Partial Biochemical Characterization Research Articles

Partial biochemical and immunological characterization of an isolated 52.1 kDa pregnancy-associated glycoprotein charge variant

Pregnancy-associated glycoproteins (PAGs) are synthesized in the placental cells of ruminants and are detectable in blood, milk, and urine. Many of these proteins have been obtained and characterized from placental extracts by precipitation with 80% ammonium sulfate. The possibility of purifying PAGs by precipitation with other concentrations of ammonium sulfate remains unexplored. We aimed to study PAG proteins obtained from extracts of ovine placenta at 100 days of gestation through precipitation with 40% ammonium sulfate (Extract 40). The main protein complex (130kDa) was obtained after Extract 40 precipitation. Under reducing SDS-PAGE conditions, the 130kDa complex dissociated in two PAG proteins with apparent molecular weights of 52.1kDa and 26.1kDa. The 130kDa protein appeared to be a molecular complex consisting of two copies of the 52.1kDa protein linked to one copy of the 26.1kDa protein, presumably by disulfide bonds. Furthermore, the 52.1kDa protein consisted of at least three isoforms with distinct isoelectric points. Amino acid microsequencing of the 52.1kDa protein revealed a chimeric structure containing amino acid sequences of PAG1, PAG4, PAG6, and PAG1-like proteins. This procedure recovered a novel 130kDa protein complex composed of 26.1kDa and two 52.1kDa PAGs. To the best of our knowledge, this has not been previously reported as heterologous polymeric molecules.

Production and Partial Biochemical Characterization of Multiple Forms of Lipases

Production and Partial Biochemical Characterization of Multiple Forms of Lipases

L-asparaginase from Aspergillus oryzae spp.: effects of production process and biochemical parameters

L-asparaginases prevent the formation of acrylamide, a substance commonly found in foods subjected to heat and that also contains reducing sugars and L-asparagine. This work aimed to select a strain of Aspergillus spp. able to produce L-asparaginase and to optimize the fermentation parameters, the partial purification and biochemical characterization were also performed. The Aspergillus oryzae IOC 3999 was selected due to its greater enzymatic activity: 1443.57 U/mL of L-asparaginase after 48 h of fermentation. The optimized conditions allowed for an increase of 223% on the L-asparaginase production: 2.9% lactose, 2.9% L-asparagine and 0.7% hydrolyzed casein, 0.152% KH2PO4, 0.052% KCl and MgSO4, 0.001% of CuNO3.3H2O, ZnSO4.7H2O and FeSO4.7H2O adjusted to pH 7.0; added a concentration of 5.05x106 spores/mL at 30 °C for 100 rpm. A purification factor of 2.11 was found and the molecular mass was estimated at 20.8 kDa. The enzyme showed optimum activity at 60 °C and pH 5 and stability at 50 °C for 1 h. The enzyme presented desirable biochemical characteristics, mainly the acid pH stability, indicating that the enzyme would work well in food matrices due to the closeness of pH, meaning that it could be a potential option for use in the food industry.

Purification and characterization of fibrinolytic protease from Streptomyces parvulus by polyethylene glycol-phosphate aqueous two-phase system.

Fibrinolytic proteases are a promising alternative in the pharmaceutical industry, they are used in the treatment of cardiovascular diseases, especially thrombosis. Microorganisms are the most interesting source of fibrinolytic proteases. The aim of this study was the production of fibrinolytic protease from Streptomyces parvulus DPUA 1573, the recovery of the protease by aqueous two-phase system and partial biochemical characterization of the enzyme. The aqueous two-phase system was performed according to a 24-full factorial design using polyethylene glycol molar mass, polyethylene glycol concentration, citrate concentration and pH as independent variables. It was analyzed the effect of different ions, surfactants, inhibitors, pH and temperature on enzyme activity. The best conditions for purifying the enzyme were 17.5% polyethylene glycol 8,000, 15% Phosphate and pH 8.0, it was obtained a partition coefficient of 7.33, a yield of 57.49% and a purification factor of 2.10-fold. There was an increase in enzyme activity in the presence of Fe2+ and a decrease in the presence of $\beta$-Mercaptoethanol, phenylmethylsulfonyl fluoride and Iodoacetic acid. The optimum pH was 7.0 and the optimum temperature was 40 ºC. The purified protease exhibited a molecular mass of 41 kDa. The fibrinolytic protease from Streptomyces parvulus proved to be a viable option for the development of a possible drug with fibrinolytic action.

