MT Isoforms Research Articles

Characterization of metallothionein isoforms by reversed-phase high-performance liquid chromatography with on-line post-column acidification and electrospray mass spectrometric detection

Post-column acidification of the chromatographic effluent was developed to eliminate artefacts in investigations of the polymorphism of metallothionein (MT) by microbore reversed-phase HPLC with detection by pneumatically assisted electrospray mass spectrometry. Metallated species (Cd, Zn and mixed Cd–Zn complexes) were decomposed on-line to produce apo metallothioneins of which the molecular masses were determined by MS. Besides the simplification of the mass spectra taken at the apexes of the chromatographic peaks, the method resulted in a 10-fold improvement of the detection limit of metallothionein and allowed a more comprehensive and less ambiguous detection and identification of the iso- and subisoforms. The method was applied to the characterization of rabbit liver metallothioneins: rabbit liver MT (purified by size-exclusion chromatography only) and MT-1 and MT-2 isoform fractions purified additionally by anion-exchange chromatography.

Quantitation of metallothionein isoforms in mouse liver on capillary zone electrophoresis

A method for determining concentration of metallothionein (MT) isoforms in mouse liver was established by using capillary zone electrophoresis (CZE), and carbonic anhydrase was used as an internal standard. The cytosol fraction was heated at 100°C for 1 min, and the supernatant was applied directly to CZE after filtration. Both MT-1 and MT-2 isoforms were identified from the migration times of purified MT isoforms, and the peaks were definitely identified from the other peaks. In addition, carbonic anhydrase was adopted as an internal standard. Based on standard curves of purified MT isoforms, concentrations of MT isoforms in the liver were determined from the estimates of peak areas. In control mouse liver, MT-1 isoform was detected at 48.5±25.4 μg/g wet weight, and MT-2 isoform was 154.0±39.7 μg/g. Twenty-four hours after zinc injection, MT-1 increased to 773.7±374.3 μg/g, and MT-2 was 487.2±207.1 μg/g. The addition of ascorbic acid to the homogenizing medium resulted in the decrease of MT isoforms in mouse liver. From these findings, we concluded that CZE analysis using a polyacrylamide-coated tube at neutral pH makes quantitation of MT isoforms in mouse liver possible.

Localization of Metallothionein-I and -III Expression in the CNS of Transgenic Mice with Astrocyte-Targeted Expression of Interleukin 6

The effect of interleukin-6 (IL-6) on metallothionein-I (MT-I) and MT-III expression in the brain has been studied in transgenic mice expressing IL-6 under the regulatory control of the glial fibrillary acidic protein gene promoter (GFAP-IL6 mice), which develop chronic progressive neurodegenerative disease.In situhybridization analysis revealed that GFAP-IL6 (G16-low expressor line, and G36-high expressor line) mice had strongly increased MT-I mRNA levels in the cerebellum (Purkinje and granular layers of the cerebellar cortex and basal nuclei) and, to a lesser degree, in thalamus (only G36 line) and hypothalamus, whereas no significant alterations were observed in other brain areas studied. Microautoradiography and immunocytochemistry studies suggest that the MT-I expression is predominantly localized to astrocytes throughout the cerebrum and especially in Bergman glia in the cerebellum. However, a significant expression was also observed in microglia of the GFAP-IL6 mice. MT-III expression was significantly increased in the Purkinje cell layer and basal nuclei of the cerebellum, which was confirmed by Northern blot analysis of poly(A)+mRNA and by ELISA of the MT-III protein. In contrast, in the G36 but not G16 mice, transgene expression of IL-6 was associated with significantly decreased MT-III RNA levels in the dentate gyrus and CA3 pyramidal neuron layer of the hippocampus and, in both G36 and G16 mice, in the occipital but not frontal cortex and in ependymal cells. Thus, both the widely expressed MT-I isoform and the CNS specific MT-III isoform are significantly affected in a MT isoform- and CNS area-specific manner in the GFAP-IL6 mice, a chronic model of brain damage.

