EspA filament-mediated protein translocation into red blood cells

Type III secretion allows bacteria to inject effector proteins into host cells. In enteropathogenic Escherichia coli (EPEC), three type III secreted proteins, EspA, EspB and EspD, have been shown to be required for translocation of the Tir effector protein into host cells. EspB and EspD have been proposed to form a pore in the host cell membrane, whereas EspA, which forms a large filamentous structure bridging bacterial and host cell surfaces, is thought to provide a conduit for translocation of effector proteins between pores in the bacterial and host cell membranes. Type III secretion has been correlated with an ability to cause contact-dependent haemolysis of red blood cells (RBCs) in vitro . As EspA filaments link bacteria and the host cell, we predicted that intimate bacteria–RBC contact would not be required for EPEC-induced haemolysis and, therefore, in this study we investigated the interaction of EPEC with monolayers of RBCs attached to polylysine-coated cell culture dishes. EPEC caused total RBC haemolysis in the absence of centrifugation and osmoprotection studies were consistent with the insertion of a hydrophilic pore into the RBC membrane. Cell attachment and haemolysis involved interaction between EspA filaments and the RBC membrane and was dependent upon a functional type III secretion system and on EspD, whereas EPEC lacking EspB still caused some haemolysis. Following haemolysis, only EspD was consistently detected in the RBC membrane. This study shows that intimate bacteria–RBC membrane contact is not a requirement for EPEC-induced haemolysis; it also provides further evidence that EspA filaments are a conduit for protein translocation and that EspD may be the major component of a translocation pore in the host cell membrane.     

New Low Molecular Weight Glycopeptide containing Triglucosylcysteine in Human Erythrocyte Membrane

ALTHOUGH protein composition of the erythrocyte membrane has been studied extensively¹, little if anything is known about membrane peptides. Among a variety of chemical agents used for the solubilization of the membrane, solutions of greater than 0.8 M NaCl have been shown to dissolve 50% of membrane proteins^2,3. As we were interested in the composition of the outer surface of the membrane, we have investigated whether lower concentrations of NaCl, which would not cause haemolysis of intact cells, would solubilize certain membrane components.     

Protection of erythrocytes by the macrophage synthesized antioxidant 7,8 dihydroneopterin

Neopterin and the reduced form, 7,8-dihydroneopterin (78NP), are pteridines released from macrophages when stimulated with γ-interferon in vivo. The role of 78NP in inflammatory response is unknown though neopterin has been used clinically as a marker of immune cell activation, due to its very fluorescent nature. Using red blood cells as a cellular model, we demonstrated that micromolar concentrations of 78NP can inhibit or reduce red blood cell haemolysis induced by 2,2′-azobis(amidinopropane)dihydrochloride (AAPH), hydrogen peroxide, or hypochlorite. One hundred μM 78NP prevented HOCl haemolysis using a high HOCl concentration of 5 μmole HOCl/10⁷ RBC. Fifty μM 78NP reduced the haemolysis caused by 2 mM hydrogen peroxide by 39% while the same 78NP concentration completely inhibited haemolysis induced by 2.5 mM AAPH. Lipid peroxidation levels measured as HPLC-TBARS were not affected by addition of 78NP. There was no correlation between lipid oxidation and cell haemolysis suggesting that lipid peroxidation is not essential for haemolysis. Conjugated diene measurements taken after 6 and 12 hour exposure to hydrogen peroxide support the TBARS data. Gel electrophoresis of cell membrane proteins indicated 78NP might inhibit protein damage. Using dityrosine as an indicator of protein damage, we demonstrated 200 μM 78NP reduced dityrosine formation in H₂O₂/Fe⁺⁺ treated red blood cell ghosts by 30%. HPLC analysis demonstrated a direct reaction between 78NP and all three oxidants. Two mM hydrogen peroxide oxidised 119 nM of 78NP per min while 1 mM AAPH only oxidised 50 nM 78NP/min suggesting that 78NP inhibition of haemolysis is not due to 78NP scavenging the primary initiating reactants. In contrast, the reaction between HOCl and 78NP was near instant. AAPH and hydrogen peroxide oxidised 78NP to 7,8-dihydroxanthopterin while hypochlorite oxidation produced neopterin. The cellular antioxidant properties of 78NP suggest it may have a role in protecting immune cells from free radical damage during inflammation.     

