Purification and kinetic properties of a castor bean seed acid phosphatase containing sulfhydryl groups

An acid phosphatase (EC 3.1.3.2) has been identified and purified from castor bean ( Ricinus communis L., IAC-80 ) seed through sulphopropyl (SP)-Sephadex, diethylaminoethyl (DEAE)-Sephadex, Sephacryl S-200, and Concanavalin A-Sepharose chromatography. The enzyme was purified 2 000-fold to homogeneity, with a final specific activity of 3.8 μkat mg⁻¹ protein. The purified enzyme revealed a single diffuse band with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis, at pH 8.3. The relative molecular mass, determined by high-performance liquid chromatography (HPLC), was found to be 60 kDa. The acid phosphatase had a pH optimum of 5.5 and an akpparent K_m value for p -nitrophenylphosphate of 0.52 m M . The enzyme-catalyzed reaction was inhibited by inorganic phosphate, fluoride, vanadate, molybdate, p -chloromercuribenzoate ( p CMB), Cu²⁺ and Zn²⁺. The strong inhibition by p CMB, Cu²⁺ and vanadate suggests the presence of sulfhydryl groups essential for catalysis. The castor bean enzyme also recognized tyrosine-phosphate and inorganic pyrophosphate (KPP_i) as substrate. The highest specificity constant (V_max/K_m) was observed with KPP_i, making it a potential physiological substrate.     

Proteinases and enzyme stability in crude extracts of castor bean endosperm

The stability of catalase, fumarase, and isocitrate lyase from deliberately broken organelles in crude extracts from endosperm tissue of castor bean seedlings has been examined. These enzymes are relatively stable at 2 C in extracts from endosperm of 2-day seedlings, but rapid losses of activity occur in extracts from older seedlings. These losses are shown to be brought about by the thiol-proteinase present in the extracts. The inclusion of 35% glycerol prevented the loss of catalase, fumarase, and isocitrate lysase activity, and various inhibitors of proteinases afforded limited protection. The most striking protectant was leupeptin, an inhibitor of serine and thiol-proteinases. Leupeptin completely inhibited the loss of activity of the three enzymes in crude extracts and improved yields when included in the grinding medium.     

The development of domestic castor bean production

Shatter resistance, earliness, high yield and plant form best adapted to mechanical harvesting have been the principal and already partly achieved objectives in breeding castor beans, which, along with improvements in harvesting and hulling machinery, increase the prospects for greater domestic production of this crop.     

Analysis of castor bean ribosome-inactivating proteins and their gene expression during seed development

Ribosome-inactivating proteins (RIPs) are enzymes that inhibit protein synthesis after depurination of a specific adenine in rRNA. The RIP family members are classified as type I RIPs that contain an RNA-N-glycosidase domain and type II RIPs that contain a lectin domain (B chain) in addition to the glycosidase domain (A chain). In this work, we identified 30 new plant RIPs and characterized 18 Ricinus communis RIPs. Phylogenetic and functional divergence analyses indicated that the emergence of type I and II RIPs probably occurred before the monocot/eudicot split. We also report the expression profiles of 18 castor bean genes, including those for ricin and agglutinin, in five seed stages as assessed by quantitative PCR. Ricin and agglutinin were the most expressed RIPs in developing seeds although eight other RIPs were also expressed. All of the RIP genes were most highly expressed in the stages in which the endosperm was fully expanded. Although the reason for the large expansion of RIP genes in castor beans remains to be established, the differential expression patterns of the type I and type II members reinforce the existence of biological functions other than defense against predators and herbivory.     

Dissociation-recombination of sunflower seed acid phosphatase

Formal genetic studies of sunflower (Helianthus annuus) seed acid phosphatase (ACP, E.C. 3.1.3.2) had suggested that the functional enzyme consists of two polypeptide subunits. The dimeric quaternary structure was demonstrated by dissociation-recombination procedures. Dissociation of electrophoretically distinct homodimers was effected upon freezing of extracts in a pH 8-9 buffer containing 1 M NaCl and 0.1 M 2-mercaptoethanol. Reassociation, as indicated by the formation of the hybrid isozyme, occurred during 12 hr dialysis against a pH 7.0 buffer.     