Partial purification and biochemical characterization of an alkaline esterase from Sorghum bicolor

Esterases are enzymes that present good potential for industrial applications since they catalyze the formation or cleavage of ester bonds in water-soluble substrates, and sorghum seeds can represent an alternative source of this enzyme. The extraction of esterase from sorghum seeds is an economical alternative to obtain an enzyme of great interest. Esterases may improve the quality or accelerate the maturation of cheeses, cured bacon and fermented sausages and may also resolve racemic mixtures. Recently, seed esterases have been the focus of much attention as biocatalysts. In some cases, these enzymes present advantages over animal and microbial lipases due to some quite interesting features such as specificity and low cost, being a great alternative for their commercial exploitation as industrial enzymes The esterase studied here was extracted from sorghum seeds and some of its biochemical properties determined using synthetic substrates (p-nitrophenyl butyrate, caprylate, laurate and palmitate). The enzyme presented optimum activity at pH 8.0 and was stable in all the pH ranges studied. The optimum temperature for its activity was 40ºC but it showed low stability at this temperature (40% relative activity). The values derived for Km and Vmax were 0.67mM and 125 U.mg-1, respectively, obtained using p-nitrophenyl butyrate as the substrate. The enzyme showed an increase in activity when K2HPO4 was added to the reaction medium, but the ions Mn2+, CO+, Hg+ and Fe2+ strongly inhibited the enzyme activity. This enzyme showed a preference for the hydrolysis of short chain fatty acids. The characteristics of sorghum esterase are very similar to those of the microbial esterases used in detergent processing.

Evaluation of hydrolytic enzyme activities from digestive fluids of Anthonomus grandis (Coleoptera: Curculionidae)

The cotton boll weevil, Anthonomus grandis, is a major pest of cotton crops in South America. In this work, partial biochemical characterizations of (hemi) cellulases and pectinases activities in the digestive system (head- and gut- extracts) of A. grandis were evaluated. Gut extract section from third instar larvae exhibited endoglucanase, xylanase, β-glucosidase, and pectinase activities. The endoglucanase and xylanase activities were localized in the foregut, whereas β-glucosidase activity was mainly detected in the hindgut. In addition, no difference in pectinase activity was observed across the gut sections. Thus, A. grandis digestive system is a potentially interesting reservoir for further lignocellulolytic enzymes research.

Keratinases from Coriolopsis byrsina as an alternative for feather degradation: applications for cloth cleaning based on commercial detergent compatibility and for the production of collagen hydrolysate.

Keratinases are proteolytic enzymes that emerge as an alternative for dealing with the disposal of chicken feathers. In this study, we aimed to investigate the keratin-degrading enzymes secreted by the fungus Coriolopsis byrsina and their partial biochemical characterization to adapt their use for keratin decomposition, detergent additive applications, and collagen degradation. We observed the secretion of different proteolytic enzymes that possessed caseinolytic activity that peaked at pH 7.0-9.0 and 60-70 °C and at pH 10.5 and 55-60°C, and keratinolytic activity that reached a maximum at pH 7.0-7.5 and 40-55ºC and at pH 9.0 and 55 °C. Keratinolytic activity was maintained at approximately 63% of residual activity for 1h at 50 °C. The caseinolytic activity at pH 10.5 remains stable until 1h at 50 °C, and this is in contrast to the activity at pH 8.5, where the residual activity was 50%. Caseinolytic activity was inhibited only by PMSF, while keratinolytic activity was inhibited by PMSF and EDTA. When investigating the application of C. byrsina peptidases as an additive to commercial detergent, we observed an egg stain removal performance that was similar to that demonstrated by the commercial detergent. Based on their activity and stability at alkaline pH, these enzymes appear to be attractive candidates for use in the detergent industry. Additionally, the collagenolytic activity of these enzymes potentially allows for their use in a wide array of industrial sectors that require collagenolytic enzymes, such as for the production of collagen hydrolysates from residues derived from the meat industry.

