Amygdalin hydrolysiswas determined by the production of HCN using the picric acid HCN assay system of Cooke et al. (9). Generation of Antibodies against ...
Plant Physiol. (1990) 93, 176-181 0032-0889/90/93/01 76/06/$01 .00/0
Received for publication November 17, 1989 and in revised form January 11, 1990
Purification, Characterization, and Localization of Linamarase in Cassava' Offiong E. Mkpong, Hua Yan, Grady Chism, and Richard T. Sayre* Departments of Botany and Biochemistry (O.E.M., H.Y., R.T.S.) and Department of Food Science and Nutrition, (G.C.), Ohio State University, Columbus, Ohio 43210 ABSTRACT
hydrolyzed in the body releasing cyanide (2, 26, 28). Chronic cyanide exposure associated with the consumption of cassava has been associated with a number of cyanide induced disorders including: goiter, dwarfism, and tropical ataxic neuropathy. This is particularly a problem in regions of the world where cassava is the major source of calories (2, 22, 26, 28). Our interest has been to develop strategies for the effective detoxification of cassava food products which would be compatible with the beneficial aspects of cyanogenesis, i.e. protection of the plant against herbivory. Recently, we have demonstrated that infiltration of cassava root tissues with excess linamarase lowers the linamarin content to levels considered safe for human consumption ( 19). This suggests that elevated levels of linamarase in cassava tissues may decrease the cyanide toxicity of the processed food products by driving cyanogenesis to completion. However, before attempts were made to modify the cyanogenic potential of cassava, a more complete understanding of the biochemistry of cyanogenesis in cassava roots and leaves was necessary. In this study we present the results of our investigations on the catalytic properties and stability of cassava linamarase, its localization in leaf cells and the relationship between linamarin content and linamarase activity in low and high cyanogenic (root) varieties of cassava. Our results suggest: (a) that linamarin is probably not hydrolyzed in intact tissues due to differential compartmentalization of the enzyme and substrate, (b) that linamarin is probably modified to a nonhydrolyzable form prior to transport from the symplast (the site of synthesis) in leaves to the roots (4, 11, 27), and (c) that varietal differences in the linamarin content of roots are probably not due to differences in linamarin catabolism in the leaves but due to differences in linamarin metabolism once it reaches the root.
We have purified cassava (Manihot esculenta) linamarase to apparent homogeneity using a simplified extraction procedure using low pH phosphate buffer. Three isozymes of cassava linamarase were identified in leaves based on differences in isoelectric point. The enzyme is capable of hydrolyzing a number of ,Bglycosides in addition to linamarin. The enzyme is unusually stable and has a temperature optimum of 550C. Immunogold labeling studies indicate that linamarase is localized in the cell walls of cassava leaf tissue. Since linamarin must cross the cell wall following synthesis in the leaf for transport to the root, it is likely that linamarin must cross the cell wall in a nonhydrolyzable form, possibly as the diglucoside, linustatin. In addition, we have quantified the levels of linamarin and linamarase activity in leaves of cassava varieties which differ in the linamarin content of their roots. We observed no substantial differences in the steady state linamarin content or linamarase activity of leaves from high or low (root) cyanogenic varieties. These results indicate that the steady state levels of linamarin and linamarase in leaves of high and low cyanogenic varieties are not correlated with the varietal differences in the steady state levels of linamarin in roots.
The generation and release of cyanide (cyanogenesis) occurs in a number of plant species following the rupture of the plant tissue and subsequent hydrolysis of cyanogenic glycoside precursors (2, 6, 7). One proposed function of cyanogenesis is to protect the plant against herbivory (1, 2, 18). However, the presence of cyanogenic glycosides in crop plants such as cassava can present health problems for peoples that subsist on these plants (2, 8, 12, 14, 17, 21). In cassava, the rate limiting step in cyanogenesis is the hydrolysis of linamarin (the predominant cyanogenic glycoside in cassava) to acetone cyanohydrin and glucose by the enzyme linamarase (f3-glucosidase, EC 3.2.1.21) (2, 8, 9). Acetone cyanohydrin dissociates spontaneously at pHs > 5.0 or enzymatically by hydroxynitrile lyase to produce HCN and acetone (6, 7, 9, 21). Generally, over 70% of the linamarin in fresh cassava is removed by enzymatic hydrolysis during processing (12, 13, 19, 20). Significantly, nearly all the cyanide which is generated is removed by volatilization or solubilization (8, 12, 14). The remaining nonhydrolyzed linamarin present in processed cassava does, however, present health problems. This is due to the fact that linamarin can be
MATERIALS AND METHODS Plant Material
Seeds and/or stem cuttings of cassava (Manihot esculenta) were obtained from Dr. Clair Hershey at the Center for
International Tropical Agriculture (CIAT), Cali, Colombia, and plants were grown under greenhouse conditions. Roots of unknown variety(s) of cassava, which were determined to have low linamarin content (see below) by gas chromatography, were obtained from local markets.
