Purification of leucoplast pyruvate kinase from developing castor bean endosperm

Leucoplast pyruvate kinase from endosperm of developing castor oil seeds (Ricinus communis L.; cv Baker) has been purified 1370-fold to a specific activity of 41.1 micromoles pyruvate produced per minute per milligram protein. Nondenaturing polyacrylamide gel electrophoresis of the purified enzyme resulted in a single protein staining band that co-migrated with pyruvate kinase activity. However, following sodium dodecyl sulfate polyacrylamide electrophoresis, two major protein staining bands of 57.5 and 44 kilodaltons, which occurred in an approximate 2:1 ratio, respectively, were observed. The native molecular mass was approximately 305 kilodaltons. Rabbit antiserum raised against the final enzyme preparation effectively immunoprecipitated leucoplast pyruvate kinase. The 57.5- and 44-kilodalton polypeptides are immunologically related as both proteins cross-reacted strongly on Western blots probed with the rabbit anti-(developing castor seed endosperm leucoplast pyruvate kinase) immunoglobulin that had been affinity-purified against the 57.5-kilodalton polypeptide. In contrast, pyruvate kinases from the following sources showed no immunological cross-reactivity with the same immunoglobulin: the cytosolic enzyme from developing or germinating castor bean endosperm; chloroplastic pyruvate kinase from expanding leaves of the castor oil plant; chloroplastic or cytosolic pyruvate kinase from the green alga, Selenastrum minutum; and mammalian or bacterial pyruvate kinases.     

Purification of pyruvate kinase from germinating castor bean endosperm

Cytosolic pyruvate kinase from endosperm of germinating castor beans (Ricinus communis L.; cv Hale) has been purified 3100-fold to apparent homogeneity and a final specific activity of 203 micromole pyruvate produced/minute per milligram protein. Purification steps included: heat treatment, polyethylene glycol fractionation, Q-Sepharose, ADP-agarose, Mono-Q and Phenyl Superose chromatography. Nondenaturing polyacrylamide gel electrophoresis of the final sample resulted in a single protein staining band which co-migrated with pyruvate kinase activity. Two protein staining bands of 57 and 56 kilodaltons were observed following SDS polyacrylamide gel electrophoresis of the final preparation. The native molecular mass was found to be about 240 kilodaltons. This enzyme appears to be a tetramer composed of two different subunits. The presence of dithioerythritol (2 millimolar) was required for optimal activity of the purified enzyme.     

Partial purification and characterization of CTP:cholinephosphate cytidylyltransferase from castor bean endosperm

CTP: cholinephosphate cytidylyltransferase (EC 2.7.7.15) has been purified approximately 600-fold from postgermination endosperm of castor bean. The enzyme was solubilized with n-octyl beta-D-glucopyranoside and then subjected to ion exchange and gel filtration chromatography. The Km's of the purified enzymatic activity were 0.37 and 1.1 mM for CTP and choline phosphate, respectively. Magnesium was required for activity. The purified cytidylyltransferase activity was inhibited by both phosphate and ATP. The extent of ATP inhibition was dependent on preincubation time, temperature, and Mg2+ and Ca2+ concentrations. The possible regulation of cytidylyltransferase in castor bean endosperm by protein phosphorylation is discussed.     

Glutamate dehydrogenase in developing endosperm, chloroplasts, and roots of castor bean

All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD⁺ and NADP⁺. The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH₄Cl and 1.5 at 12.5 millimolar NH₄Cl. K_m values for NH₄⁺ and NAD(P)H are reduced at low salt concentrations. The low K_m values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations.     

Polygalacturonase from Rhizopus stolonifer, an Elicitor of Casbene Synthetase Activity in Castor Bean (Ricinus communis L.) Seedlings

Apparently homogeneous polygalacturonase-elicitor purified from the filtrates of Rhizopus stolonifer cultures stimulates germinating castor bean seedlings to produce greatly increased levels of casbene synthetase activity. The purification procedure involved gel-filtration chromatography on Sephadex G-25 and G-75 columns followed by cation-exchange chromatography on a Sephadex CM C-50 column. Homogeneity of the purified preparation was indicated by the results of cationic polyacrylamide disc gel electrophoresis and isoelectric focusing (pI = 8.0). The identity of the casbene elicitor activity and polygalacturonase were indicated by the coincidence of the two activities at all stages of purification, the coincidence of both activities with the single protein-staining band detected on a cationic polyacrylamide disc gel and an isoelectric focusing gel, and the identical behavior of both activities on an agarose gel affinity column. The purified polygalacturonase-elicitor is a glycoprotein with approximately 20% carbohydrate content and an estimated molecular weight of 32,000 by polyacrylamide disc gel electrophoresis.     