Utilization of agroindustrial byproducts for the production of lipase by a new strain of Pseudomonas sp.

This study aimed to evaluate the production of lipases by a new strain of Pseudomonas sp. Using fermentation medium containing byproducts of poultry meat or soybean oil industry. The results indicate that chicken fat and soybean gum induced 48.3 U/mL and 93.3 of lipase activity, respectively. However, the higher lipase production was obtained when the crude lecithin gum was used, archiving 272.6 U/ml of activity after 24 hours. The partial biochemical characterization of the enzyme showed that the optimum reaction conditions were pH 9.0 and 35 °C. The enzyme was stable at temperatures between 25 to 75 °C and at pH from 6 to 9. The enzyme also showed good stability in organic solvents, such as acetronitrile, hexane, ethanol and isopropanol. This study indicates that the byproducts tested are promising for the production of lipase and can contribute to the reduction of enzymatic production costs on a large scale, increase the value of these byproducts and reduce potential environmental impacts caused by its accumulation in nature.

Acanthamoeba mauritaniensis genotype T4D: An environmental isolate displays pathogenic behavior

Acanthamoeba spp. are free-living amoebae with a worldwide distribution. These amoebae can cause granulomatous amoebic encephalitis and amoebic keratitis in humans. Proteases are considered virulence factors in pathogenic Acanthamoeba. The objective of this study was to evaluate the behavior of Acanthamoeba mauritaniensis, a nonpathogenic amoeba. We analyzed the cytopathic effect of A. mauritaniensis on RCE1(5 T5) and MDCK cells and compared it to that of Acanthamoeba castellanii. A partial biochemical characterization of proteases was performed in total crude extracts (TCE) and conditioned medium (CM). Finally, we evaluated the effect of proteases on tight junction (TJ) proteins and the transepithelial electrical resistance of MDCK cells. The results showed that this amoeba can induce substantial damage to RCE1(5T5) and MDCK cells. Moreover, the zymograms and Azocoll assays of amoebic TCE and CM revealed different protease activities, with serine proteases being the most active. Furthermore, A. mauritaniensis induced the alteration and degradation of MDCK cell TJ proteins with serine proteases. After genotyping this amoeba, we determined that it is an isolate of Acanthamoeba genotype T4D. From these data, we suggest that A. mauritaniensis genotype T4D behaves similarly to the A. castellanii strain.

Purification and partial biochemical characterization of recombinant lactate dehydrogenase 1 (LDH-1) of the white shrimp Litopenaeus vannamei

Lactate dehydrogenase (LDH) is a key enzyme to produce energy during hypoxia by anaerobic glycolysis. In the white shrimp Litopenaeus vannamei, two protein subunits (LDH-1 and LDH-2) were previously identified, deduced from two different transcripts that come from the same LDH gene by processing via mutually exclusive alternative splicing. LDH-1 contains exon five and LDH-2 contains exon six and the two proteins differ only in 15 amino acid residues. Both subunits were independently cloned and overexpressed in E. coli as a fusion protein containing a chitin binding domain. Previously, recombinant LDH-2 was successfully purified and characterized, but LDH-1 was insoluble and aggregated forming inclusion bodies. We report the production of soluble LDH-1 by testing different pHs in the buffers used to lyse the bacterial cells before the purification step and the characterization of the purified protein to show that the cDNA indeed codes for a functional and active protein. The recombinant native protein is a homotetramer of approximately 140 kDa composed by 36 kDa subunits and has higher affinity for pyruvate than for lactate. LDH-1 has an optimum pH of 7.5 and is stable between pH 8.0 and 9.0; pH data analysis showed two pKa values of 6.1 ± 0.15 and 8.8 ± 0.15 suggesting a histidine and asparagine, respectively, involved in the active site. The enzyme optimal temperature was 44 °C and it was stable between 20 and 60 °C. LDH-1 was slightly activated by NaCl, KCl and MgCl2 and fully inhibited by ZnCl2.

Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion

BackgroundAnimal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads’ secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules.MethodsTotal RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman’s degradation.ResultsWe were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion.ConclusionsWe identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored.

