Calcitonin receptor 1 (AedaeGPCRCAL1) hindgut expression and direct role in myotropic action in females of the mosquito Aedes aegypti (L.)

In anautogenous mosquitoes such as Aedes aegypti females the calcitonin-like diuretic hormone 31 (DH₃₁) stimulates natriuretic fluid excretion from the Malpighian tubules (MTs) after a blood meal. We previously cloned and functionally characterized AedaeGPCRcal1 from A. aegypti, the ortholog of the Drosophila melanogaster DH₃₁ receptor and immunolocalized it in the MTs. However, localization of the calcitonin receptor-like receptor 1 (GPCRCAL1) in the hindgut of any insect is unknown, and specifically, knowledge on its role in hindgut contraction in response to Aedae-DH₃₁ peptide is lacking. We analyzed the expression of AedaeGPCRCAL1 in hindgut by western blot and immunohistochemisty, and evaluated its role in hindgut contractility by application of Aedae-DH₃₁ before and after receptor RNA interference (RNAi). The receptor was detected as a 73 kDa band in western blots of hindgut and immunofluorescence revealed the receptor was expressed in hindgut circular and longitudinal muscles but not in the hindgut epithelial cells. In vitro, incubation in 1 μM solution of Aedae-DH₃₁ peptide significantly increased the hindgut contraction frequency in normal mosquitoes. Hindguts from females treated with AedaeGPCRcal1 dsRNA and incubated with DH₃₁ showed a reduction of 50% percent in their contraction frequency with respect to controls. These results suggest that DH₃₁ hormone released from the brain post-blood meal has a direct and coordinative action on the excretory system, MTs and hindgut, by which AedaeGPCRCAL1 signaling stimulates MT primary urine secretion and hindgut contraction resulting in rapid postprandial fluid excretion.     

The corticotropin-releasing factor-like diuretic hormone 44 (DH44) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster

Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in insects, and are under neuroendocrine control by multiple neuropeptides secreted by identified neurons. Here, we demonstrate roles for CRF-like diuretic hormone 44 (DH₄₄) and Drosophila melanogaster kinin (Drome-kinin, DK) in desiccation and starvation tolerance.Gene expression and labelled DH₄₄ ligand binding data, as well as highly selective knockdowns and/or neuronal ablations of DH₄₄ in neurons of the pars intercerebralis and DH₄₄ receptor (DH₄₄-R2) in Malpighian tubule principal cells, indicate that suppression of DH₄₄ signalling improves desiccation tolerance of the intact fly.Drome-kinin receptor, encoded by the leucokinin receptor gene, LKR, is expressed in DH₄₄ neurons as well as in stellate cells of the Malpighian tubules. LKR knockdown in DH₄₄-expressing neurons reduces Malpighian tubule-specific LKR, suggesting interactions between DH₄₄ and LK signalling pathways.Finally, although a role for DK in desiccation tolerance was not defined, we demonstrate a novel role for Malpighian tubule cell-specific LKR in starvation tolerance. Starvation increases gene expression of epithelial LKR. Also, Malpighian tubule stellate cell-specific knockdown of LKR significantly reduced starvation tolerance, demonstrating a role for neuropeptide signalling during starvation stress.     

Isolation and Functional Characterization of Calcitonin-Like Diuretic Hormone Receptors in Rhodnius prolixus

