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Bestenbalt products were used in the following studies:

Metallothionein induces a regenerative reactive astrocyte phenotype via JAK/STAT and RhoA signalling pathways
Y.K.J. Leung^a, M. Pankhurst ^a, S.A. Dunlop ^b, S. Ray ^a, J. Dittmann ^a, E.D. Eaton ^a, P. Palumaa ^c, R. Sillard ^c, M.I. Chuah ^a, A.K. West ^a and R.S. Chung ^a.

a) Menzies Research Institute, University of Tasmania, Private Bag 58, Hobart, Tasmania 7001, Australia
b) School of Animal Biology, University of Western Australia, Nedlands, Western Australia 6907, Australia
c) Department of Gene Technology, Tallinn Technical University. Akadeemia tee 15, Tallinn 12618, Estonia
 

Journal:   Experimental Neurology, Vol 221 (1), 98-106, 2010.

Abstract:  Following central nervous system injury, astrocytes rapidly respond by undergoing a stereotypical pattern of molecular and morphological alterations termed “reactive” astrogliosis. We have reported previously that metallothioneins (MTs) are rapidly expressed by reactive astrocytes and that their secretion and subsequent interaction with injured neurons leads to improved neuroregeneration. We now demonstrate that exogenous MT induces a reactive morphology and elevated GFAP expression in cultured astrocytes. Furthermore, these astrogliotic hallmarks were mediated via JAK/STAT and RhoA signalling pathways. However, rather than being inhibitory, MT induced a form of astrogliosis that was permissive to neurite outgrowth and which was associated with decreased chondroitin sulphate proteoglycan (CSPG) expression. The results suggest that MT has an important role in mediating permissive astrocytic responses to traumatic brain injury.

Keywords: Traumatic brain injury; Astrogliosis; Regeneration
 

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Metallothionein II-A improves healing after a burn wound
 

Morellini N.^1,2,3, Giles N. ^3,4, Rea S. ^3,5,6, King C. ^1,2, Dunlop S. ^1,2, Beazley L. ^1,2, West A. ⁷, Wood F. ^1,5,6, Fear M. ³

1School of Animal Biology, University of Western Australia,
2Western Australian Institute for Medical Research, Australia,
3The McComb Research Foundation, Perth, Australia,
4Department of Anatomy and Human Biology, University of Western Australia,
5Royal Perth Hospital, Perth, Australia,
6Princess Margaret Hospital for Children, Perth, Australia,
7NeuroRepair Group, Menzies Research Institute, University of Tasmania
 

The epidermal barrier prevents infection and dehydration. Its rapid repair is essential after injury. Severe injuries often result in scarring and life-long functional deficits, the outcome worsening with longer times to heal. We investigated the potential of Metallothionein II-A (Apo-MT-IIA: Bestenbalt, Tallinn, Estonia, rabbit-derived, >98% pure by HPLC in zinc sulphate solution, PBS, pH 7.4), a naturally occurring small cysteine-rich protein, to accelerate healing after burn wounds. In vitro assays of a human keratinocyte cell line (HaCaT) indicated that at 1 g/ml and 2 g/ml MT-IIA significantly increased cell proliferation (p<0.05). Annexin V and propidium iodide FACS analysis of keratinocytes with increasing amount of MTII-A reduced the percentage of cells undergoing apoptosis in response to both a UV insult and to Staurosporine in a dose-dependent manner with the effect at 1 g/ml reducing apoptosis by >50% p<0.05)). After a full thickness burn to the dorsal skin of adult mice, immunohistochemistry revealed that endogenous MT-I/II expression increased in basal keratinocytes during healing. Increases are seen in the wound margin at early stages (3 and 7 days; p<0.05) and in its centre by 11 days (p<0.05). Topical administration of exogenous MT-IIA immediately post-burn accelerated the return of MTI/II expression to normal values by day 14 in the wound margin (Normal: 39 ± 3% vs MT: 49 ± 7%, p>0.05; vs PBS: 68 ± 7%, p<0.05) and improved healing as assessed by reduced epidermal thickness (MTII-A: 45 ± 4 μm vs control: 101 ± 19 μm, p<0.05) and faster wound closure at Day 3 post-injury 8.9 mm ± 0.27 mm in controls compared to 7.1 mm ± 0.7 mm in treated wounds, by day 7 5.8 mm ± 0.98mm in controls versus 3.6 mm ± 1.0 mm in treated wounds, p<0.05).. Our data suggest that MT-IIA may prove a valuable therapeutic for patients with burns and other skin injuries.