Protection of erythrocytes by the macrophage synthesized antioxidant 7,8 dihydroneopterin

Neopterin and the reduced form, 7,8-dihydroneopterin (78NP), are pteridines released from macrophages when stimulated with γ-interferon in vivo. The role of 78NP in inflammatory response is unknown though neopterin has been used clinically as a marker of immune cell activation, due to its very fluorescent nature. Using red blood cells as a cellular model, we demonstrated that micromolar concentrations of 78NP can inhibit or reduce red blood cell haemolysis induced by 2,2'-azobis(amidinopropane)dihydrochloride (AAPH), hydrogen peroxide, or hypochlorite. One hundred μM 78NP prevented HOCl haemolysis using a high HOCl concentration of 5 μmole HOCl/10⁷ RBC. Fifty μM 78NP reduced the haemolysis caused by 2 mM hydrogen peroxide by 39% while the same 78NP concentration completely inhibited haemolysis induced by 2.5 mM AAPH. Lipid peroxidation levels measured as HPLC-TBARS were not affected by addition of 78NP. There was no correlation between lipid oxidation and cell haemolysis suggesting that lipid peroxidation is not essential for haemolysis. Conjugated diene measurements taken after 6 and 12 hour exposure to hydrogen peroxide support the TBARS data. Gel electrophoresis of cell membrane proteins indicated 78NP might inhibit protein damage. Using dityrosine as an indicator of protein damage, we demonstrated 200 μM 78NP reduced dityrosine formation in H₂O₂/Fe⁺⁺ treated red blood cell ghosts by 30%. HPLC analysis demonstrated a direct reaction between 78NP and all three oxidants. Two mM hydrogen peroxide oxidised 119 nM of 78NP per min while 1 mM AAPH only oxidised 50 nM 78NP/min suggesting that 78NP inhibition of haemolysis is not due to 78NP scavenging the primary initiating reactants. In contrast, the reaction between HOCl and 78NP was near instant. AAPH and hydrogen peroxide oxidised 78NP to 7,8-dihydroxanthopterin while hypochlorite oxidation produced neopterin. The cellular antioxidant properties of 78NP suggest it may have a role in protecting immune cells from free radical damage during inflammation.     

Glucose-dependent Protection by Dietary Selenium against Haemolysis of Rat Erythrocytes <Emphasis Type="Italic">in vitro</Emphasis>

THE occurrence in man of drug-induced haemolysis in glucose-6-phosphate dehydrogenase (G6PD) deficient erythrocytes¹ suggested the possibility of an analogy to the haemolysis which occurs in vitamin E deficient red blood cells. Cohen and Hochstein² have shown that haemolysis in G6PD deficient cells is associated with the inability of the cell to generate adequate reduced glutathione (GSH) through GSSG reductase because of the impaired generation of NADPH. Moreover, there is evidence that glucose protects red blood cells from haemolysis by its ability to provide NADPH through G6PD which subsequently generates GSH³. The G6PD deficient cell, however, cannot maintain an adequate concentration of GSH in the cell, even in the presence of glucose⁴, whereas the normal cell can maintain a normal concentration of GSH in the presence of glucose, preserving the integrity of the red blood cell. Vitamin E protects red blood cells from haemolysis whether supplied in vivo or in vitro and its effect has usually been demonstrated without glucose in the incubation medium. Although selenium prevents many of the same deficiency symptoms as vitamin E, it has not been uniformly effective in preventing the in vitro haemolysis of red blood cells. If a protective action of selenium against haemolysis were dependent on the presence of GSH, or if selenium were involved in the generation of GSH, selenium would not be expected to prevent haemolysis unless glucose was present in the incubation medium to provide a constant source of NADPH for the generation of GSH from GSSG through GSSG reductase (Fig. 1).     

Determination of intracellular conductivity from electrical breakdown measurements

The intracellular resistivity (conductivity) of cells can be easily calculated with high accuracy from electrical membrane breakdown measurements. The method is based on the determination of the size distribution of a cell suspension as a function of the electrical field strength in the orifice of a particle volume analyser (Coulter counter). The underestimation of the size distribution observed beyond the critical external field strength leading to membrane breakdown represents a direct access to the intracellular resistivity as shown by the theoretical analysis of the data. The potential and the accuracy of the method is demonstrated for red blood cells and for ghost cells prepared by electrical haemolysis. The average value of 180 omega X cm for the intracellular resistivity of intact red blood cells is consistent with the literature.     

