The most abundant ,B-amylase (EC 3.2.1.2) in pea (Pisum sativum L.) was purified greater than 880-fold from epicotyls of etiolated germinating seedlings by anion exchange and gel filtration chromatography, glycogen precipitation, and preparative electrophoresis. The electrophoretic mobility and relative abundance of this ,#-amylase are the same as that of an exoamylase previously reported to be primarily vacuolar. The enzyme was determined to be a ,#-amylase by end product analysis and by its inability to hydrolyze #-limit dextrin and to release dye from starch azure. Pea #B-amylase is an approximate 55 to 57 kilodalton monomer with a pi of 4.35, a pH optimum of 6.0 (soluble starch substrate), an Arrhenius energy of activation of 6.28 kilocalories per mole, and a Km of 1.67 milligrams per milliliter (soluble starch). The enzyme is strongly inhibited by heavy metals, p-chloromercuriphenylsulfonic acid and N-ethylmaleimide, but much less strongly by iodoacetamide and iodoacetic acid, indicating cysteinyl sulfhydryls are not directly involved in catalysis. Pea ,B-amylase is competitively inhibited by its end product, maltose, with a K, of 11.5 millimolar. The enzyme is partially inhibited by Schardinger maltodextrins, with a-cyclohexaamylose being a stronger inhibitor than jl-cycloheptaamylose. Moderately branched glucans (e.g. amylopectin) were better substrates for pea jB-amylase than less branched or non-branched (amyloses) or highly branched (glycogens) glucans. The enzyme failed to hydrolyze native starch grains from pea and glucans smaller than maltotetraose. The mechanism of pea j-amylase is the multichain type.Possible roles of pea ,-amylase in cellular glucan metabolism are discussed.ofthese enzymes are located in the same cellular compartment as is particulate starch. Several studies with pea indicate that most of the cell's 3-amylase activity is extrachloroplastic and that chloroplasts contain very low or no fl-amylase activity (12, 13, and refs. contained therein). In contrast, one study with pea (34) indicates relatively high f,-amylase activity in chloroplasts; however, even in this study most of the f3-amylase was found to be extrachloroplastic with 50 to 60% localized in vacuoles. As higher plant particulate starch is contained within plastids, the role of vacuolar f3-amylase in starch degradation is uncertain. Furthermore, the role of nonvacuolar ,B-amylase has not been established, and in some storage tissues f3-amylase appears to be inessential for starch degradation (29).To understand the possible roles f3-amylase may have in cellular glucan metabolism, it is necessary to elucidate the physical and kinetic properties of the enzyme. While the characteristics of,B-amylase from storage tissues such as barley (15) and wheat (30) grains, soybean seeds (17, 19), and sweet potato tubers (29) have been well documented, much less information is known about ,3-amylases from tissues containing transitory starch, such as leaves. We present here the purification and characterization of,3-amylase fr...
Mature black cherry (Prunus serotina Ehrh.) seeds accum ulate high levels of the cyanogenic disaccharide (R)-amygdalin. Extracts from these seeds contain two β-glycosidases which have been identified and completely resolved by DEAE-cellulose ion-exchange chromatography. Amygdalin hydrolase hydrolyzed (R)-am ygdalin at an optimum pH of 5.5, releasing (R)-prunasin and D-glucose. This enzyme showed highest activity towards (R)-am ygdalin and failed to hydrolyze (R)-prunasin. linamarin, β-gentiobiose and cellobiose. A distinct β-glycosidase, prunasin hydrolase, displayed a pronounced preference for (R)-prunasin, hydrolyzing this cyanogenic monosaccharide at an optimum pH of 6.5 to mandelonitrile and D-glucose. Prunasin hydrolase was inactive towards (R)-am ygdalin, linamarin, and β-gentiobiose. Both enzymes showed significant activity towards the artificial substrates β-ONPGlu and β-PNPGlu but did not hydrolyze α-PNPGlu. In view of the pronounced specificity of these enzymes towards endogenous cyanogens, it is concluded that upon disruption of black cherry seeds (R)- amygdalin is catabolized to mandelonitrile in a stepwise manner (the sequential mechanism) by amygdalin hydrolase and prunasin hydrolase with (R)-prunasin serving as intermediate. Young fronds of Davallia trichomanoides are rich sources of (R)-vicianin (the β-vicianoside of (R)-mandelonitrile). A β-glycosidase, vicianin hydrolase, has been partially purified from frond extracts by ion-exchange chromatography. At the optimum pH of 6.0, this enzyme showed highest hydrolytic activity with (R)-vicianin, although both (R)-am ygdalin and (R)-prunasin could be hydrolyzed at approximately 15% of the rate observed with (R)-vicianin. It failed to hydrolyze β-gentiobiose, cellobiose, linamarin and α-PNPGlu. Closer exam ination revealed that (R)-vicianin and (R)-amygdalin were hydrolyzed at the aglycone-disaccharide bond (the simultaneous mechanism) yielding mandelonitrile and the respective disaccharides vicianose and β-gentiobiose
The cyanogenic glycoside of young fronds and fiddleheads of the fern Davallia trichomanoides Blume was identified as (R)-vicianin (the β-vicianoside of (R)-mandelonitrile) by acid and enzymic hydrolysis, 1H-NMR and 13C-NMR spectroscopy, and by comparison with an authentic sample isolated from Vicia angustifolia seeds.
