously [4]. However, the in vitro hydrolysis of the gliadin started after the first day of germination of wheat seeds, in association with the appearance of proteinase activity against this stor- age protein [6]. Preliminary studies conducted with crude extracts from germinated (4 days) wheat seeds in the presence of proteinase inhibitors showed that about 89% of the proteinase activity responsible for the increase in gliadin hydrolysis was of cysteine-type (EC 3.4.22) [6]. Although cysteine proteinases have been *Author to whom correspondence should be addressed. previously found in germinating wheat seeds [4, 7], they have not been isolated to a sufficiently high degree of purification for the study of their characteristics and physiological role. The present work describes the isolation and the partial characterization of a cysteine proteinase that is probably involved in the initial cleavage of gliadin to polypeptides in germinated Triticum durum seeds. RESULTS AND DISCUSSION Purification of the proteinase The purification of the proteinase from endosperms of germinating durum wheat is summarized in Table 1. Endosperms of 4-day-old seedlings were used as the starting material for the purification of the enzyme, since our previous work [6] showed that gliadin mobili- zation and gliadin activity were most prominent during this period. The fractionation of the proteins on DEAE- cellulose allowed us to eliminate most of the con- taminating proteins. Initial attempts to fractionate the crude protein extract with the Rotofor cell, either by using ampholytes with a broad pH range (3-10), or with a narrow pH range (4-6) were unsuccessful. In both cases there was considerable precipitation, which started in the end compartments and then spread inward during the run. Use of glycerol and pre-focus of the pH gradient before loading the extract did not alleviate the problem. On the other hand, protein precipitation did not happen when samples fractionated on DEAE-cellu- lose were applied to the Rotofor cell. However, the 39