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Simultaneous iron, zinc, sulfur and phosphorus speciation analysis of barley grain tissues using SEC-ICP-MS and IP-ICP-MS

Daniel P. Persson, Thomas H. Hansen, Kristian H. Laursen, Jan K. Schjoerring and Søren Husted

Journal:    Metallomics, 2009, 1, 418 - 426.

Abstract: The increasing prevalence of iron (Fe) and zinc (Zn) deficiencies in human populations worldwide has stressed the need for more information about the distribution and chemical speciation of these elements in cereal products. In order to investigate these aspects, barley grains were fractionated into awns, embryo, bran and endosperm and analysed for Fe and Zn. Simultaneously, phosphorus (P) and sulfur (S) were determined since these elements are major constituents of phytic acid and proteins, respectively, compounds which are potentially involved in Fe and Zn binding. A novel analytical method was developed in which oxygen was added to the octopole reaction cell of the ICP-MS. This approach greatly improved the sensitivity of sulfur, measured as 48SO+. Simultaneously, Fe was measured as 72FeO+, P as 47PO+, and Zn as 66Zn+, enabling sensitive and simultaneous analysis of these four elements. The highest concentrations of Zn, Fe, S and P were found in the bran and embryo fractions. Further analysis of the embryo using SEC-ICP-MS revealed that the speciation of Fe and Zn differed. The majority of Fe co-eluted with P as a species with the apparent mass of 12.3 kDa, whereas the majority of Zn co-eluted with S as a 3 kDa species, devoid of any co-eluting P. Subsequent ion pairing chromatography of the Fe/P peak showed that phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate: IP6) was the main Fe binding ligand, with the stoichiometry Fe4(IP6)18. When incubating the embryo tissue with phytase, the enzyme responsible for degradation of phytic acid, the extraction efficiency of both Fe and P was doubled, whereas that of Zn and S was unaffected. Protein degradation on the other hand, using protease XIV, boosted the extraction of Zn and S, but not that of Fe and P. It is concluded that Fe and Zn have a different speciation in cereal grain tissues; Zn appears to be mainly bound to peptides, while Fe is mainly associated with phytic acid.

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Metallothionein Treatment Attenuates Microglial Activation and Expression of Neurotoxic Quinolinic Acid Following Traumatic Brain Injury

R. S. Chung¹, Y. K. Leung¹, C. W. Butler¹, Y. Chen², E. D. Eaton¹, M. W. Pankhurst¹, A. K. West¹ and G. J. Guillemin²

(1) NeuroRepair Group, Menzies Research Institute, University of Tasmania, Private Bag 58, Hobart, TAS, 7001, Australia
(2) Centre for Immunology, University of New South Wales, Sydney, 2052, Australia

Journal:     Neurotoxicity Research, Volume 15, Number 4 / May, 2009.

Abstract: The kynurenine pathway has been implicated as a major component of the neuroinflammatory response to brain injury and neurodegeneration. We found that the neurotoxic kynurenine pathway intermediate quinolinic acid (QUIN) is rapidly expressed, within 24 h, by reactive microglia following traumatic injury to the rodent neocortex. Furthermore, administration of the astrocytic protein metallothionein attenuated this neuroinflammatory response by reducing microglial activation (by approximately 30%) and QUIN expression. The suppressive effect of MT was confirmed upon cultured cortical microglia, with 1 μg/ml MT almost completely blocking interferon–gamma induced activation of microglia and QUIN expression. These results demonstrate the neuroimmunomodulatory properties of MT, which may have therapeutic applications for the treatment of traumatic brain injury.

