Ribose-5-phosphate Isomerase Research Articles

Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes

SnRK2.8 is a member of the sucrose nonfermenting-related kinase family that is down-regulated when plants are deprived of nutrients and growth is reduced. When this kinase is over expressed in Arabidopsis, the plants grow larger. To understand how this kinase modulates growth, we identified some of the proteins that are phosphorylated by this kinase. A new phosphoproteomic method was used in which total protein from plants overexpressing the kinase was compared with total protein from plants in which the kinase was inactivated. Protein profiles were compared on two-dimensional gels following staining by a dye that recognizes phosphorylated amino acids. Candidate target proteins were confirmed with in vitro phosphorylation assays, using the kinase and target proteins that were purified from Escherichia coli. Seven target proteins were confirmed as being phosphorylated by SnRK2.8. Certain targets, such as 14-3-3 proteins, regulate as yet unidentified proteins, whereas other targets, such as glyoxalase I and ribose 5-phosphate isomerase, detoxify byproducts from glycolysis and catalyze one of the final steps in carbon fixation, respectively. Also, adenosine kinase and 60S ribosomal protein were confirmed as targets of SnRK2.8. Using mass spectrometry, we identified phosphorylated residues in the SnRK2.8, the 14-3-3kappa, and the 14-3-3chi. These data show that the expression of SnRK2.8 is correlated with plant growth, which may in part be due to the phosphorylation of enzymes involved in metabolic processes.

Ribose 5-phosphate isomerase type B from Trypanosoma cruzi: kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism

Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phosphate) with Km values of 4 mM (R5P) and 1.4 mM (Ru5P). 4-phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competitively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the know-ledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease.

A mutation in an Arabidopsis ribose 5‐phosphate isomerase reduces cellulose synthesis and is rescued by exogenous uridine

The Arabidopsis radial swelling mutant rsw10 showed ballooning of root trichoblasts, a lower than wild-type level of cellulose and altered levels of some monosaccharides in non-cellulosic polysaccharides. Map-based cloning showed that the mutated gene (At1g71100) encodes a ribose 5-phosphate isomerase (RPI) and that the rsw10 mutation replaces a conserved glutamic acid residue with lysine. Although RPI is intimately involved with many biochemical pathways, media supplementation experiments suggest that the visible phenotype results from a defect in the production of pyrimidine-based sugar-nucleotide compounds, most likely uridine 5'-diphosphate-glucose, the presumed substrate of cellulose synthase. Two of three RPI sequences in the nuclear genome are cytoplasmic, while the third has a putative chloroplast transit sequence. The sequence encoding both cytoplasmic enzymes could complement the mutation when expressed behind the CaMV 35S promoter, while fusion of the RSW10 promoter region to the GUS reporter gene established that the gene is expressed in many aerial tissues as well as the roots. The prominence of the rsw10 phenotype in roots probably reflects RSW10 being the only cytosolic RPI in this tissue and the gene encoding the plastid RPI being relatively weakly expressed. We could not, however, detect a decrease in total RPI activity in root extracts. The rsw10 phenotype is prominent near the root tip where cells undergo division, endoreduplication and cell expansion and so are susceptible to a restriction in de novo pyrimidine production. The two cytosolic RPIs probably arose in an ancient duplication event, their present expression patterns representing subfunctionalization of the expression of the original ancestral gene.

Structure of ribose 5-phosphate isomerase from Plasmodium falciparum.

The structure of ribose 5-phosphate isomerase from Plasmodium falciparum, PFE0730c, has been determined by molecular replacement at 2.09 angstroms resolution. The enzyme, which catalyzes the isomerization reaction that interconverts ribose 5-phosphate and ribulose 5-phosphate, is a member of the pentose phosphate pathway. The P. falciparum enzyme belongs to the ribose 5-phosphate isomerase A family, Pfam family PF06562 (DUF1124), and is structurally similar to other members of the family.

