Vol. 182, No. 2, 1992 January 31, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 579-582 D-LREQUHWMENT OF ATP AND EXTRA-RIBOSOMAL PROTEINS FOR RIBOSOME INACTIVATION BY EIGHT RNA N-GLYCOSIDASES Domenica Camicelli, Maurizio Brigotti, Lucia Montanaro and Simonetta Sperti Dipartimento di Patologia sperimentale dell’Universita di Bologna, Via S. Giacomo 14, 40126 Bologna, Italy Received December 6, 1991 SUMMARY. The requirement of ATP and extra-ribosomal proteins for the inactivation of ribosomes by eight plant RNA N-glycosidases [ribosome-inactivating proteins (RIPS)] was investigated. Tritin, pokeweed antiviral protein and barley RIP depend, as gelonin [Sperti, S., Brigotti, M., Zamboni, M., Camicelli, D. and Montanaro, L. (1991) B&hem. J., 277, 281- 2841, on the presence of ATP and extra-ribosomal proteins for full inactivation of ribosomes, while bryodin, lychnin, momordin, momorcochin and saporin inactivate isolated Arfemiu sulina ribosomes suspended in buffer saline. 0 1992 Academic Press, Inc. Gelonin, the ribosome-inactivating protein (RIP) from Gelonium multiflorum, requires the presence of ATP and high molecular weight factor(s) from the rabbit reticulocyte post- ribosomal supematant in order to inactivate isolated Artemia salina or rabbit reticulocyte ribosomes (1) which are otherwise strikingly resistant to this RIP (2,3). Earlier evidence indicated that two other RIPS, tritin from Trificum uestivum (4) and pokeweed antiviral protein (PAP) from Phyfoluccu umericuna (5) required ATP and some extra-ribosomal protein for inactivation of ribosomes. In the present paper six other RIPS, all with N-glycosidase activity on 28s RNA, were investigated for these requirements. MATERIALS AND METHODS. Tritin (M, 30000) from Triticum aesfivum was purified as described by Roberts and Stewart (6) with a further chromatographic step on Blue-Sepharose CL-6B (1.4 x 2 cm column) from which the RIP was eluted with a 20-ml gradient of O-O.2 M KC1 in 20 mM Hepes-KOH, pH 7.6. Bryodin-R (M, 30000) from the roots of Bryoniu dioicu, lychnin (M, 26600) from the seeds of Lychnis chalcedonica, momorcochin-S (M, 30700) from the seeds of Momordicu cochinchinensis, momordin (M, 31000) from the seeds of Momordica chararuia, saporin 6 (M, 29500) from the seeds of Saponaria o$‘icinalis, barley RIP (M, 3 1000) from the seeds of Hordezun vu&are and PAP-S (M, 31000) from the seeds of Phytolacca americana were generous gifts of Prof. F. Stirpe of this Department and had been prepared by previously described methods (7,8,9,10). The procedures for the preparation of the gel-filtered rabbit reticulocyte post-ribosomal supematant (gel-filtered ‘S-140’), of 80s A. sulinu ribosomes and of ‘S-105’ (proteins precipitated from the A. salina post-ribosomal supematant by 75%~satd. ammonium sulphate) were as previously described (1). Protein was measured by the method of Lowry et al. (11); 0006-291x/92 $1.50 579 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.