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A mandelonitrile lyase (EC 4.1.2.10) that catalyzes the dissociation of (S)-(-)-mandelonitrile to benzaldehyde and hydrogen cyanide has been purified to apparent homogeneity from leaves ofXimenia americana L. (Olacaceae). The lyase was purified 122-fold with 38% yield by chromatography on carboxymethyl-celiulose and chromatofocusing. The enzyme had a pH optimum of 5.5, with a K. value of 280 ,uM.Activity toward 4-hydroxy-(R,S)-mandelonitrile was 77% of that observed with the endogenous substrate; no activity was observed toward the aliphatic substrate acetone cyanohydrin. The enzyme was stable at 40C and at room temperature for at least 1 month. Native and subunit molecular weights of 38,000 and 36,500, respectively, suggest the enzyme is a monomer. The isoelectric point was pH 3.9 as determined by isoelectric focusing. Staining with periodic acid-Schiff and fluoresceinlabeled concanavalin A reagents indicate this enzyme is a glycoprotein. In contrast to (R)-mandelonitrile lyases isolated from Prunus species, the Ximenia lyase does not appear to be a flavoprotein. A second enzyme that eluted from the chromatofocusing column at pH 4.0 was also active toward mandelonitrile. However, this form accounted for less than 10% of the total activity, and its specific activity was only 6% of that of the major component. Additional physical and kinetic studies suggested this activity may be due to a nonspecific enzyme that is active toward mandelonitrile.(R)-(+)-Mandelonitrile, produced by hydrolysis of cyanogenic glucosides that occur in members of the family Rosaceae, is reversibly dissociated into benzaldehyde and HCN by the enzyme mandelonitrile lyase, an a-hydroxynitrile Iyase (EC 4.1.2.10) (1). Pfeil and associates (2-4) reported that these enzymes, when isolated from the genus Prunus, were flavoproteins and speculated on the role of the flavin cofactor in a reaction not involving oxidation/reduction.Other workers (5-8) have examined the role of the flavin moiety and this has been the subject of considerable debate.Although the (R)-(+)-mandelonitrile lyases of rosaceous species appear to be flavoproteins (1), the analogous enzyme in Sorghum bicolor that utilizes 4-hydroxy-(S)-mandelonitrile as a substrate does not contain a flavin cofactor (9).Moreover, lyases in cassava (10) and flax (11), which utilize the aliphatic substrate acetone cyanohydrin, do not appear to be flavoproteins. Thus the (R)-(+)-mandelonitrile lyase of rosaceous species is atypical in its requirement for a flavin cofactor.The occurrence of the glucoside of (S)-(-)-mandelonitrile in Ximenia americana (family Olacaceae) (12) provided an opportunity to examine whether the lyase that acts on the enantiomer of (R)-(+)-mandelonitrile is a flavoprotein. Accordingly the a-hydroxynitrile Iyase of X. americana has been purified to apparent electrophoretic homogeneity and its properties have been examined. MATERIALS AND METHODS Materials. Leaves of X. americana were collected at Fort Desoto Park (Saint Petersburg, FL) and shipped to Davis, California...

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Discs excised from Solanum tuberosum L. cv White Rose tubers demonstrated a 4.5-fold increase in chorismate mutase activity 48 hours after excision. Incubation in the presence of cycloheximide (25 micromolar Chorismic acid is located at the branchpoint between tryptophan biosynthesis on one hand, and phenylalanine and tyrosine biosynthesis on the other. CM is involved in the regulation of flux among the three aromatic amino acids. CM was reported to be present in multiple forms in a number of plant species (8,9,21,22 Disc Incubation Conditions. Tubers were removed from cold storage 24 h prior to disc preparation and placed in the dark at room temperature. Tubers were sliced into 2 to 3 mm sections. Discs (approximately 1.2 gfw each) were excised from the center tissue with a number 15 cork borer and rinsed with distilled water followed by streptomycin sulfate (25 mg/L). Three discs were placed in sterile Petri dishes (10 x 1.5 cm) containing sterile Whatman No. 3 filter paper (7.0 cm diameter), 9 ml distilled water, and streptomycin sulfate (25 mg/L). Wound response inhibition studies were performed essentially as described above with the inclusion of either cycloheximide (25 ,um) or actinomycin D (100 ,um). The Petri dishes were covered with aluminum foil and placed in the dark at room temperature. Enzyme activities were monitored 12 h and every 24 h after excision for 10 d. Studies of light activation were performed by incubating discs under white fluorescent lights (156 uE m-2 s-1), with assays being performed every 8 h after excision for 48 h.Enzyme Assays. Unless otherwise noted, all steps were carried out at 4°C. Discs were homogenized in a mortar containing 5.0 ml of 0.5 M citrate-phosphate buffer (pH 8.0), 1.5% (v/v),-mercaptoethanol, 1.0 mM tryptophan, 7.5 g (w/v) hydrated PVPP, 30 mm thiourea, and 2.5 g quartz sand. Higher buffer concentrations were necessary since homogenization typically resulted in a five-fold dilution. The homogenate was filtered through Miracloth moistened with homogenization buffer and centrifuged for 20 min at 10,000g. The supernatant was decanted and aliquots (2.5 ml) applied onto a Sephadex G-25 column (Pharmacia PD-10) equilibrated with either 0.01 M Tris-HCI buffer (pH 8.0) (for CM assays) or 0.1 M borate-NaOH buffer (pH 8.8), containing 0.1% (v/v), 3-mercaptoethanol (for PAL assays). Proteins were eluted with 3.0 ml of the respective buffer.The standard assay for CM activity contained 0.317 mm chorismic acid, 0.1 M citrate-phosphate buffer (pH 7.0) and up to 40 ,ul of enzyme in a final volume of 0.167 ml. Assays were performed either in the presence or absence of 0.1 mM tryptophan. After incubation for 10 min at 25°C, reactions were terminated by adding 0.