Determination of intracellular conductivity from electrical breakdown measurements

The intracellular resistivity (conductivity) of cells can be easily calculated with high accuracy from electrical membrane breakdown measurements. The method is based on the determination of the size distribution of a cell suspension as a function of the electrical field strength in the orifice of a particle volume analyser (Coulter counter). The underestimation of the size distribution observed beyond the critical external field strength leading to membrane breakdown represents a direct access to the intracellular resistivity as shown by the theoretical analysis of the data. The potential and the accuracy of the method is demonstrated for red blood cells and for ghost cells prepared by electrical haemolysis. The average value of 180 Ω - cm for the intracellular resistivity of intact red blood cells is consistent with the literature.     

The effect of the growth medium composition on the fatty acids of Rhodothermus marinus and ''Thermus thermosphilus'' HB-8

Abstract We have investigated the mechanisms used by Trichomonas vaginalis to damage cellular membranes, using human erythrocytes as target cells. Haemolysis is a contact- and temperature-dependent phenomenon, and is inhibited in 4 mM EGTA. Osmotic protection experiments using carbohydrates with different molecular diameters as protectants demonstrated that the cytolytic activity of T. vaginalis is inhibited in 75 mM stachyose. On the basis of our data, we hypothesize a cytopathic mechanism mediated by the formation of functional pores into the target membrane. Some of the Trichomonas protein involved in haemolysis have been immunologically characterized.     

Structural characterization of platelets and platelet microvesicles

Platelets are blood cells without nuclei, which, in conjunction with fibrin, cause bleeding to stop (hemostasis). Cellular microvesicles are microscopic particles released into extracellular space under activation and/or apoptosis of cells of different types. Platelet microvesicles form the main population of blood circulating through microvesicles and play an important role in the reactions of hemostasis, thrombosis, and many other (patho)physiological processes. Despite the large number of studies that have been devoted to the function of platelet microvesicles, the mechanisms of their formation and structural details remain poorly understood. The ultrastructure of the initial platelets and microvesicles formed in vitro from resting cells and platelets activated by arachidonic acid, ADP, thrombin, and calcium ionophore A23187 is investigated in this study. The intracellular origin, stages of formation, structural diversity, and size of microvesicles were analyzed according to the results of transmission electron microscopy of human platelets and isolated microvesicles. It was shown that thrombin, unlike other activators, not only stimulates microvesiculation of the plasma membrane, but also causes decomposition of cells with the formation of subcellular particles that have sizes comparable with the size of the microvesicles from the outer membrane of the cells. Some of these microparticles are cellular organelles surrounded by a thin membrane. The size of isolated microvesicles ranges from 30 to 500 nm, but their size distribution depends on the nature of the activating stimulus. The obtained results contain new data on the formation of platelet microvesicles and their structural diversity, which are important for understanding of their multiple functions in health and disease.     

Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli

Many mucosal pathogens use type III secretion systems for the injection of effector proteins into target cells. The type III-secreted proteins EspB and EspD of enteropathogenic Escherichia coli (EPEC) are inserted into the target cell membrane. Together with EspA, these proteins are supposed to constitute a molecular syringe, channelling other effector proteins into the host cell. In this model, EspB and EspD would represent the tip of the needle forming a pore into target cell membranes. Although contact-dependent and Esp-mediated haemolytic activity by EPEC has already been described, the formation of a putative pore resulting in haemolysis has not been demonstrated so far. Here, we show that (i) diffusely adhering (DA)-EPEC strains exhibit a type III-dependent haemolytic activity too; (ii) this activity resides in the secreted proteins and, for DA-EPEC strains, in contrast to EPEC strains, does not require bacterial contact; and (iii) pores are introduced into the target cell membrane. Osmoprotection revealed a minimal pore size of 3–5 nm. The pores induced by type III-secreted proteins of DA-EPEC were characterized by electron microscopy techniques. Analysis by atomic force microscopy demonstrated the pores to be composed of six to eight subunits with a lateral extension of 55–65 nm and to be raised 15–20 nm above the membrane plane. We could also demonstrate an association of EspB and EspD with erythrocyte membranes and an interaction of both proteins with each other in vitro . These results, together with the homologies of EspB and EspD to proposed functional domains of other pore-forming proteins (Yop/Ipa), strongly support the idea that both proteins are directly involved in pore formation, which might represent the type III secretion system translocon.     