Developmental changes in the activities of enzymes of free multienzyme complexes involved in the Benson-Calvin cycle

The electrophoretically homogenous preparations of free multienzyme complexes involved in the Benson-Calvin cycle with mol wt of 520 ± 20 and 240 ± 10 kD were isolated from 15–25-day-old cotton (Gossypium hirsutum L.) leaves of the same story (the third and the fourth upper leaves). Enzyme preparations were obtained at three stages of plant development: at the stage of 6–8 true leaves, during flower-bud formation, and during flowering. Comparison studies of developmental changes in activities of ribose phosphate isomerase (RPI), phosphoribulokinase (PRK), and Rubisco were carried out. RPI and PRK activities of multienzyme complexes differed insignificantly (by 2.3% on the average). Significant changes were observed in Rubisco activity (by 30% on the average). The optimum enzymatic activities of these complexes as well as the highest photosynthetic rate were revealed at the stage of reproductive organ formation. This correlation indicates the major role of multienzyme complexes involved in the Benson-Calvin cycle in the developmental control of photosynthesis and epigenesis.

Fatty acid synthesis and the oxidative pentose phosphate pathway in developing embryos of oilseed rape (Brassica napus L.)

The potential role of the plastidial oxidative pentose phosphate pathway (OPPP) in providing the NADPH for fatty acid synthesis in plastids from developing embryos of Brassica napus (L.) has been investigated. Measurements of distributions of enzyme activities in fractions obtained from homogenates of isolated embryos have revealed that the glucose 6-phosphate and 6-phosphogluconate dehydrogenases are present in both cytosol and plastid, as is ribose 5-phosphate isomerase. However, transketolase and transaldolase are most probably confined to the plastid, while ribulose 5-phosphate epimerase is essentially cytosolic, although a very small proportion of plastid-localized activity cannot be ruled out. The activity of the OPPP in intact plastids was measured by the release of (14)CO(2) from [1-(14)C]glucose 6-phosphate. Activity was detectable in the absence of electron sinks created by the addition of metabolites to the incubation media and was stimulated 1.3-, 3.2-, and 7.9-fold by the respective additions of glutamine plus 2-oxoglutarate, cofactors and substrates for fatty acid synthesis, or methyl viologen. An increase in OPPP activity in response to additions that are absolutely required for fatty acid synthesis in these isolated plastids provides direct evidence that these two processes are connected, most probably by NADP/NADPH metabolism. The OPPP activity with methyl viologen was more than twice that during fatty acid synthesis, suggesting that the latter is not limited by OPPP capacity. Light energy may also contribute to reductant provision and, consistent with the possibility of maintenance of a balance of NADPH from light and the OPPP, glucose 6-phosphate dehydrogenase activity in the isolated plastids was decreased by light or by DTT.

A continuous process for production of -ribulose-1,5-bisphosphate from -glucose

A continuous bioprocess for production of d-ribulose-1,5-bisphosphate (RuBP) using d-glucose as starting material is described. Based on ideal plug-flow reactor modeling using kinetic parameters, a series of plug-flow reactors were designed. A cohort of enzymes namely, hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase (6PGDH), ribose-5-phosphate isomerase (RPI) and phosphoribulokinase, purified, immobilized and resident in five successive plug-flow reactors enables a series of conversion resulting in an overall 60% yield of RuBP from d-glucose. Continuous conversion using this series of reactor provides a convenient route to produce RuBP in large quantities that could be used as starting material in other bioprocesses.

A continuous process for production of d-ribulose-1,5-bisphosphate from d-glucose

A continuous bioprocess for production of d-ribulose-1,5-bisphosphate (RuBP) using d-glucose as starting material is described. Based on ideal plug-flow reactor modeling using kinetic parameters, a series of plug-flow reactors were designed. A cohort of enzymes namely, hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase (6PGDH), ribose-5-phosphate isomerase (RPI) and phosphoribulokinase, purified, immobilized and resident in five successive plug-flow reactors enables a series of conversion resulting in an overall 60% yield of RuBP from d-glucose. Continuous conversion using this series of reactor provides a convenient route to produce RuBP in large quantities that could be used as starting material in other bioprocesses.

Ribose-5-Phosphate Isomerase Deficiency: New Inborn Error in the Pentose Phosphate Pathway Associated with a Slowly Progressive Leukoencephalopathy

The present article describes the first patient with a deficiency of ribose-5-phosphate isomerase (RPI) (Enzyme Commission number 5.3.1.6) who presented with leukoencephalopathy and peripheral neuropathy. Proton magnetic resonance spectroscopy of the brain revealed highly elevated levels of the polyols ribitol and D-arabitol, which were subsequently also found in high concentrations in body fluids. Deficient activity of RPI, one of the pentose-phosphate-pathway (PPP) enzymes, was demonstrated in fibroblasts. RPI gene-sequence analysis revealed a frameshift and a missense mutation. Recently, we described a patient with liver cirrhosis and abnormal polyol levels in body fluids, related to a deficiency of transaldolase, another enzyme in the PPP. RPI is the second known inborn error in the reversible phase of the PPP, confirming that defects in pentose and polyol metabolism constitute a new area of inborn metabolic disorders.

