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Triosephosphate isomerase (TIM) is an enzyme of the glycolysis pathway which exists in almost all types of cells. Its structure is the prototype of a motif called TIM-barrel or (α/β) 8 barrel, which is the most common fold of all known enzyme structures. The simplest form in which TIM is catalytically active is a homodimer, in many species of bacteria and eukaryotes, or a homotetramer in some archaea. Here we show that the purified homodimeric TIMs from nine different species of eukaryotes and one of an extremophile bacterium spontaneously form higher order aggregates that can range from 3 to 21 dimers per macromolecular complex. We analysed these aggregates with clear native electrophoresis with normal and inverse polarity, blue native polyacrylamide gel electrophoresis, liquid chromatography, dynamic light scattering, thermal shift assay and transmission electron and fluorescence microscopies, we also performed bioinformatic analysis of the sequences of all enzymes to identify and predict regions that are prone to aggregation. Additionally, the capacity of TIM from Trypanosoma brucei to form fibrillar aggregates was characterized. Our results indicate that all the TIMs we studied are capable of forming oligomers of different sizes. This is significant because aggregation of TIM may be important in some of its non-catalytic moonlighting functions, like being a potent food allergen, or in its role associated with Alzheimer's disease.

Protein structure determination by X-ray crystallography guides structure-function and rational drug design studies. Helminths cause devastating diseases, including schistosomiasis that affects over one-third of the human population. Trematodes from the genus Schistosoma heavily depend on glycolysis; thus enzymes involved in this metabolic pathway are potential drug targets. Here we present a protocol to obtain crystal structures of recombinantly expressed triosephosphate isomerase from S. mansoni (SmTPI) that diffracted in house to a resolution of 2 Å.

Triosephosphate isomerase (TPI), the glycolytic enzyme that catalyzes the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P), has been frequently identified as a target of S-nitrosylation by proteomic studies. However, the effect of S-nitrosylation on its activity has only been explored in plants and algae. Here, we describe the in vitro S-nitrosylation of human TPI (hTPI), and the effect of the modification on its enzymatic parameters. NO-incorporation into the enzyme cysteine residues occurred by a time-dependent S-transnitrosylation from both, S-nitrosocysteine (CySNO) and S-nitrosoglutathione (GSNO), with CySNO being the more efficient NO-donor. Both X-ray crystal structure and mass spectrometry analyses showed that only Cys217 was S-nitrosylated. hTPI S-nitrosylation produced a 30% inhibition of the Vmax of the DHAP conversion to G3P, without affecting the Km for DHAP. This is the first study describing features of human TPI S-nitrosylation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis , we detected 2 strains of T. vaginalis ; the virus-infected (V + ) and uninfected (V - ) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V + compared with V - isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V + isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V + and V - isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

Triose phosphate isomerase (TIM) is a cytoplasmic enzyme of prime importance in the mammalian glycolytic pathway. It has a major role in the conversion of dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. We have successfully purified a stable complex of TIM with β-globin subunit from the sheep kidney using a simple two-step chromatography procedure. It is seen for the first time that TIM is forming a stable complex with β-globin. The purified protein-protein complex was crystallized and preliminary diffraction data were collected at 2.1Å resolution. We further studied guanidinium chloride (GdmCl)-induced denaturation of TIM-β-globin complex by monitoring changes in the mean residue ellipticity at 222nm ([θ] 222 ) and difference absorption coefficient at 406nm (Δε 406 ) at pH 7.5 and 25°C. We have observed that GdmCl-induced denaturation is reversible. Coincidence of normalized transition curves of both physical properties ([θ] 222 and Δε 406 ) suggests that folding/unfolding of TIM and β-subunit proteins is a two-state process. Denaturation curves of [θ] 222 and Δε 406 were used to estimate the stability parameters of the protein-protein complex. This is the first report on the isolation, purification, crystallization and biophysical characterization of the naturally occurring complex of TIM with the β-globin subunit., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background & Objectives: Diarrhoea is the main clinical manifestation caused by intestinal parasitic infections in patients, with special reference to transplant recipients who require careful consideration to reduce morbidity and mortality. Further, molecular characterization of some important parasites is necessary to delineate the different modes of transmission to consider appropriate management strategies. We undertook this study to investigate the intestinal parasitic infections in transplant recipients with or without diarrhoea, and the genotypes of the isolated parasites were also determined., Methods: Stool samples from 38 transplant recipients comprising 29 post-renal, two liver and seven bone marrow transplant (BMT) recipients presenting with diarrhoea and 50 transplant recipients (42 post-renal transplant, eight BMT) without diarrhoea were examined for the presence of intestinal parasites by light microscopy using wet mount, modified Ziehl-Neelsen staining for intestinal coccidia and modified trichrome staining for microsporidia. Genotypes of Cryptosporidium species were determined by multilocus genotyping using small subunit ribosomal (SSUrRNA), Cryptosporidium oocyst wall protein (COWP) and dihydrofolate reductase (DHFR) as the target genes. Assemblage study for Giardia lamblia was performed using triose phosphate isomerase (TPI) as the target gene. Samples were also screened for bacterial, fungal and viral pathogens., Results: The parasites that were detected included Cryptosporidium species (21%, 8/38), Cystoisospora (Isospora) belli (8%, 3), Cyclospora cayetanensis (5%, 2), G. lamblia (11%, 4), Hymenolepis nana (11%, 4), Strongyloides stercoralis (3%, 1) and Blastocystis hominis (3%, 1). Multilocus genotyping of Cryptosporidium species at SSUrRNA, COWP and DHFR loci could detect four isolates of C. hominis; two of C. parvum, one of mixed genotype and one could not be genotyped. All the C. hominis isolates were detected in adult post-renal transplant (PRT) recipients, whereas the C. parvum isolates included a child with BMT and an adult with PRT. Clostridium difficle, cytomegalovirus and Candida albicans were found in 2, 3 and 2 patients, respectively., Interpretation & Conclusions: In the present study, C. hominis was observed as an important parasite causing intestinal infections in transplant recipients. Multilocus genotyping of Cryptosporidium species could detect four isolates of C. hominis; two of C. parvum, one of mixed genotype and one could not be genotyped. Genotyping of G. lamblia revealed that assemblage B was most common., Competing Interests: None.

