Differential expression of 3ß-HSD and mlncRNAs in response to abiotic stresses in Digitalis nervosa.

Digitalis nervosa is an important medicinal plant species belonging to the family of Scrophulariaceae that has the potential to be used for heart failure. 3ß-hydroxysteroid dehydrogenase (3ß-HSD) is a key gene in the biosynthesis of cardenolides for making digitalis effective compounds, hence identification of this gene is important for genetic engineering purposes towards increasing the yield of cardiac glycosides. In addition, mRNA-like non-coding RNAs (mlncRNAs), a class of long non coding RNAs, play key roles in various biological processes and may affect cardenolides pathway in digitalis plants.  In the present work, full sequence of 3ß-HSD was isolated from Digitalis nervosa. Gene expression patterns of 3ß-HSD along with three mlncRNAs including mlncRNA23, mlncRNA28 and mlncRNA30 were studied and the results indicated that they are differentially expressed in different tissues including roots, stems and leaves, with the highest expression levels in leaves.  Moreover, the transcript levels of these genes affected by the cold and drought stresses. The results obtained from the present study is important in order to understand the potential role of mlncRNAs in digitalis plants, especially in response to abiotic stresses.

PRISEs (progesterone 5ß-reductase and/or iridoid synthase-like 1,4-enone reductases): Catalytic and substrate promiscuity allows for realization of multiple pathways in plant metabolism.

PRISEs (progesterone 5ß-reductase and/or iridoid synthase-like 1,4-enone reductases) are involved in cardenolide and iridoid biosynthesis. We here investigated a PRISE (rAtSt5ßR) from Arabidopsis thaliana, a plant producing neither cardenolides nor iridoids. The structure of rAtSt5ßR was elucidated with X-ray crystallography and compared to the known structures of PRISEs from Catharanthus roseus (rCrISY) and Digitalis lanata (rDlP5ßR). The three enzymes show a high degree of sequence and structure conservation in the active site. Amino acids previously considered to allow discrimination between progesterone 5ß-reductase and iridoid synthase were interchanged among rAtSt5ßR, rCrISY and rDlP5ßR applying site-directed mutagenesis. Structural homologous substitutions had different effects, and changes in progesterone 5ß-reductase and iridoid synthase activity were not correlated in all cases. Our results help to explain fortuitous emergence of metabolic pathways and product accumulation. The fact that PRISEs are found ubiquitously in spermatophytes insinuates that PRISEs might have a more general function in plant metabolism such as, for example, the detoxification of reactive carbonyl species.

Effects of salicylic acid on thermotolerance and cardenolide accumulation under high temperature stress in Digitalis trojana Ivanina.

Long periods of high temperature or transitory increased temperature, a widespread agricultural problem, may lead to a drastic reduction in economic yield, affecting plant growth and development in many areas of the world. Heat stress causes many anatomical and physiological changes in plants. Its unfavorable effects can be alleviated by thermotolerance induced by exogenous application of plant growth regulators and osmoprotectants or by gradual application of temperature stress. Digitalis trojana Ivanina is an important medicinal plant species well known mainly for its cardenolides. The production of cardenolides via traditional agriculture is commercially inadequate. In this study, elicitation strategies were employed for improving crop thermotolerance and accumulation of cardenolides. For these purposes, the effects of salicylic acid (SA) and/or high temperature treatments in inducing cardenolide accumulation and thermotolerance were tested in callus cultures of D. trojana. Considerable increases in the production of cardenolides (up to 472.28 µg.g(-1) dry weight, dw) and induction of thermotolerance capacity were observed when callus cultures were exposed to high temperature for 2 h after pretreating with SA. High temperature treatments (2 h and 4 h) caused a marked reduction in superoxide dismutase (SOD; EC 1.15.1.1) and catalase (CAT; EC 1.11.1.6) activities, while SA pretreatment increased their activities. High temperature and/or SA appeared to increase the levels of proline, total phenolic, and flavonoid content. Elevated phenolic accumulation could be associated with increased stress protection. These results indicated that SA treatments induced synthesis of antioxidants and cardenolides, which may play a significant role in resistance to high temperature stress.

Purification of Δ(5)-3-ketosteroid isomerase from Digitalis lanata.

