Immunocytochemical Localization of Mandelonitrile Lyase in Mature Black Cherry (Prunus serotina Ehrh.) Seeds

Abstract

Mandelonitrile lyase (MDL, EC 4.1.2.10), which catalyzes the reversible dissociation of (R)-(+)-mandelonitrile to benzaldehyde and hydrogen cyanide, was purified to apparent homogeneity from mature black cherry (Prunus serotina Ehrh.) seeds by conventional protein purification techniques. This flavoprotein is monomeric with a subunit molecular mass of 57 kilodaltons. Glycoprotein character was shown by its binding to the affinity matrix concanavalin A-Sepharose 4B with subsequent elution by a-methyl-D-glucoside. Upon chemical deglycosylation by trifluoromethanesulfonic acid, the molecular mass was reduced to 50.9 kilodaltons. Two-dimensional gel analysis of deglycosylated MDL revealed the presence of several subunit isoforms of similar molecular mass but differing slightly in isoelectric point. Polyclonal antibodies were raised in New Zealand white rabbits against deglycosylated and untreated MDL. Antibody titers were determined by enzyme linked immunosorbent and dot immunobinding assays, while their specificities were assessed by Western immunoblot analysis. Antibodies raised against untreated Iyase recognized several proteins in addition to MDL. In contrast, antisera raised against deglycosylated MDL were monospecific and were utilized for developmental and immunocytochemical localization studies. SDS-PAGE and immunoblotting analysis of seed proteins during fruit maturation showed that MDL first appeared in seeds shortly after cotyledons began development. In cotyledon cells of mature seeds, MDL was localized primarily in the cell wall with lesser amounts in the protein bodies, whereas in endosperm cells, this labeling pattern was reversed. N-terminal sequence data was gathered for future molecular approaches to the question of MDL microheterogeneity.Cyanogenesis, the production of the respiratory poison HCN3 by biological organisms, was first described in plants almost two Enzyme Assay MDL activity was measured spectrophotometrically by monitoring the conversion of (R,S)-mandelonitrile to benzaldehyde and HCN by the increase in absorbance at 249.6 nm (3 1). Protein EstimationProtein content was estimated by the Bradford procedure (2) with BSA serving as standard. Deglycosylation of MDLChemical deglycosylation of MDL was performed using TFMS as described by Edge et al. (5). The deglycosylated protein was recovered by extraction into aqueous phase followed by overnight dialysis against 4 L of 2 mm pyridine acetate buffer, pH 5.5. The dialyzed preparation was concentrated by ultrafiltration (Amicon Minicon) to approximately 1 mL and stored at -20°C. Generation of Polyclonal Antibodies against MDLBefore immunization ofanimals, homogeneous MDL, with or without prior deglycosylation, was subjected to two-dimensional PAGE (IEF range pH 4-6.5, followed by 10% SDS-PAGE). Areas containing MDL were excised and electroeluted into 10 mm Tris-acetate buffer (pH 6.8 LOCALIZATION OF PRUNUS SEROTINA MANDELONITRILE LYASE alkaline phosphatase-conjugated goat anti-rabbit IgG (diluted 1:1000 with blocking solution) for ...

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

“…Previous attempts (27) (Fig. 2C) of endosperm tissue strips failed to detect any greater MDL levels than might easily be explained by contamination by adhering cotyledon (or testa) cells.…”

“…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.…”

“…Prior to immunoblotting analysis, homogenates were centrifuged for 5 min in a Beckman Microfuge, and supernatants were diluted to contain equal protein concentrations as determined by the Bradford (1) procedure Proteins were subjected to SDS-PAGE on 10% gels (10), electroblotted to nitrocellulose membranes using a Bio-Rad Trans-Blot apparatus (25), and challenged with monospecific polyclonal ant bodies (12,27). Anti-AH, anti-PH, and anti-MDL antisera were diluted 1:500, 1:500, and 1:750, respectively, before use.…”

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

Utilization of Amygdalin during Seedling Development of Prunus serotina

“…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).…”