Proteases and Peptidases of Castor Bean Endosperm

The endosperm of castor bean seeds (Rkinus comunuis L.) contains two -SH-dependent amptidases, one hydrolyzing L-leucine-,Bnaphthylamide optimally at pH 7.0, and the other hydrolyzing L-proline,B-naphthylamide optimally at pH 7.5. After germination the endosprm contains in addition an -SH-dependent hemoglobin protease, a serinedependent carboxypeptidase, and at least two -SH-dependent enzymes hydrolyzing the model substrate a-N-benzoyl-DL-arginne-I8-naphthylamide (BANA). The carboxypeptidase is active on a variety of N-carbobenzoxy dipeptides, especially N-carbobenzoxy-L-phenylalanine-L-alanine and Ncarbobenzoxy-L-tyrosine-L-leucine. The pH optima for the protease, carboxypeptidase, and BANAase activities are 3.5 to 4.0, 5.0 to 5.5, and 6 to 8, respectively. The two amn_opeptidases increased about 4-fold in activity during the first 4 days of growth, concurrent with the period of rapid depletion of storage protein. Activities then declined as the endosperm senesced, but were stiDl evident after 6 days. Senescence was complete by day 7 to 8. Hemoglobin protease, carboxypeptidase, and BANAase acivities appeared in the endosperm at day 2 to 3, and reached peak activity at day 5 to 6. The data indicate that the ptidases are involved in the early mobilization of endosperm storage protein, whereas protease, carboxypeptidase, and BANAase may take part in later turnover and/or senescence. The endosperm tissue of castor bean seeds (Ricinus communis L.) contains storage proteins sequestered in protein bodies (29, 32). During germination, these proteins are hydrolyzed to free amino acids, which support protein synthesis in the endosperm and embryo. Massive proteolysis also occurs throughout the endosperm later during senescence. These hydrolytic events are mediated by a system of proteases and peptidases, with varying substrate specificities and activities during germination. This study was undertaken to characterize some of the proteolytic nzymes of castor bean endosperm, and to correlate their changes in activity during germination with the breakdown of storage protein, and with the onset and completion of endosperm senescence. The activities examined include Hb-ase,3 CPase, BANAase, LeuNAase, and ProNAase. ' This work was supported by National Science Foundation Grant PCM 75-23566 to H. B., and by a National Science Foundation Graduate Fellowship to R. T. 2 Present address: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824. 3 Abbreviations: BANA: a-N-benzoyl-DL-arginine-,8-naphthylamide; BAPNA: a-N-benzoyl-DL-arginine-p-nitroaniline; CPase: carboxypeptidase; Hb-ase: hemoglobin protease; lAc: iodoacetate; IAc-NH2: iodoacetamide; LeuNA: L-leucyl-fl-naphthylamide; NEM: N-ethylmaleimide; pCMB: p-chloromercuribenzoate; PMSF: phenylmethylsulfonyl fluoride; ProNA: L-prolyl-f8-naphthylamide; Z: N-carbobenzoxy-. MATERIALS AND METHODS Castor bean seeds (R. communis L. var. Hale) were soaked overnight in running tap water. They were sown in moist Vermiculite (day 0) and allowed to germinate at 30 C. The seedlings were harvested at the desired age, and the endosperm tissue collected for extraction. PROTEIN AND ENZYME CHANGES DURING GERMINATION Endosperm tissue from 10 seedlings was ground with a mortar and pestle in 10 ml of 0.2 M K-phosphate (pH 7.0) at 0 C. The crude homogenates were quantitatively transferred to a ground glass homogenizer, where the extractions were completed. The extracts were brought to a final volume of 20 ml with the grinding buffer. After removing 2.5 ml for protein determination, the homogenates were centrifuged at 10,000g for 10 min. An additional 2.5 ml of the supernatant solutions were removed for protein assays, and the remainder dialyzed against grinding buffer to remove free amino acids, which interfered with the ninhydrin assays. The extracts were assayed for protein and enzymes as follows. ,8-Naphthylamidases. The method used is essentially that of Elleman (8). Fifty ,ul of extract were added to 0.5 ml of substrate solution, and the reaction was carried out at 37 C. The substrate for LeuNAase was 0.5 mM L-LeuNA in 0.1 M Tris-HCI (pH 8.0); for ProNAase, 0.5 mM L-ProNA in the same buffer; and for BANAase, 0.5 mM DL-BANA in 0.1 M K-phosphate (pH 7.0). The pH optima for LeuNAase and ProNAase were subsequently found to be about 7.0 to 7.5 in borate-citrate (Fig. 1). Reaction times for LeuNAase, ProNAase, and BANAase were 10, 14, and 30 min, respectively. Optical densities of the colored reaction product were normalized to 30 min. Acid Protease (Hb-ase). Fifty ,ul of extract were added to 1.0 ml of 1% bovine hemoglobin in 0.1 M K-malate (pH 3.5) and incubated for 30 min at 37 C. The reaction time course was linear for at least 3 hr. The reaction was stopped and undigested protein precipitated by adding 1.0 ml of 10%o trichloroacetic acid. After removing the precipitate by centrifugation, the supernatant solutions were assayed for free amino groups by the ninhydrin method of Rosen (21). CPase. Fifty ,tl of extract were added to 1.0 ml of 1 mm Z-PheAla in 0.1 M K-acetate (pH 4.5) and incubated at 37 C for 30 min. The reaction was stopped with 1.0 ml of 10%o trichloroacetic acid, and the alanine released was assayed with ninhydrin. The reaction was first order with respect to enzyme concentration. Protein. Each of the previously described 2.5-ml samples of crude homogenate and supernatant solution were mixed with 7.5 ml of I N NaOH to dissolve the protein. After standing for at least 1 hr at room temperature, the insoluble materials were sedimented by centrifugation. One ml of each supernatant solution was added to 3 ml of 1O0o trichloroacetic acid to precipitate the protein. The protein was recovered by centrifugation and washed with 3 ml of 95% ethanol. The protein pellets were dried overnight at 37 C. 746 www.plantphysiol.org on November 11, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. RICINUS PROTEASES AND PEPTIDASES