' Financial support from the Colleges of Agriculture and Biological Sciences, Ohio State University.
176
CYANOGENESIS IN CASSAVA
Isolation of Linamarase Linamarase was isolated from leaves homogenized in a low pH Na-phosphate buffer (0.1 M, pH 3.5). The homogenate was filtered (Miracloth) and centrifuged (24,000g, 30 min) to remove cell debris and brought to 40% saturation with solid ammonium sulfate to precipitate proteins. The precipitated proteins were pelleted and dialyzed extensively against 0.05 M Na-phosphate buffer (pH 4.5). The protein solution was then treated twice with 0.3 g activated charcoal/10 mL solution, filtered, reprecipitated with 40% ammonium sulfate and dialyzed against 0.10 M Na-phosphate buffer (pH 8.0) prior to chromatography on a Sephadex G-200 column (2.5 x 50 cm). Fractions having linamarase activity eluted near the void volume. For some preparations a second passage over the column was necessary to purify the enzyme. All steps were carried out at 4°C. Determination of Linamarase Activity
177
Immunohistochemical Localization of Linamarase in Leaves Leaf tissue was cut into 1 x 2 mm pieces and fixed in 2% (v/v) glutaraldehyde and 1% (w/v) paraformaldehyde in 50 mm K-phosphate buffer (pH 6.8) for 1 to 2 h at room temperature. Leaf pieces were then rinsed in buffer and dehydrated in a graded methanol series at 4°C. The samples were infiltrated and embedded with LR Gold resin (EMS, Ft. Washington, PA) and polymerized using indirect UV light for 24 to 28 h at -15°C. Ultrathin sections were cut with a diamond knife on a Reichert ultramicrotome and placed on formvar coated 150-mesh nickel grids. The grids were floated, section side down, on a drop of blocking buffer consisting of 30 mm Tris-HCl buffer (pH 7.2); 1% bovine serum albumin, 150 mm NaCl, 0.1% Tween 20 and 0.1% gelatin to prevent nonspecific binding of the antibodies. The sections were then incubated for 1 h in linamarase antibody diluted 1:200 in the aforementioned blocking buffer. The sections were then rinsed in blocking buffer and incubated for 1 h with goat antirabbit IgG conjugated to 15 nm colloidal gold (Jansen Life Sciences, Olen, Belgium) diluted 1:50 with blocking buffer. Sections were rinsed in blocking buffer followed by water and finally post-stained with 2% uranyl acetate and aqueous lead citrate for 15 min each. Controls were done in parallel using preimmune rabbit IgG. Electron microscopy was carried out on a Zeiss-EM 10 electron microscope at 60 Kv.
Linamarase activity was routinely measured using the chromogenic substrate p-nitrophenol-3-glucopyranoside (20 mM) in 0.05 M Na-phosphate assay buffer at pH 5.5 for 1 h at 25°C. The reaction was stopped by the addition of an equal volume of 0.2 M sodium borate (pH 9.8) and p-nitrophenol was quantified spectrophotometrically at 400 nm (13). The following substrates: linamarin, cellobiose, and prunasin were assayed in a coupled reaction using glucose oxidase, horse radish peroxidase and the chromogenic substrate, o-diansidine to determine glucose production catalyzed by linamarase (13). Briefly, an aliquot of enzyme is incubated with the substrate (10-20 mM) in 50 mm Na-phosphate buffer (pH 7.5) for 1 h followed by boiling for 2 min to inactivate linamarase. Glucose production is then determined via the glucose oxidase/ horseradish peroxidase coupled assay system according to the method of Hosel and Barz (13). The linamarase activity of various cassava varieties was determined from crude extracts of tissues homogenized in low pH phosphate buffer as described above. Dilutions of the crude extracts were used which gave linear rates of enzyme activity with linamarin. Linamarase activity was also detected in isoelectric focusing gels using the fluorescent substrate 4-methylumbelliferyl-f-glucopyranoside (0.2 mM) according to the procedure of Selmar et al. (24). Amygdalin hydrolysis was determined by the production of HCN using the picric acid HCN assay system of Cooke et al. (9).