Isoform separation of metallothioneins by capillary zone electrophoresis with Tris–tricine buffer in the presence or absence of methanol

A simple capillary electrophoretic method for analysis of metallothionein (MT) isoforms has been developed. Rabbit liver metallothionein (MT) isoforms are readily separated by capillary zone electrophoresis (CZE) with 150 mM Tris(hydroxymethyl)aminomethane (Tris)–150 mM N-Tris(hydroxymethyl)methylglycine (Tricine) buffer (pH 7.50) using an uncoated polyimide cladded fused-silica capillary column thus offering an alternative method for MT separations under original conditions. The effects of buffer concentration, buffer pH and buffer composition on the separation of MT isoforms were investigated. The effect of the addition of organic solvent, MeOH, as buffer modifier on the separation was also studied. Addition of MeOH to the buffer system enhanced the separation and additional information of the heterogeneity within each isoform group was gained. Also the effect of pH on the separation is enhanced when using organic solvent modified buffer. Enhanced information is gained by using photodiode array detection allowing recording of UV spectra of the putative isoform peaks thus offering the possibility of characterisation of the peaks obtained by comparing them to the unique characteristics of metallothionein metal–thiol bonds. The Tris–tricine method presented in this paper allows the separation of peaks that were found to need two separate runs under different conditions for separation to occur when using Tris–borate buffer. Rabbit liver MT, theoretically a mixture of MT-1 and MT-2, exhibited two major peaks, one medium sized peak and several smaller peaks. The experiments indicate that rabbit liver MT exhibited peaks which were not found in either MT-1 or MT-2 isoforms or they were found only as small residues. Both MT-1 and MT-2 samples exhibited one major peak and several smaller peaks. Residues of MT-2 were found in the MT-1 sample and vice versa.

Speciation of metal complexes with biomolecules by reversed-phase HPLC with ion-spray and inductively coupled plasma mass spectrometric detection

Complementarity of ion-spray MS and ICP-MS as detection techniques in reversed-phase HPLC for the characterization of metals complexed by biomacromolecules is discussed by the example of the speciation of metallothionein-bound cadmium. The commercially purified rabbit liver MT-2 isoform is eluted from a microbore C8 column with a gradient of up to 50% methanol in acetate buffer (pH 6.0) to give one major and three minor peaks detected at 254 nm. The preparation is further characterized by using an ICP mass spectrometer interfaced with HPLC via a direct injection nebulizer which allows for the specific detection of cadmium down to the 10 ng mL–1 level. On-line detection by mass spectrometry with an ion-spray (pneumatically-assisted electrospray) ion source further allows the determination of the molecular masses of the eluted compounds.

Separation of metallothionein isoforms of mouse liver cytosol by capillary zone electrophoresis

Metallothionein (MT) isoforms of mouse liver cytosol were separated by capillary zone electrophoresis (CZE) using a polyacrylamide-coated tube at neutral pH, samples prepared from non-treated, heat-treated, and ethanol-precipitated specimens were compared. The liver was homogenized in three kinds of media, 0.25 M sucrose containing 100 mM Tris–HCl buffer at pH 7.4 (BS), BS containing 1% ascorbic acid (BS-C), and BS containing 5 mM β-mercaptoethanol (BS-M). Mouse liver was used 24 h after subcutaneous injection of 50 mg Zn kg −1. In the non-treated specimen of the cytosol fraction, the MT-2 isoform was separated in all three media, while the MT-1 isoform was difficult to identify. In the ethanol-precipitated specimen, MT isoforms were separated well using either BS or BS-C. However, when BS-M was used, a small MT-2 peak was obtained the MT-1 peak could not be identified. MT-1 isoform in the heat-treated specimen was difficult to identify. In contrast, MT-2 isoform was separated well in all three kinds of media. In the non-treated specimen of the control liver cytosol, the MT-2 isoform was detected using all three media, the MT-1 peak was undetected. Based on these results, MT isoforms can be detected in the crude cytosol fraction of liver using CZE combined with a polyacrylamide-coated tube at neutral pH.