A monoclonal anticarbohydrate antibody detecting superfast myosin in the masseter muscle

Adrenal medullary chromaffin cells secrete catecholamines through exocytosis of their intracellular chromaffin granules. Osmotic granule swelling has been implicated to play a role in the generation of membrane stress associated with the fusion of the granule membrane. However, controversy exists as to whether swelling occurs before or after the actual fusion event. Using morphometric methods we have determined the granule diameter distributions in rapidly frozen, freeze-substituted chromaffin cells. Our measurements show that intracellular chromaffin granules increase in size from an average of 234 nm to 274 nm or 277 nm in cells stimulated to secrete with nicotine or high external K⁺, respectively. Granule swelling occurs before the formation of membrane contact. Ammonium chloride, an agent which inhibits stimulated catecholamine secretion by approximately 50% by altering the intragranular pH, also inhibits granule swelling. In addition, ammonium chloridetreated secreting cells show more granule-plasma membrane contacts than untreated secreting cells. Sodium propionate induces granule swelling in the absence of secretagogue and has been shown to enhance nicotine- and high K⁺- induced catecholamine release. These results indicate that in adrenal chromaffin cells granule swelling is an essential step in exocytosis before fusion pore formation, and is related to the pH of the granule environment.     

How do the polyene macrolide antibiotics affect the cellular membrane properties?

In the 1970's great strides were made in understanding the mechanism of action of amphotericin B and nystatin: the formation of transmembrane pores was clearly demonstrated in planar lipid monolayers, in multilamellar phospholipid vesicles and in Acholeplasma laidlawii cells and the importance of the presence and of the nature of the membrane sterol was analyzed. For polyene antibiotics with shorter chains, a mechanism of membrane disruption was proposed. However, recently obtained data on unilamellar vesicles have complicated the situation. It has been shown that: membranes in the gel state (which is not common in cells), even if they do not contain sterols may be made permeable by polyene antibiotics, several mechanisms may operate, simultaneously or sequentially, depending on the antibiotic/lipid ratio, the time elapsed after mixing and the mode of addition of the antibiotic, there is a rapid exchange of the antibiotic molecules between the vesicles. Although pore formation is apparently involved in the toxicity of amphotericin B and nystatin, it is not the sole factor which contributes to cell death, since K+ leakage induced by these antibiotics is separate from their lethal action. The peroxidation of membrane lipids, which has been demonstrated for erythrocytes and Candida albicans cells in the presence of amphotericin B, may play a determining role in toxicity concurrently with colloid osmotic effect. On the other hand, it has been shown that the action of polyene antibiotics on cells is not always detrimental: at sub-lethal concentrations these drugs stimulate either the activity of some membrane enzymes or cellular metabolism. In particular, some cells of the immune system are stimulated. Furthermore, polyene antibiotics may act synergistically with other drugs, such as antitumor or antifungal compounds. This may occur either by an increased incorporation of the drug, under the influence of a polyene antibiotic-induced change of membrane potential, for example, or by a direct interaction of both drugs. That fungal membranes contain ergosterol while mammalian cell membranes contain cholesterol, has generally been considered the basis for the selective toxicity of amphotericin B and nystatin for fungi. Actually, in vitro studies have not always borne out this assumption, thereby casting doubt on the use of polyene antibiotics as antifungal agents in mammalian cell culture media.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calmodulin binding to the Fas-mediated death-inducing signaling complex in cholangiocarcinoma cells

We have previously demonstrated that the antagonists of calmodulin (CaM) induce apoptosis of cholangiocarcinoma cells partially through Fas-mediated apoptosis pathways. Recently, CaM has been shown to bind to Fas, which is regulated during Fas or CaM antagonist-mediated apoptosis in Jurkat cells and osteoclasts. Accordingly, the present studies were designed to determine whether Fas interacts with CaM in cholangiocarcinoma cells and to elucidate its role in regulating Fas-mediated apoptosis. CaM bound to Fas in cholangiocarcinoma cells. CaM was identified in the Fas-mediated death inducing signaling complex (DISC). The amount of CaM recruited into the DISC was increased after Fas-stimulation, a finding confirmed by immunofluorescent analysis that demonstrated increased membrane co-localization of CaM and Fas upon Fas-stimulation. Consistently, increased Fas microaggregates in response to Fas-stimulation were found to bind to CaM. Fas-induced recruitment of CaM into the DISC was inhibited by the Ca(2+) chelator, EGTA, and the CaM antagonist, trifluoperazine (TFP). TFP decreased DISC-induced cleavage of caspase-8. Further, inhibition of actin polymerization, which has been demonstrated to abolish DISC formation, inhibited the recruitment of CaM into the DISC. These results suggest an important role of CaM in mediating DISC formation, thus regulating Fas-mediated apoptosis in cholangiocarcinoma cells. Characterization of the role of CaM in Fas-mediated DISC formation and apoptosis signaling may provide important insights in the development of novel therapeutic targets for cholangiocarcinoma.     