For the quantification of linamarin, cassava leaf, or root material was frozen and ground in liquid nitrogen to a fine powder using a mortar and pestle followed by lyophilization. To S mg of the freeze dried material, 10 ,uL of phenyl-fl-Dglucopyranoside (internal standard) was added, followed by addition of 165 ,uL of acetonitrile and 25 ,uL of BSTFA.2 The samples were then heated in a capped serum vial at 90°C for 30 min to derivatize linamarin. One ,L of sample was chromatographed on 2 % OV- 17 on 100 to 120 mesh Chromsorb AWHP in a 6' x 1/8" column using a nitrogen flow rate of 62 mLs/min and detected by a flame ionization detector. The temperature program was 140 to 250 °C at 8°C/min with a 3 min delay. Injector and detector temperatures were 280°C. Peak areas were determined using an HP3392A integrator.
Generation of Antibodies against Linamarase
Additional Analytical Procedures
Purified linamarase (Sephadex G-200 fraction) was subjected to SDS-PAGE electrophoresis and electroeluted from the gel prior to immunization. An aliquot (150 tg) of gel purified linamarase was dissolved in 0.5 mL of 0.15 M Naphosphate buffer (pH 8.0) and mixed with an equal volume of Freund's complete adjuvant for subcutaneous injection in rabbits (23). Serum was collected after 3 weeks and used directly without further purification. Antibody specificity was confirmed by Western blots of total leaf extracts (23).
Analytical SDS-PAGE and Western blots were carried out according to previously published procedures (23). Isoelectric focusing was carried out using a Phast gel system (Pharmacia) on pH 3-5 isoelectric focussing gels according to the manufacturers directions. Protein was quantified using the method of Bradford (3).
Linamarin Determination
2
Abbreviation:
acetamide.
BSFTA;
N,O-bis-(trimethylsilyl)-trifluoro-
178
MKPONG ET AL.
RESULTS Purification and Catalytic Properties of Cassava Leaf Linamarase We have developed a simple purification procedure for the isolation of cassava linamarase based on extraction of leaf tissues using a low pH buffer (Table I). The specific activity of linamarase increased 100-fold during purification with a final yield of 4.0% of the total linamarase activity. The low yield can be attributed to problems with effectively removing all contaminating proteins. Analysis of the polypeptide profiles of various fractions by SDS-PAGE indicated that extraction with low pH (3.5) Na-phosphate buffer essentially solubilized only two proteins. The final fraction in the purification consisted of a single polypeptide which had a molecular mass of 65 kD (Fig. 1). The experimentally determined molecular mass was identical to that reported by Eksittikul and Chulavatnatol (10) for purified cassava linamarase. In addition, we found three different linamarase isozymes on isoelectric foTable I. Cassava Linamarse Purification Total
Fraction
Activity m/h
1.44 Crude 40% Ammonium sulfate 0.18 Activated charcoal 0.09 Sephadex G-200 0.058
5.S
Total Specific Protein Activity mg mmol/mg protein/h 2.3 x 104 0.06
214 43.9 9.74
0.84
Plant Physiol. Vol. 93,1990
Table II. Kinetic Properties of Linamarases from Various Species Property Cassava Butter Bean Rubber Tree Vmax (linamarin)a (mmol/mg 29.4 11.5 14.6 protein/h) Km (mM)a 1.9 7.6 5.6 55 Temperature optimum (0C) 62 pH Optimumb 7.0 5.6 5.9 b a Measured at 55, 30, and 370C, respectively. PNBPG as a substrate. References 15 and 24.
