Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase／Oxygenase in Wheat Leaves

The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.     

Degradation of Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves During Dark-induced Senescence

The degradation of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves during dark-induced senescence was studied. An in vivo degradation product of Rubisco large subunit (LSU) with molecular weight of 50 kD was detected by SDS-PAGE and immunoblotting with antibody against tobacco Rubisco. This fragment could also be detected in natural senescence. The result also suggested that the Rubisco holoenzyme had not dissociated when LSU hydrolyzed from 53 kD to 50 kD. And LSU could be fragmented to 50 kD at 30-35 ℃ and at pH 7.5 in crude enzyme extracts of wheat leaves dark-induced for 48 h, which suggested that maybe LSU was degraded to 50 kD by an unknown protease in chloroplast.     

A novel 51-kDa fragment of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase formed in the stroma of chloroplasts in dark-induced senescing wheat leaves

The degradation of large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in wheat ( Triticum aestivum L. cv. Yangmai 158) leaves was studied. A novel 51-kDa fragment was detected in leaf crude extracts and in chloroplast lysates from leaves with dark-induced senescence. Further studies showed that the 51-kDa fragment was found in the reaction solution with stroma fraction but not in that with the chloroplast membrane fraction and in the chloroplast lysates from mature wheat leaves. The reaction of producing the 51-kDa fragment was inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 1,10-phenanthroline and EDTA. The N-terminal sequence analysis indicated that the LSU was cleaved at the peptide bond between Lys-14 and Ala-15. In addition, a 50-kDa fragment of LSU formed obviously at pH 6.0–6.5 was detected in the crude extracts of leaves with dark-induced senescence but was not found in lysates of chloroplasts. The degradation was prevented by AEBSF, leupeptin and transepoxysuccinyl- l -leucylamido (4-guanidino) butane (E-64). The results obtained in this study imply that the appearance of the 51-kDa fragment could be because of the involvement of a new senescence-associated protease that is located in the stroma of chloroplasts in senescing wheat leaves.     

Senescence in wheat leaves: is a cysteine endopeptidase involved in the degradation of the large subunit of Rubisco?

In wheat (Triticum aestivum L.), leaf senescence can be initiated by different factors. Depending on the plant system (intact plants or detached leaves) or the environmental conditions (light, nutrient availability), the symptoms of senescence differ. The aim of this work was to elucidate the catabolism of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC. 4.1.1.39) under various senescence-inducing conditions. Leaf senescence was initiated in intact plants by darkness or by N-deprivation and in leaf segments by exposure to light or darkness. Depending on the treatment, a 50 kDa fragment of Rubisco was observed. The formation of this fragment was enhanced by leaf detachment and low light. In segments exposed to high light and in intact plants induced to senesce by N-deprivation, the fragment was essentially absent. Since an antibody against the N-terminus of a large subunit of Rubisco (LSU) did not cross-react with the fragment, it appears likely that a smaller fragment was removed from the N-terminus of LSU. Inhibitor studies suggest that a cysteine endopeptidase was involved in the formation of the 50 kDa fragment. Non-denaturing-PAGE followed by SDS-PAGE revealed that the fragment was produced while LSU was integrated in the holoenzyme complex, and that it remained there after being produced. It remains open how the putative endopeptidase reaches the stromal protein Rubisco. The results indicate that depending on the senescence-inducing conditions, different proteolytic enzymes may be involved. The involvement of vacuolar proteases must be considered as occurring during LSU degradation, which takes place in darkness, low light or under carbon limitation.     

The Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase is Fragmented into 37-kDa and 16-kDa Polypeptides by Active Oxygen in the Lysates of Chloroplasts from Primary Leaves of Wheat

Lysates of chloroplasts isolated from wheat (Triticum aestivumL. cv. Aoba) leaves were incubated on ice (pH 5.7) for 0 to60 min in light (15 µmol quanta m^–2 s^–1),and degradation of the large subunit (LSU) of ribulose-l,5-bis-phosphatecarboxylase/oxygenase (Rubisco: EC 4.1.1.39 [EC] ) was analyzed byapplying immunoblotting with site-specific antibodies againstthe N-terminal, internal, and C-terminal amino acid sequencesof the LSU of wheat Rubisco. The most dominant product of thebreakdown of the LSU and that which was first to appear wasan apparent molecular mass of 37-kDa fragment containing theN-terminal region of the LSU. A 16-kDa fragment containing theC-terminal region of the LSU was concomitantly seen. This fragmentationof the LSU was inhibited in the presence of EDTA or 1,10-phenanthroline.The addition of active oxygen scavengers, catalase (for H₂O₂)and n-propyl gallate (for hydroxyl radical) to the lysates alsoinhibited the fragmentation. When the purified Rubisco fromwheat leaves was exposed to a hydroxyl radical-generating systemcomprising H₂O₂, FeSO₄ and ascorbic acid, the LSU was degradedin the same manner as observed in the chloroplast lysates. Theresults suggest that the large subunit of Rubisco was directlydegraded to the 37-kDa fragment containing the N-terminal regionand the 16-kDa fragment containing the C-terminal region ofthe LSU by active oxygen, probably the hydroxyl radical, generatedin the lysates of chloroplasts. (Received October 28, 1996; Accepted February 7, 1997)     

Light-dependent fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in chloroplasts isolated from wheat leaves

The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) is degraded into an N-terminal side fragment of 37 kDa and a C-terminal side fragment of 16 kDa by the hydroxyl radical in the lysates of chloroplasts in light (H. Ishida et al. 1997, Plant Cell Physiol 38: 471–479). In the present study, we demonstrate that this fragmentation of the LSU also occurs in the same manner in intact chloroplasts, and discuss the mechanisms of the fragmentation. The fragmentation of the LSU was observed when intact chloroplasts from wheat leaves were incubated under illumination in the presence of KCN or NaN₃, which is a potent inhibitor of active oxygen-scavenging enzyme(s). The properties, such as molecular masses and cross-reactivities against the site-specific anti-LSU antibodies, of the fragments found in the chloroplasts were the same as those found in the lysates. These results indicate that, as in the lysates, the fragmentation of the LSU in the intact chloroplasts was also caused by the hydroxyl radical generated in light. The fragmentation of the LSU was completely inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), and only partially inhibited by methyl viologen in the lysates. The addition of hydrogen peroxide to the lysates stimulated LSU fragmentation in light, but did not induce any fragmentation in darkness. Thus, we conclude that both production of hydrogen peroxide and generation of the reducing power at thylakoid membranes in light are essential requirements for fragmentation of the LSU. Received: 14 June 1997 / Accepted: 28 August 1997     

小麦Rubisco 活化酶基因的克隆和表达特性

Rubisco活化酶是广泛存在于光合生物中调节Rubisco活性的酶, 我们利用PCR技术, 从小麦(Triticum aestivum)叶片cDNA文库中克隆得到Rubisco活化酶基因cDNA片段, 该片段长度为850 bp, 编码201个氨基酸。Northern blot表明, 小麦叶片在暗诱导衰老的条件下, 叶片中活化酶基因表达水平逐渐下降; 同时, 小麦叶片的光合特性、叶绿素含量和Rubisco活性呈现下降趋势。这些结果表明, 衰老时小麦叶片Rubisco活化酶基因表达水平下降与光合速率下降密切相关。     

小麦Rubisco活化酶基因的克隆和表达特性

Rubisco活化酶是广泛存在于光合生物中调节Rubisco活性的酶,我们利用PCR技术,从小麦(Triticum aestivum)叶片cDNA文库中克隆得到Rubisco活化酶基因cDNA片段,该片段长度为850 bp,编码201个氨基酸.Northern blot表明,小麦叶片在暗诱导衰老的条件下,叶片中活化酶基因表达水平逐渐下降;同时,小麦叶片的光合特性、叶绿素含量和Rubisco活性呈现下降趋势.这些结果表明,衰老时小麦叶片Rubisco活化酶基因表达水平下降与光合速率下降密切相关.     