The isomerization of 5-pregnene-3,20-dione into 4-pregnene-3,20-dione was investigated to shed further light on cardenolide biosynthesis and to characterize the enzymes involved in cardenolide formation. It was shown that the Δ(5)-3-ketosteroid isomerase of Digitalis lanata, which catalyzes this isomerization, is an individual enzyme and not, as previously thought, associated with Δ(5)-3ß-hydroxysteroid dehydrogenase. The enzyme was purified by fractionated ammonium sulfate precipitation, hydrophobic interaction chromatography and gel filtration. The purification protocol resulted in a 68.1-fold enriched specific enzyme activity with a yield of 2.2%. After an additional chromatofocusing step the 3KSI activity appeared as a single protein band at 17kDa in SDS-PAGE. Plant 3KSI displayed similar properties to microbial 3-ketosteroid isomerases.

Truncation of N-terminal regions of Digitalis lanata progesterone 5ß-reductase alters catalytic efficiency and substrate preference.

N-Terminal truncated forms of progesterone 5ß-reductase (P5ßR) were synthesized taking a full-length cDNA encoding for Digitalis lanata P5ßR with a hexa-histidine tag attached at the C-terminus (rDlP5ßRc) as the starting point. Four pETite-c-His/DlP5ßR constructs coding for P5ßR derivatives truncated in the N-terminal region, termed rDlP5ßRcn-10, rDlP5ßRcn-20, rDlP5ßRcn-30, and rDlP5ßRcn-40 were obtained by site-directed mutagenesis. The cDNAs coding for full-length rDlP5ßRc, rDlP5ßRcn-10 and rDlP5ßRcn-20 were over-expressed in Escherichia coli and the respective enzymes were soluble and catalytically active (progesterone and 2-cyclohexen-1-one as substrates). GST-tagged recombinant DlP5ßR (rDlP5ßR-GST) and rDlP5ßR-GSTr, with the GST-tag removed by protease treatment were produced as well and served as controls. The Km values and substrate preferences considerably differed between the various DlP5ßR derivatives. As for the C-terminal His-tagged rDlP5ßR the catalytic efficiency for progesterone was highest for the full-length rDlP5ßRc whereas the N-terminal truncated forms preferred 2-cyclohexen-1-one as the substrate. Affinity tags and artifacts resulting from the cloning strategy used may alter substrate specificity. Therefore enzyme properties determined with recombinant proteins should not be used to infer in vivo scenarios and should be considered for each particular case.

Vein Patterning 1-encoded progesterone 5ß-reductase: activity-guided improvement of catalytic efficiency.

Progesterone 5ß-reductases (P5ßR; EC 1.3.99.6) encoded by Vein Patterning 1 (VEP1) genes are capable of reducing the CC double-bond of a variety of enones enantioselectively. Sequence and activity data of orthologous P5ßRs were used to define a set of residues possibly responsible for the large differences in enzyme activity seen between rAtSt5ßR and rDlP5ßR, recombinant forms of P5ßRs from Arabidopsis thaliana and Digitalis lanata, respectively. Tyrosine-156, asparagine-205 and serine-248 were identified as hot spots in the rDlP5ßR responsible for its low catalytic efficiency. These positions were individually substituted for amino acids found in the strong rAtSt5ßR in the corresponding sites. Kinetic constants were determined for rDlP5ßR and its mutants as well as for rAtSt5ßR using progesterone and 2-cyclohexen-1-one as substrates. Enzyme mutants in which asparagine-205 was substituted for methionine or alanine showed considerably lower km and higher K(cat)/k(m) values than the wild-type DlP5ßR, approaching the catalytic efficiency of strong P5ßRs. The introduced mutations not only lead to an improved capability to reduce progesterone but also to altered substrate preference. Our findings provided structural insights into the differences seen among the natural P5ßRs with regard to their substrate preferences and catalytic efficiencies.

Expression of 3beta-HSD and P5betaR, genes respectively coding for Delta5-3beta-hydroxysteroid dehydrogenase and progesterone 5beta-reductase, in leaves and cell cultures of Digitalis lanata EHRH.