Triosephosphate isomerase (Tpi) is a glycolytic enzyme that is essential for efficient energy production in many pathogens. However, its function in Mycoplasma gallisepticum has not been fully elucidated. In this study, the mga0357 gene of M. gallisepticum, which encodes TpiA (MGTpiA), was amplified and expressed in Escherichia coli by IPTG induction. The purified recombinant MGTpiA protein exhibited catalytic activity that was similar to TPI from rabbit muscle, reducing NAD(+) to NADH. The MGTpiA was also found to be a surface-exposed protein by western blotting and immunofluorescence assays. In addition, cytadherence inhibition assays confirmed that the cytadherence of M. gallisepticum to the DF-1 cells was significantly inhibited by the anti-MGTpiA serum. The results of the study suggested that MGTpiA plays an important role in the metabolism and closely related to the M. gallisepticum pathogenicity., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10(9) mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.

Triosephosphate isomerase (TIM) is a major enzyme in the glycolytic pathway, which catalyzes the interconversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. Here, we report cloning, expression and purification of a catalytically active recombinant TIM of Leishmania donovani (LdTIM). The recombinant LdTIM had a pH optimum in the range of 7.2-9.0, found stable at 25°C for 30 min and K(m) and V(max) for the substrate glyceraldehyde 3-phosphate was 0.328±0.02mM and 10.05mM/min/mg, respectively. The cysteine-reactive agent methylmethane thiosulphonate (MMTS) was used as probe, in order to test its effect on enzyme activity. The MMTS induced 75% enzyme inactivation within 15 min at 250 μM concentration. The biochemical characterization of LdTIM described in this work is the essential step towards deeper understanding of its role in parasite survival. The purification of LdTIM in bioactive form provides important tools for further functional and structural studies., (Published by Elsevier Inc.)

Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 μmol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The effect of treatments with four herbicides and a safener on the activity of triosephosphate isomerase (TPI) extracted from shoots of Italian ryegrass was investigated. It was found that atrazine and fluorodifen, herbicides which interfere with photosynthesis, caused a decrease in measured enzyme activity. In addition, the in vitro effect of oxidized glutathione (GSSG), a compound produced in situations of oxidative stress, on TPI activity was investigated. It was shown that GSSG was a strong inhibitor of enzyme activity, at low concentrations in a dose-time-dependent manner. The enzyme extracts were submitted to chromatographic purifications and to two-dimensional electrophoresis. Some spots had molecular masses ranging between 20 and 30 kDa and were characterized and identified by LC-ESI-MS/MS as TPIs. The mass spectrometry also made it possible to identify the presence of cysteine residues that could be subjected to S-glutathionylation, which regulate the enzyme activity.

The glycolytic enzyme triosephosphate isomerase (TPI; EC 5.3.1.1) of Staphylococcus aureus is a candidate adhesion molecule for the interaction between the bacterium and the fungal pathogen Cryptococcus neoformans. TPI may recognize the mannan backbone of glucuronoxylomannan (GXM) of C. neoformans. We purified TPI from extracts of S. aureus surface proteins to investigate its binding by surface plasmon resonance analysis. The immobilized TPI reacted with GXM in a dose-dependent manner. Furthermore, the interactions between staphylococcal TPI and alpha-(1-->3)-mannooligosaccharides derived from GXM were examined. The oligosaccharides exhibited binding with TPI; however, monomeric mannose did not. Differences in the slopes of the sensorgrams were observed between oligosaccharides with an even number of residues versus those with an odd number. A heterogeneous ligand-parallel reaction model revealed the existence of at least two binding sites on TPI. The enzymic activities of TPI were inhibited in a dose-dependent manner by alpha-(1-->3)-mannooligosaccharides larger than triose. The binding of TPI and alpha-(1-->3)-mannotriose near the substrate-binding site was predicted in silico (AutoDock 3.05). An oligosaccharide of size equal to or greater than triose could bind to the site, affecting enzymic activities. Moreover, affinities were indicated, especially for biose and tetraose, to another binding pocket, which would not affect enzymic activity. These data suggest a novel role for TPI, in addition to glycolysis, on the surface of S. aureus.

Triosephosphate isomerase from methicillin-resistant Staphylococcus aureus (MRSA252) was cloned in pQE30 vector, overexpressed in Escherichia coli M15 (pREP4) cells and purified to homogeneity. The protein was crystallized from 1.6 M trisodium citrate dihydrate pH 6.5 using the hanging-drop vapour-diffusion method. The crystals belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 79.15, c = 174.27 A. X-ray diffraction data were collected and processed to a maximum resolution of 1.9 A. The presence of two molecules in the asymmetric unit gave a Matthews coefficient (V(M)) of 2.64 A(3) Da(-1), with a solvent content of 53.63%.

Background: Watermelon is a worldwide consumed Cucurbitaceae fruit that can elicit allergic reactions. However, the major allergens of watermelon are not known. The aim of this study is to identify and characterize major allergens in watermelon., Methods: Twenty-three patients allergic to watermelon took part in the study. The diagnosis was based on a history of symptoms and positive skin prick-prick tests to watermelon, confirmed by positive open oral challenge testing to watermelon pulp. Allergenic components were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by N-terminal amino acid sequencing and mass spectrometry. Allergens were purified combining several chromatographic steps., Results: Several IgE binding bands (8-120 kDa) were detected in watermelon extract. Three major allergens were identified as malate dehydrogenase (36 kDa), triose phosphate isomerase (28 kDa) and profilin (13 kDa). Purified allergens individually inhibited IgE binding to the whole watermelon extract., Conclusions: All in all these results indicate that malate dehydrogenase, triose phosphate isomerase and profilin are major allergens involved in watermelon allergy., (Copyright (C) 2009 S. Karger AG, Basel.)