Purification and Properties of Nonproteolytic Degraded ADPglucose Pyrophosphorylase from Maize Endosperm

ADPglucose pyrophosphorylase from developing endosperm tissue of starchy maize (Zea mays) was purified 88-fold to a specific activity of 34 micromoles α-glucose-1-P produced per minute per milligram protein. Rabbit antiserum to purified spinach leaf ADPglucose pyrophosphorylase was able to inhibit pyrophosphorolysis activity of the purified enzyme by up to 90%. The final preparation yielded four major protein staining bands following sodium dodecyl sulfate polyacrylamide gel electrophoresis. When analyzed by Western blot hybridization only the fastest migrating, 54 kilodaltons, protein staining band cross-reacted with affinity purified rabbit antispinach leaf ADPglucose pyrophosphorylase immunoglobulin. The molecular mass of the native enzyme was estimated to be 230 kilodaltons. Thus, maize endosperm ADPglucose pyrophosphorylase appears to be comprised of four subunits. This is in contrast to the respective subunit and native molecular masses of 96 and 400 kilodaltons reported for a preparation of maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). Proteolytic degradation of maize endosperm ADPglucose pyrophosphorylase appears to occur during incubation of crude extracts at 30°C or during the partial purification of the enzyme according to a previously reported procedure (DB Dickinson, J Preiss 1969 Arch Biochem Biophys 130: 119-128). The progressive appearance of a 53 kilodalton antigenic peptide suggested the loss of a 1 kilodalton proteolytic fragment from the 54 kilodalton subunit. The complete conservation of the 54 kilodalton subunit structure following extraction of the enzyme in the presence of phenylmethylsulfonyl fluoride and/or chymostain was observed. The allosteric and catalytic properties of the partially purified proteolytic degraded versus nondegraded enzyme were compared. The major effect of proteolysis was to enhance enzyme activity in the absence of added activator while greatly decreasing its sensitivity to the allosteric effectors 3-P-glycerate and inorganic phosphate.     

Enzymes of the Primary Phosphatidylethanolamine Biosynthetic Pathway in Postgermination Castor Bean Endosperm (Developmental Profiles and Partial Purification of the Mitochondrial CTP:Ethanolaminephosphate Cytidylyltransferase)

Ethanolamine kinase, CTP:ethanolaminephosphate cytidylyltransferase (ECT), and ethanolaminephosphotransferase, which sequentially catalyze the primary pathway for phosphatidylethanolamine synthesis, were measured in castor bean (Ricinus communis L. var Hale) endosperm for 6 d after the onset of imbibition. Ethanolamine kinase (EC 2.7.1.82) activity was cytosolic, increasing slowly during the first 5 d and then declining. Total ECT (EC 2.7.7.14) activity increased until the 4th d, but the endoplasmic reticulum fraction of the activity peaked at d 3, and the mitochondrial activity peaked at d 4. Diacylglycerol:CDPethanolamine ethanolaminephosphotransferase (EC 2.7.8.1) increased during the first 2 d after imbibition began, after which it declined. The lowest activity of ethanolamine kinase during postgermination was more than 5-fold higher than the maximum activity of ECT, and the total activity of diacylglycerol:CDPethanolamine ethanolaminephosphotransferase at d 2 was at least triple that of ECT of the endoplasmic reticulum. We have partially purified ECT from mitochondrial fractions of postgermination castor bean endosperm starting with mitochondria purified by sucrose (Suc) density gradient centrifugation and broken by osmotic shock and homogenization. The membrane-bound ECT was solubilized with 1.5% 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate and purified approximately 118-fold by polyethylene glycol precipitation, chromatography on Sephacryl S-200, and then Suc gradient centrifugation. The continuous presence of both salt (0.5 M NaCl) and detergent (1% [w/v] 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) was necessary to prevent aggregation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final activity peak resulted in a prominent protein band at 35 kD, which correlated with bands from peak ECT activity fractions after both Suc gradient centrifugation and gel filtration on Sephacryl S-200. The activity of this enzyme was enhanced by the addition of several phospholipids.     