Metallothionein isoforms from horse, rabbit and rat separated by capillary zone electrophoresis at low pH

A comparative study of MT isoforms in rat liver and in commercial Sigma MT preparations from rabbit liver and horse kidney was performed using capillary zone electrophoresis (CZE). Electropherograms revealed the co-migration of MT forms from these species. A special form, the a-form (not binding Cd), occurred in various MT samples in different amounts, depending on the method used for MT purification. In the rabbit liver electropherogram a main form appeared (the b-form), which might be a modified MT form. A band of unknown composition, running ahead of the rat liver MT-I and -II forms on polyacrylamide gels, not having Cd binding affinity, probably had its counterpart in a yet unidentified CZE peak. CZE electropherograms of purified MT samples may contain main peaks that do not represent genuine and functional MT isoforms. Results are also presented which indicate that at low pH the MT-II form is more unstable than MT-I.

Polymorphism and identification of metallothionein isoforms by reversed-phase HPLC with on-line ion-spray mass spectrometric detection.

Microbore reversed-phase HPLC with on-line ion-spray mass spectrometric detection is proposed for a study of polymorphism of rabbit liver metallothionein (MTRL) and its major isoforms MT-1 and MT-2. Separation conditions had been optimized until each chromatographic peak could be attributed to a single metalloprotein species, of which the molecular mass could be determined by ionspray MS. At the optimized conditions (elution with the gradient 5-8% of acetonitrile within 50 min using a 5 mM acetate buffer at pH 6), the chromatogram of MTRL showed five peaks, whereas those of MT-1 and MT-2 showed nine and seven different peaks, respectively. The on-line determination of the molecular masses (+/- 1 Da) of the compounds eluted permits the unambiguous cataloguing and further referencing of putative and true MT subisoforms. The results obtained are compared with those obtained by direct ion-spray MS of the MT preparations at different pHs, with a goal to identify possible chromatographic artifacts. Metals (Cd, Cu) in the eluted complexes were studied by HPLC with on-line ICP MS detection.

Role of placental metallothionein in maternal to fetal transfer of cadmium in genetically altered mice

The role of placental metallothionein (MT) as a barrier for maternal to fetal transfer of cadmium (Cd) was investigated using mice which overexpressed the MT-1 isoform (MT-1*), mice which did not express the MT-1 and 2 isoforms (MT-null) and control mice (C57BL/6). In addition, immunohistochemical localization of MT in the placenta was determined in these mice. Two days prior to parturition, the mice were injected with radioactive 109Cd chloride (4 μCi, 0.6 ng Cd/mouse) and killed 24 h later. Organs and fetuses were collected and radioactivity, MT and metal levels were measured. Cd accumulated mainly in the liver and kidney (80% of administered dose) with very low levels (0.1–0.3%) detected in fetuses. When analyzed on a per organ or per gram basis, MT-null fetuses accumulated significantly more Cd (3–10-fold) than the control fetuses and there was no significant difference in fetal Cd accumulation in the MT-1* and control fetuses. As expected, MT and zinc levels were higher in MT-1* than C57BL/6 mice and no MT was detected in MT-null mice. Most striking was the high hepatic MT levels in MT-1* dams (4 mg/g). Immunohistochemical analysis showed MT staining in spongiotrophoblasts, glycogen cells, visceral yolk sac, trophoblast giant cells and maternal decidual cells with the MT-1* placenta staining much more intensely as compared to control placenta. The results suggest that placental MT reduces maternal to fetal Cd transfer, however the low doses of Cd administered in the present experiment resulted in high levels of Cd accumulation in liver and kidney in all groups of mice with a low concentration of Cd reaching the placenta. Thus, the role of placental MT as a barrier for Cd is inconclusive.

Characterization of horse kidney metallothionein isoforms by electrospray MS and reversed-phase HPLC-electrospray MS.