Effect of lysophosphatidylcholine on salt permeability through the erythrocyte membrane under haemolytic conditions

Human erythrocytes were incubated in haemolytic salt or sucrose media and the amount of potassium and haemoglobin released were monitored. In hypotonic NaCl and KCl solutions potassium release and haemolysis increased with time showing that the cell membrane had been injured and became permeable to intra- and extracellular cations which, due to intracellular haemoglobin, causes water influx and continuous haemolysis. Both potassium release and haemolysis remained, however, at their 2-minute level in the presence of LPC. Thus, LPC could reseal the membrane and prevent continuous salt fluxes. It protected erythrocytes from hypotonic haemolysis and the protection was more efficient in NaCl than in sucrose media. This suggests that the increase in the critical volume of erythrocytes caused by LPC occurs both in electrolyte and sucrose media, and the additional protection observed in electrolyte media is due to the resealing of the injured cell membrane by LPC. The repairing mechanism was mediated via the membrane lipids or integral proteins, since the time-course of haemolysis of erythrocytes swollen in NaCl media at the spectrin-denaturing temperature of 49.5 degrees C was similar to that at room temperature with and without LPC. LPC did not protect erythrocytes from colloid osmotic haemolysis caused by ammonia influx in an isotonic NH4Cl medium, but protected the cells from colloid osmotic haemolysis caused by sodium influx through nystatin-channels in NaCl media without any area or volume increase. Hence, LPC could not prevent ammonia influx through the lipid bilayer, but suppressed sodium influx through nystatin-channels presumably via LPC interference with cholesterol.     

Metabolic responses of Neisseria gonorrhoeae to human serum and myeloid cells. Adaptation to host defenses?

The interaction of gonococci with host defenses including serum and phagocytic cells has been extensively studied. We have shown that a small molecular weight factor in serum stimulates gonococcal metabolism. This factor has now been isolated by column chromatography and may be released from mammalian cells including phagocytes. Exposure of gonococci to serum decreases membrane fluidity as demonstrated by EPR, seems to reduce OMP shedding, and reduces uptake by PMNS. Serum stimulated gonococci consume O₂ to an extent adequate to interfere with PMN formation of reactive oxygen intermediates. We propose that all of these responses are adaptive and favor survival of this pathogen.     

Heterogeneity and timing of translocation and membrane-mediated assembly of different annexins

Many cell types, including neurons and epithelial cells, express a variety of annexins. Although the overall function has only been partially unravelled, a dominant feature is the formation of two-dimensional assemblies under the plasma membrane in a calcium-dependent manner. Here we show that fluorescently tagged annexins A1, A2, A4, A5, and A6 translocate and assemble at the plasma membrane and the nuclear envelope, except annexin A2, which only attaches to the plasma membrane. All annexins have different response times to elevated calcium levels as was shown by the translocation of co-expressed proteins. Fluorescence recovery after photobleaching revealed the static nature of all annexin assemblies. Analysis of the assemblies by Foerster resonance energy transfer (FRET) using acceptor bleaching demonstrated mostly annexin-specific self-assembly. Heterogeneous assembly formation was shown between annexins A5 and A1, and A5 and A2. The formation of homo- and heterogeneous annexin assemblies may play an important role when high increases in calcium occur, such as after disruption of the plasma membrane.     