In this article we report on the characterization of the enzymatic synthesis of D-xylulose 5-phosphate using triosephosphate isomerase and transketolase. Two potential starting substrates are possible with this scheme. The data presented here allow a comparison of both routes for the synthesis, based on experimental information on reaction kinetics. Operational guidelines are proposed which should assist in the scale-up of such syntheses.

In this study, a tpi1 gene encoding for the enzyme triose phosphate isomerase in Klebsiella pneumoniae DSM2026 was knocked out in an effort to metabolically engineer this strain as a model system for the production of 1,3-propanediol. Investigations of the tpi1 knockout mutant led to the discovery of a second tpi gene (tpi2) in this organism. The new tpi2 gene was cloned and sequenced. The coding region of the tpi2 gene contains 795bp (base pairs) and the deduced protein consists of 265 amino acids. Sequence comparison of TPI2 proteins in different organisms revealed the presence of a highly conserved signature A-Y-E-P-V-W-A-I-G-[EDVS]-[GKNASH], which is nearly the same as the reported TPI consensus signature. The tpi1 gene of K. pneumoniae DSM2026 shows a high sequence similarity to that of E. coli, whereas, the tpi2 gene resembles more its relatives in the alpha-proteobacteria, suggesting that they evolve from different ancestors. The overexpression of the tpi2 gene restores the growth deficiency of tpi1 knockout mutant on the minimal medium containing glucose or glycerol. Furthermore, the catalytic activity of this new triose phosphate isomerase was confirmed in both tpi1 knockout mutant and tpi2 over-expressing strain by enzyme assays. For the first time, the co-existence of two tpi genes in an enteric bacterium is experimentally confirmed.

The thermostability of hyperthermophile proteins is not easily studied because such proteins tend to be extremely recalcitrant to unfolding. Weeks of exposure to structurally destabilizing conditions are generally required to elicit any evidence of conformational change(s). The main reason for this extreme kinetic stability would appear to be the dominance of local unfolding transitions that occur within different parts of the structures of these molecules; put differently, local sub structural unfolding transitions that occur autonomously and reversibly are thought to fail to cooperate to bring about global unfolding in a facile manner, leading to a low overall observed rate of unfolding. For reasons that are not yet fully understood, unfolding is also reported to occur irreversibly in hyperthermophile proteins. Therefore, conventional experimental approaches are often unsuited to the study of their unfolding. Here, we describe a novel electrophoretic approach that facilitates separation, direct visualization, and quantitation of the folded, partially folded, and unfolded forms of the hyperthermophile protein triosephosphate isomerase from Pyrococcus furiosus, produced in the course of its irreversible structural destabilization by the combined action of heat and chemical agents. Our approach exploits (i) the irreversibility of global unfolding effected by heat and denaturants such as urea or guanidine hydrochloride, (ii) the stability of the native form of the protein to unfolding by the anionic detergent sodium dodecyl sulfate, (iii) the differential susceptibilities of various protein conformations to being bound by SDS, and (iv) the differential electrophoretic migration behavior displayed as a consequence of differential SDS binding.

The susceptibility of a peptide bond to cleavage by a protease is determined by: (a) the flexibility of the protein chain region in which it is located, (b) the extent to which the bond is exposed, and (c) the nature of the local interactions made by the sidechains of its flanking residues. Each of these parameters is known to be influenced by the overall structural stability of the protein; thus, proteins of higher structurally stability commonly show higher resistance to proteolysis. Extrapolating this relationship to 'ultrastable' proteins, our intention here was to investigate whether a hyperthermophile protein expressed and folded within Escherichia coli could prove to be so resistant to proteolysis as to allow direct purification from complex mixtures of E. coli cytoplasmic and/or membrane proteins, through proteolytic means. Thus, we cloned the gene encoding the triosephosphate isomerase enzyme of Pyrococcus furiosus (PfuTIM) and overexpressed it in E. coli in fusion with glutathione S-transferase (GST). The GST-PfuTIM fusion product partitioned mainly into the insoluble fraction of the whole cell lysate. Upon exposure of the E. coli cell lysate precipitate fractions to the non-specific protease, subtilisin, all polypeptides barring PfuTIM (including the GST affinity tag cloned in fusion with PfuTIM) were found to be degraded to undetectable levels. Trace residual amounts of an E. coli protein, OmpF, survived proteolytic digestion, together with an extremely pure population of PfuTIM. Either autonomously or in combination with the more conventional method of heating solutions to enrich heat-stable proteins through the thermal unfolding and aggregation of all other proteins, such proteolysis-based purification could prove to be useful.

Subunit dissociation of dimeric rabbit muscle triosephosphate isomerase (TIM) by hydrostatic pressure has previously been shown not to follow the expected dependence on protein concentration [Rietveld and Ferreira (1996) Biochemistry 35, 7743-7751]. This anomalous behavior was attributed to persistent conformational heterogeneity (i.e., the coexistence of long-lived conformational isomers) in the ensemble of TIM dimers. Here, we initially show that subunit dissociation/unfolding of TIM by guanidine hydrochloride (GdnHCl) also exhibits an anomalous dependence on protein concentration. Dissociation/unfolding of TIM by GdnHCl was investigated by intrinsic fluorescence and circular dichroism spectroscopies and was found to be a highly cooperative transition in which the tertiary and secondary structures of the protein were concomitantly lost. A procedure based on size-exclusion chromatography in the presence of intermediate (0.6 M) GdnHCl concentrations was developed to isolate two conformational isomers of TIM that exhibit significantly different stabilities and kinetics of unfolding by GdnHCl. Complete unfolding of the two isolated conformers at a high GdnHCl concentration (1.5 M), followed by refolding by removal of the denaturant, completely abolished the differences in their unfolding kinetics. These results indicate that such differences stem from conformational heterogeneity of TIM and are not related to any chemical modification of the protein. Furthermore, they add support to the notion that long-lived conformational isomers of TIM coexist in solution and provide a basis for the interpretation of the persistent heterogeneity of this protein.