Synthesis and evaluation of new 4-phospho- d-erythronic acid derivatives as competitive inhibitors of spinach ribose-5-phosphate isomerase

This paper reports the synthesis of new 4-phospho- d-erythronic acid derivatives, namely 4-phospho- d-erythronohydroxamic acid ( 1 ), 4-phospho- d-erythronohydrazide ( 2 ), and 4-phospho- d-erythronamide ( 3 ), and their kinetic evaluation as new competitive inhibitors of the isomerization reaction between d-ribose 5-phosphate and d-ribulose 5-phosphate catalyzed by spinach ribose-5-phosphate isomerase (RPI). By comparison to the only known RPI inhibitor, 4-phospho- d-erythronate ( 4 , K i=28 μM, K m/ K i=270), the hydroxamic acid 1 , obtained by an eight-step synthesis from d-arabinose, appears as a new potent high-energy intermediate analogue inhibitor of the isomerization reaction ( K i=29 μM, K m/ K i=260).

Alternative pathway for the formation of 4,5-dihydroxy-2,3-pentanedione, the proposed precursor of 4-hydroxy-5-methyl-3(2H)-furanone as well as autoinducer-2, and its detection as natural constituent of tomato fruit.

The formation of 4-hydroxy-5-methyl-3(2H)-furanone (HMF, norfuraneol) by spinach ribosephosphate isomerase was reinvestigated. Incubation experiments using D-ribose-5-phosphate and D-ribulose-5-phosphate clearly revealed a spontaneous nonenzymatic formation of the hydroxy-furanone from the ketose-phosphate under physiological conditions at 35 degrees C and pH 7.5, whereupon up to 1.3% of D-ribulose-5-phosphate was transformed to HMF within 15 h. 4,5-Dihydroxy-2,3-pentanedione was deduced as ultimate precursor of HMF, since addition of o-phenylenediamine to the incubation mixture led to lower amounts of HMF and to the formation of 3-(1,2-dihydroxyethyl)-2-methylquinoxaline, which was identified by means of high pressure liquid chromatography with diode array detection (HPLC-DAD), HPLC-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) and NMR spectroscopy. Additionally, the spontaneous formation of 4,5-dihydroxy-2,3-pentanedione was demontrated by its conversion to the respective alditol acetate using either NaBH(4) or NaBD(4) for the reduction. Comparative gas chromatography-mass spectrometry (GC-MS) analysis revealed the incorporation of two deuterium atoms and confirmed the dicarbonyl structure. Application of 1-13C-D-ribulose-5-phosphate as well as 5-13C-D-ribulose-5-phosphate and analysis of the derived quinoxaline derivatives by HPLC-ESI-MS/MS demonstrated the formation of the methyl-group at C-5 of the carbohydrate phosphate in consequence of a nonenzymatic phosphate elimination. Application of o-phenylenediamine into ripe tomatoes led to the detection of 3-(1,2-dihydroxyethyl)-2-methylquinoxaline by means of HPLC-MS/MS analysis implying the genuine occurrence of 4,5-dihydroxy-2,3-pentanedione in this fruit.

Mutation of rpiA in Enterobacter cloacae decreases seed and root colonization and biocontrol of damping-off caused by Pythium ultimum on cucumber.

Strains of Enterobacter cloacae show promise as biocontrol agents for Pythium ultimum-induced damping-off on cucumber and other crops. E. cloacae A145 is a mini-Tn5 Km transposon mutant of strain 501R3 that was significantly reduced in suppression of damping-off on cucumber caused by P. ultimum. Strain A145 was deficient in colonization of cucumber, sunflower, and wheat seeds and significantly reduced in colonization of corn and cowpea seeds relative to strain 501R3. Populations of strain A145 were also significantly lower than those of strain 501R3 at all sampling times in cucumber, wheat, and sunflower rhizosphere. Populations of strain A145 were not detectable in any rhizosphere after 42 days, while populations of strain 501R3 remained at substantial levels throughout all experiments. Molecular characterization of strain A145 indicated mini-Tn5 Km was inserted in a region of the E. cloacae genome with a high degree of DNA and amino acid sequence similarity to rpiA, which encodes ribose-5-phosphate isomerase. In Escherichia coli, RpiA catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate and is a key enzyme in the pentose phosphate pathway. Ribose-5-phosphate isomerase activity in cell lysates from strain A145 was approximately 3.5% of that from strain 501R3. In addition, strain A145 was a ribose auxotroph, as expected for an rpiA mutant. Introduction of a 1.0-kb DNA fragment containing only the rpiA homologue into strain A145 restored ribose phosphate isomerase activity, prototrophy, seedling colonization, and disease suppression to levels similar to those associated with strain 501R3. Experiments reported here indicate a key role for rpiA and possibly the pentose phosphate pathway in suppression of damping-off and colonization of subterranean portions of plants by E. cloacae.