Several families of diuretic hormones exist in insects, one of which is the calcitonin-like diuretic hormone (CT/DH) family. CT/DH mediates its effects by binding to family B G-protein coupled receptors (GPCRs). Here we isolate and functionally characterize two R. prolixus CT/DH receptor paralogs (Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2) using a novel heterologous assay utilizing a modified human embryonic kidney 293 (HEK293) cell line. Rhopr-CT/DH-R1 is orthologous to the previously characterized D. melanogaster CT/DH receptor (CG17415) while Rhopr-CT/DH-R2 is orthologous to the D. melanogaster receptor (CG4395), an orphan receptor whose ligand was unknown until now. We determine the cDNA sequences of three splice variants encoding Rhopr-CT/DH-R1 (Rhopr-CT/DH-R1-A, Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C) and two splice variants encoding Rhopr-CT/DH-R2 (Rhopr-CT/DH-R2-A and Rhopr-CT/DH-R2-B). Rhopr-CT/DH-R1-A and Rhopr-CT/DH-R2-A encode truncated receptors that lack six and seven of the characteristic seven transmembrane domains, respectively. Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C, which only differ by 2 amino acids in their C-terminal domain, can both be activated by Rhopr-CT/DH at equal sensitivities (EC₅₀ = 200-300nM). Interestingly, Rhopr-CT/DH-R2-B is much more sensitive to Rhopr-CT/DH (EC₅₀ = 15nM) compared to Rhopr-CT/DH-R1-B/C and also yields a much greater response (amplitude) in our heterologous assay. This is the first study to reveal that insects possess at least two CT/DH receptors, which may be functionally different. Quantitative PCR demonstrates that Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2 have distinct expression patterns, with both receptors expressed centrally and peripherally. Moreover, the expression analysis also identified novel target tissues for this neuropeptide, including testes, ovaries and prothoracic glands, suggesting a possible role for Rhopr-CT/DH in reproductive physiology and development.     

The control of Malpighian tubule secretion in a predacious hemipteran insect, the spined soldier bug Podisus maculiventris (Heteroptera, Pentatomidae)

Spined soldier bugs, Podisus maculiventris, are heteropteran insects that feed voraciously on other insects, particular the soft bodied larval forms of Lepidoptera and Coleoptera. The response of P. maculiventris Malpighian tubules (MTs) to serotonin and known diuretic and antidiuretic peptides has been investigated, and is compared with that of MT from the hematophagous and phytophagous heteropteran bugs Rhodnius prolixus and Acrosternum hilare, respectively. A CRF-related peptide diuretic hormone (DH) from the termite Zootermopsis nevadensis (Zoone-DH) stimulated MT secretion, which was reversed by a member of the CAP_2b family of peptides from A. hilare (Acrhi-CAP_2b-2), an antidiuretic effect. Serotonin had no effect on secretion, neither did a representative calcitonin-like DH, kinin, tachykinin-related peptide, and an antidiuretic factor from the mealworm Tenebrio molitor (Tenmo-ADFb) in both P. maculiventris or A. hilare. Serotonin is a DH in R. prolixus, and its lack of effect on MT from P. maculiventris and A. hilare suggests this is an adaptation to hematophagy. On the other hand, the antidiuretic activity of members of the CAP_2b family in all three bugs is consistent with this being a heteropteran feature rather than a specialism for hematophagy.     

Mechanism and function of Drosophila capa GPCR: a desiccation stress-responsive receptor with functional homology to human neuromedinU receptor

The capa peptide receptor, capaR (CG14575), is a G-protein coupled receptor (GPCR) for the D. melanogaster capa neuropeptides, Drm-capa-1 and -2 (capa-1 and -2). To date, the capa peptide family constitutes the only known nitridergic peptides in insects, so the mechanisms and physiological function of ligand-receptor signalling of this peptide family are of interest. Capa peptide induces calcium signaling via capaR with EC₅₀ values for capa-1 = 3.06 nM and capa-2 = 4.32 nM. capaR undergoes rapid desensitization, with internalization via a b-arrestin-2 mediated mechanism but is rapidly re-sensitized in the absence of capa-1. Drosophila capa peptides have a C-terminal -FPRXamide motif and insect-PRXamide peptides are evolutionarily related to vertebrate peptide neuromedinU (NMU). Potential agonist effects of human NMU-25 and the insect -PRLamides [Drosophila pyrokinins Drm-PK-1 (capa-3), Drm-PK-2 and hugin-gamma [hugg]] against capaR were investigated. NMU-25, but not hugg nor Drm-PK-2, increases intracellular calcium ([Ca²⁺]i) levels via capaR. In vivo, NMU-25 increases [Ca²⁺]i and fluid transport by the Drosophila Malpighian (renal) tubule. Ectopic expression of human NMU receptor 2 in tubules of transgenic flies results in increased [Ca²⁺]i and fluid transport. Finally, anti-porcine NMU-8 staining of larval CNS shows that the most highly immunoreactive cells are capa-producing neurons. These structural and functional data suggest that vertebrate NMU is a putative functional homolog of Drm-capa-1 and -2. capaR is almost exclusively expressed in tubule principal cells; cell-specific targeted capaR RNAi significantly reduces capa-1 stimulated [Ca²⁺]i and fluid transport. Adult capaR RNAi transgenic flies also display resistance to desiccation. Thus, capaR acts in the key fluid-transporting tissue to regulate responses to desiccation stress in the fly.     

