Prunus serotina Amygdalin Hydrolase and Prunasin Hydrolase

Abstract

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...

Utilization of Amygdalin during Seedling Development of Prunus serotina

“…In 1992, fruits were obtained from a different tree located approximately 50 m from the tree used previously. Their seeds were processed and stored for at least 3 months at 4OC as described by Li et al (1992). Seeds were germinated by soaking in aerated distilled H 2 0 at 22OC before planting in Jiffy Mix potting soil.…”

“…106, 1994 cellulose (Whatman Chemical Separation Ltd., Kent, UK). Monospecific polyclonal anti-AH, anti-PH, and anti-MDL antisera were produced and characterized in our laboratory (Wu and Poulton, 1991;Li et al, 1992).…”

Tissue and Subcellular Localization of Enzymes Catabolizing (R)-Amygdalin in Mature Prunus serotina Seeds

“…All Grids were examined using an Hitachi H-7000 electron microscope at 50 kV. The specificity of labeling was determined on the basis of the following control treatments: (a) sections were incubated with preimmune sera instead of affinity-purified antisera, (b) sections were incubated with secondary antibody without prior exposure to a primary antibody, and (c) sections were incubated with affinity-purified antisera previously exposed to nitrocellulose paper preloaded with either AH, PH, or MDL (1-2 mg homogeneous enzyme [12,27] per ml of respective antiserum). The third control was evaluated against each immune serum, which had been similarly incubated with nitrocellulose paper to which PBS rather than enzyme had been applied.…”

“…fruits were collected and processed, and their pits (endocarp enclosing seed) were stored as described previously (12).…”

“…characterized (12,27). The present study describes their use in immunolocalization of these enzymes at the tissue, cellular, and subcellular levels.…”