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Two proteolytic enzymes with carboxypeptidase activity have been isolated from a germinated wheat extract and partially characterized. Both enzymes rapidly released amino acids from hemoglobin and gluten and hydrolyzed carbobenzoxy-phenylalanylalanine. The enzymes were inhibited by diisopropylphosphofluoridate, but unaffected by salts, ethylenediaminetetraacetate, and sulflydryl reagents at lower concentrations, and had molecular weights of approximately 55,000 and 61,000. Analysis of the hydrolysis products of hemoglobin and gluten indicated that both enzymes had broad specificities, including the ability to release proline.During the germination of wheat. proteolytic enzymes rapidly hydrolyze the storage (gluten) proteins, thus providing important nutrients to the growing seedling (7). A number of peptidases and proteases of potential importance in this process have been identified utilizing various substrates (6. 10. 13, 16). However, few studies involving the purification and properties of these enzymes are available (17. 22. 23. 27).Recently we have become interested in a group of wheat proteases which are cap2ble of hydrolyzing hemoglobin or gluten (a native substrate) to amino acids and/or small peptides (18,19). Their relatively high levels of activity in mature wheat kernels, following removal of inhibitors, and their location in the endosperm, the main site of storage protein, suggested that these enzymes may be of importance in providing amino acids and/or small peptides to the growing embryo. particularly during the early stages of germination. In this communication the purification and partial characterization of two of these enzymes from germinated wheat is reported. was added slowly with stirring to the dialyzed crude extract to 35 % saturation and the resulting precipitate removed by centrifugation. The supernatant was brought to 70 % saturation and the precipitate, following centrifugation, was dissolved in 50 ml of 0.05 M acetate buffer (pH 4) and dialyzed against the same buffer, giving a final volume of 100 ml. MATERIALS AND METHODSAffinity Chromatography. Hemoglobin substrate powder (Worthington), following exhaustive dialysis against water, was coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia) as described by Chua and Bushuk (3). The ammonium sulfate-treated extract (25 ml) was applied to a hemoglobinSepharose column (1.5 x 15 cm) and eluted with 0.05 M acetate buffer (pH 4) followed by 0.5 M acetate buffer (pH 5). The active peak was dialyzed against 0.025 M acetate buffer (pH 4.4).Ion Exchange Chromatography. Ion exchange chromatography was carried out on a column (2.6 x 40 cm) of CM-32 cation exchanger (Whatman) equilibrated with 0.025 M acetate buffer (pH 4.4). The active peak from affinity chromatography was introduced into the column and eluted with a 2 liter linear gradient of 0.025 to 0.45 M acetate (pH 4.4). Fractions containing proteolytic activity were pooled, concentrated by ultrafiltration on a PM 10 membrane (Amicon), and dialyzed against 0.15 M acetate bu...

The effects of gibberellic acid, abscisic acid, cyclohexinide, actinomycin D, and cordycepin upon exo-and endoproteolytic activities and storage (gluten) protein hydrolysis in germinating wheat and in incubated embryoless wheat seeds have been studied. Early increases in endoproteolytic activity were insensitive to the addition of gibberellic acid and inhibitors of protein and RNA synthesis. Later Wheat endosperm storage proteins consist of two major groups of proteins, the gliadins and glutenins (16). These proteins are responsible for the unique viscoelastic properties of wheat flour which allows the production of bread. However, during germination (or field-sprouting) these proteins are hydrolyzed by increasing levels of proteolytic activity leading to a reduction in breadmaking quality (3,11,22). A number of studies have been concerned with determining the enzymes responsible for this process (14,21,26 For studies with embryoless seeds, whole seeds were softened for 1 h in water and the embryo portion carefully removed by scalpel under a microscope. Following treatment with Javex as described above, the embryoless seeds (50 seeds per sample) were incubated in a germination cabinet at 18.5 C on silica sand saturated with water containing the various compounds. Samples were withdrawn at various times, extracted, and analyzed.Extraction and Determination of Exo-and Endoproteolytic Activity. All procedures were done at 4 C. For extraction of activity in whole seeds, 100 seed samples (in duplicate) were extracted in a VirTis homogenizer for 2 min (6-to 20-s intervals) with 40 ml of 0.1 M sodium acetate (pH 4.4) buffer and then stirred 1 h. Samples were then centrifuged at 40,000g (15 min) and the precipitate was resuspended in 30 ml of buffer and stirred an additional hour. Following centrifugation at 40,000g, supernatants were combined and analyzed for activity.For extraction of activity from embryoless seeds, 50 seeds (in duplicate) including the sand were ground with a mortar and pestle in 20 ml of 0.1 M sodium acetate (pH 4.4) buffer and stirred for I h. Following centrifugation for 15 min (100,OOOg), the supernatants were analyzed for activity.Endoproteolytic (azocaseinase) activity (19) was determined in duplicate by incubating the extracts (0.5 ml) in 3.

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