Partial biochemical characterization of digestive proteases present in the gastric juices of two Chilean crustaceans, Lithodes santolla (Molina, 1782) and Cancer edwardsii (Bell, 1835)

The objectives of the present study were to characterize biochemically the proteases present in gastric juices from adults of the southern king crab ( Lithodes santolla ) and the Chilean rock crab ( Cancer edwardsii ), to determine the classes of proteases using specific inhibitors, as well as to evaluate the effect of temperature and pH that affect the proteolytic activity. The enzyme activity analysis revealed non-significant differences between the activities of serine proteases (trypsin and chymotrypsin) in both species. On the other hand, acid protease activity in C. edwardsii was significantly higher than in L. santolla . The activity of aspartate proteases was inhibited by Pepstatin A in both species, with no significant differences in the extent of inhibition between them. The maximal enzyme activity detected for both species occurred at alkaline pH. The optimum pH for proteolytic activity was ranged between 7 and 9 for C. edwardsii , and between 7 and 11 for L. santolla . Moreover, the highest proteolytic activity was recorded at 60°C in both species. However, at this temperature, enzyme activity was unstable after 20 min. Finally, collagenase-like activity in the gastric juices was detected for both species; this activity deserves further investigation, considering its biotechnological potential.

First insights into insecticidal activity against Aedes aegypti and partial biochemical characterization of a novel low molecular mass chymotrypsin-trypsin inhibitor purified from Lonchocarpus sericeus seeds.

Arboviroses such as dengue, Zika and chikungunya represent a serious public health issue as a consequence of the absence of approved vaccines or specific antiviral drugs against the arboviruses that cause them. One way to prevent these diseases is by combating the vector mosquito, Aedes aegypti (Diptera), which has serine proteases in the midgut. Protease inhibitors are molecules that can block enzyme activity, impairing digestion and nutrition, which can lead to death. Thus, we purified and characterized a novel chymotrypsin-trypsin inhibitor (LsCTI) from Lonchocarpus sericeus seeds and investigated its effect upon Ae. aegypti egg hatching, larval development and digestive proteases. LsCTI showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mass determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was 8870.45 Da. Kinetics analyses revealed a noncompetitive type of inhibition and low inhibition constant (Ki ) for chymotrypsin (8.24 x 10-8 m). The thermal resistance was remarkable, even at 100 °C for 180 min. The inhibitor concentration required for 50-percent enzyme inhibition (IC50 ) of LsCTI was 4.7 x 10-7 m for Ae. aegypti midgut larval enzymes. LsCTI did not affect egg hatchability at 0.3 mg mL-1 , but caused a high larval mortality rate (77%) and delayed development (37%). LsCTI is a novel protease inhibitor with remarkable biochemical characteristics and is a potential tool to control Ae. aegypti development. © 2017 Society of Chemical Industry.

Differential Inhibition of Helicoverpa armigera (Lep.: Noctuidae) Gut Digestive Trypsin by Extracted and Purified Inhibitor of Datura metel (Solanales: Solanaceae).

The cotton bollworm, Helicoverpa armigera Hubner (Lep: Noctuidae), is an economically important pest of numerous major food crops worldwide. Protease inhibitors from plants, expressed constitutively in transgenic crops, have potential for pest management as an alternative to chemical pesticides. In this study, a protease inhibitor was isolated, purified, and characterized from Datura metel L. seeds. The purity of the isolated inhibitor was confirmed by reverse-phase high-performance liquid chromatography, and activity staining showed one major peak and one clear activity band for the protein. Electrophoretic studies following gel filtration and ion-exchange chromatography revealed two and one bands for purified proteins, respectively. Partial biochemical characterizations of the purified inhibitor were determined. Maximum inhibitory activity was observed at 40-45°C (optimal temperature) when tested against gut extracts of fourth to sixth instar H. armigera larvae. Thermo-stability of the trypsin inhibitor against sixth instar larval midgut trypsin was observed up to 50°C when incubated for 30 min and 2 h. Among metal ions tested, Fe2+, Cu2+, and Mn2+ were found to decrease the trypsin inhibitory activity, whereas Hg2+, Mg2+, K+, Zn2+, Na+, Ca2+, and Cd2+ were found to significantly increase the inhibitory effect. This trypsin inhibitor showed competitive inhibition where the apparent value of Michaelis-Menten Km increased, but the value of Vmax remained unchanged.

