Cotyledons of mature black cherry (Prunus serotina Ehrh.) seeds contain the cyanogenic diglucoside @)-amygdalin. The levels of amygdalin, its corresponding monoglucoside (R)-prunasin, and the enzymes that metabolize these cyanoglycosides were measured during the course of seedling development. During the first 3 weeks following imbibition, cotyledonary amygdalin levels declined by more than 80%, but free hydrogen cyanide was not released to the atmosphere. Concomitantly, prunasin, which was not present in mature, ungerminated seeds, accumulated in the seedling epicotyls, hypocotyls, and cotyledons to levels approaching 4 pmol per seedling. Whether this prunasin resulted from amygdalin hydrolysis remains unclear, however, because these organs also possess UDPChandelonitrile glucosyltransferase, which catalyzes de novo prunasin biosynthesis. The reduction in amygdalin levels was paralleled by declines in the levels of amygdalin hydrolase (AH), prunasin hydrolase (PH), mandelonitrile lyase (MDL), and j3-cyanoalanine synthase. At all stages of seedling development, AH and PH were localized by immunocytochemistry within the vascular tissues. In contrast, MDL occurred mostly in the cotyledonary parenchyma cells but was also present in the vascular tissues. Soon after imbibition, AH, PH, and MDL were found within protein bodies but were later detected in vacuoles derived from these organelles.

Air-dried soybeans absorb water rapidly for the first 2 hr followed by a slower rate of uptake. The beans take up an equal weight of water (100% hydration) after approximately 2.5 hr at 37°C to 5.5 hr at 20°C and reach complete hydration (140% hydration) after 6 hr at 37°C and 16 hr at 20°C. Soluble solids are leached out of the beans at a fairly steacly rate throughout the hydration, and the amount is greater with higher temperatures. Temperature is the most important factor in determining the rate of water absorption and of solids losses. Of the total solids lost, 7-16% is protein. The proportion of protein loss increases as the soaking time and temperature increase. About 30-50% of soluble sugars, including fructose, sucrose, raffinose and stachyose, are removed from the beans after overnight soaking at 25°C. On the other hand, the amounts of trypsin inhibitor and hemagglutinin found in the soybean soak are relatively small as compared to that present in the air-dried beans. Hydrating soybeans to 100% prior to cooking reduces the cooking time, increases the tenderness and weight of the cooked beans, and improves their appearance. Complete hydration results in no further improvement in cooking rate or cooking quality of the beans.

Biochemical changes related to cyanogenesis (hydrogen cyanide production) were monitored during maturation of black cherry (Prunus serotina Ehrh.) fruits. At weekly intervals from flowering until maturity, fruits (or selected parts thereof) were analyzed for (a) fresh and dry weights, (b) prunasin and amygdalin levels, and (c) levels of the catabolic enzymes amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. During phase 1 (0-28 days after flowering [DAF]), immature fruits accumulated prunasin (mean: 3 micromoles/fruit) but were acyanogenic because they lacked the above enzymes. Concomitant with cotyledon development during mid-phase 11, the seeds began accumulating both amygdalin (mean: 3 micromoles/seed) and the catabolic enzymes and were highly cyanogenic upon tissue disruption. Meanwhile, prunasin levels rapidly declined and were negligible by maturity. During phases 11(29-65 DAF) and III (66-81 DAF), the pericarp also accumulated amygdalin, whereas its prunasin content declined toward maturity. Lacking the catabolic enzymes, the pericarp remained acyanogenic throughout all developmental stages.

In black cherry (Prunus serotina Ehrh.) homogenates, (R)-amygdalin is catabolized to HCN, benzaldehyde, and D-glucose by the sequential action of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. The tissue and subcellular localizations of these enzymes were determined within intact black cherry seeds by direct enzyme analysis, immunoblotting, and colloidal gold immunocytochemical techniques. Taken together, these procedures showed that the two d-glucosidases are restricted to protein bodies of the procambium, which ramifies throughout the cotyledons. Although amygdalin hydrolase occurred within the majority of procambial cells, prunasin hydrolase was confined to the peripheral layers of this meristematic tissue. Highest levels of mandelonitrile lyase were observed in the protein bodies of the cotyledonary parenchyma cells, with lesser amounts in the procambial cell protein bodies. The residual endosperm tissue had insignificant levels of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase.

In black cherry (Prunus serotina Ehrh.) seed homogenates, amygdalin hydrolase (AH) participates with prunasin hydrolase (PH) and mandelonitrile lyase in the sequential degradation of (R)-amygdalin to HCN, benzaldehyde, and glucose. Four isozymes of AH (designated AH I, I', II, II') were purified from mature cherry seeds by concanavalin A-Sepharose 4B chromatography, ion-exchange chromatography, and chromatofocusing. All isozymes were monomeric glycoproteins with native molecular masses of 52 kD. They showed similar kinetic properties (pH optima, K., Vmax) but differed in their isoelectric points and N-terminal amino acid sequences. Analytical isoelectric focusing revealed the presence of subisozymes of each isozyme. The relative abundance of these isozymes and/or subisozymes varied from seed to seed. Three isozymes of PH (designated PH I, Ila, and lIb) were purified to apparent homogeneity by affinity, ion-exchange, and hydroxyapatite chromatography and by nondenaturing polyacrylamide gel electrophoresis. PH I and PH lIb are 68-kD monomeric glycoproteins, whereas PH lla is dimeric (140 kD). The N-terminal sequences of all PH and AH isozymes showed considerable similarity. Polyclonal antisera raised in rabbits against deglycosylated AH I or a mixture of the three deglycosylated PH isozymes were not monospecific as judged by immunoblotting analysis, but also cross-reacted with the opposing glucosidase. Monospecific antisera deemed suitable for immunocytochemistry and screening of expression libraries were obtained by affinity chromatography. Each antiserum recognized all known isozymes of the specific glucosidase used as antigen.the sequential action of the enzymes AH2, PH, and MDL (12,29). One of the complexities of the Prunus cyanogenic system yet to be satisfactorily unraveled is the microheterogeneity shown by these catabolic enzymes. Earlier studies (13,14,29) established that AH and PH occur in cherry homogenates as two and three isozymes, respectively, whose existence was not attributable to partial proteolysis during isolation. The nature and physiological significance of this multiplicity might best be approached by N-terminal sequencing followed by construction and screening of a black cherry cDNA expression library. As with other cyanogenic species, large-scale cyanogenic glucoside catabolism in P. serotina occurs only after tissue disruption, implicating some crucial compartmentation of amygdalin and its catabolic enzymes in undamaged seeds. However, such spatial regulation of cyanogenesis remains poorly understood (27,28).In this paper, we describe how previous purification procedures have been improved to achieve the isolation of four AH and three PH isozymes for use in N-terminal sequencing. Monospecific polyclonal antibodies have been generated against AH and PH for future use in immunocytochemical localization studies and screening of expression libraries. Additionally, to gain further insight into the multiplicity shown by AH, we have devised a simple protocol involving affinity chromatog...