Impact of membrane curvature on amyloid aggregation

The misfolding, amyloid aggregation, and fibril formation of intrinsically disordered proteins/peptides (or amyloid proteins) have been shown to cause a number of disorders. The underlying mechanisms of amyloid fibrillation and structural properties of amyloidogenic precursors, intermediates, and amyloid fibrils have been elucidated in detail; however, in-depth examinations on physiologically relevant contributing factors that induce amyloidogenesis and lead to cell death remain challenging. A large number of studies have attempted to characterize the roles of biomembranes on protein aggregation and membrane-mediated cell death by designing various membrane components, such as gangliosides, cholesterol, and other lipid compositions, and by using various membrane mimetics, including liposomes, bicelles, and different types of lipid-nanodiscs.We herein review the dynamic effects of membrane curvature on amyloid generation and the inhibition of amyloidogenic proteins and peptides, and also discuss how amyloid formation affects membrane curvature and integrity, which are key for understanding relationships with cell death. Small unilamellar vesicles with high curvature and large unilamellar vesicles with low curvature have been demonstrated to exhibit different capabilities to induce the nucleation, amyloid formation, and inhibition of amyloid-β peptides and α-synuclein. Polymorphic amyloidogenesis in small unilamellar vesicles was revealed and may be viewed as one of the generic properties of interprotein interaction-dominated amyloid formation. Several mechanical models and phase diagrams are comprehensively shown to better explain experimental findings. The negative membrane curvature-mediated mechanisms responsible for the toxicity of pancreatic β cells by the amyloid aggregation of human islet amyloid polypeptide (IAPP) and binding of the precursors of the semen-derived enhancer of viral infection (SEVI) are also described. The curvature-dependent binding modes of several types of islet amyloid polypeptides with high-resolution NMR structures are also discussed.     

Inhibitors of metalloendoproteases block spiculogenesis in sea urchin primary mesenchyme cells

Metalloendoproteases have been implicated in a variety of fusion processes including plasma membrane fusion and exocytosis. As a prerequisite to skeleton formation in the sea urchin embryo, primary mesenchyme cells undergo fusion via filopodia to form syncytia. The spicule is formed within the syncytial cable by matrix and mineral deposition. To investigate the potential involvement of a metalloendoprotease in spiculogenesis, the effect of inhibitors of this enzyme on skeleton formation was studied. Experiments with primary mesenchyme cells in vitro and in normal embryos revealed that skeleton formation was blocked by these inhibitors. These findings implicate a metalloendoprotease in spiculogenesis; such an enzyme has been demonstrated in homogenates of primary mesenchyme cells. The most likely site of action of the metalloendoprotease is at the cell membrane fusion stage and/or at subsequent events requiring membrane fusion.     

Penetration and entrapment of large particles in erythrocytes by electrical breakdown techniques

Human erythrocytes suspended in isotonic solutions were subjected to haemolysis by application of an electric field pulse to the cell suspension. The field strengths used were 12 and 16kV/cm, respectively; the pulse duration 40 microseconds. The lysed cells showed resealing properties. The permeability change of the membrane generated by the field pulse and by the subsequent osmotic processes were large enough to facilitate the penetration and entrapment of ferritin and Latex particles (diameter: 0.091 and 0.176 micron, respectively) as revealed by electron microscopy. Correct identification of the Latex particles in the electron-micrographs indicated that LOYTER et al. [J. Cell Biol. 66, 292 (1975)], who recently demonstrated the entrapment of Latex spheres in erythrocytes prepared by osmotic haemolysis mistook electron-dense bodies probably consisting of denaturated protein for Latex particles. Under conditions of osmotic haemolysis, carried out according to BODEMANN and PASSOW, particles could only occasionally be detected within the membrane itself and never within the cell interior, suggesting that the electrical haemolysis method is much more effective in the generation of large holes in the membrane.     

Cloning and expression of a cDNA encoding a maize glutathione-S-transferase in E. coli.

The isolation and characterization of a family of maize glutathione-S-transferases (GST's) has been described previously. These enzymes are designated GSTs I, II and III based on size, substrate specificity and responsiveness to safeners. GST III has been shown to act on the herbicide alachlor as well as the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Clones were isolated from a maize cDNA library in lambda gt10. Three clones contained the entire coding region for GST III. The sequences of these clones were consistent with the known amino terminal GST III protein sequence. Moreover, expression of one of these clones in E. coli resulted in a GST activity as measured with both CDNB and alachlor, proving that at least one of the clones encodes an active GST III species. With the enzyme expressed in E. coli it will become possible to study enzyme structure-function relationships ex planta. While a number of different GST proteins are present in maize tissue the GST III gene is present in single or low copy in the genome.