Pneumatically assisted electrospray mass spectrometry (ESMS) in the direct mode and as a chromatographic detection technique was developed for the characterization of horse kidney metallothionein isoforms. Direct analysis in an acidic medium showed the presence of three major and five minor isoforms, the molecular masses of which were determined. The presence of the major isoforms (two of which matched the molecular masses calculated according to the published sequences) was confirmed by complexation with Cd at pH 4.0 and the determination of the stoichiometry of the complexes formed. Reversed-phase chromatography of Cd7-MT complexes (pH 6.0) gave two signals corresponding to the MT-1A and MT-1B isoforms. A post-column acidification procedure was developed to eliminate the possibility of artefacts associated with the formation of mixed-metal (Cd, Zn) complexes during chromatography in neutral media, and to improve the accuracy of the determination of the molecular mass of MT isoforms.

Astrocyte metallothioneins (MTs) and their neuroprotective role.

I have briefly detailed in this review the role of astrocytes in MeHg neurotoxicity, emphasizing the mechanisms and significance of astrocytic swelling in neuropathological conditions. I have also described the functions of brain MTs and have reported recent observations on their propensity to attenuate cytotoxicity. While it is unclear why three different MT genes are expressed in the brain, this redundancy should allow for greater accumulation of MTs under stressful conditions compared to its accumulation if only a single gene was present. Another explanation may be that genes encoding functionally identical MTs might be regulated independently, thus permitting cell-specific MT expression. Finally, each of the three MT isoforms may have distinct functions. As discussed herein, astrocytic MTs afford protection from the acute cytotoxic effects of MeHg, reversing the effect of this organometal on RVD and inhibition of taurine release. Whether other vital cellular functions are protected by MTs will have to await future studies, as will the mechanisms associated with MT-induced cellular protection. That the resistance to heavy metal toxicity is closely related to the cellular ability to synthesize MTs, raises interesting questions regarding the potential involvement of heavy metals in neurodegenerating (amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease) under conditions of compromised MT synthesis. Future studies on the expression and regulation of MT genes are likely to culminate in novel strategies for manipulating intracellular MT levels, providing insight to their role in both health and disease.

Acylphosphatase is involved in differentiation of K562 cells

The level of both isoforms of acylphosphatase was evaluated in the human erythroleukemia K562 cell line during differentiation. K562 cells were treated with PMA, which induces megakaryocytic differentiation, and with aphidicolin or hemin, which stimulate erythrocytic differentiation. While the MT isoform showed an average 10-fold increase independently of the differentiating agent used, only hemin treatment caused a similar increase of the CT isoform, suggesting a different role of the two isoforms in the cell. Treatment with either hemin or aphidicolin of K562 cells overexpressing the two acylphosphatase isoforms suggested the possibility that acylphosphatases play a role in the onset of differentiation.

Metalloprotein analysis by capillary isoelectric focusing.

Capillary isoelectric focusing (cIEF) was used to analyze three metalloproteins: conalbumin, transferrin and metallothionein (MT). Two different ampholyte mixtures were employed that generated linear pH gradients of 3-10 and 5-8. Several different proteins and one peptide with known isoelectric points (pIs) were used to establish linear relationships between peak migration time and pI. These standards were also used as internal markers to estimate peak pI values of the metalloproteins subjected to cIEF. Conalbumin (iron-free) subjected to cIEF with a pH gradient of 3-10 yielded a single major component (pI 7.17). When the protein was saturated with iron (2 Fe3+/mol protein), a shift to lower pI was observed with a major peak (pI 6.24) and a lesser peak (pI 6.09). Mixing iron-free with iron-saturated conalbumin or adding iron to iron-free conalbumin prior to cIEF produced an additional peak (pI 6.68) that was presumed to be conalbumin containing a single iron atom (monoferric form). Human transferrin subjected to cIEF with a pH range of 3-10 gave a similar separation pattern to conalbumin with four major peaks at pI values of 6.25 (apotransferrin), 5.96 (monoferric form), 5.48 and 5.34 (diferric forms). Additional resolution of the molecular forms of both conalbumin and transferrin was achieved using a narrower pH gradient (5-8). Rabbit liver MT subjected to cIEF with a pH gradient of 3-10 gave a complex separation pattern with two prominent peaks (pI values of 3.73 and 3.56) that were presumed to be the fully metal-saturated MT-1 and MT-2 isoforms. When individual MT isoforms (MT-1 and MT-2) were separately subjected to cIEF with a pH gradient of 3-10, heterogeneous peaks with higher pI values (4.12-4.74) were observed. In contrast, horse kidney MT gave a single predominant peak with a pI of 4.09. MT samples could be separated using a pH gradient of 5-8 despite the fact that their apparent pI values were below the limits of the pH gradient established. In general, the heterogeneity observed for conalbumin, transferrin and MT proteins subjected to cIEF reflects the presence or absence of bound metal. Thus, cIEF represents a potentially useful analytical method which can provide information concerning the metal-binding characteristics of these and perhaps other metalloproteins.