The absence of Rubisco activase activity in total wheat leaf extracts is recovered in the purified protein

When desalted extracts of soluble protein from dark-adaptedwheat leaves were assayed for ribulose-1, 5-bisphosphate carboxylase/oxygenase(Rubisco) activase activity in the presence of 1 mM ATP andan ATP-regenerating system, very little ATP-dependent activationof RuBP-inactivated Rubisco was found. In extracts from light-adaptedleaves a very similar pattern of Rubisco activation was observedexcept that the overall level of Rubisco activity was much lowerthan in the extracts from dark-adapted leaves. These featureswere apparent both at low (120µg per ml) and high (640µg per ml) protein concentrations. We were unable to demonstrateRubisco activase activity in crude leaf extracts. Consequently,in order to establish that Rubisco activase was present in wheatleaf extracts the wheat leaf protein was purified to homogeneity.The identity of the protein was confirmed with antibodies tothe spinach enzyme, ATPase activity and activase-mediated releaseof the inhibitor, carboxyara-binitol-1-phosphate (CA1P) fromthe tertiary Rubisco complex. The pure wheat Rubisco activaserelieved the CA1P-induced inhibition of Rubisco activity. Rubiscoactivase had no significant effect on the affinity of wheatRubisco for the substrate, ribulose-1, 5-bisphosphate (RuBP). Key words: Rubisco activase, Rubisco, regulation     

Modification of Rubisco and Altered Proteolytic Activity in O3-Stressed Hybrid Poplar (Populus maximowizii x trichocarpa)

Exposing hybrid poplar (Populus maximowizii x trichocarpa) plants to ozone (O3) resulted in an acceleration of the visual symptoms of senescence and a decrease in the activity and quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Whole plants, crude leaf extracts, and isolated intact chloroplasts of hybrid poplar clone 245 were used to test the hypothesis that O3-induced structural modifications of Rubisco affect the activity of this key photosynthetic enzyme. Proteolytic activity, per se, could not account for losses in Rubisco; acidic and alkaline protease activities declined or were unaffected in foliage of O3-treated poplar saplings. In vitro treatment of leaf extracts with O3 decreased total Rubisco activity and binding of the enzyme's transition-state analog, 2-carboxyarabinitol bisphosphate. Additionally, O3 increased the loss of Rubisco large subunit (LSU) when extracts were incubated at 37[deg]C. Treatment of isolated intact chloroplasts with O3 accelerated both the loss of the 55-kD Rubisco LSU and the accumulation of Rubisco LSU aggregates, as visualized by immunoblotting. The time-dependent modification in Rubisco structure was the primary response of the isolated organelles to O3 treatment, with little proteolytic degradation of the LSU detected.     

Victorin induction of an apoptotic/senescence-like response in oats.

Victorin is a host-selective toxin produced by Cochliobolus victoriae, the causal agent of victoria blight of oats. Previously, victorin was shown to be bound specifically by two proteins of the mitochondrial glycine decarboxylase complex, at least one of which binds victorin only in toxin-sensitive genotypes in vivo. This enzyme complex is involved in the photorespiratory cycle and is inhibited by victorin, with an effective concentration for 50% inhibition of 81 pM. The photorespiratory cycle begins with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and victorin was found to induce a specific proteolytic cleavage of the Rubisco large subunit (LSU). Leaf slices incubated with victorin for 4 hr in the dark accumulated a form of the LSU that is cleaved after the 14th amino acid. This proteolytic cleavage was prevented by the protease inhibitors E-64 and calpeptin. Another primary symptom of victorin treatment is chlorophyll loss, which along with the specific LSU cleavage is suggestive of a victorin-induced, senescence-like response. DNA from victorin-treated leaf slices showed a pronounced laddering effect, which is typical of apoptosis. Calcium appeared to play a role in mediating the plant response to victorin because LaCl3 gave near-complete protection against victorin, preventing both leaf symptoms and LSU cleavage. The ethylene inhibitors aminooxyacetic acid and silver thiosulfate also gave significant protection against victorin-induced leaf symptoms and prevented LSU cleavage. The symptoms resulting from victorin treatment suggest that victorin causes premature senescence of leaves.     