Endogenous abscisic acid signaling towards storage reserve filling in developing seed tissues of castor bean (Ricinus communis L.)

Abscisic acid (ABA; free form) is a naturally occurring physiological growth hormone of higher plants. A detailed study involving the time course growth of developing seed tissues associated with endogenous levels of free ABA were investigated using a novel enzyme-linked immunosorbent assay. Seed filling in castor (Ricinuc communis L.) endosperm, embryo, and pod is marked with a rapid increase in fresh weight during the mid-developmental stages [21–42 days after pollination (DAP)], followed by a steady decline at the maturation stages (42–63 DAP) accompanied with a rapid lipid synthesis (in endosperm and embryo) during the same period, except for in pod. Endogenous ABA levels in endosperm (0.001–0.32 μg/g) and embryo (0.003–0.13 μg/g) followed a concurrent pattern with seed reserve filling, showing a rapid increase during the mid-developmental stages 21–42 DAP, whereas ABA levels in seed pod (0.2–22.9 μg/g) showed a different accumulation pattern with rapid increase and decline during the early-mid developmental stages, preceded by the maximal increase during the maturation stage (63 DAP). Together, our results provide evidence for the association of endogenous ABA in seed filling as well as in reserve deposition and provides clue for the effective usage of exogenous ABA concentrations in developing seeds with a focus, on improving seed reserve complex in castor.     

The action of exogenous gibberellic acid on protein and mRNA in germinating castor bean seeds

Gibberellic acid (GA₃) stimulates water uptake in castor beans and increases the activity of certain enzymes associated with lipid mobilisation.The effect of the GA₃ on the enzymes is possibly due to a general effect of the growth substance on protein synthesis. Gibberellic acid advanced the appearance of rRNA and poly (A⁺)RNA in castor bean endosperms without specifically stimulating the synthesis of particular mRNA species. Thus these increased levels of mRNA and rRNA may act synergistically to affect the rate of a predetermined pattern of protein synthesis.Abbreviations SDS sodium dodecyl sulphate - GA₃ gibberellic acid - PAGE polyacrylamide gel electrophoresis     

Amino acid sequences of two nonspecific lipid-transfer proteins from germinated castor bean

The amino acid sequence of two nonspecific lipid-transfer proteins (nsLTP) B and C from germinated castor bean seeds have been determined. Both the proteins consist of 92 residues, as for nsLTP previously reported, and their calculated Mr values are 9847 and 9593 for nsLTP-B and nsLTP-C, respectively. The sequences of nsLTP-B and nsLTP-C, compared to the known sequence of nsLTP-A from the same source, are 68% and 35% similar, respectively. No variation was found at the positions of the cysteine residues, indicating that they might be involved in disulfide bridges.     

Nucleic acid (cDNA) and amino acid sequences of isocitrate lyase from castor bean

A cDNA library was constructed to mRNA enriched for isocitrate-lyase mRNA from castor-bean (Ricinus communis var. zanzibarensis) endosperms. Nine clones for isocitrate lyase (EC 4.1.3.1) were identified. The insert of 2.2 kb from clone ICL4 was sequenced and proved to contain the entire coding region, 1731 bp, for isocitrate lyase. The amino acid sequence of isocitrate lyase was deduced from the nucleic acid sequence. By analogy with muscle aldolase a lysine residue that possibly takes part in the binding of the substrate was identified. The 3 untranslated region contained three putative polyadenylation addition signals and two direct repeats.     

Properties of a soluble ATPase from castor bean endosperm mitochondria

An ATPase was extracted and purified from castor bean endospermmitochondria. The enzyme is stable at 60°C only in the presenceof ATP in the incubation medium. It is less stable at 0°Cthan at 30°C but is stabilized by ammonium sulfate or glycerol.Activity is dependent on the presence of Mg⁺⁺, and in the presenceof Mg⁺⁺ is enhanced by 2,4-dinitrophenol, but is not inhibitedby oligomycin. The enzyme hydrolyzes ITP in addition to ATP,but ITPase activity is hardly enhanced by 2,4-dinitrophenol.This preparation has many properties in common with the ATPase(coupling factor 1) from beef heart mitochondria. (Received November 8, 1969; )     

Control of the citric acid cycle in mitochondria from germinating castor bean endosperm

Only a part of the citric acid cycle seems to be functional in the endosperm of germinating castor bean seeds. Mitochondria isolated from the endosperm can oxidize all of the citric acid cycle substrates. This was investigated further by studying the enzymic activities of isolated mitochondria during germination. Whilst all enzymic activities increase during germination there is an imbalance in the absolute levels of activities, with very low activities of those enzymes involved in converting pyruvate to succinate. It is suggested that the enzymic activity represents a coarse control of the cycle in this tissue.     