Association of phosphoenolpyruvate phosphatase activity with the cytosolic pyruvate kinase of germinating mung beans

The procedure of Malhotra and Kayastha ([1990] Plant Physiology 93: 194-200) for the purification to homogeneity of a phosphoenolpyruvate-specific alkaline phosphatase (PEP phosphatase) from germinating mung beans (Vigna radiata) was followed. Although a higher specific activity of 1.4 micromoles pyruvate produced per minute per milligram protein was obtained, the final preparation was less than 10% pure as judged by polyacrylamide gel electrophoresis. Attempts to further purify the enzyme resulted in loss of activity. The partially purified enzyme contained significant pyruvate kinase activity (0.13 micromole pyruvate produced per minute per milligram protein) when assayed at pH 7.2, but not at pH 8.5. The PEP phosphatase activity of the final preparation exhibited hysteresis; a lag time of 5 to 6 minutes was required before a steady-state reaction rate was attained. A western blot of the final preparation revealed an immunoreactive 57 kilodalton polypeptide when probed with monospecific rabbit polyclonal antibodies prepared against germinating castor bean cytosolic pyruvate kinase. No antigenic cross-reaction of the final preparation was observed with antibodies against castor bean leucoplast pyruvate kinase, or black mustard PEP-specific acid phosphatase. Nondenaturing polyacrylamide gel electrophoresis of the final preparation resulted in a single PEP phosphatase activity band; when this band was excised and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting, a 57 kilodalton silver-staining polypeptide was obtained that strongly cross-reacted with the anti-(cytosolic pyruvate kinase) immunoglobulin G. It is suggested that mung bean PEP-specific alkaline phosphatase activity is due to cytosolic pyruvate kinase, in which pyruvate and ortho-phosphate are formed in the absence of ADP.     

Characterization of ATP-Dependent Fructose 6-Phosphate 1-Phosphotransferase Isozymes from Leaf and Endosperm Tissues of Ricinus communis

Plastid and cytosolic isozymes of ATP:fructose 6-phosphate 1-phosphotransferase (PFK_p and PFK_c, respectively) have been isolated from leaves and developing endosperm tissues of the castor oil plant (Ricinus communis L). Endosperm PFK_p has been purified to apparent homogeneity. Polyclonal antibodies raised against one of the four polypeptides associated with potato tuber PFK (molecular mass, 46 kilodaltons) immunoprecipitated developing endosperm and leaf PFK_p, but not PFK_c isozymes. Western blots, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and analytical gel filtration show that PFK_p from developing endosperm is a 220 kilodalton homotetramer composed of 57 kilodalton subunits. Kinetic studies of leaf PFK_p and PFK_c isozymes reveal both similarities and differences to the characteristics of the respective endosperm isozymes studied previously (WJ Garland, DT Dennis [1980] Arch Biochem Biophys 204: 302-317). The immunological and kinetic data suggest that leaf and developing endosperm PFK_p are different but structurally related proteins.     

Purification of Spinach Leaf ADPglucose Pyrophosphorylase

ADPglucose pyrophosphorylase from spinach leaves has been purified to homogeneity by hydrophobic chromatography carried out in 1 molar phosphate buffer. After polyacrylamide gel electrophoresis, the preparation showed only one protein staining band that coincided with a single activity stain. The enzyme appears to be composed of two subunits with molecular weights of 44,000 and 48,000, respectively, as determined by SDS polyacrylamide gel electrophoresis. Thus ADPglucose pyrophosphorylase of spinach appears to be comprised of subunits which are similar in size to the subunits of ADPglucose pyrophosphorylase isolated from bacterial sources. In contrast, a subunit molecular weight of 96,000 has been reported for the maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). The purified enzyme retains similar allosteric and catalytic properties as reported previously and is more sensitive to phosphate inhibition under “dark”-simulated conditions than under “light”-simulated conditions.     

Characterization of the major integral protein of vacuolar membrane

The vacuolar membrane of radish (Raphanus sativus) taproot contained a large quantity of a protein of 23 kilodaltons that accounted for more than 25% of the total membrane proteins. The protein, tentatively named VM 23, was purified and characterized. VM 23 tends to aggregate at high temperature even in the presence of 1% sodium dodecyl sulfate. The apparent molecular size of VM 23 was estimated to be about 400 kilodaltons by polyacrylamide gel electrophoresis in the presence of 0.1% Triton X-100. VM 23 was partially extracted from the vacuolar membranes with chloroform:methanol, indicating its high hydrophobicity. The hydrophobic carboxyl modifier N,N′-dicyclohexylcarbodiimide bound covalently to VM 23. The results suggest that VM 23 may act as a secondary transport system coupled with the proton transport. The antibody against radish VM 23 reacted with the major proteins in the vacuolar membranes of mung bean (Vigna radiata) and castor bean (Ricinus communis) hypocotyls and pumpkin (Cucurbita moschata) epicotyl, but not with that of sugar beet (Beta vulgaris) taproot. VM 23 comigrated with vacuolar H⁺-pyrophosphatase on sucrose density gradient centrifugation after sonication of membranes, indicating that it is associated with the vacuolar membrane.     