We produced the Taenia solium triosephosphate isomerase (TPI) in Escherichia coli and compared its biochemical and immunological properties with those of the commercial TPI from Sus scrofa. Taenia solium TPI is a homodimer composed of two 27-kDa monomers, with a specific activity of 5,683 U/mg and a Km value of 0.758, and S. scrofa TPI is also dimeric with similar monomeric molecular weight, specific activity of 4,227 U/mg, and a Km value of 0.51. The catalytic parameters for the isomerization of glyceraldehyde 3-phosphate, affinity between TPI monomers, and kinetic thermal denaturation and inactivation were similar for both enzymes. Anti-T. solium TPI antibodies cross-react weakly with Schistosoma mansoni TPI but do not cross-react with S. scrofa, human, or protozoan TPIs. These antibodies inhibited T. solium TPI activity but did not affect S. scrofa enzymatic activity. Immunizations with 1 microg of the T. solium TPI reduced 52% of cysticerci in a mouse-Taenia crassiceps model 1 mo after challenge. Our findings show that T. solium and S. scrofa TPIs possess similar biochemical and enzymatic properties but do not share immunological properties because anti-T. solium TPI antibodies did not recognize S. scrofa TPI. Inhibition of enzyme activity by anti-TPI antibodies suggests that they can be used as inhibitors of the enzyme.

Plasmodium falciparum triosephosphate isomerase (PfTIM) contains two tryptophan residues, W11 and W168. One is positioned in the interior of the protein, and the other is located on the active-site loop 6. Two single-tryptophan mutants, W11F and W168F, were constructed to evaluate the contributions of each chromophore to the fluorescence of the wild-type (wt) protein and to probe the utility of the residues as spectroscopic reporters. A comparative analysis of the fluorescence spectra of PfTIMwt and the two mutant proteins revealed that W168 possesses an unusual, blue-shifted emission (321 nm) and exhibits significant red-edge excitation shift of fluorescence. In contrast, W11 emits at 332 nm, displays no excitation dependence of fluorescence, and behaves like a normal buried chromophore. W168 has a much shorter mean lifetime (2.7 ns) than W11 (4.6 ns). The anomalous fluorescence properties of W168 are abolished on unfolding of the protein in guanidinium chloride (GdmCl) or at low pH. Analysis of the tryptophan environment using a 1.1-A crystal structure established that W168 is rigidly held by a complex network of polar interactions including a strong hydrogen bond from Y164 to the indole NH group. The environment is almost completely polar, suggesting that electrostatic effects determine the unusually low emission wavelength of W168. To our knowledge this is a unique observation of a blue-shifted emission from a tryptophan in a polar environment in the protein. The wild-type and mutant proteins show similar levels of enzymatic activity and secondary and tertiary structure. However, the W11F mutation appreciably destabilizes the protein to unfolding by urea and GdmCl. The fluorescence of W168 is shown to be extremely sensitive to binding of the inhibitor, 2-phosphoglycolic acid.

Plasmodium falciparum triose-phosphate isomerase, a homodimeric enzyme, contains four cysteine residues at positions 13, 126, 196, and 217 per subunit. Among these, Cys-13 is present at the dimer interface and is replaced by methionine in the corresponding human enzyme. We have investigated the effect of sulfhydryl labeling on the parasite enzyme, with a view toward developing selective covalent inhibitors by targeting the interface cysteine residue. Differential labeling of the cysteine residues by iodoacetic acid and iodoacetamide has been followed by electrospray ionization mass spectrometry and positions of the labels determined by analysis of tryptic fragments. The rates of labeling follows the order Cys-196 > Cys-13 Cys-217/Cys-126, which correlates well with surface accessibility calculations based on the enzyme crystal structure. Iodoacetic acid labeling leads to a soluble, largely inactive enzyme, whereas IAM labeling leads to precipitation. Carboxyl methylation of Cys-13 results in formation of monomeric species detectable by gel filtration. Studies with an engineered C13D mutant permitted elucidation of the effects of introducing a negative charge at the interface. The C13D mutant exhibits a reduced stability to denaturants and 7-fold reduction in the enzymatic activity even under the concentrations in which dimeric species are observed.

In a Hungarian family with severe decrease in triosephosphate isomerase (TPI) activity, 2 germ line-identical but phenotypically differing compound heterozygote brothers inherited 2 independent (Phe240Leu and Glu145stop codon) mutations. The kinetic, thermodynamic, and associative properties of the recombinant human wild-type and Phe240Leu mutant enzymes were compared with those of TPIs in normal and deficient erythrocyte hemolysates. The specific activity of the recombinant mutant enzyme relative to the wild type was much higher (30%) than expected from the activity (3%) measured in hemolysates. Enhanced attachment of mutant TPI to erythrocyte inside-out vesicles and to microtubules of brain cells was found when the binding was measured with TPIs in hemolysate. In contrast, there was no difference between the binding of the recombinant wild-type and Phe240Leu mutant enzymes. These findings suggest that the missense mutation by itself is not enough to explain the low catalytic activity and "stickiness" of mutant TPI observed in hemolysate. The activity of the mutant TPI is further reduced by its attachment to inside-out vesicles or microtubules. Comparative studies of the hemolysate from a British patient with Glu104Asp homozygosity and with the platelet lysates from the Hungarian family suggest that the microcompartmentation of TPI is not unique for the hemolysates from the Hungarian TPI-deficient brothers. The possible role of cellular components, other than the mutant enzymes, in the distinct behavior of TPI in isolated form versus in hemolysates from the compound heterozygotes and the simple heterozygote family members is discussed.