Spectroscopic characterization of a fruit-specific metallothionein: M. acuminata MT3

Metallothioneins (MTs) are ubiquitous low molecular mass, cysteine-rich proteins with the ability to bind d¹⁰ metal ions in the form of metal-thiolate clusters. In contrast to the vertebrate forms, knowledge about the properties of members of the plant metallothionein family is still scarce. The amino acid sequences of plant MTs are distinctively different to the sequences of other MT species. The protein under investigation, Musa acuminata (banana) MT3, belongs to the plant MT fruit-specific p3 subfamily. With a total of 10 cysteine residues, MT3 features a cysteine content and percentage that is more comparable to fungal and prokaryotic MTs than to the well characterized mammalian iso-forms. The gene sequence encoding MT3 was cloned into a suitable vector and the protein was recombinantly overexpressed in Escherichia coli. MT3 is able to coordinate a maximum of four divalent d¹⁰ metal ions under the formation of metal-thiolate clusters. The hitherto unknown spectroscopic behavior of MT3 in combination with the metal ions Zn²⁺, Cd²⁺, Pb²⁺, and Hg²⁺ will be presented and gives rise to the existence of a weaker metal ion coordination site. The pH stability of the investigated zinc and cadmium clusters is comparable to the values found for other plant metallothioneins though significantly lower than for the mammalian iso-forms. Possible metal-thiolate cluster structures will additionally be discussed.     

Serotonin has kinin-like activity in stimulating secretion by Malpighian tubules of the house cricket Acheta domesticus

Serotonin stimulates secretion by Malpighian tubules (MT) of a number of insects, and functions as a diuretic hormone in Rhodnius prolixus and in larval Aedes aegypti. Serotonin is here shown to be a potent stimulant of secretion by MT of the house cricket, Acheta domesticus, with an apparent EC₅₀ of 9.4 nmol L⁻¹, although its diuretic activity is just 25% of the maximum achievable with either the native CRF-related peptide, Achdo-DH, or a crude extract of the corpora cardiaca. In this respect, the diuretic activity of serotonin is similar to that of the cricket kinin Achdo-KI, and when tested together their actions are not additive, which suggests they target the same transport process. Consistent with this suggestion, the activity of serotonin is chloride-dependent and is associated with a non-selective stimulation of NaCl and KCl transport. In common with Achdo-KI, serotonin has no effect on cAMP production by isolated MT, and both act synergistically with exogenous 8bromo-cAMP in stimulating fluid secretion, most likely by promoting the release of Ca²⁺ from intracellular stores. A number of serotonin agonists and antagonists were tested to determine the pharmacological profile of receptors on cricket MT. The results are consistent with the diuretic activity of serotonin being mediated through a 5-HT₂-like receptor.     

Human Microtubule-Associated-Protein Tau Regulates the Number of Protofilaments in Microtubules: A Synchrotron X-Ray Scattering Study