Table Ill. Linamarase Substrate Specificity PNBPG = p-nitrophenol-l-glucopyranoside, PNAPG = p-nitrophenol-A-glucopyranoside, ONPGal = o-nitrophenol-fl-galactoside. Enzymes assays were carried out at 300C. with 10 mm substrate in 50 mm Na-phosphate buffer (pH 6.5). Linamarase Substrate Rate Acivity Activity
Linamarin PNBPG PNAPG ONPGal Prunasin
Amygdalin Cellobiose
mmol/mg protein/h 16.5 10.8 0 3.1 10.7 0 0.19
%
100 65 0 19 65 0 1.1
2.1
6.0
KL ,
Figure 1. SDS-PAGE of purified cassava linamarase and Western blot of crude leaf extracts screened with linamarase antibodies. Five fractions in the purification of linamarase are shown in lanes 1 to 5. (1) 20 jug of purified cassava linamarase from the Sephadex G-200 fraction; (2) 5 Ag of trypsin digested linamarase; (3) 50 jig of linamarase fraction treated with activated charcoal; (4)30 IAg of crude extract of linamarase precipitated with 40% ammonium sulfate; and (5) 50 Ag of crude extract of cassava leaves. Proteins were stained with Coomassie blue. Lane A is a Western blot of crude cassava leaf extract (25 jig) detected with antibodies generated against linamarase (1 :100 dilution).
cusing gels of the Sephadex G-200 fraction. The isozymes have pIs of4.4, 3.4, and 3.0 (data not shown) (10). The kinetic properties of the leaf enzyme(s) (Sephadex G-200 fraction) are listed in Table II. The Km and Vmax for linamarin were 1.9 mm and 29.4 mM/mg protein/h, respectively (measured at 55°C, the optimum temperature). These values were significantly higher than those measured previously for the 4.3 isoelectric point cassava isozyme (10) (Km = 0.57 mm and Vmax = 3.5 mM/mg protein/h measured at 37°C). They are more similar to values obtained for butter bean and rubber tree linamarase (15, 24). The differences in Vmax and Km values between our work and that of Eksittikul and Chulavatnatol (10) may be attributed either to differences in the kinetic properties of the isozymes or assay temperature (see below). As shown in Table III, cassava linamarase hydrolyzes a number of other glucosides in addition to linamarin including PNBPG and prunasin but does not efficiently hydrolyze a-glucopyranosides or diglucosides such as amygdalin or cellobiose (9, 10, 29). Interestingly, the galactoside, o-nitrophenyl-,B-galactopyranoside was hydrolyzed by cassava linamarase but at much lower rate than linamarin. Based on its ability to hydrolyze prunasin, cassava linamarase is more similar to that of butter bean linamarase than the more closely related rubber tree ( 15, 24). Last of all, several lines of evidence indicate that cassava linamarase has unusually high stability. Similar to butter bean linamarase, the enzyme has an unusually high temperature optimum of 55°C (Table I) (15). Second, we have determined that active enzyme can be isolated from desiccated (exfoliated) leaves. Finally, we found that linamarase (activity) is quite
CYANOGENESIS IN CASSAVA
179 lo W"
P
iI 'i .1
1.
.?4M
Figure 2. Localization of linamarase in the cell walls of cassava leaves. The thin section of cassava leaf was treated with antibodies against linamarase (1:100). Bound linamarase antibodies were localized with immunogold IgG. Magnification is x45,000.
insensitive to trypsin digestion (1.0 Ag trypsin/1.0 ,g linamarase for 1 h, 37°C, Fig. 1). Immunohistochemical Localization of Linamarase
Western blots of crude cassava leaf extracts are shown in Figure 1. Linamarase was the predominant protein detected using polyclonal antibodies generated against gel (SDSPAGE) purified linamarase from the final Sephadex G-200 fraction. The identity of a second lesser reactive protein (50 kD) is not known. Immunofluorescent labeling studies indicated that linamarase was localized either in the inner cell wall or the plasmalemma of leaf tissue. In order to determine the localization of linamarase at greater resolution linamarase was localized in thin sections by immunogold labeling. As shown in Figure 2, linamarase is localized in the cell walls of cassava leaf tissue. In addition, the golgi apparatus was also labeled by immunogold particles (Fig. 3). Since cell wall proteins are typically processed and packaged in the golgi apparatus prior to delivery to the cell wall labeling of the golgi apparatus is consistent with the localization of linamarase in the cell wall (5).