Plants of the genus Digitalis produce 5 beta-cardenolides that are used in the therapy of cardiac insufficiency in humans. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and progesterone 5 beta-reductase (P5 betaR) are both supposed to be important enzymes in the biosynthesis of these natural products. Activity and gene expression were demonstrated for both enzymes in cardenolide-accumulating leaves of Digitalis lanata but also in cardenolide-free permanent cell suspension cultures initiated from D. lanata leaf tissue. Enzyme activities were determined and quantified by HPLC and GC-MS methods. Expression of the respective genes, namely AY585867.1 (P5betaR gene) and DQ466890.1 (3beta-HSD gene), was made evident by real-time polymerase chain reaction (qPCR) analysis. We demonstrate for the first time that the P5betaR gene, encoding an enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in cardenolide-free tissues of cardenolide-containing plants.

Digitalis purpurea P5 beta R2, encoding steroid 5 beta-reductase, is a novel defense-related gene involved in cardenolide biosynthesis.

The stereospecific 5 beta-reduction of progesterone is a required step for cardiac glycoside biosynthesis in foxglove plants. Recently, we have isolated the gene P5 beta R, and here we investigate the function and regulation of P5 beta R2, a new progesterone 5 beta-reductase gene from Digitalis purpurea. P5 beta R2 cDNA was isolated from a D. purpurea cDNA library and further characterized at the biochemical, structural and physiological levels. Like P5 beta R, P5 beta R2 catalyzes the 5 beta-reduction of the Delta(4) double bond of several steroids and is present in all plant organs. Under stress conditions or on treatment with chemical elicitors, P5 beta R expression does not vary, whereas P5 beta R2 is highly responsive. P5 beta R2 expression is regulated by ethylene and hydrogen peroxide. The correlation between P5 beta R2 expression and cardenolide formation demonstrates the key role of this gene in cardenolide biosynthesis, and therefore in the chemical defense of foxglove plants.

Purification and characterization of malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase (Dp21MaT) from leaves of Digitalis purpurea L.

With respect to the cardenolide pathway and the characterization of enzymes involved in the formation of cardenolides, a malonyltransferase, termed malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase (Dp21MaT) has been purified. The enzyme catalyses the transfer of the malonyl moiety from malonyl-coenzyme A to 21-hydroxypregnane substrates. Malonyltransferase activity was checked in several potential starting materials including fresh leaves and cell suspension cultures from different plants. Fresh Digitalis purpurea L. leaves turned out to be the best enzyme source. The purification protocol included ammonium sulphate precipitation, hydrophobic interaction chromatography on Phenylsepharose 6 FF, ion exchange chromatography on Source 30 Q, affinity chromatography on Cibacron Blue 3GA and gel filtration on Superdex 75. Gel filtration and native SDS-PAGE analysis showed that Dp21MaT exists as a monomer with a molecular mass of 27kDa. Its pI, as determined by isoelectric focusing, was 4.66. The enzyme showed maximal activity at pH 6.5 when incubated at 42 degrees C. The energy of activation was 29.28kJmol(-1), whereas that of inactivation was 48.57kJmol(-1). Dp21MaT was purified 252-fold with a yield of about 1%. Hanes plots of kinetic data indicated K(m) values of 99microM (V(max) 47.57microkatkg(-1)) and 28.44microM (V(max) 39.4microkatkg(-1) protein) for 3beta-benzoyloxy-5beta-pregnane-14beta,21-dihydroxy-20-one and malonyl-CoA, respectively.

Delta 5-3beta-hydroxysteroid dehydrogenase (3 beta HSD) from Digitalis lanata. Heterologous expression and characterisation of the recombinant enzyme.