Microtubules (MTs), a major component of the eukaryotic cytoskeleton, are 25 nm protein nanotubes with walls comprised of assembled protofilaments built from αβ heterodimeric tubulin. In neural cells, different isoforms of the microtubule-associated-protein (MAP) tau regulate tubulin assembly and MT stability. Using synchrotron small angle x-ray scattering (SAXS), we have examined the effects of all six naturally occurring central nervous system tau isoforms on the assembly structure of taxol-stabilized MTs. Most notably, we found that tau regulates the distribution of protofilament numbers in MTs as reflected in the observed increase in the average radius 〈R^MT〉 of MTs with increasing Φ, the tau/tubulin-dimer molar ratio. Within experimental scatter, the change in 〈R^MT〉 seems to be isoform independent. Significantly, 〈R^MT〉 was observed to rapidly increase for 0 < Φ < 0.2 and saturate for Φ between 0.2-0.5. Thus, a local shape distortion of the tubulin dimer on tau binding, at coverages much less than a monolayer, is spread collectively over many dimers on the scale of protofilaments. This implies that tau regulates the shape of protofilaments and thus the spontaneous curvature C_o^MT of MTs leading to changes in the curvature C^MT (=1/R^MT). An important biological implication of these findings is a possible allosteric role for tau where the tau-induced shape changes of the MT surface may effect the MT binding activity of other MAPs present in neurons. Furthermore, the results, which provide insight into the regulation of the elastic properties of MTs by tau, may also impact biomaterials applications requiring radial size-controlled nanotubes.     

Metal binding properties and structure of a type III metallothionein from the metal hyperaccumulator plant Noccaea caerulescens

Metallothioneins (MT) are low molecular weight proteins with cysteine-rich sequences that bind heavy metals with remarkably high affinities. Plant MTs differ from animal ones by a peculiar amino acid sequence organization consisting of two short Cys-rich terminal domains (containing from 4 to 8 Cys each) linked by a Cys free region of about 30 residues. In contrast with the current knowledge on the 3D structure of animal MTs, there is a striking lack of structural data on plant MTs. We have expressed and purified a type III MT from Noccaea caerulescens (previously Thlaspi caerulescens). This protein is able to bind a variety of cations including Cd²⁺, Cu²⁺, Zn²⁺ and Pb²⁺, with different stoichiometries as shown by mass spectrometry. The protein displays a complete absence of periodic secondary structures as measured by far-UV circular dichroism, infrared spectroscopy and hydrogen/deuterium exchange kinetics. When attached onto a BIA-ATR biosensor, no significant structural change was observed upon removing the metal ions.     

Identification of microtubule growth deceleration and its regulation by conserved and novel proteins

Microtubules (MTs) are cytoskeletal polymers that participate in diverse cellular functions, including cell division, intracellular trafficking, and templating of cilia and flagella. MTs undergo dynamic instability, alternating between growth and shortening via catastrophe and rescue events. The rates and frequencies of MT dynamic parameters appear to be characteristic for a given cell type. We recently reported that all MT dynamic parameters vary throughout differentiation of a smooth muscle cell type in intact Caenorhabditis elegans. Here we describe local differences in MT dynamics and a novel MT behavior: an abrupt change in growth rate (deceleration) of single MTs occurring in the cell periphery of these cells. MT deceleration occurs where there is a decrease in local soluble tubulin concentration at the cell periphery. This local regulation of tubulin concentration and MT deceleration are dependent on two novel homologues of human cylicin. These novel ORFs, which we name cylc-1 and -2, share sequence homology with stathmins and encode small, very basic proteins containing several KKD/E repeats. The TOG domain–containing protein ZYG-9^TOGp is responsible for the faster polymerization rate within the cell body. Thus we have defined two contributors to the molecular regulation for this novel MT behavior.     

Arsenic-metalation of triple-domain human metallothioneins: Support for the evolutionary advantage and interdomain metalation of multiple-metal-binding domains