Keywords: Traumatic brain injury - Neuroinflammation - Neuron-glia interactions
 

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Potential Role of a-Synuclein and Metallothionein in Lead-Induced Inclusion Body Formation
Peijun Zuo*, Wei Qu*, Ryan N. Cooper*, Robert A. Goyer*, Bhalchandra A. Diwan and Michael P. Waalkes*

* Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Basic Research Program, SAIC-Frederick, Inc., NCI at Frederick, Maryland 21702
 

Journal:   Toxicological Sciences 2009 111(1):100-108;

Abstract: Lead (Pb) produces aggresome-like inclusion bodies (IBs) in target cells as a toxic response. Our prior work shows metallothionein (MT) is required for this process. We used MT-I/II double knockout (MT-null) and parental wild-type (WT) cell lines to further explore the formation process of Pb-induced IBs. Unlike WT cells, MT-null cells did not form IBs after Pb exposure. Western blot of cytosol showed soluble MT protein in WT cells was lost during Pb exposure as IBs formed. Transfection of MT-I into MT-null cells allowed IBs formation after Pb exposure. Considering Pb-induced IBs may be like disease-related aggresomes, which often contain alpha-synuclein (Scna), we investigated Scna expression in cells capable (WT) and incapable (MT-null) of producing IBs after Pb exposure. Scna protein showed poor basal expression in MT-null cells. Pb exposure increased Scna expression only in WT cells. MT transfection increased Scna transcript to WT levels. In WT or MT-transfected MT-null cells, Pb-induced Scna expression rapidly increased and then decreased over 48 h as Pb-induced IBs were formed. A direct interaction between Scna and MT was confirmed ex vivo by antibody pulldown assay where the proteins coprecipitated with an antibody to MT. Pb exposure caused increased colocalization of MT and Scna proteins with time only in WT cells. In WT mice after chronic Pb exposure Scna was localized in renal cells containing forming IBs, whereas MT-null mice did not form IBs. Thus, Scna could be component of Pb-induced IBs and, with MT, may play a role in IBs formation.

Key Words: lead; inclusion bodies; alpha-synuclein; metallothionein; MT-null.
 

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Redefining the Role of Metallothionein within the Injured Brain
EXTRACELLULAR METALLOTHIONEINS PLAY AN IMPORTANT ROLE IN THE ASTROCYTE-NEURON RESPONSE TO INJURY.

Roger S. Chung1, Milena Penkowa2, Justin Dittmann1, Carolyn E. King3, Carole Bartlett3, Johanne W. Asmussen2, Juan Hidalgo4, Javier Carrasco4, Yee Kee J. Leung1, Adam K. Walker1, Samantha J. Fung1, Sarah A. Dunlop3, Melinda Fitzgerald3, Lyn D. Beazley3, Meng I. Chuah1, James C. Vickers1, and Adrian K. West1

1)NeuroRepair Group, Menzies Research Institute, University of Tasmania, Private Bag 58, Hobart, Tasmania 7001, Australia,
2)Section of Neuroprotection, Faculty of Health Sciences, University of Copenhagen, Copenhagen DK2200, Denmark,
3)School of Animal Biology, University of Western Australia, Nedlands, Western Australia 6907, Australia, and
4)Institute of Neurosciences and Department of Cellular Biology, Physiology and Immunology, Animal Physiology Unit, Faculty of Sciences, Autonomous University of Barcelona, Bellaterra, Barcelona 08193, Spain

Journal: Journal of Biological Chemistry, Vol. 283, Issue 22, 15349-15358, May 30, 2008.

Abstract: A number of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.
 

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Exogenous metallothionein-IIA promotes accelerated healing after a burn wound
 

Natalie M. Morellini^1,2,3; Natalie L. Giles^3,4; Suzanne Rea^3,5,6; Katharine F. Adcroft^3,4; Sian Falder^3,5,6; Carolyn E. King^1,2; Sarah A. Dunlop^1,2; Lyn D. Beazley^1,2; Adrian K. West⁷; Fiona M. Wood^3,5,6; Mark W. Fear³
 

1. School of Animal Biology, University of Western Australia, Crawley, WA, Australia,
2. Western Australian Institute for Medical Research, Perth, WA, Australia,
3. The McComb Research Foundation, Perth, WA, Australia,
4. Department of Anatomy and Human Biology, University of Western Australia, Crawley, WA, Australia,
5. Royal Perth Hospital, Perth, WA, Australia,
6. Princess Margaret Hospital for Children, Perth, WA, Australia, and
7. NeuroRepair Group, Menzies Research Institute, University of Tasmania, Hobart, Tas., Australia
 

Journal:    Wound Repair and Regeneration, Volume 16 Issue 5, Pages 682 - 690, 2008.