Proteomic analysis of pupal gut serine protease of Silkworm, Bombyx mori : Partial purification and biochemical characterization

Abstract In the present study, one-dimensional gel electrophoresis (1-DE) and two-dimensional gel electrophoresis (2-DE) coupled zymography were performed for pupal gut proteins which revealed a single prominent proteolytic band/spot at molecular weight of 37 kDa and an isoelectric point (pI) at 5–6 approximately. The proteolytic spot was identified as 37 k-protease of B. mori using mass spectrometry. This 37k-Pupal gut serine protease (PGSP) was purified by anionic exchange and gel filtration chromatography. The biochemical characteristics of PGSP were evaluated using quantitative protease assay. PGSP has substrate specificity with gelatin and cannot be inhibited by various protease inhibitors. However, it was sensitive to reducing agents, dithiothreitol and β-mercaptoethanol. The proteolytic activity was not affected by metal ions (10 mM) except Cu2+ and Hg2+. The optimum pH and temperature of PGSP was determined as pH 9.0 and 60 °C. The purified protease revealed significant stability and compatibility towards surfactants, oxidizing agent and commercial detergents at 25% concentration. The PGSP (90 µg) with 1% detergent (Rin) showed complete destaining of blood stained cloth. It has significant clot lysis (> 40%) activity within 3 h. In addition, the PGSP shows stable activity in presence of 25% concentration of organic solvents. The overall results suggest that PGSP of B. mori could be utilized as an additive agent in detergent industry.

Biochemical properties of a new thermo- and solvent-stable xylanase recovered using three phase partitioning from the extract of Bacillus oceanisediminis strain SJ3

The present study investigates the production and partial biochemical characterization of an extracellular thermostable xylanase from the Bacillus oceanisediminis strain SJ3 newly recovered from Algerian soil using three phase partitioning (TPP). The maximum xylanase activity recorded after 2 days of incubation at 37 °C was 20.24 U/ml in the presence of oat spelt xylan. The results indicated that the enzyme recovered in the middle phase of TPP system using the optimum parameters were determined as 50% ammonium sulfate saturation with 1.0:1.5 ratio of crude extract: t-butanol at pH and temperature of 8.0 and 10 °C, respectively. The xylanase was recovered with 3.48 purification fold and 107% activity recovery. The enzyme was optimally active at pH 7.0 and was stable over a broad pH range of 5.0–10. The optimum temperature for xylanase activity was 55 °C and the half-life time at this temperature was of 6 h. At this time point the enzyme retained 50% of its activity after incubation for 2 h at 95 °C. The crude enzyme resist to sodium dodecyl sulfate and β-mercaptoethanol, while all the tested ions do not affect the activity of the enzyme. The recovered enzyme is, at least, stable in tested organic solvents except in propanol where a reduction of 46.5% was observed. Further, the stability of the xylanase was higher in hydrophobic solvents where a maximum stability was observed with cyclohexane. These properties make this enzyme to be highly thermostable and may be suggested as a potential candidate for application in some industrial processes. To the best of our knowledge, this is the first report of xylanase activity and recoverey using three phase partitioning from B. oceanisediminis.

Purification, partial biochemical characterization and inactivation of polyphenol oxidase from Mexican Golden Delicious apple (Malus domestica )

Polyphenol oxidase (PPO) is a critical molecule because of its participation in enzymatic browning in fruits and vegetables during ripening, handling, storage, and processing. In this contribution, PPO was extracted, purified, and characterized from Golden Delicious apples, PPO activity was increased 319-fold with a yield of 1.6%. A monomer of 58 kDa was obtained by gel filtration and SDS-PAGE; the optimum temperature and pH were 35°C and 6.0, respectively; the best catalytic efficiencies (Vmax/Km ratios [U/(mg·mM)]) were 15,402 for 4-methylcatechol and 5,941 for pyrogallol. The most efficient inhibitors were sodium metabisulfite (≥1 mM) and tropolone (≥10 mM). PPO activity decreased as the temperature increased and thermal inactivation was achieved at 80°C in 5 min. An Ea of 69.2 kJ/mol was estimated from the kinetic inactivation at different temperatures. Microwave irradiation inactivates PPO after 120 s in crude extract and after 60 s in fresh apple juice. Practical applications According to FAO (Food and Agricultural Organization) data, Mexico produced about 858,608 tons of apple in 2013, Mexico is in 30th place in the world overall apple production. It is important to characterize the Polyphenol Oxidase (PPO) from Golden Delicious apple and provide data on its inactivation. PPO has been considered a crucial molecule because of its participation in enzymatic browning in several fruit and vegetables during ripening, handling, storage, and processing, being responsible for economical loses for producers and industry. In this contribution PPO was extracted, purified, and partially characterized from Mexican Golden Delicious apples by fast protein liquid chromatography. Also some inactivation methods like the use of several inhibitors and antibrowning agents, thermal and microwave inactivation where tested. The information obtained in this research is important for better understanding of PPO from Golden Delicious apple and gives information on ways to optimize inactivation process of the purified enzyme or in apple juice.