Metallothionein (MT) is a prominent metal-binding protein and in mammalian systems contains a two-domain βα motif, while in lower life forms MT often consists of only a single-domain structure. There are also unusual MTs from American oysters that consist of multiple domains (from one to four α domains). This report details the study of the As³⁺-metalation to two different concatenated triple β and α domain MTs using time-resolved electrospray ionization mass spectrometry (ESI MS). Analysis of kinetic ESI MS data show that ααα human MT and βββ human MT bind As³⁺ in a noncooperative manner and involves up to 11 sequential bimolecular reactions. We report the complete progress of the reactions for the As³⁺-metalation of both triple-domain MTs from zero and up to 9 (βββ) or 10 As³⁺ ions (ααα). The rate constants for the As³⁺-metalation are reported for both the βββ and ααα human MT. At room temperature (298 K) and pH 3.5, the sequential individual rate constants, k_n (n = 1-9) for the As³⁺-metalation of βββhMT starting at k_1βββ are 40, 36, 37, 26, 27, 17, 12, 6, and 1 M⁻¹ s⁻¹; while at room temperature (298 K) and pH 3.5, the sequential individual rate constants, k_n (n = 1-10) for the As³⁺-metalation of αααhMT starting at k_1ααα are 52, 45, 46, 42, 38, 36, 29, 25, 14, and 6 M⁻¹ s⁻¹. The trend in the rate constant values reported for these two triple-domain MT proteins supports our previous proposal that the rate constant values are proportionally related to the total number of equivalent binding sites. The rate of binding for the 1st As³⁺ is the fastest we have determined for any MT to date. Additionally, we propose that the data show for the first time for any MT species, that interdomain metalation occurs in the binding of the 10th and 11th As³⁺ to αααhMT.     

Stimulus Bias Provides Evidence for Conformational Constraints in the Structure of a G Protein-coupled Receptor

A key characteristic of G protein-coupled receptors (GPCRs) is that they activate a plethora of signaling pathways. It is now clear that a GPCR coupling to these pathways can be regulated selectively by ligands that differentially drive signaling down one pathway in preference to another. This concept, termed stimulus bias, is revolutionizing receptor biology and drug discovery by providing a means of selectively targeting receptor signaling pathways that have therapeutic impact. Herein, we utilized a novel quantitative method that determines stimulus bias of synthetic GPCR ligands in a manner that nullifies the impact of both the cellular background and the “natural bias” of the endogenous ligand. By applying this method to the M₂ muscarinic acetylcholine receptor, a prototypical GPCR, we found that mutation of key residues (Tyr-80^2.61 and Trp-99^3.28) in an allosteric binding pocket introduces stimulus bias in response to the atypical ligands AC-42 (4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)piperidine HCl) and 77-LH-28-1 (1-(3-(4-butyl-1-piperidinyl)propyl)- 3,3-dihydro-2(1H)-quinolinone). By comparing stimulus bias factors among receptor internalization, G protein activation, extracellular-regulated protein kinase 1/2 (ERK1/2) signaling, and receptor phosphorylation, we provide evidence that Tyr-80^2.61 and Trp-99^3.28 act either as molecular switches or as gatekeeper residues that introduce constraints limiting the active conformation of the M₂ muscarinic acetylcholine receptor and thereby regulate stimulus bias. Furthermore, we provide evidence that downstream signaling pathways previously considered to be related to each other (i.e. receptor phosphorylation, internalization, and activation of ERK1/2) can act independently.     

Identification of four evolutionarily related G protein-coupled receptors from the malaria mosquito Anopheles gambiae

The mosquito Anopheles gambiae is an important vector for malaria, which is one of the most serious human parasitic diseases in the world, causing up to 2.7 million deaths yearly. To contribute to our understanding of A. gambiae and to the transmission of malaria, we have now cloned four evolutionarily related G protein-coupled receptors (GPCRs) from this mosquito and expressed them in Chinese hamster ovary cells. After screening of a library of thirty-three insect or other invertebrate neuropeptides and eight biogenic amines, we could identify (de-orphanize) three of these GPCRs as: an adipokinetic hormone (AKH) receptor (EC₅₀ for A. gambiae AKH, 3 × 10⁻⁹ M), a corazonin receptor (EC₅₀ for A. gambiae corazonin, 4 × 10⁻⁹ M), and a crustacean cardioactive peptide (CCAP) receptor (EC₅₀ for A. gambiae CCAP, 1 × 10⁻⁹ M). The fourth GPCR remained an orphan, although its close evolutionary relationship to the A. gambiae and other insect AKH receptors suggested that it is a receptor for an AKH-like peptide. This is the first published report on evolutionarily related AKH, corazonin, and CCAP receptors in mosquitoes.     