The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants.

Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate. Therefore, the translocator was named Xul-5-P/phosphate translocator (XPT). The XPT shares only approximately 35% to 40% sequence identity with members of both the triose phosphate translocator and the phosphoenolpyruvate/phosphate translocator classes, but a higher identity of approximately 50% to glucose 6-phosphate/phosphate translocators. Therefore, it represents a fourth group of plastidic phosphate translocators. Database analysis revealed that plant cells contain, in addition to enzymes of the oxidative branch of the oxidative pentose phosphate pathway, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase in both the cytosol and the plastids, whereas the transketolase and transaldolase converting the produced pentose phosphates to triose phosphates and hexose phosphates are probably solely confined to plastids. It is assumed that the XPT function is to provide the plastidic pentose phosphate pathways with cytosolic carbon skeletons in the form of Xul-5-P, especially under conditions of a high demand for intermediates of the cycles.

Cloning and characterization of Edwardsiella ictaluri proteins expressed and recognized by the channel catfish Ictalurus punctatus immune response during infection.

An Edwardsiella ictaluri expression library was screened for clones expressing antigenic E. ictaluri proteins using anti-E. ictaluri serum, which resulted in the isolation of 32 clones. The clones were partially characterized and 4 were selected for complete analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2-dimensional PAGE, Western blotting, and DNA sequencing were used to analyze expressed antigenic proteins and encoded genes. Sequence analysis identified 4 putative open reading frames (ORFs) in the insert of Clone 4d6, which corresponded to antigenic acidic proteins of 55, 20 and 18 kDa expressed by both the clone and E. ictaluri cells. The predicted gene products of these ORFs were similar to several products of the imp locus of Rhizobium leguminosarum bv. trifolii. The imp locus of R. leguminosarum contains 14 genes that encode proteins involved in a putative temperature-dependent protein secretion system. In addition there was significant amino acid identity for a variety of hypothetical proteins from R. solanacearum, Ps. aeruginosa, A. tumefaciens, Y. pestis, and Salmonella typhimurium. Overlapping inserts of Clones 1.4, 5d2, and 5d3 encoded ORFs similar to Escherichia coli partial genes serA and pgk, and complete genes rpiA, iciA, yggE, yggB and fda. These genes encode D-3-phosphoglycerate dehydrogenase (serA), ribose 5-phosphate isomerase (rpiA), a specific inhibitor of chromosomal initiation of replication (iciA), a hypothetical protein (yggE), a protein involved in responses to osmotic stress (yggB), fructose 1,6-bisphosphate aldolase (fda), and phosphoglycerate kinase (pgk). Cloned antigenic E. ictaluri proteins of 33, 27, 35 and 45 kDa appeared to be products of the ORFs similar to yggE, rpiA, iciA, and fda respectively. All the cloned antigenic proteins were recognized by antiserum from catfish that had recovered from enteric septicemia of catfish (ESC), indicating that these antigens are expressed during the infectious process. The cloned antigenic proteins were subsequently evaluated as subunit vaccines for protection against wild-type E. ictaluri. All vaccine treatments were protective against E. ictaluri in catfish, but results were inconclusive due to high levels of cross-reactive protection afforded by the E. coli host strain of the cloning vector.

Free and Membrane-Bound Multienzyme Complexes with Calvin Cycle Activities in Cotton Leaves

Free and membrane-bound forms of Calvin-cycle multienzyme complexes with a mol wt of 520 ± 20 kD and 640 ± 25 kD, respectively, were isolated from the cotton (Gossypium hirsutum L.) leaves. Both complexes exhibited the following enzymatic activities: ribose phosphate isomerase, phosphoribulokinase, ribulose bisphosphate carboxylase (Rubisco), phosphoglycerate kinase, and glyceraldehyde phosphate dehydrogenase. The activities of the membrane-bound multienzyme complex were significantly higher than the activities of the free complex. This difference was especially pronounced in the case of carboxylase activity. An increase in the enzymatic activity of membrane-bound multienzyme complex in comparison with the free complex is presumably due to the different number of their constituent parts. Another possible cause is the membrane-level regulation of the functional activity of the enzymes composing the complex.