Evidence that the castor bean allergens are the albumin storage proteins in the protein bodies of castor bean

The well characterized castor bean (Ricinus communis L.) allergens were identified as the low molecular weight albumin storage proteins in the matrix of the protein bodies in the endosperm. The methods of identification involved molecular weight estimation, amino acid composition, stability at 100° C, solubility in various solvents, gel electrophoresis, and immunological techniques. The finding explains the wide distribution of allergens in various seeds.     

Characterization of glyoxysomes from castor bean endosperm

Electron micrographs are presented which establish the identity of the components of the 3 major bands observed after sucrose density centrifugation of the crude particulate fraction from the endosperm of germinating castor bean seedlings. These are: mitochondria (density 1.19 g/cc), proplastids (density 1.23 g/cc) and glyoxysomes (density 1.25 g/cc). Further evidence is provided on the enzymatic composition of the glyoxysomes. Essentially all of the particulate malate synthetase, isocitrate lyase, catalase, and glycolic oxidase is present in these organelles. The distribution of glyoxysomal enzymes on sucrose density gradients is contrasted with that of the strictly mitochondrial enzymes fumarase, NADH oxidase, and succinoxidase. Malate dehydrogenase and citrate synthetase are present in both organelles. The functional role of glyoxysomes and their relationship to cytosomes from other tissues is discussed.     

Purification and characterization of homogeneous sunflower seed acid phosphatase

Acid phosphatase (EC 3.1.3.2) from sunflower seed was purified 1800-fold to homogeneity using both conventional and affinity chromatographic methods. The purified enzyme was a mixture of two enzyme forms distinguishable by polyacrylamide gel electrophoresis (PAGE). Gel exclusion chromatography, which did not distinguish between the two forms, gave an apparent M, of 103 000. Preparative PAGE permitted the separation of the two forms, and SDS-PAGE showed that they contained equivalent peptide subunits of apparent M, 56 000 and 52 000. Amino acid analysis indicated that both enzyme forms have similar amino acid compositions. Data on substrate specificity and pH dependence is presented. The kinetic constants for hydrolysis of p-nitrophenyl phosphate as catalysed by sunflower seed acid phosphatase were independent of pH in the range 3-5. The enzyme was competitively inhibited by inorganic phosphate and non-competitively inhibited by phosphomycin.     

Long chain fatty acid synthesis in developing castor bean seeds IV. The synthetic system in proplastids

The proplastid fraction containing no cytosol and mitochondrionwas isolated from developing castor bean endosperm by stepwisesucrose density centrifugation. This fraction possesses thecapacity to synthesize LFAs from [u-14C]sucrose, [u-¹⁴C]-glucose,[u-¹⁴C]G-1-P, [u-¹⁴C]G-6-P, [2-¹⁴C]pyruvate and [1-¹⁴C]acetate.Little was incorporated from [1-¹⁴C]pyruvate into LFAs, butmuch into ¹⁴CO_a. Addition of cytosol to the proplastid fractiondid not enhance the LFA synthesis. From these data, the wholepath from sucrose to LFAs through glycolytic path and pyruvatedecarboxylation seems to be located within the proplastid indeveloping castor bean endosperm. The difference in utilizationof substrates indicates that the rate of LFA synthesis in castorbean proplastids is limited at a step between sucrose and hexosephosphate. In addition, experiments with CO₂ output and LFAsynthesis from [1-¹⁴C]glucose, [6-¹⁴C]glucose and [u-¹⁴C]G-6-Pstrongly suggest that the path flow branches actively throughG-6-P to the pentose phosphate path and little through acetylCoAto the TCA cycle. (Received May 12, 1975; )