Figure 3. Localization of linamarase in the golgi apparatus of cassava leaves. The thin section was treated with antibodies against cassava linamarase (1:200). Bound linamarase antibodies were localized with immunogold IgG. Magnification is x64,000. Arrows indicate the position of immunogold particles in the Golgi apparatus.
Relationship between Leaf Linamarin and Linamarase Activities in Low Cyanide and High Cyanide Cassava Varieties Cassava varieties can be classified into two groups based on the linamarin content of their roots (2, 17). These groups are the so-called low (-50 mg/Kg fresh weight) and high (2 100 mg/Kg fresh weight) cyanide (linamarin) varieties (low cyanide varieties have been derived from high cyanide varieties by selective breeding.). Previously it has been demonstrated that linamarin is synthesized in the leaf and petiole prior to transport to the root (2). To determine whether the steady state pool sizes of linamarin in roots was regulated by either the steady state level of linamarin or linamarase activity in leaves, we measured the linamarin content and linamarase activity of leaves and roots from low and high cyanide (cyanogenic) varieties of cassava. Significantly, the average steady state linamarin content of leaves from low and high cyanide varieties was nearly identical. Furthermore, the average linamarase activities in leaves of low and high cyanide varieties was also quite similar or only slightly higher in high cyanide varieties (Table IV). However, most importantly, there was no relationship between linamarin content and linamarase activity in leaves of low cyanide varieties. Compared with roots, both the linamarin content and linamarase activity of leaves were approximately 18-fold higher (low cyanide variety). These results suggest that varietal differences in root
MKPONG ET AL.
180
Table IV. Linamarin Content and Linamarase Activitya in Low and High Cyanide Varieties of Cassava Variety and Source
Leaves, High Cyanide CM 849 MCOL 1648 SM 301-3 MTAI-1 MVEN-25 Average Leaves, Low
Cyanide CM 3306-9 CM 52.37 MCOL 1522 MCOL 1468 MCOL 2215 Average Roots, Low
Linamarin Content .smol/g dry wt
Linamarase
Activity mmol/g dry wt/h
49.0 46.9 46.9 72.1 67.6 56.5
1.46 1.53 1.43 1.27 2.06 1.55
58.7 37.1 48.9 56.0 72.1 54.6
1.22 1.41 0.19 1.33 1.00 1.03
Cyanide 2.96 (5.4%) Unknown 0.06 (5.8%) aValues are the average of 2 to 3 measurements. Values in parentheses are the amounts present in roots expressed as a percentage of amounts present in low cyanide leaves.
linamarin content cannot be attributed to the differences in the catabolism of linamarin in leaves.
DISCUSSION The pattern of substrate specificity observed for cassava linamarase is similar to linamarases isolated from a number of species but is most like that of butter bean based on its ability to hydrolyze prunasin (15). Interestingly, the rubber tree linamarase, which is evolutionarily more closely related, does not hydrolyze prunasin (24). However, rubber tree linamarase may be unusual in other aspects. It appears to be the only ,B-glucosidic enzyme present in that species and may catalyze a number of reactions in addition to cyanogenesis (24). At present we do not known whether cassava linamarase is likewise the sole f,-glucosidase present in the plant or whether the enzyme has multiple functions. Based on this study as well as others it appears that there are a number of different linamarase isozymes present in cassava (15, 24). There are at least three difference isozymes of 65 kD enzyme in cassava leaves which can be distinguished on the basis of their isoelectric points (10). Eksittikul and Chulavatnatol (10) have determined the amino acid composition for linamarase isozymes isolated from several tissues including: leaves, petioles, and roots but found little variation in amino acid sequence between the different forms. Our preliminary analyses of the N-terminal amino acid sequence of the leaf isozymes indicate that there is a two amino acid displacement in the position of the N-terminal amino acid between two of the isozymes, suggesting that some of the differences between the isozymes may be due to posttransla-
Plant Physiol. Vol. 93,1990