Streptomyces thermocerradoensis I3 secretes a novel bifunctional xylanase/endoglucanase under solid-state fermentation

Lignocellulosic wastes can be potentially converted into several bioproducts such as glucose, xylo-oligosaccharides, and bioethanol. Certain processes, such as enzymatic hydrolysis, are generally needed to convert biomass into bioproducts. The present study investigated the production of xylanases and cellulases by Streptomyces thermocerradoensis I3 under solid-state fermentation (SSF), using wheat bran as a low-cost medium. The activities of xylanase and carboxymethyl cellulase (CMCase) were evaluated until 96 hr of incubation. The highest enzyme activity was observed after 72 hr of incubation. The crude enzyme extract was sequentially filtered, first using a 50 kDa filter, followed by a 30 kDa filter. Fraction 3 (F3) exhibited activities of both xylanase and CMCase. Xylanase and CMCase showed optimum activity at 70°C and pH 6.0 and 55°C and pH 6.0, respectively. The zymogram analysis showed a single activity band with a molecular mass of approximately 17 kDa. These findings provide strong evidence that the enzyme is a bifunctional xylanase/endoglucanase. This enzyme improved the saccharification of sugarcane bagasse by 1.76 times that of commercial cellulase. This enzyme has potential applications in various biotechnological procedures.     

RNA binding activity of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit from Chlamydomonas reinhardtii

Transfer of the green algae Chlamydomonas reinhardtii from low light to high light generated an oxidative stress that led to a dramatic arrest in the synthesis of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The translational arrest correlated with transient changes in the intracellular levels of reactive oxygen species and with shifting the glutathione pool toward its oxidized form (Irihimovitch, V., and Shapira, M. (2000) J. Biol. Chem. 275, 16289-16295). Here we examined how the redox potential of glutathione affected the RNA-protein interactions with the 5'-untranslated region of rbcL. This RNA region specifically binds a group of proteins with molecular masses of 81, 62, 51, and 47 kDa in UV-cross-linking experiments under reducing conditions. Binding of these proteins was interrupted by exposure to oxidizing conditions (GSSG), and a new protein of 55 kDa was shown to interact with the RNA. The 55-kDa protein comigrated with Rubisco LSU in one- and two-dimensional gels, and its RNA binding activity was further verified by using the purified protein in UV-cross-linking experiments under oxidizing conditions. However, the LSU of purified and oxidized Rubisco bound to RNA in a sequence-independent manner. A remarkable structural similarity was found between the amino-terminal domain of Rubisco LSU in C. reinhardtii and the RNA binding domain, a highly prevailing motif among RNA-binding proteins. It appears from the crystal structure of Rubisco that the amino terminus of LSU is buried within the holoenzyme. We propose that under oxidizing conditions it is exposed to the surface and can, therefore, bind RNA. Accordingly, a recombinant form of the polypeptide domain that corresponds to the amino terminus of LSU was found to bind RNA in vitro with or without GSSG.     

Some structural properties of plant serine:glyoxylate aminotransferase?