ABSTRACT: Severe injury to the epidermal barrier often results in scarring and life-long functional deficits, the outcome worsening with a number of factors including time taken to heal. We have investigated the potential of exogenous metallothionein IIA (Zn7-MT-IIA), a naturally occurring small cysteine-rich protein, to accelerate healing of burn wounds in a mouse model. Endogenous MT-I/II expression increased in basal keratinocytes concurrent with reepithelialization after a burn injury, indicating a role for MT-I/II in wound healing. In vitro assays of a human keratinocyte cell line indicated that, compared with saline controls, exogenous Zn7-MT-IIA significantly increased cell viability by up to 30% (p<0.05), decreased apoptosis by 13% (p<0.05) and promoted keratinocyte migration by up to 14% (p<0.05), all properties that may be desirable to promote rapid wound repair. Further in vitro assays using immortalized and primary fibroblasts indicated that Zn7-MT-IIA did not affect fibroblast motility or contraction (p>0.05). Topical administration of exogenous Zn7-MT-IIA (2 μg/mL) in vivo, immediately postburn accelerated healing, promoted faster reepithelialization (3 days: phosphate-buffered saline (PBS), 8.9±0.3 mm diameter vs. MT-I/II, 7.1±0.7 mm; 7 days: PBS 5.8±0.98 mm vs. MT-I/II, 3.6±1.0 mm, p<0.05) and reduced epidermal thickness (MT-I/II: 45±4 μm vs. PBS: 101±19 μm, p<0.05) compared with controls. Our data suggest that exogenous Zn7-MT-IIA may prove a valuable therapeutic for patients with burns and other skin injuries.

 

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Simple Method for Identification of Metallothionein Isoforms in Cultured Human Prostate Cells by MALDI-TOF/TOF Mass Spectrometry
Rongying Wang,†‡ Donald A. Sens,† Amy Albrecht,† Scott Garrett,† Seema Somji,† Mary Ann Sens,† and Xiaoning Lu*†‡

Department of Pathology, and Proteomics Core Facility, School of Medicine and Health Science, University of North Dakota, Grand Forks, North Dakota 58202

Journal:    Anal. Chem., 2007, 79 (12), pp 4433–4441.

Abstract: The present paper describes a rapid method for identification and characterization of human metallothionein (MT) isoforms in complex cell cultures using high-resolution matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF). In the proposed method, the sample preparation of MTs from cultured cells is both simple and fast. It is accomplished by trypsin cleavage of cell proteins into small peptide species, the majority of which are subsequently removed by gel filtration using beads with an exclusion limit of 4000 Da. In contrast to most cell proteins, MTs remain intact (undigested) upon being treated with trypsin, being excluded by the gel beads and thus recovered by low-speed centrifugation. To identify the protein constitutes of the MT preparation, the MT sample is divided into two parts, one for intact protein accurate mass measurement, the other for tryptic digestion followed by MS and MS/MS analyses. In the latter case, the MT proteins are denatured by the addition of EDTA which strips heavy metals from MTs and renders them susceptible to tryptic digestion. The obtained accurate mass with the unique peptide sequences of each MT isoform allows for unambiguous identification of MT isoforms in the prepared mixture. The method has been applied to RWPE-1 cells derived from normal human prostate epithelium. Four MT isoforms, 1E, 1G, 1X, and 2A, have been confidently identified, being primarily acetylated at N-termini. These results are in agreement with the expression of MT mRNAs in RWPE-1 cells determined by real-time reverse-transcription polymerase chain reaction (RT-PCR).
 