During the biosynthesis of cardiac glycosides, Delta (5)-3beta-hydroxysteroid dehydrogenase (3 beta HSD, EC 1.1.1.51) converts pregnenolone (5-pregnen-3beta-ol-20-one) to isoprogesterone (5-pregnene-3,20-dione). A 3 beta HSD gene was isolated from leaves of Digitalis lanata. It consisted of 870 nucleotides containing a 90 nucleotide long intron. A full-length cDNA clone that encodes 3 beta HSD was isolated by RT-PCR from the same source. A SPH I /KPN I 3 beta HSD cDNA was cloned into the pQE30 vector and then transferred into E. COLI strain M15[pREP4]. 3 beta HSD cDNA was functionally expressed as a His-tagged fusion protein (pQ3 beta HSD) composed of 273 amino acids (calculated molecular mass 28,561 Da). pQ3 beta HSD was purified by metal chelate affinity chromatography on Ni-NTA. Pregnenolone and other 3beta-hydroxypregnanes but not cholesterol were 3beta-oxidised by pQ3 beta HSD when NAD was used as the co-substrate. Testosterone (4-androsten-17beta-ol-3-one) was converted to 4-androstene-3,17-dione indicating that the pQ3 beta HSD has also 17beta-dehydrogenase activity. pQ3 beta HSD was able to reduce 3-keto steroids to their corresponding 3beta-hydroxy derivatives when NADH was used as the co-substrate. For comparison, 3 beta HSD genes were isolated and sequenced from another 6 species of the genus DIGITALIS. Gene structure and the deduced 3 beta HSD proteins share a high degree of similarity.

Plant progesterone 5beta-reductase is not homologous to the animal enzyme. Molecular evolutionary characterization of P5betaR from Digitalis purpurea.

Plants of the genus Digitalis produce cardiac glycosides, i.e. digoxin, which are widely used for congestive heart failure. Progesterone 5beta-reductase (P5betaR) is a key enzyme in the biosynthesis of these natural products. Here, we have carried out the purification and partial amino acid sequencing of the native P5betaR from foxglove (Digitalis purpurea), and isolated a cDNA encoding this enzyme. Similarly to other steroid 5beta-reductases, the recombinant P5betaR catalyzes the stereospecific reduction of the Delta(4)-double bond of several steroids with a 3-oxo,Delta(4,5) structure. The gene encoding P5betaR is expressed in all plant organs, and maximally transcribed in leaves and mature flowers. P5betaR belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, bearing no structural homology to its mammalian counterpart, which is a member of the aldo-keto reductase (AKR) superfamily. A similar situation occurs with 3beta-hydroxy-Delta(5)-steroid dehydrogenase (3betaHSD), the gene immediately preceding P5betaR in the cardenolide pathway, which suggests that the entire route has evolved independently in animals and plants. P5betaR is retained only in plants, where it is ubiquitous, and a few distantly related bacterial lineages after its diversification from the last universal common ancestor. Evolutionary conserved changes in its putative active site suggest that plant P5betaR is a member of a novel subfamily of extended SDRs, or a new SDR family.

Expression of recombinant Digitalis lanata EHRH. cardenolide 16'-O-glucohydrolase in Cucumis sativus L. hairy roots.

The coding sequence for the Digitalis lanata EHRH. cardenolide 16'-O-glucohydrolase was inserted downstream of the 35S promoter in the binary vector pBI121 resulting in plant expression vector pBI121cgh. Cotyledon explants excised from 10-day-old seedlings of Cucumis sativus L. were transformed using Agrobacterium rhizogenes 15834 harbouring pBI121cgh. Hairy roots were obtained from infected cotyledon explants in vitro 10 days after inoculation. PCR amplification of coding sequences for cgh I, rolB and rolC from Ri plasmid showed that the aimed sequences were inserted into the genome of transformed cucumber hairy roots. Glycolytic activity of the transgenic CGH I was measured by HPLC using Lanatoside glycosides as substrate. Therefore, the cgh I transformed cucumber hairy roots may provide a valuable model for biotransformation of natural compounds by recombinant enzymes.

Crystallization and preliminary crystallographic analysis of selenomethionine-labelled progesterone 5beta-reductase from Digitalis lanata Ehrh.

Progesterone 5beta-reductase (5beta-POR) catalyzes the reduction of progesterone to 5beta-pregnane-3,20-dione and is the first stereospecific enzyme in the putative biosynthetic pathway of Digitalis cardenolides. Selenomethionine-derivatized 5beta-POR from D. lanata was successfully overproduced and crystallized. The crystals belong to space group P4(3)2(1)2, with unit-cell parameters a = 71.73, c = 186.64 A. A MAD data set collected at 2.7 A resolution allowed the identification of six out of eight possible Se-atom positions. A first inspection of the MAD-phased electron-density map shows that 5beta-POR is a Rossmann-type reductase and the quality of the map is such that it is anticipated that a complete atomic model of 5beta-POR will readily be built.