Isoform‐specific responses of metallothioneins in a marine pollution biomonitor,the green‐lipped mussel Perna viridis,towards different stress stimulations

Metallothioneins (MTs) are commonly used as biomarker for metal pollution assessment in marine ecosystems. Using integrated genomic and proteomic analyses, this study characterized two types of MT isoform in the digestive gland of a common biomonitor, the green‐lipped mussel Perna viridis, towards the challenges of a metal (cadmium; Cd) and a non‐metal oxidant (hydrogen peroxide; H₂O₂) respectively. The two isoforms differed in their deduced protein sequences, with 73 amino acids for MT10‐I and 72 for MT10‐II (a novel type), but both consisted of a high percentage (27.4 to 29.2%) of cysteine. Two‐dimensional gel and Western blot showed that the MT proteins were present in multiple isoform spots, and they were further validated to be MT10‐I and MT10‐II using MS analysis coupled with unrestricted modifications searching. Expression of mRNA revealed that MT10‐I responded promptly to Cd but had a lagged induction to H₂O₂ treatments, while MT10‐II was exclusively induced by Cd treatment over the course of exposure. Expression of the MT proteins also showed a delayed response to H₂O₂, compared to Cd treatments. This study uncovered the potential different functional roles of various MTs isoforms in P. viridis and thus advances the resolution of using MTs as biomarkers in future applications.     

Direct Regulation of Microtubule Dynamics by KIF17 Motor and Tail Domains

KIF17 is a kinesin-2 family motor that interacts with EB1 at microtubule (MT) plus-ends and contributes to MT stabilization in epithelial cells. The mechanism by which KIF17 affects MTs and how its activity is regulated are not yet known. Here, we show that EB1 and the KIF17 autoinhibitory tail domain (KIF17-Tail) interacted competitively with the KIF17 catalytic motor domain (K370). Both EB1 and KIF17-Tail decreased the K_0.5MT of K370, with opposing effects on MT-stimulated ATPase activity. Importantly, K370 had independent effects on MT dynamic instability, resulting in formation of long MTs without affecting polymerization rate or total polymer mass. K370 also inhibited MT depolymerization induced by dilution in vitro and by nocodazole in cells, suggesting that it acts by protecting MT plus-ends. Interestingly, KIF17-Tail bound MTs and tubulin dimers, delaying initial MT polymerization in vitro and MT regrowth in cells. However, neither EB1 nor KIF17-Tail affected K370-mediated MT polymerization or stabilization significantly in vitro, and EB1 was dispensable for MT stabilization by K370 in cells. Thus, although EB1 and KIF17-Tail may coordinate KIF17 catalytic activity, our data reveal a novel and direct role for KIF17 in regulating MT dynamics.     

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics^1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization¹. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs^1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker⁴, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones⁵. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types^6-8.     

Heterologous Expression and Metal-Binding Characterization of a Type 1 Metallothionein Isoform (OsMTI-1b) from Rice (Oryza sativa)

Metallothioneins (MTs) are ubiquitous, low molecular mass and cysteine-rich proteins that play important roles in maintaining intracellular metal homeostasis, eliminating metal toxification and protecting the cells against oxidative damages. MTs are able to bind metal ions through the thiol groups of their cysteine residues. Plants have several MT isoforms which are classified into four types based on the arrangement of cysteine residues. In the present study, a rice (Oryza sativa) gene encoding type 1 MT isoform, OsMTI-1b, was inserted in vector pET41a and overexpressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST). The recombinant protein GST-OsMTI-1b was purified using affinity chromatography and its ability to bind with Ni²⁺, Cd²⁺, Zn²⁺ and Cu²⁺ ions was analyzed. The results demonstrated that this isoform has ability to bind Ni²⁺, Cd²⁺ and Zn²⁺ ions in vitro, whereas it has no substantial ability to bind Cu²⁺ ions. From competitive reaction with 5,5′-dithiobis(2-nitrobenzoic acid), DTNB, the affinity of metal ions for recombinant form of GST-OsMTI-1b was as follows: Ni²⁺/Cd²⁺ > Zn²⁺ > Cu²⁺