tional processing (data not shown). Further evidence for the heterogeneity of cassava linamarases comes from comparisons of the catalytic properties of leaf and root linamarase. Yeoh (29) demonstrated that cassava root linamarase (partially purified enzyme) has a Vmax (linamarin) which is 25-fold lower than that of the leaf enzyme. The significance of these differences in terms of linamarin catabolism in roots and leaves remains to be determined but it is apparent that there are several distinctly different linamarases which are localized in an organ specific manner. The localization of linamarase in the cell walls of cassava is consistent with the observation that cyanogenesis is induced only during cell rupture assuming that linamarin is localized in the symplast (2, 1 1). Linamarase has also been shown to be localized in the cell walls of white clover and lima bean (1 1, 16). However, the enzyme dhurrinase, which hydrolyzes the cyanogenic glycoside, dhurrin, has been shown to be localized in the chloroplasts of sorghum (27). Unfortunately, less is known about the cellular localization of linamarin. However, investigations by Fehrner and Conn (11) have shown that linamarin is localized in protoplasts (symplastic) of Costa Rican wild lima beans and is not apoplastic. Our results indicating that linamarin steady state pool sizes are not correlated with the level of linamarase activity in leaves versus roots supports the argument that linamarin is symplastic and linamarase is apoplastic. For linamarin to be transported from leaves and petioles to target tissues (roots) it must avoid hydrolysis by linamarase either by conversion to a nonhydrolyzable form or via transport by a nonapoplastic route. The latter possibility is unlikely since linamarin has been shown to move through the vascular system of the petiole to the root (2). Alternatively, there is evidence indicating that linamarin may be transported in a nonhydrolyzable form in some plants (1 1, 25). Selmar et al. (25) have proposed that in rubber tree linamarin is glycosylated to linustatin prior to crossing the apoplast for transport to expanding leaves in emerging rubber tree seedlings. Since rubber tree linamarase is unable to hydrolyze cyanogenic diglucosides, i.e. linustatin (as well as the diglycoside, amygdalin) it can cross the apoplast for transport to target tissues. Upon reaching the target tissue, linustatin is hydrolyzed to gentiobiose and acetone cyanohydrin which in turn dissociates to cyanide. The cyanide is then fixed by ,B-cyanoalaninesynthase and the nitrogen incorporated into amino acids (25). By this mechanism linamarin/linustatin transport nitrogen from the seed to the expanding leaves (25). As shown in Table III, cassava linamarase is also unable to hydrolyze the cyanogenic diglucoside, amygdalin as well as the diglucoside, cellobiose (1 1, 24, 29). Since the linamarase activity of cassava leaves is more than sufficient to effectively hydrolyze any linamarin which is to be transported to roots we suggest that the linamarin must also be converted to a nonhydrolyzable form prior to transport to the roots in cassava. Furthermore, varietal differences in the cyanogenic glycoside content of cassava roots must be attributed to differences in catabolism of cyanogenic glycosides in roots since leaf steady state levels are similar in low and high cyanide varieties. Further analyses on the metabolism of linamarin in roots are necessary before a clearer understanding of the process of
CYANOGENESIS IN CASSAVA
linamarin metabolism and cyanogenesis in roots can emerge. However, it appears that in cassava leaves cyanogenesis is regulated by the hydrolytic enzyme linamarase which is compartmentalized in the cell wall separate from its substrate. Therefore, we propose that manipulations which would elevate linamarase levels in leaf cell walls should not reduce the cyanogenic potential of cassava but perhaps enhance it. If the induction of cyanogenesis in roots is analogous to that in leaves, then similarly elevated levels of linamarase in roots should not diminish cyanogenesis but more importantly, should facilitate detoxification of cassava food products during processing as a result of more complete hydrolysis of linamarin (19).
13. 14. 15.
16. 17. 18. 19.