The crystal structure of leishmania triosephosphate isomerase (TIM) complexed with 2-(N-formyl-N-hydroxy)-aminoethyl phosphonate (IPP) highlights the importance of Asn11 for binding and catalysis. IPP is an analogue of the substrate D-glyceraldehyde-3-phosphate, and it is observed to bind with its aldehyde oxygen in an oxyanion hole formed by ND2 of Asn11 and NE2 of His95. Comparison of the mode of binding of IPP and the transition state analogue phosphoglycolohydroxamate (PGH) suggests that the Glu167 side chain, as well as the triose part of the substrate, adopt different conformations as the catalysed reaction proceeds. Comparison of the TIM-IPP and the TIM-PGH structures with other liganded and unliganded structures also highlights the conformational flexibility of the ligand and the active site, as well as the conserved mode of ligand binding.

Several trees with expected heterozygous phenotype for triose-phosphate isomerase (TPI) were discovered in a population of dwarf mountain pine (Pinus mugo Turra) from southern Poland. As the inheritance of this enzyme in pines has not been reported, segregation of allelic variants was tested in eight trees with putative heterozygous phenotypes for two loci, TpiA and TPIB: Linkage between these and some other isozyme loci were studied and evidence for linkage has been found between TpiA and PgdA (r = 0.10) and between TpiB and DiaD (r = 0.36), but in single trees only. The subcellular localization of TPI isozymes was determined by comparing isoenzymes from the total extract with those found in fraction enriched in plastids, prepared by differential gradient centrifugation of cellular organelles. The more slowly migrating TPI-B isozyme is located in plastids.

Background: Wheat flour proteins are allergens for 60% to 70% of bakers with workplace-related respiratory symptoms., Objective: The aim of the study was to investigate the variability of IgE antibody patterns of wheat flour-sensitized bakers and to identify the most frequently recognized allergens., Methods: Water/salt-soluble wheat flour proteins from the cultivar Bussard were separated by using 2-dimensional gel electrophoresis with immobilized pH gradients. IgE-reactive proteins were identified by means of immunoblotting with sera of 10 subjects with baker's asthma. Mass spectrometric fingerprinting was used to identify the proteins most frequently recognized by IgE., Results: The IgE immunoblots obtained with 10 different sera exhibited a remarkable heterogeneity. Each patient showed an individual IgE-binding pattern with 4 to 50 different allergen spots. Altogether, more than 100 IgE-binding protein spots were detected. Nine of the predominant IgE-binding protein spots were identified by using mass spectrometric fingerprinting. The obtained masses matched 2 different isoforms of glycerinaldehyde-3-phosphate dehydrogenase from Hordeum vulgare, triosephosphate isomerase from H vulgare, and serpin, a serine proteinase inhibitor from Triticum aestivum., Conclusions: The results show a great interindividual variation of IgE-binding patterns of wheat flour proteins in baker's asthma. The clinical relevance of the identified 4 new allergens will be further investigated in the near future.

Two-dimensional electrophoresis and modern image analysis systems have made it possible to study protein expression and regulation of proteins in biological systems. Proteins in the molecular mass region of 20-120 kDa are well investigated and described. However, proteins with masses below 20 kDa are the least investigated as they are rarely seen on 2D-PAGE due to fast migrations in the electric field and lack of staining efficiency. This paper describes a technique that enriches proteins in the lower mass region using solid-phase extraction. The purification step is carried out using C18 functionalised "restricted access" affinity chromatography whereby simultaneous trace enrichment and sample clean up is achieved. In this study expression patterns of TGF-beta stimulated and non-stimulated fibroblasts were compared after the solid-phase fractionation procedure. An increased expression pattern was obtained whereby 400 protein spots could be detected by image analysis in the <20-kDa region. Out of these, specific regulations of 14 spots were found by quantitative image analysis and spots of interest were identified with MALDI TOF-MS. The regulated and identified proteins were triosephosphate isomerase, cofilin and heat shock 27-kDa protein.

The dimeric enzyme triosephosphate isomerase (TPI) converts glyceraldehyde-3-phosphate to dehydroxyacetone phosphate, a key reaction in glycolysis. Previous studies of the native enzyme in the human blood-flukes belonging to the genus Schistosoma have indicated that TPI is a promising anti-schistosome vaccine antigen. However, a recombinant form of the enzyme is required as an alternative to the impractical option of using biochemically purified TPI obtained from worm tissue for large-scale vaccine use. We previously cloned and sequenced a full-length cDNA encoding the TPI of the Asian (Chinese strain) schistosome Schistosoma japonicum (SjcTPI). We now report very high level bacterial expression of this cDNA and the subsequent purification of the recombinant protein to >98% homogeneity under nondenaturing conditions. The recombinant SjcTPI (re-SjcTPI) was shown to be enzymatically active with a specific activity of 7687 units/mg protein, an activity higher than that of commercially obtained porcine TPI tested concurrently under the same assay conditions. The K(m) value for the re-SjcTPI using glyceraldehyde-3-phosphate as substrate was 406.7 microM, which is similar to the K(m) values reported for the yeast enzyme and various mammalian TPIs. With the availability of substantial amounts of enzymatically active and readily purified re-SjcTPI made in bacteria we can now test whether the recombinant protein can induce a similar level of protection in vaccination/challenge experiments as the native, biochemically purified enzyme., (Copyright 1999 Academic Press.)

Lack of triose phosphate isomerase activity (TIM) is of special interest because this enzyme works at an important branch point of glycolytic flux. In this paper, we report the cloning and sequencing of the Kluyveromyces lactis gene encoding TIM. Unlike Saccharomyces cerevisiae DeltaTPI1 mutants, the K. lactis mutant strain was found to be able to grow on glucose. Preliminary bioconversion experiments indicated that, like the S. cerevisiae TIM-deficient strain, the K. lactis TIM-deficient strain is able to produce glycerol with high yield.