The structural properties of photorespiratory serine:glyoxylate aminotransferases (SGAT, EC 2.6.1.45) from maize (Zea mays L.) and wheat (Triticum aestivum L.) leaves were examined. By means of molecular sieving on Zorbax SE-250 column and filtration through centrifugal filters it was shown that dimers of wheat enzyme (molecular mass of about 90 kDa) dissociate into component monomers (molecular mass of about 45 kDa) upon decrease in pH value (from 9.1 or 7.0 to 6.5). At pH 9.1 a 50-fold decrease of ionic strength elicited a similar effect. Under the same conditions homodimers of the maize enzyme (molecular mass similar to that of the wheat enzyme) remained stable. Immunoblot analysis with polyclonal antiserum against wheat seedling SGAT on leaf homogenates or highly purified preparations of both enzymes showed that the immunogenic portions of the wheat enzyme are divergent from those of the maize enzyme. The sequence of 136 amino acids of the maize enzyme and 78 amino acids of the wheat enzyme was established by tandem mass spectrometry with time of flight analyzer. The two enzymes likely share similarity in tertiary and quaternary structures as well as high level of hydrophobicity on their molecular surfaces. They likely differ in the mechanism of transport from the site of biosynthesis to peroxisomes as well as in some aspects of secondary structure.     

烟草Rubisco活化酶的纯化及其特性

利用35％饱和硫酸铵分部、DEAE－Sephacel和FPIC－MonoQ柱层析等步骤从烟草叶片中纯化了Rubisco活化酶,并制备了其专一性抗体。此法不仅快速,而且比活力高。以往认为菠菜和拟南芥Rubisco活化酶由两种亚基组成。通过快速制备的粗提液分析．发现烟草Rubisco活化酶由一种42kD的亚基组成。即使在有多种蛋白酶抑制剂存在的情况下,此亚基仍很易降解为39kD的亚基。ATP不仅对酶的活性所必需,而且也有利于维持酶的稳定性。该酶的热稳定性远比Rubisco差。     

Purification and characterization of a subtilisin-like serine protease induced during the senescence of wheat leaves

A senescence-specific protease accounting for almost 70% of the total peptide hydrolytic activity of protein extracts, was isolated from detached wheat leaves induced to senescence by incubation in the dark for 72 h. Purification to apparent homogeneity was performed by ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The enzymatic activity was followed by its ability to hydrolyse the synthetic peptide Suc-AAPF-pNA. SDS/PAGE and gel filtration analysis indicated that the enzyme was a dimer composed of two identical subunits of 59 kDa. The apparent K _m and V _max for the peptide were 1.18 m m and 2.27 mmol pNA mg⁻¹ h⁻¹, respectively. The enzyme was active at pH values above 8.0 and remained active after heat treatment at 60°C for 10 min. It was inhibited by chymostatin, indicating that the enzyme possesses a chymotrypsin-like activity. Rubisco was readily hydrolysed by the purified protease. A sequenced internal fragment of 17 amino acids showed a high level of similarity (65–75% identity) with a highly conserved region of several plant subtilisin-like serine proteases. The absence of this enzymatic activity in fractionated extracts from non-senescent tissues suggests that it might play a role in the senescing process.     

Degradation of ribulose-bisphosphate carboxylase by vacuolar enzymes of senescing French bean leaves: Immunocytochemical and ultrastructural observations

Summary The possible involvement of vacuolar cysteine proteinases in degradation of ribulose-bisphosphate carboxylase (Rubisco) in senescing French bean leaves was studied by ultrastructural and immunocytochemical analyses with antibodies raised against the large subunit (LSU) of Rubisco and SH-EP, a cysteine proteinase fromVigna mungo that is immunologically identical to one of the major proteinases of French bean plants. Primary leaves of 10-day-old plants were detached and placed at 25 °C in darkness for 0, 4, and 8 days to allow their senescence to proceed. The leaves at each senescence stage were subjected to the conventional electron microscopic and immunocytochemical studies. The results indicated that the chloroplasts of senescing French bean leaves were separated from the cytoplasm of the cell periphery and taken into the central vacuole and that the Rubisco LSU in the chloroplasts was degraded by vacuolar enzymes such as an SH-EP-related cysteine proteinase that developed in senescing leaves. The present results together with the results of previous biochemical studies using vacuolar lysates support the view that Rubisco is degraded through the association of chloroplasts with the central vacuole during the senescence of leaves that were detached and placed in darkness.     