LITERATURE CITED 1. Arias B, Bellotti A (1984) Aspectos ecologicos y de manejo de Cytomenus bergi, Chinche de la viruela en el cultivo de la yuca Manihot esculenta. CIAT publication, Cali, Colombia 2. Balagopalan C, Padmaja G, Nanda S, Morthy S (1988) Cassava in Food, Feed and Industry. CRC Press, Boca Raton, FL 3. Bradford M (1976) A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-drug binding. Anal Biochem 72: 248-254 4. Butler G, Conn E (1964) Biosynthesis of cyanogenic glycosides linamarin and lotaustralin. J Biol Chem 239: 1674-1679. 5. Cassab G, Varner J (1988) Cell wall proteins. Annu Rev Plant
Physiol Plant Mol Biol 39: 321-353 6. Conn E (1969) Cyanogenic glycosides. J Agric Food Chem 17: 519-526 7. Conn E (1981) Cyanogenic glycosides. Biochem Plants 7: 479-500 8. Cooke R (1978) An enzymatic assay for the total cyanide content of cassava. J Sci Food Agric 29: 345-352 9. Cooke R, Blake G, Battershill J (1978) Purification of cassava linamarase. Phytochemistry 17: 381-383 10. Eksittikul T, Chulavatnatol M (1988) Characterization of cyanogenic B-glucosidase (linamarase) from cassava (Manihot esculenta Crantz). Archiv Biochem Biophys 266: 263-269 1 1. Frehner M, Conn E (1987) The linamarin B-glucosidase in Costa Rican wild lima beans (Phaseolus lunatus L.) is apoplastic. Plant Physiol 84: 1296-1300 12. Gomez G, Valdivieso M (1985) Cassava foliage: chemical com-
20. 21. 22. 23.
24. 25. 26. 27.
28. 29.
181
position, cyanide content and effect of drying on cyanide elimination. J Sci Food Agric 36: 433-441 Hosel W, Barz W (1975) B-glucosidases from Cicer arientum L. Eur J Biochem 57: 607-616 Ikediobi C, Onyia G, Eluwah C (1980) A rapid inexpensive enzymatic assay for total cyanide in cassava and cassava products. Agric Biol Chem 44: 2803-2809 Itoh-Nashida T, Hiraiwa M, Uda Y (1987) Purification and properties of B-D-glucosidase (linamarase) from the butter bean Phaseolus lunatus. J Biochem 101: 847-854 Kakes P (1985) Linamarase and other B-glucosidases are present in the cell walls of Trifolium repens L. leaves. Planta 166: 156160 Laing D, Cock J, Roca W (1989) Report on the founding workshop for the advanced cassava research network. CIAT document No. 52, Cali, Colombia. Miller J, Conn E (1980) Metabolism of hydrogen cyanide by higher plants. Plant Physiol 65: 1199-1202 Mkpong 0, Chism G, Sayre RT (1989) Isolation of cassava linamarase: evidence that endogenous levels are insufficient for effective hydrolysis of linamarin. Report on the founding workshop for the cassava biotechnology research network, page 25, CIAT document No. 52. Cali, Colombia. Nambisan B, Sundaresan S (1985) Effect of processing on the cyanoglucoside content of cassava. J Sci Food Agric 36: 11971203 Nartley F (1968) Studies on cassava, Cyanogenesis: the biosynthesis of linamarin and lotaustralin in etiolated seedlings. Phytochemistry 7: 1307-1312 Oke 0 (1980) Toxicity of cyanogenic glycosides. Food Chem 6: 97-109 Sayre RT, Andersson B, Bogorad L (1986) The topology of a membrane protein: the orientation of the 32 Kd Qb-binding chloroplast thylakoid membrane protein. Cell 47: 601-608 Selmar D, Lieberei R, Biehl B, Voight J (1987) Hevea linamarase-a nonspecific f,-glucosidase. Plant Physiol 83: 557-563 Selmar D, Liebrei R, Biehl B (1988) Mobilization and utilization of cyanogenic glycosides: The linustatin pathway. Plant Physiol 86:711-716 Tewe 0 (1984) Cyanogenic glycoside, protein interaction in cassava peel based rations. Nutr Rep Int 30: 425-431 Thayer S, Conn E (1981) Subcellular localization of dhurrin ,Bglucosidase and hydroxynitrile lyase in the mesophyll cells of Sorghum leaf blades. Plant Physiol 67: 617-622 Umoh I, Ogunkoya F, Oke 0 (1985) Effect of thiamin status on the metabolism of linamarin in rats. Ann Nutr Metab 29: 312324 Yeoh H-H (1989) Kinetic properties of B-glucosidae from cassava. Phytochemistry 28: 721-724