In previous work we tested what three amino acid sequences could serve as a protein hinge in triosephosphate isomerase [Sun, J., and Sampson, N. S. (1998) Protein Sci. 7, 1495-1505]. We generated a genetic library encoding all 8000 possible 3 amino acid combinations at the C-terminal hinge and selected for those combinations of amino acids that formed active mutants. These mutants were classified into six phylogenetic families. Two families resembled wild-type hinges, and four families represented new types of hinges. In this work, the kinetic characteristics and thermal stabilities of mutants representing each of these families were determined in order to understand what properties make an efficient protein hinge, and why all of the families are not observed in nature. From a steady-state kinetic analysis of our mutants, it is clear that the partitioning between protonation of intermediate to form product and intermediate release from the enzyme surface to form methylglyoxal (a decomposition product) is not affected. The two most impaired mutants undergo a change in rate-limiting step from enediol formation to dihydroxyacetone phosphate binding. Thus, it appears that k(cat)/K(m)'s are reduced relative to wild type as a result of slower Michaelis complex formation and dissociation, rather than increased loop opening speed.

The conformational stability of Plasmodium falciparum triosephosphate isomerase (TIMWT) enzyme has been investigated in urea and guanidinium chloride (GdmCl) solutions using circular dichroism, fluorescence, and size-exclusion chromatography. The dimeric enzyme is remarkably stable in urea solutions. It retains considerable secondary, tertiary, and quaternary structure even in 8 M urea. In contrast, the unfolding transition is complete by 2.4 M GdmCl. Although the secondary as well as the tertiary interactions melt before the perturbation of the quaternary structure, these studies imply that the dissociation of the dimer into monomers ultimately leads to the collapse of the structure, suggesting that the interfacial interactions play a major role in determining multimeric protein stability. The Cm(urea)/Cm(GdmCl) ratio (where Cm is the concentration of the denaturant required at the transition midpoint) is unusually high for triosephosphate isomerase as compared to other monomeric and dimeric proteins. A disulfide cross-linked mutant protein (Y74C) engineered to form two disulfide cross-links across the interface (13-74') and (13'-74) is dramatically destablized in urea. The unfolding transition is complete by 6 M urea and involves a novel mechanism of dimer dissociation through intramolecular thiol-disulfide exchange.

Recombinant triosephosphate isomerase (TIM) from a hyperthermophilic Archaeon, Pyrococcus woesei, has been crystallized. Three crystal forms have been obtained: monoclinic, orthorhombic and hexagonal. The monoclinic crystals belong to space group P21 with cell dimensions a = 79.1, b = 89.2, c = 145.4 A and beta = 92.8 degrees, and diffract to at least 2.6 A. The orthorhombic crystals belong to space group P21212 with a = 89.4, b = 155.9, c = 79.5 A, and diffract to 2.9 A. Diffraction from the hexagonal form showed extensive disorder. The monoclinic form contains two tetramers in the asymmetric unit, which are in the same orientation but related by a pseudo-centering. The orthorhombic form contains one tetramer in the asymmetric unit which is in approximately the same orientation as in the monoclinic form. Knowledge of the structure of this hyperthermostable TIM, which is tetrameric in contrast to dimeric forms previously observed, will add to our understanding of protein thermostability.

The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins.

Objectives: The objective of this study was to establish the identity of a protein found in high concentrations in squamous metaplasia of the bladder., Design and Methods: The protein was isolated and subjected to a series of physical, chemical, and catalytic studies., Results: In the normal urothelium the protein was confined to a juxtanuclear pattern on the luminal side of the umbrella cells; in squamous metaplasia and squamous cell carcinoma the protein was increased and exhibited a more diffuse intracellular distribution. The protein was found to be identical to triosephosphate isomerase (EC 5.3.1.1; TPI) with respect to its immunological properties, native and subunit molecular weights, electrophoretic mobility, catalytic activity, and amino acid sequence., Conclusions: While the basis for the altered distribution of TPI remains to be established, the increased amounts of the protein in urine or bladder tissue may be indicative of squamous metaplasia, squamous cell carcinoma, or other bladder injuries.

Triosephosphate isomerase (TIM) is a very stable dimer. In order to understand better the importance of dimerization for stability and catalytic activity, we have constructed a monomeric double-mutation variant. The dimer interface residues Thr75 and Gly76, which are at the tip of loop 3, have been substituted by an arginine and a glutamate, respectively. In wild type TIM, these two residues are at a distance of 27 A from the active site (as measured within the same subunit). This new variant, RE-TIM, was expressed in Escherichia coli, purified to homogeneity, and biochemically characterized. Sedimentation equilibrium ultracentrifugation runs showed that RE-TIM is a monomer in solution. Far-UV CD spectra indicate that this new variant is folded properly and that the secondary structure contents of RE-TIM are similar to those of wild type TIM. The monomeric RE-TIM has residual TIM activity. The thermal stability of RE-TIM is lower than that for wild type TIM. CD melting curves for RE-TIM and wild type TIM show Tm values of 52 and 57 degrees C, respectively, in the presence of the active site ligand 2-phosphoglycolate at 1 mM. Previously, we have characterized two other monomeric forms of TIM: monoTIM and H47N-TIM. The properties of RE-TIM, H47N-TIM, and monoTIM are compared, and it is argued that the properties of RE-TIM will be very similar to those of wild type monomeric subunits. This implies that wild type monomeric subunits have some stability and are catalytically active. It is also inferred that these monomeric subunits have flexible loops which rigidify at the dimer interface on dimerization, causing a 1000-fold increase of kcat and a 10-fold decrease of Km.

We have isolated a cDNA clone of the glycolytic enzyme, triosephosphate isomerase (TPI) from Entamoeba histolytica. Degenerate oligonucleotides obtained by reverse translation of conserved polypeptide sequences, derived from TPIs of other organisms, were used to amplify a 450-bp fragment using E. histolytica cDNA as a template. The fragment was used to screen a cDNA library. The isolated cDNA, encoding a protein of 261 amino acids, shares 43-52.6% positional identity with other known protozoan TPIs. The catalytic residues were conserved; nevertheless, several indels occurred at other regions in the protein sequence. The complete coding sequence of the E. histolytica TPI gene was cloned into the expression vector pRSET and expressed as a wild-type TPI enzyme (E. histolytica TPI) and as a fusion protein with an N-terminal tail of six histidine residues E. histolytica TPI-His6); both recombinant proteins were purified. Molecular modeling of E. histolytica TPI showed an identical topology to the known structures of other TPI molecules, but with a remarkable feature; more than 10 inserted residues are located in the same region of the molecular surface. Studies were performed to detect possible changes that might be caused by the inserted amino acids. The catalytic activity and oligomeric state of the purified protein were similar to that reported for TPI from other sources. In contrast, stability towards dilution, as well as thermal inactivation and unfolding assays, showed that E. histolytica TPI is significantly more stable towards denaturation than Trypanosoma brucei TPI.