Biosynthesis of nonspecific lipid transfer proteins in germinating castor bean seeds

The biosynthesis of nonspecific lipid transfer proteins (ns-LTPs) in germinating castor bean (Ricinus communis L.) seeds were investigated. Lipid transfer activities of ns-LTPs in the cotyledons, axis, and endosperm increased with growth after germination. The activity increases were accompanied by increased amounts of ns-LTPs in each tissue, as measured by immunoblot using anti-ns-LTP serum. These results suggest that the ns-LTPs are synthesized de novo in each tissue after germination and not activated from inactive proteins synthesized before germination. Comparison of the immunoblot products in each tissue from 4-day-old seedlings indicate the occurrence of tissue-specific isoforms of ns-LTPs; 9 kilodaltons (major) and 7 kilodaltons (minor) in the cotyledons, and 7 kilodaltons (major) and 9 kilodaltons (minor) in the axis, whereas only the 8-kilodalton ns-LTP is present in the endosperm. In vitro translation from poly(A)⁺ RNAs from three tissues of castor bean seedlings and the detection of immunoprecipitated products indicate that translatable mRNAs for ns-LTPs exist in the three tissues a day before the synthesis of ns-LTPs; the translation products, which are 3.5 to 4.0 kilodaltons larger than ns-LTPs, were processed to the mature ns-LTPs. The production of mature ns-LTPs from translatable mRNAs without any delay suggests that gene expression of ns-LTPs in castor bean seedlings is controlled at a step before the formation of translatable mRNAs.     

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.     

Purification and characterization of a cysteine endopeptidase in cotyledons of germinated mung bean seeds

A cysteine endopeptidase (EC 3.4.22.-) present in cotyledons of mung bean (Vigna radiata) seedlings was purified to homogeneity, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This proteinase has an apparent molecular mass of 33 kilodaltons as estimated by SDS-PAGE and belongs to the class of cysteine proteinases as judged by the effects of various proteinase inhibitors on the activity of the enzyme. When proangiotensin is used as a substrate, the enzyme preferentially hydrolyzes the peptide bonds formed by the amino group of Leu or lle in this oligopeptide chain; for the enzyme to cleave those bonds, peptide sequences consisting of at least three amino acid residues on the amino side of Leu or lle must be present. The proteinase readily digests globulin present in mung bean cotyledons to smaller polypeptides.     

The action of exogenous gibberellic acid on isocitrate lyase —mRNA in germinating castor bean seeds

Gibberellic acid (GA₃) stimulates isocitrate lyase activity of the endosperm during germination of castor bean seeds. Isocitrate lyase from castor bean was purified and an antibody to it was prepared from rabbit serum. This antibody was used to measure the amounts of isocitrate lyase-mRNA using an in vitro translation system. No specific stimulation of isocitrate lyase-mRNA by application of GA₃ was detected. The stimulation of isocitrate lyase activity by exogenous GA₃ may be accounted for by the action of the growth substance in advancing the overall production of rRNA and mRNA which accelerates the rate of total protein synthesis during germination. The application of Amo 1618 retards the production of isocitrate lyase activity but also retards protein synthesis in general. This suggests that endogenous gibberellins also act non-specifically in the regulation of protein synthesis during castor bean germination.Abbreviations SDS sodium dodecyl sulphate - GA₃ gibberellic acid - PAGE polyacrylamide gel electrophoresis     

蓖麻β-半乳糖苷酶的纯化及其基本性质

蓖麻籽黄化苗中存在高活性β-半乳糖苷酶。经硫酸铵分级分离、DEAE-纤维素离子交換层析、Sephadex G-100、CM-Sephadex和DEAE-Sephadex层析纯化。活性收率为6.4％,纯化倍数达107倍。纯化了的酶经聚丙烯酰胺凝胶电泳显示单一蛋白带,SDS-PAGE显示两条蛋白带,其相应分子量分别为3.25×10~4和2.94×10~4。用Sephadex G-200分子筛层析法测得分子量为6.7×10~4。综合上述结果推测该酶是由两个不同的亚基构成。以邻硝基苯酚-β-半乳糖苷为底物测得该酶的表观Km为5.9×10~(-3)mol/L。最适pH和最适温度分别为4.5和50℃。酸碱稳定区域在pH4.6—7.5之间。不同浓度缓冲液以及不同种类缓冲液、不同金属离子对酶活性影响均进行了讨论。     