Formula omitted]-Ribose-5-Phosphate Isomerase from Spinach: Heterologous Overexpression, Purification, Characterization, and Site-Directed Mutagenesis of the Recombinant Enzyme

A cDNA encoding spinach chloroplastic ribose-5-phosphate isomerase (RPI) was cloned and overexpressed in Escherichia coli, and a purification scheme for the recombinant enzyme was developed. The purified recombinant RPI is a homodimer of 25-kDa subunits and shows kinetic properties similar to those of the homodimeric enzyme isolated from spinach leaves (A. C. Rutner, 1970, Biochemistry 9, 178–184). Phosphate, used as a buffer in previous studies, is a competitive inhibitor of RPI with a Ki of 7.9 mM. d-Arabinose 5-phosphate is an effective inhibitor, while d-xylulose-5 phosphate is not, indicating that the configuration at carbon-3 contributes to substrate recognition. Although d-arabinose 5-phosphate binds to RPI, it is not isomerized, demonstrating that the configuration at carbon-2 is crucial for catalysis. Alignment of RPI sequences from diverse sources showed that only 11 charged amino acid residues of the 236-residue subunit are conserved. The possible function of four of these residues was examined by site-directed mutagenesis. D87A, K100A, and D90A mutants show greatly diminished kcat values (0.0012, 0.074, and 0.38% of the wild type, respectively), while E91A retains substantial activity. Only insignificant or moderate changes in Km of d-ribose 5-phosphate are observed for D87A, K100A, and D90A, indicating a direct or indirect catalytic role of the targeted residues.

Subcellular distribution of enzymes of the oxidative pentose phosphate pathway in root and leaf tissues

sketolase, ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase) appear to be restricted to between phosphorylated 3-, 4-, 5-, 6-, and 7-carbon sugars catalysed by the enzymes ribose 5-phosphate isomerase the plastid. In tobacco root and leaf, however, the non-oxidative enzymes were found in the cytosolic as (Rib5P isomerase), ribulose 5-phosphate 3-epimerase (Rul5P 3-epimerase), transaldolase ( TA), and transketolwell as the plastidic compartments. In the absence of ribose 5-phosphate isomerase and ribulose ase ( TK ). With the exception of the transaldolase, these reversible enzymes are amphibolic, and also play a part 5-phosphate 3-epimerase in the cytosol, the product of the oxidative limb of the pathway (ribulose in the Calvin cycle. Their function as part of the pentose phosphate pathway is to provide carbon skeletons for the 5-phosphate) must be transported into a compartment capable of utilizing it. Ribulose 5-phosphate was sup- shikimate pathway via erythrose 4-phosphate, and nucleotide synthesis utilizing ribose 5-phosphate, as well as plied to isolated intact pea root plastids and was shown to be capable of supporting nitrite reduction. recycling of sugar phosphate intermediates for use in the glycolytic pathway (ap Rees, 1985). The kinetics of ribulose 5-phosphate-driven nitrite reduction in isolated pea root plastids suggested that The enzymes of the oxidative portion of the pathway are commonly found in both the cytosolic and plastidic the metabolite was translocated across the plastid envelope in a carrier-mediated transport process, fractions of photosynthetic (Herbert et al., 1979) and non-photosynthetic cells (Ashihara and Komamine, 1976; indicating the presence of a translocator capable of transporting pentose phosphates.

Ribose catabolism of Escherichia coli: characterization of the rpiB gene encoding ribose phosphate isomerase B and of the rpiR gene, which is involved in regulation of rpiB expression.