The gene that encodes for triosephosphate isomerase from Trypanosoma cruzi was cloned and sequenced. In T. cruzi, there is only one gene for triosephosphate isomerase. The enzyme has an identity of 72% and 68% with triosephosphate isomerase from Trypanosoma brucei and Leishmania mexicana, respectively. The active site residues are conserved: out of the 32 residues that conform the interface of dimeric triosephosphate isomerase from T. brucei, 29 are conserved in the T. cruzi enzyme. The enzyme was expressed in Escherichia coli and purified to homogeneity. Data from electrophoretic analysis under denaturing techniques and filtration techniques showed that triosephosphate isomerase from T. cruzi is a homodimer. Some of its structural and kinetic features were determined and compared to those of the purified enzymes from T. brucei and L. mexicana. Its circular dichroism spectrum was almost identical to that of triosephosphate isomerase from T. brucei. Its kinetic properties and pH optima were similar to those of T. brucei and L. mexicana, although the latter exhibited a higher Vmax with glyceraldehyde 3-phosphate as substrate. The sensitivity of the three enzymes to the sulfhydryl reagent methylmethane thiosulfonate (MeSO2-SMe) was determined; the sensitivity of the T. cruzi enzyme was about 40 times and 200 times higher than that of the enzymes from T. brucei and L. mexicana, respectively. Triosephosphate isomerase from T. cruzi and L. mexicana have the three cysteine residues that exist in the T. brucei enzyme (positions 14, 39, 126, using the numbering of the T. brucei enzyme); however, they also have an additional residue (position 117). These data suggest that regardless of the high identity of the three trypanosomatid enzymes, there are structural differences in the disposition of their cysteine residues that account for their different sensitivity to the sulfhydryl reagent. The disposition of the cysteine in triosephosphate isomerase from T. cruzi appears to make it unique for inhibition by modification of its cysteine.

Triosephosphate isomerase (TIM) of the hyperthermophilic Archaea Pyrococcus woesei and Methanothermus fervidus have been purified to homogeneity. The enzymes from the two hyperthermophiles represent homo-tetramers of 100 kDa, contrary to all known bacterial and eukaryotic TIMs, which are dimers of 48-60 kDa. Molecular size determination of the TIM from the mesophilic methanogen Methanobacterium bryantii yielded the usual molecular mass of only 57 kDa, indicating that the tetrameric aggregation state does not represent an archaeal feature but rather correlates with thermoadaptation. A similar preference for higher protein aggregates in hyperthermophilic Archaea has previously been demonstrated for 3-phosphoglycerate kinases. The gene of the P. woesei TIM was cloned and sequenced. The archaeal TIM proved to be homologous to its bacterial and eukaryotic pendants. Most strikingly, the deduced protein sequence comprises only 224 residues and thus represents the shortest TIM sequence known as yet. Taking the three-dimensional structure of the eucaryal TIM as a basis, from the shortenings of the chain considerable rearrangements at the bottom of the alpha/beta barrel and at its functionally inactive flank are expected, which are interpreted in terms of the formation of new subunit contacts.

Triosephosphate isomerase (TPI) provides an excellent model for terminal marking and protein degradation. Mammalian TPI is terminally modified by deamidation at Asn71-Gly, resulting in unfolding, dissociation, and proteolysis. In contrast, chicken TPI, which contains a lysine at position 71, is terminally modified by the oxidation of Cys126. Thus, both deamidation and oxidation initiate degradation of TPI from different species. To explore the terminal marking in other avians, we have purified the turkey TPI to homogeneity and determined its characteristics. Although the molecular properties of the turkey and chicken TPI were very similar, their tolerances to temperature, oxidants, and alkaline pH were very different. For example, chicken TPI was inactivated 80% in either 10 mM oxidized glutathione or H2O2, whereas 120 mM GSSG had no effect on turkey TPI, and > 120 mM H2O2 was needed for 80% inactivation. Under alkaline conditions that cause rapid deamidation of the mammalian TPI, neither avian enzyme deamidated. Chicken TPI, however, aggregated. Aggregation was reversed by 2-mercaptoethanol. Under prolonged exposure to milder conditions the turkey enzyme was completely inactivated and deamidated at Asn15-Gly. Thus, there are marked differences in the susceptibility of these two avian enzymes to oxidation and deamidation, and their terminal marking mechanisms appear to be different.

Phosphoglycerate kinase (PGK) from the hyperthermophilic bacterium Thermotoga maritima has been purified to homogeneity. A second larger enzyme with PGK activity and identical N-terminal sequence was also found. Surprisingly, this enzyme displayed triosephosphate isomerase (TIM) activity. No other TIM is detectable in T. maritima crude extracts. As shown by ultracentrifugal analysis, PGK is a 43 kDa monomer, whereas the bifunctional PGK-TIM fusion protein is a homotetramer of 240-285 kDa. SDS-PAGE indicated a subunit size of 70 kDa for the fusion protein. Both enzymes show high thermostability. Measurements of the catalytic properties revealed no extraordinary results. pH optima, Km values and activation energies were found to be in the range observed for other PGKs and TIMs investigated so far. The corresponding pgk and tpi genes are part of the apparent gap operon of T. maritima. This gene segment contains two overlapping reading frames, where the 43 kDa PGK is encoded by the upstream open reading frame, the pgk gene. On the other hand, the 70 kDa PGK-TIM fusion protein is encoded jointly by the pgk gene and the overlapping downstream open reading frame of the tpi gene. A programmed frameshift may be responsible for this fusion. A comparison of the amino acid sequence of both the PGK and the TIM parts of the fusion protein with those of known PGKs and TIMs reveals high similarity to the corresponding enzymes from different procaryotic and eucaryotic organisms.