Vicianin hydrolase, which catalyzes the hydrolysis of vicianin (K.,,, 4.9 millimolar) to (R)-mandelonitrile and vicianose at an optimum pH of 5.5, was extensively purified from the young fronds and fiddleheads of the squirrel's foot fern (Davallia trichomanoides Blume) using DEAE-cellulose and Ultrogel HA chromatography. The native molecular weight of the enzyme was 340,000, and the isoelectric point was 4.6 to 4.7. SDS-PAGE analysis yielded three polypeptides with molecular weights of 56,000,49,000, and 32,500. The enzyme hydrolyzed only a narrow range ofglycosides and, among cyanogenic glycosides, exhibited a strict requirement for (R)-epimers and a preference for disaccharides over monosaccharides. (R)-Amygdalin, (R)-prunasin and p-nitrophenyl-ft-Dglucoside were hydrolyzed at 27, 14, and 3%, respectively, of the rate of vicianin hydrolysis. Mixed substrate studies showed that (R)-vicianin, (R)-prunasin, and p-nitrophenyl-,%-Dglucoside competed for the same active site.The enzyme was significantly inhibited by castanospermine, &glucono-lactone, and p-chloromercuriphenylsulfonate. Failure to recognize concanavalin A-Sepharose 4B and to stain with periodic acid-Schiff reagent indicated that the enzyme was not a glycoprotein.Cyanogenesis, the production ofHCN by biological organisms, has been recognized in over 2000 plant species distributed throughout 110 different families (4). While the biochemistry of cyanogenesis has been well studied in several economically important dicots (e.g. cassava, flax) and monocots (e.g. sorghum), relatively little is known about HCN production in ferns. Among fern species sampled, 5% were cyanogenic (6), but only rarely has the source of HCN been identified. The cyanogenic monosaccharide (R)-prunasin occurs in Cystopterisfragilis and Pteridium aquilinum (Polypodiaceae) (14). The role of HCN release from (R)-prunasin in determining the palatibility ofP. aquilinum to insect and mammalian herbivores has been investigated (5). Cyanogenic dissacharides may be hydrolyzed by two distinct pathways (15). As exemplified by the catabolism of (R)-amygdalin in black cherry (Prunus serotina) seeds (16,17) and of linustatin in flax (Linum usitatissimum) seeds (8), the two sugar ' Supported by National Science Foundation grant PCM-8314330.residues might be removed singly by stepwise hydrolysis with a cyanogenic monosaccharide acting as intermediate (the sequential mechanism). In each species, two distinct (3-glycosidases are required for complete hydrolysis. Alternatively, hydrolysis at the aglycone-disaccharide bond would yield the a-hydroxynitrile and a disaccharide (the simultaneous mechanism). We recently showed that cell-free extracts of D. trichomanoides displayed a f3-glycosidase activity which rapidly hydrolyzed (R)-vicianin and (R)-amygdalin to (R)-mandelonitrile by removal of the intact disaccharides vicianose and ,3-gentiobiose, respectively (15). This activity, which was also detected in crude preparations from V. angustifolia seeds (13) but not further purified, was referred...
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