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Metallothionein-IIA promotes neurite growth via the megalin receptor

Melinda Fitzgerald (1) , Pia Nairn (1), Carole A. Bartlett (1), Roger S. Chung (3), Adrian K. West (3) and Lyn D. Beazley (1, 2)

(1) Experimental and Regenerative Neurosciences, School of Animal Biology, University of Western Australia, Hackett Drive, Crawley, 6009, WA, Australia
(2) Western Australian Institute of Medical Research, University of Western Australia, Hackett Drive, Crawley, 6009, WA, Australia
(3) Neurorepair Group, Menzies Research Institute, University of Tasmania, Private Bag 58, Hobart, TAS, 7001, Australia

Journal:    Experimental Brain Research, Volume 183, Number 2 / November, 2007, p. 171-180

Open: Entire document  (Subscription to the journal required).

Abstract: Metallothionein (MT)-I/II has been shown to be neuroprotective and neuroregenerative in a model of rat cortical brain injury. Here we examine expression patterns of MT-I/II and its putative receptor megalin in rat retina. At neonatal stages, MT-I/II was present in retinal ganglion cells (RGCs) but not glial or amacrine cells; megalin was present throughout the retina. Whilst MT-I/II was absent from adult RGC in normal animals and after optic nerve transection, the constitutive megalin expression in RGCs was lost following optic nerve transection. In vitro MT-IIA treatment stimulated neuritic growth: more RGCs grew neurites longer than 25 μm (P < 0.05) in dissociated retinal cultures and neurite extension increased in retinal explants (P < 0.05). MT-IIA treatment of mixed retinal cultures increased megalin expression in RGCs, and pre-treating cells with anti-megalin antibodies prevented MT-IIA-stimulated neurite extension. Our results indicate that MT-IIA stimulates neurite outgrowth in RGCs and may do so via the megalin receptor; we propose that neurite extension is triggered via signal transduction pathways activated by the NPxY motifs of megalin’s cytoplasmic tail.

Keywords:  Metallothionein - Retinal ganglion cells - Neuroregeneration

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Metal binding of metallothionein-3 versus metallothionein-2: lower affinity and higher plasticity

Peep Palumaa (1), , Indrek Tammiste (2), Keiu Kruusel (1), Liina Kangur (1), Hans Jörnvall (3) and Rannar Sillard (3)

(1) Department of Gene Technology, Tallinn Technical University Akadeemia tee 23, 12618 Tallinn, Estonia
(2) National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia
(3) Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden

Journal:    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics. Volume 1747, Issue 2, 14 March 2005, Pages 205-211

Abstract: Mammalian metallothioneins (MTs) are involved in cellular metabolism of zinc and copper and in cytoprotection against toxic metals and reactive oxygen species. MT-3 plays a specific role in the brain and is down-regulated in Alzheimer's disease. To evaluate differences in metal binding, we conducted direct metal competition experiments with MT-3 and MT-2 using electrospray ionization mass spectroscopy (ESI-MS). Results demonstrate that MT-3 binds Zn2+ and Cd2+ ions more weakly than MT-2 but exposes higher metal-binding capacity and plasticity. Titration with Cd2+ ions demonstrates that metal-binding affinities of individual clusters of MT-2 and MT-3 are decreasing in the following order: four-metal cluster of MT-2>three-metal cluster of MT-2≈four-metal cluster of MT-3>three-metal cluster of MT-3>extra metal-binding sites of MT-3. To evaluate the reasons for weaker metal-binding affinity of MT-3 and the enhanced resistance of MT-3 towards proteolysis under zinc-depleted cellular conditions, we studied the secondary structures of apo-MT-3 and apo-MT-2 by CD spectroscopy. Results showed that apo-MT-3 and apo-MT-2 have almost equal helical content (approximately 10%) in aqueous buffer, but that MT-3 had slightly higher tendency to form α-helical secondary structure in TFE–water mixtures. Secondary structure predictions also indicated some differences between MT-3 and MT-2, by predicting random coil for common MTs, but 22% α-helical structure for MT-3. Combined, all results highlight further differences between MT-3 and common MTs, which may be related with their functional specificities.