Partial biochemical characterization of crude extract extracellular chitinase enzyme from Bacillus subtilis B 298

Extraction and characterization of extracellular chitinase from Bacillus subtilis B 298 have been done. Growth curve determination of B. subtilis B 298, production curve determination of crude extract chitinase from B. subtilis B 298, and partial biochemical characterization of crude extract chitinase have been achieved in this study. Optimum growth of B. subtilis B 298 was achieved at logarithmic phase within 9 hours incubation time, so it was used as inoculum for enzyme production. According to production curve of the enzyme, it was known that incubation time which gave the highest chitinase activity of 15 hours with activity of 6.937 U/mL respectively. Effect of various temperatures on chitinase activity showed that optimum activity was achieved at 40°C with an activity of 5.764 U/mL respectively. Meanwhile, the optimum pH for chitinase activity was achieved at pH of 5.0 with an activity of 6.813 U/mL respectively. This enzyme was then classified as metalloenzyme due to the decline of the activity by EDTA addition. All divalent cations tested acted as inhibitors.

Partial biochemical characterization of Cu,Zn-superoxide dismutase extracted from eggs of hens (Gallus gallus domesticus).

Biochemical characteristics of Cu,Zn-SOD derived from hen egg white and egg yolk were determined, and compared with those of enzymes from erythrocytes of hens and SOD standard. The presence of dimer with a molecular weight of 33.38±0.34kDa, and pI of 6.30±0.15 was confirmed in samples of SOD extracted from egg yolk. Cu,Zn-SOD isolated from egg yolk had an optimum at pH 6. Average SOD activity in egg yolk was 98.5±19.5U·g-1 while in egg white reached 6.1±0.8U·g-1. Changes in SOD activity of the egg yolk during its storage for 200days were also described. FTIR analysis confirmed that the enzymatic protein described in this study was SOD, while MALDI-TOF analysis confirmed only SOD from erythrocytes. Since eggs are a cheap and easily obtainable source of SOD, this enzymatic protein could be used in food, cosmetic or pharmaceutical industries.

Probing allocrite preferences of 2 naturally occurring variants of the wheat LR34 ABC transporter.

For almost a century, the wheat Lr34 gene has conferred durable resistance against fungal rust diseases. While sequence homology predicts a putative ATP binding cassette transporter, the molecules that are transported (allocrites) by the encoded LR34 variants, and any associated mechanism of resistance, remain enigmatic. Here, the in vitro transport characteristics of 2 naturally occurring Lr34 variants (that differ in their ability to mediate disease resistance; Lr34sus and Lr34res) are investigated. Initially, a method to express and purify recombinant LR34Sus and LR34Res pseudo half-molecules from Saccharomyces cerevisiae, is described. Subsequently, a semi-targeted chlorophyll catabolite (CC) extraction from Lr34res-expressing wheat plants was performed based on previous reports highlighting increased accumulation of CCs in Lr34res-expressing flag leaves. Following partial biochemical characterization, this extract was applied to an LR34 in vitro proteoliposome transport assay. Mass spectroscopic analyses of transported metabolites revealed that LR34Sus imported a wheat metabolite of 618 Da and that the LR34Res transporter did not. While the identity of the LR34Sus transported metabolite remains to be confirmed and any allocrites of LR34Res remain to be detected, this work demonstrates that these variants have different allocrite preferences, a finding that may be relevant to the mechanism of disease resistance.