Molecular cloning and heterologous expression of progesterone 5beta-reductase from Digitalis lanata Ehrh.

A full-length cDNA clone that encodes progesterone 5beta-reductase (5beta-POR) was isolated from Digitalis lanata leaves. The reading frame of the 5beta-POR gene is 1170 nucleotides corresponding to 389 amino acids. For expression, a Sph1/Sal1 5beta-POR fragment was cloned into the pQE vector and was transformed into Escherichia coli strain M15[pREP4]. The recombinant gene was functionally expressed and the recombinant enzyme was characterized. The K(m) and v(max) values for the putative natural substrate progesterone were calculated to be 0.120 mM and 45 nkat mg(-1) protein, respectively. Only 5beta-pregnane-3,20-dione but not its alpha-isomer was formed when progesterone was used as the substrate. Kinetic constants for cortisol, cortexone, 4-androstene-3,17-dione and NADPH were also determined. The molecular organization of the 5beta-POR gene in D. lanata was determined by Southern blot analysis. The 5beta-POR is highly conserved within the genus Digitalis and the respective genes and proteins share considerable homology to putative progesterone reductases from other plant species.

Cloning and expression of two novel aldo-keto reductases from Digitalis purpurea leaves.

The aldo-keto reductase (AKR) superfamily comprises proteins that catalyse mainly the reduction of carbonyl groups or carbon-carbon double bonds of a wide variety of substrates including steroids. Such types of reactions have been proposed to occur in the biosynthetic pathway of the cardiac glycosides produced by Digitalis plants. Two cDNAs encoding leaf-specific AKR proteins (DpAR1 and DpAR2) were isolated from a D. purpurea cDNA library using the rat Delta4-3-ketosteroid 5beta-reductase clone. Both cDNAs encode 315 amino acid proteins showing 98.4% identity. DpAR proteins present high identities (68-80%) with four Arabidopsis clones and a 67% identity with the aldose/aldehyde reductase from Medicago sativa. A molecular phylogenetic tree suggests that these seven proteins belong to a new subfamily of the AKR superfamily. Southern analysis indicated that DpARs are encoded by a family of at most five genes. RNA-blot analyses demonstrated that the expression of DpAR genes is developmentally regulated and is restricted to leaves. The expression of DpAR genes has also been induced by wounding, elevated salt concentrations, drought stress and heat-shock treatment. The isolated cDNAs were expressed in Escherichia coli and the recombinant proteins purified. The expressed enzymes present reductase activity not only for various sugars but also for steroids, preferring NADH as a cofactor. These studies indicate the presence of plant AKR proteins with ketosteroid reductase activity. The function of the enzymes in cardenolide biosynthesis is discussed.

Functional replacement of the essential ESS1 in yeast by the plant parvulin DlPar13.

A functionally Pin1-like peptidyl-prolyl cis/trans isomerase (PPIase(1)) was isolated from proembryogenic masses (PEMs) of Digitalis lanata according to its enzymatic activity. Partial sequence analysis of the purified enzyme (DlPar13) revealed sequence homology to members of the parvulin family of PPIases. Similar to human Pin1 and yeast Ess1, it exhibits catalytic activity toward substrates containing (Thr(P)/Ser(P))-Pro peptide bonds and comparable inhibition kinetics with juglone. Unlike Pin1-type enzymes it lacks the phosphoserine or phosphothreonine binding WW domain. Western blotting with anti-DlPar13 serum recognized the endogenous form in nucleic and cytosolic fractions of the plant cells. Since the PIN1 homologue ESS1 is an essential gene, complementation experiments in yeast were performed. When overexpressed in Saccharomyces cerevisiae DlPar13 is almost as effective as hPin1 in rescuing the temperature-sensitive phenotype caused by a mutation in ESS1. In contrast, the human parvulin hPar14 is not able to rescue the lethal phenotype of this yeast strain at nonpermissive temperatures. These results suggest a function for DlPar13 rather similar to parvulins of the Pin1-type.