SDS-dependent proteases induced by ABA and its relation to Rubisco and Rubisco activase contents in rice leaves

Protease activities and its relation to the contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase were investigated in detached leaves of rice (Oryza sativa L.) floated on the solutions containing abscisic acid (ABA) or benzyladenine (BA). Rubisco and Rubisco activase contents were decreased during the time course and the decreases were enhanced by ABA and suppressed by BA. The decrease in Rubisco activase was faster than that in Rubisco. SDS-dependent protease activities at 50–70 kDa (rice SDS-dependent protease: RSP) analyzed by the gelatin containing PAGE were significantly enhanced by ABA. RSPs were also increased in attached leaves during senescence. RSPs had the pH optimum of 5.5, suggesting that RSPs are vacuolar protease. Both decrease in Rubisco and Rubisco activase contents and increase in RSPs activities were suppressed by cycloheximide. These findings indicate that the activities of RSPs are well correlated with the decrease in these protein contents. Immunoblotting analysis showed that Rubisco in the leaf extracts was completely degraded by 5 h at pH 5.5 with SDS where it was optimal condition for RSPs. However, the degradation of Rubisco did not proceed at pH 7.5 without SDS where it is near physiological condition for stromal proteins. Rubisco activase was degraded at similar rate under both conditions. These results suggest that RSPs can functions in a senescence related degradation system of chloroplast protein in rice leaves. Rubisco activase would be more susceptible to proteolysis than Rubisco under physiological condition and this could affect the contents of these proteins in leaves.     

Tree stem phosphoenolpyruvate carboxylase (PEPC): lack of biochemical and localization evidence for a C4-like photosynthesis system

Here, the kinetic properties and immunolocalization of phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in young stems of Fagus sylvatica were investigated. The aim of the study was to test the hypothesis that there is a C4-like photosynthesis system in the stems of this C3 tree species. The activity, optimal pH and L-malate sensitivity of PEPC, and the Michaelis-Menten constant (Km) for phosphoenolpyruvate (PEP), were measured in protein extracts from current-year stems and leaves. A gel blot experiment and immunolocalization studies were performed to examine the isozyme complexity of PEPC and the tissue distribution of PEPC and Rubisco in stems. Leaf and stem PEPCs exhibited similar, classical values characteristic of C3 PEPCs, with an optimal pH of c. 7.8, a Km for PEP of c. 0.3 mM and a IC50 for L-malate (the L-malate concentration that inhibits 50% of PEPC activity at the Km for PEP) of c. 0.1 mM. Western blot analysis showed the presence of two PEPC subunits (molecular mass c. 110 kDa) both in leaves and in stems. Immunogold labelling did not reveal any differential localization of PEPC and Rubisco, neither between nor inside cells. This study suggests that C4-type photosynthesis does not occur in stems of F. sylvatica and underlines the importance of PEPC in nonphotosynthetic carbon fixation by most stem tissues (fixation of respired CO2 and fixation via the anaplerotic pathway).     

Purification and characterisation of adenosine nucleosidase from Coffea arabica young leaves

An adenosine nucleosidase (ANase) (EC 3.2.2.7) was purified from young leaves of Coffea arabica L. cv. Catimor. A sequence of fractionating steps was used starting with ammonium sulphate salting-out, followed by anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme was purified 5804-fold and a specific activity of 8333 nkat mg-1 protein was measured. The native enzyme is a homodimer with an apparent molecular weight of 72 kDa estimated by gel filtration and each monomer has a molecular weight of 34.6 kDa, estimated by SDS-PAGE. The enzyme showed maximum activity at pH 6.0 in citrate-phosphate buffer (50 mM). The calculated Km is 6.3 microM and Vmax 9.8 nKat.