Keywords: MT-3; Alzheimer's disease; ESI-MS; Secondary structure prediction; CD spectroscopy

Abbreviations: MT, metallothionein; DTT, dithiothreitol; TFE, 2,2,2,-trifluoroethanol; CD, circular dichroism; ESI MS, electrospray ionization mass spectroscopy
 

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Metal binding to brain-specific metallothionein-3 studied by electrospray ionization mass spectrometry.

Palumaa P (1), Eriste E (2), Kruusel K (1), Kangur L, (1) Jörnvall H (2), Sillard R (2).

(1) Department of Gene Technology, Tallinn Technical University, Ehitajate tee 5, EE-19086 Tallinn, Estonia.

(2) Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.

Journal:    Cell Mol Biol (Noisy-le-grand). 2003 Jul;49(5):763-768.

Abstract: Metallothionein-3 (MT-3) is a brain-specific isoform of metallothioneins, which is down-regulated in Alzheimer's disease (AD), inhibits the growth of neurons in vitro, and differs from common MTs also in gene regulation. To elucidate the differences in structure and function between MT-3 and common MTs, Zn2+ and Cd2+ binding to MT-3 and MT-1 were studied using electrospray ionization time of flight mass spectrometry (ESI TOF MS) at pH values between 7.5 and 2.7. The metal binding properties of MT-3 differ considerably from those of MT-1. After reconstitution with a metal excess, metallated MT-3 exists as a mixture of Zn7MT-3 (or Cd7MT-3, respectively) and several metalloforms with stoichiometries below and above seven. In contrast, MT-1 exists as a single Zn7MT-1 (or Cd7MT-1). Lowering of pH leads to a stepwise release of metals from metallated MT-3, first from extra sites, then from the 3-metal cluster and finally from the 4-metal cluster. At acidic pH values the 4-metal cluster of MT-3 is slightly more stable than that of MT-1. The results demonstrate higher structural plasticity, dynamics and metal binding capacity of MT-3 than of MT-1, which makes MT-3 suitable as a zinc buffer-transfer molecule in zinc-enriched neurons functioning at conditions of fluctuating zinc concentrations.

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Brain-Specific Metallothionein-3 Has Higher Metal-Binding Capacity than Ubiquitous Metallothioneins and Binds Metals Noncooperatively

Peep Palumaa, Elo Eriste, Olga Njunkova, Lesja Pokras, Hans Jörnvall, and Rannar Sillard

Centre for Gene Technology, Tallinn Technical University, Ehitajate tee 5, EE-19086 Tallinn, Estonia,

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden,

National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, EE-12618 Tallinn, Estonia

Journal:     Biochemistry 41(19), 6158-6163; 2002.

Abstract:  Zinc metabolism in the cells is largely regulated by ubiquitous small proteins, metallothioneins (MT). Metallothionein-3 is specifically expressed in the brain and is down regulated in Alzheimer's disease. We demonstrate by mass spectrometry that MT-3, in contrast to common MTs, binds Zn2+ and Cd2+ in a noncooperative manner and can also bind higher stoichiometries of metals than seven. MT-3 reconstituted with seven metals exists in a dynamic equilibrium of different metalloforms, where the prevalent metalloform is Me7MT-3, but metalloforms with 6, 8, and even 9 metals are also present. The results from pH and stability studies demonstrate that the heterogeneity of metalloforms originates from the N-terminal -cluster, whereas the C-terminal -cluster of MT-3 binds four metal ions such as that of common MTs. Experiments with EDTA demonstrate that the -cluster of ZnMT-3 has a higher metal transfer potential than the -cluster of Zn7MT-2. Moreover, ZnMT-3 loses metals during ultrafiltration. MT-3, reconstituted with an excess of Zn2+ or Cd2+, exists as a dynamic mixture of metalloforms with higher than 7 metal stoichiometries (8-11). Such forms of ZnMT-3 are unstable and decompose partly already during a rapid gel filtration, whereas CdMT-3 forms are more stable. Extra metal ions may bind to the -cluster region as well as to the carboxylates of MT-3. The specific metal-binding properties of MT-3 could be functionally implemented for buffering of fluctuating concentrations of zinc in zincergic neurons and for transfer of zinc to synaptic vesicles.
 

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