Cloning and functional expression in Escherichia coli of a cDNA encoding cardenolide 16'-O-glucohydrolase from Digitalis lanata Ehrh.

A clone of cardenolide 16'-O-glucohydrolase cDNA (CGH I) was obtained from Digitalis lanata which encodes a protein of 642 amino acids (calculated molecular mass 73.2 kDa). The amino acid sequence derived from CGH I showed high homology to a widely distributed family of beta-glucohydrolases (glycosyl hydrolases family 1). The recombinant CGH I protein produced in Escherichia coli had CGH I activity. CGH I mRNA was detected in leaves, flowers, stems and fruits of D. lanata.

Delta(5)-3beta-hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. - a multifunctional enzyme in steroid metabolism?

Delta(5)-3beta-Etaydroxysteroid dehydrogenase (Delta(5)-3beta-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3beta-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column chromatography on Fractogel-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Delta(5)-3beta-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Delta(5)-3beta-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed.

Stress-induced expression of cyclophilins in proembryonic masses of Digitalis lanata does not protect against freezing/thawing stress.

Using proembryonic masses (PEMs) of Digitalis lanata Erh., it was demonstrated that cold, hormonal or osmotic stress, which increased freezing tolerance during cryopreservation, induced an increasing level of two peptidyl-prolyl-cis/transisomerases (PPIases). The difference in pI (9.2 +/- 0.2 and 9.5 +/- 0.2, +/- SD; n = 3) allowed the separation of the two enzymes by free-flow isoelectrophoresis. Both were inhibited by cyclosporin A and thus belong to the cyclophilin family of PPIases. The enzymes differed slightly in their substrate specificity and their relative molecular masses of 18038 +/- 4 Da (D. lanataCyp18.0) and 18132 +/- 3 Da (D. lanataCyp18.1). Both cyclophilins were blocked N-terminally. Partial internal amino acid sequences from the two cyclophilins, with a length of 34 amino acids, displayed 82% sequence identity to each other. Pretreatment of PEMs with abscisic acid, sorbitol or a combination of both substances led to a 270 +/- 30% elevation of the total cytosolic cyclophilin concentration determined with a cyclophylin affinity sensor. During the first 4 d of pretreatment, the total PPIase activity was enhanced up to 230 +/- SD% compared with the control culture. The lag phase between maximal PPIase concentration after 4 d of pretreatment and maximal effect of freezing tolerance after 10 d of pretreatment indicated that increasing levels of cytosolic PPIases may be necessary to overcome the stress induced by hormones and osmotica during pretreatment but not to protect against freezing/thawing stress.

Cardenolide 16'-O-glucohydrolase from Digitalis lanata. Purification and characterization.

A three-step chromatographic procedure was developed for purification of cardenolide 16'-O-glucohydrolase (CGH) from Digitalis lanata Ehrh. leaves, including Phenyl-Sepharose hydrophobic interaction chromatography followed by SP-Sepharose cation exchange and Q-Sepharose anion-exchange chromatography. Starting with acetone dry powder the purification resulted in an 760-fold enrichment of CGH. Molecular weight, substrate specificity, pH optimum and temperature stability of CGH were determined. Antibodies against CGH were prepared in rabbits. The SDS gel electrophoresis of protein extracts from leaves of D. lanata and other D. species showed bands at 70 kDa and 36 kDa reacting with the antibodies. The 70-kDa protein is the main protein stained with CGH antibodies in freshly prepared extracts of D. lanata. It may represent undegraded CGH. The 36-kDa protein is enriched in aged CGH preparations. It is probably a degradation product. Proteins related to 70-kDa and 36-kDa bands also occur in crude protein preparations from leaves of D. heywoodii P. et M. Silva, D. mariana Boiss., D. purpurea L., and D. thapsi L. indicating that CGH is also present in these species. Purified CGH was digested with proteases V8 and Lys-C and the resulting fragments obtained were sequenced. One fragment had the typical amino-acid sequence of the catalytic center of family-1 glycosyl hydrolases (EC 3.2.1.x). Cardenolide 16'-O-glucohydrolase, like the other members of this enzyme family, appeared to have a glutamic acid residue directly involved in glycosidic bond cleavage as a nucleophile.