Escherichia coli strains defective in the rpiA gene, encoding ribose phosphate isomerase A, are ribose auxotrophs, despite the presence of the wild-type rpiB gene, which encodes ribose phosphate isomerase B. Ribose prototrophs of an rpiA genetic background were isolated by two different approaches. Firstly, spontaneous ribose-independent mutants were isolated. The locus for this lesion, rpiR, was mapped to 93 min on the linkage map, and the gene order zje::Tn10-rpiR-mel-zjd::Tn10-psd-purA was established. Secondly, ribose prototrophs resulted from the cloning of the rpiB gene on a multicopy plasmid. The rpiB gene resided on a 4.6-kbp HindIII-EcoRV DNA fragment from phage lambda 10H5 (642) of the Kohara gene library and mapped at 92.85 min. Consistent with this map position, the cloned DNA fragment contained two divergent open reading frames of 149 and 296 codons, encoding ribose phosphate isomerase B (molecular mass, 16,063 Da) and a negative regulator of rpiB gene expression, RpiR (molecular mass, 32,341 Da), respectively. The 5' ends of rpiB- and rpiR-specified transcripts were located by primer extension analysis. No significant amino acid sequence similarity was found between ribose phosphate isomerases A and B, but ribose phosphate isomerase B exhibited high-level similarity to both LacA and LacB subunits of the galactose 6-phosphate isomerases of several gram-positive bacteria. Analyses of strains containing rpiA, rpiB, or rpiA rpiB mutations revealed that both enzymes were equally efficient in catalyzing the isomerization step in either direction and that the construction of rpiA rpiB double mutants was a necessity to fully prevent this reaction.

The ribose 5-phosphate isomerase-encoding gene is located immediately downstream from that encoding murine immunoglobulin κ

The immunoglobulin κ locus ( Igκ) is active only in the B-lymphocyte cell lineage. By exon-trapping we found a gene situated downstream from the murine Igκ locus. This gene encodes a protein with 53% sequence identity to the ribose 5-phosphate isomerase A (RPI-A) of Escherichia coli and is therefore likely to be the murine homologue (m RPI) of this enzyme. We confirmed this assumption by showing that a glutathione S-transferase (GST)::m RPI fusion protein has enzymatic activity and that an anti-m RPI antibody detects a protein of the predicted mass of RPI (33 kDa). Cloning and sequencing of the human counterpart show that the RPI gene is evolutionarily conserved. The expression of m RPI is not influenced by the rearrangement status of the Igκ locus in B cells and m RPI is expressed in all tissues. We thus show that two genes with very different expression patterns, a housekeeping gene and a gene expressed in a tissue-specific manner, can be located on a chromosome in close proximity to each other

Structural and functional properties of a multi‐enzyme complex from spinach chloroplasts

Antibodies have been raised specifically against chloroplast phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase and ribulose 1,5-bisphosphate carboxylase-oxygenase. Each of these antibodies recognizes the same macromolecular entity isolated and purified from chloroplasts. This entity is a multi-enzyme complex, previously isolated and made up of ribose-phosphate isomerase, phosphoribulokinase, ribulose 1,5-bisphosphate carboxylase-oxygenase, phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase. Under denaturing conditions the multi-enzyme complex contains two polypeptides of 54 kDa and 15 kDa corresponding to the large and the small subunits of ribulose 1,5-bisphosphate carboxylase-oxygenase, the two polypeptides of the glyceraldehyde-3-phosphate dehydrogenase of 39 kDa and 37 kDa, one polypeptide of 40 kDa pertaining to phosphoribulokinase and one polypeptide of 30 kDa very likely pertaining to ribose-phosphate isomerase. The combined use of immunochemical and densitometric techniques allows one to determine the number and the stoichiometry of the various types of polypeptide chains and to compare them with the quaternary structure of the corresponding isolated enzymes. Ribulose 1,5-bisphosphate carboxylase-oxygenase of higher plants consists of eight large and eight small subunits. Glyceraldehyde-3-phosphate dehydrogenase is made up of two types of polypeptide chains called A and B and its simplest quaternary structure is A2B2. Finally, phosphoribulokinase is a dimer made up of two identical subunits. Therefore, for the three isolated enzymes, the stoichiometry of the polypeptide chains is always 1:1. Within this multi-enzyme complex, there are two subunits of phosphoribulokinase, two A and B subunits of glyceraldehyde-3-phosphate dehydrogenase and two large and four small subunits of ribulose 1,5-bisphosphate carboxylase-oxygenase. Therefore the number and the stoichiometry of the polypeptide chains of phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase are the same in the multi-enzyme complex and in the free enzymes, but those of ribulose 1,5-bisphosphate carboxylase-oxygenase are completely different. This conclusion that the multi-enzyme complex contains two active sites for ribulose 1,5-bisphosphate may be confirmed independently by kinetic inhibition studies using 6-phosphogluconate.