Triosephosphate isomerase (EC 5.3.1.1) from Lactococcus lactis was purified to electrophoretic homogeneity. Approximately 3 mg purified enzyme (specific activity 3300 U mg-1) was obtained from 70 g (wet wt) cells. In solution, triosephosphate isomerase (pI 4.0-4.4) was observed to exist as a homodimer (M(r) 57,000) of noncovalently linked subunits. The sequence of the first 37 amino acid residues from the NH2-terminus were determined by step-wise Edman degradation. This sequence, and that of a region conserved in all known bacterial triosephosphate isomerases, was used to design oligonucleotide primers for the synthesis of a lactococcal tpi probe by PCR. The probe was used to isolate a molecular clone of tpi from a lambda GEM11 library of L. lactis LM0230 DNA. The nucleotide sequence of tpi predicted a protein of 252 amino acids with the same NH2-terminal sequence as that determined for the purified enzyme and a subunit M(r) of 26,802 after removal of the NH2-terminal methionine. Escherichia coli cells harbouring a plasmid containing tpi had 15-fold higher triosephosphate isomerase activity than isogenic plasmid-free cells, confirming the identity of the cloned gene. Northern analysis of L. lactis LM0230 RNA showed that a 900 base transcript hybridized with tpi. The 5' end of the transcript was determined by primer extension analysis to be a G located 65 bp upstream from the tpi start codon. These transcript analyses indicated that in L. lactis, tpi is expressed on a monocistronic transcript. Nucleotide sequencing indicated that the DNA adjacent to tpi did not encode another Embden-Meyerhoff-Parnas pathway enzyme. The location of tpi on the L. lactis DL11 chromosome map was determined to be between map coordinates 1.818 and 1.978.

Apparent physical interaction between pea chloroplast (Pisum sativum L.) glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase (EC 4.1.2.13) is seen in phase-partitioning, fluorescent-anisotropy and isoelectric-focusing experiments. Similarly, results obtained in phase-partitioning and isoelectric-focusing experiments indicate physical interaction between aldolase and triose-phosphate isomerase (EC 5.3.1.1). Kinetic experiments suggest that both aldolase-bound glyceraldehyde-3-phosphate can act as substrate for glyceraldehyde-3-phosphate dehydrogenase. These results are consistent with the notion that there is interaction between these three enzymes both during photosynthetic CO2 fixation and during glycolysis in the chloroplast.

The enzyme triosephosphate isomerase (TPI) was purified to homogeneity from the mosquito Culex tarsalis. Anti-C. tarsalis TPI antibodies cross-reacted with TPIs from other organisms but bands on western blots were most intense with proteins from closely related Dipterans. Using a degenerate primer corresponding to the amino-terminal sequence of the protein in a polymerase chain reaction (PCR), a cDNA corresponding to the TPI gene (Tpi) was isolated and sequenced. Subsequently, a genomic sequence including 305 bp to the 5'-end of the coding sequence was obtained. Comparison of C. tarsalis Tpi to that of Drosophila melanogaster revealed that although the two genes had little similarity in the intron and 5' flanking sequences, they were highly similar (73% identity) in their coding sequence. The rate of synonymous substitution in insect genes may be slower than that of vertebrates, but the nonsynonymous substitution rate, and hence the rate of TPI evolution, appears to be faster in insects than in vertebrates.

Chloroplast and cytosolic triosephosphate isomerases from spinach were separated and purified to homogeneity. Both enzymes were partially sequenced by Edman degradation. Using degenerate primers designed against the amino acid sequences, a homologous probe for the chloroplast enzyme was amplified and used to isolate several full-size cDNA clones. Chloroplast triosephosphate isomerase is encoded by a single gene in spinach. Analysis of the chloroplast cDNA sequence in the context of its homologues from eukaryotes and eubacteria reveals that the gene arose through duplication of its preexisting nuclear counterpart for the cytosolic enzyme during plant evolution.

The gene of triose-phosphate isomerase in Leishmania mexicana has been cloned and characterized. The gene encodes a polypeptide of 251 amino acids, with a calculated molecular mass of 27,561 Da and a net charge of +2. Only one gene could be detected, although the enzyme is present in two different compartments of the cell, in microbody-like organelles called glycosomes and in the cytosol. The primary structure of the enzyme has many features in common with that of triose-phosphate isomerase in the related organism Trypanosoma brucei. Their sequences are 68% identical. The residues constituting the subunit interface are highly conserved between the enzyme of L. mexicana and T. brucei, but are mostly different from those in the enzyme of other organisms. One major substitution was detected in the interface region of the L. mexicana protein: a glutamate was found at position 66, instead of glutamine in all other available 20 sequences. The glutamine is thought to be important for the stability of the dimeric enzyme. L. mexicana triose-phosphate isomerase has been overexpressed in Escherichia coli. Growth conditions were established to obtain high levels of soluble and active protein. The enzyme has been purified to near homogeneity. It appears a stable dimeric protein with a specific activity of 5500 units/mg protein, a subunit mass of 28 kDa and an isoelectric point of 9.0. The enzyme has also been partially purified from glycosomes of cultured L. mexicana promastigotes. Some kinetic properties of the recombinant protein have been compared with those of the promastigote enzyme and with the values previously reported for the T. brucei enzyme. The kinetics of the different enzyme preparations were very similar. For the recombinant enzyme the following values were measured: with glyceraldehyde 3-phosphate as substrate Km = 0.30 +/- 0.05 mM and kcat = 2.5 x 10(5) min-1; with dihydroxyacetone phosphate as substrate Km = 1.3 +/- 0.3 mM and kcat = 2.8 x 10(4) min-1.