Comparison of homoserine dehydrogenase from different plant sources

Homoserine dehydrogenase (HSD) was partially purified from castor bean, pea and wheat seedlings. The enzyme from pea had a MW of 75 000 and no sensitivity to threonine when measured in the direction of homoserine formation (forward reaction). The enzyme purified from castor bean had a MW of 290 000–350 000 and exhibited an almost complete inhibition by 1 mM threonine. Furthermore, this enzyme exhibited a polymeric nature as shown by polyacrylamide electrophoresis of the desensitized preparation and by SDS electrophoresis of the native enzyme. In wheat two isoenzymes were separated by gel filtration on Sephadex G 200. The fast-moving fraction (HSD I) was completely inhibited by threonine and exhibited a MW of 280 000, while the slow-moving fraction (HSD II) was insensitive to threonine and had a MW of 75 000. The sensitive enzyme from wheat and castor bean showed an almost absolute requirement for K⁺. The enzyme from pea and the insensitive form from wheat did not show a requirement for K⁺. For the wheat enzyme the effect of several amino acids and the main kinetic constants were studied.     

Aspartic proteinase from wheat seeds: isolation,properties and action on gliadin

Wheat endosperm was shown to contain an aspartic proteinase capable of hydrolyzing the wheat storage protein, gliadin, in vitro. The enzyme was purified to homogeneity by affinity chromatography on bacilliquin-silochrome, diethylaminoethyl-Toyopearl ion-exchange chromatography, chromatofocusing, and preparative polyacrylamide gel electrophoresis. The sedimentation constant of the enzyme was 3.4 S and the relative molecular mass (M_r), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 58000 dalton (Da). The purified enzyme was completely inhibited by pepstain whereas other enzyme inhibitors did not affect its activity. The enzyme was found to hydrolyze mainly - and -gliadins with M_r's of 67000–95000 Da, with maximal activity at pH 4.5. The data make it possible to suggest that the enzyme has an endogenous function by initiating proteolysis of storage proteins in germinating wheat seeds.Abbreviations BSA bovine serum albumin - Da dalton - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Phosphatidylethanolamine synthesis by castor bean endosperm. Intracellular distribution and characteristics of CTP:ethanolaminephosphate cytidylyltransferase.

The intracellular distribution and catalytic properties of CTP: ethanolaminephosphate cytidylyltransferase from endosperm of castor bean (Ricinus communis L. var. Hale) have been studied. This enzyme was confined to membranes, with about 80% of the activity occurring in mitochondria and the rest in endoplasmic reticulum (ER) following sucrose density gradient centrifugation. The mitochondrial location of this enzyme was supported by further purifying mitochondria on Percoll density gradients. The mitochondrial cytidylyltransferase was detected largely in outer membrane fractions, and lost its activity after trypsin treatment, indicating that the active sites are exposed to the cytoplasm. Both mitochondrial and ER cytidylyltransferase required cations for activity; Mg2+ was preferred over Mn2+ and Ca2+. The pH optima both were 6.5. The apparent Km values for ethanolamine phosphate were 143 and 83 microM and those for CTP were 125 and 1010 microM, respectively, for the mitochondrial and ER activities. The mitochondrial cytidylyltransferase reached a maximal velocity of 3.0 nmol/min/mg protein, whereas ER cytidylyltransferase was 0.424 nmol/min/mg protein. These findings reveal that the majority of the cytidylyltransferase activity in castor bean endosperm is not closely associated with ethanolaminephosphotransferase (predominantly in ER) which catalyzes the subsequent reaction in the synthesis of phosphatidyl-ethanolamine by a nucleotide pathway. The possible roles of these enzymes in phosphatidylethanolamine synthesis in plants are discussed.     

Localization and properties of ribulose diphosphate carboxylase from castor bean endosperm

A substantial portion of the ribulose 1,5-diphosphate carboxylase activity in the endosperm of germinating castor beans (Ricinus communis var. Hale) is recovered in the proplastid fraction. The partially purified enzyme shows homology with the enzyme from spinach (Spinacia oleracea) leaves, as evidenced by its reaction against antibodies to the native spinach enzyme and to its catalytic subunit. The enzyme from the endosperm of castor beans has a molecular weight of about 500,000 and, with the exception of a higher affinity for ribulose 1,5-diphosphate, has similar kinetic properties to the spinach enzyme. The castor bean carboxylase is inhibited by oxygen and also displays ribulose 1,5-diphosphate oxygenase activity with an optimum at pH 7.5.