Metallothioneins 1 and 2 Are Expressed in the Olfactory Mucosa of Mice in Untreated Animals and during the Regeneration of the Epithelial Layer

We have examined the expression of the MT1 and MT2 isoforms of metallothionein in the mouse olfactory mucosa. In untreated mice, metallothionein was strongly expressed in supporting cells, acinar cells of the Bowman's glands, and olfactory neurons. Expression was however restricted to a subset of cells within each type, and to zones within the olfactory system. Irrigation with ZnSO4solution caused exfoliation of the olfactory epithelium and during the resultant regeneration, metallothionein immunoreactivity was associated with the proliferating basal cells. The ability to express MTs 1 and 2 did not appear to be obligatory for the early stages of regeneration since mice which do not express these isoforms responded similarly to wild type mice. Strong nuclear expression of metallothionein was noted in the untreated olfactory chamber following unilateral irrigation.

Role of Metallothionein in Cadmium-Induced Hepatotoxicity and Nephrotoxicity

MT, a cysteine-rich, metal-binding protein, exists in most tissues and is easily induced by many stimuli. There are four major MT isoforms in mammalian tissues, with MT-I and -II present in all tissues, MT-III only in brain, and MT-IV located in epithelium. Many factors regulate MT synthesis, such as age, species, hormones, inflammation, and various chemical treatments. Not only is MT synthesis important, but degradation of MT is also an important mechanism of MT regulation. The importance of MT in Cd toxicology has been extensively investigated. MT does not have a major effect on absorption and tissue distribution of Cd, but it does play a major role in binding Cd in the cell, thus decreasing its elimination from the body, especially into the bile. MT is at least partially responsible for the retention of Cd in tissues and the long biological half-life of the metal. MT plays an important role in Cd tolerance and Cd-induced hepatotoxicity. MT binds Cd in the hepatic cytosol and renders it "inert." Therefore, MT is beneficial to the liver. However, the Cd-MT complex is nephrotoxic and is proposed to be responsible for chronic Cd poisoning. MT appears to play less of a protective role in Cd-MT-induced acute nephrotoxicity, and Zn-induced protection against CdMT acute renal injury is not mediated by MT. The role of MT in chronic Cd nephrotoxicity needs to be further clarified.

Identification and characterization of metallothionein III (growth inhibitory factor) from porcine brain

Porcine metallothionein III (MTIII) was isolated from brain tissue by the combination of gel filtration and anion-exchange chromatographies. The identity of MTIII was confirmed by comparing the chromatographic characteristics to other MT isoforms isolated from porcine liver. Porcine MTIII has a low molecular weight and a pl of 4.1. This protein contains both zinc and copper, and the zinc can be replaced by cadmium. Using reverse transcriptase-polymerase chain reaction, a 0.2 kb DNA fragment can be amplified from the porcine brain mRNA. This DNA fragment was demonstrated to contain MTIII coding region after cloning and sequencing. The revealed DNA sequence can be translated into 68 amino acids and shows a common structural characteristic of MTIII from other species. Northern blot analysis indicated that MTIII inRNA is expressed in every specified region of the porcine brain examined here.

Analysis of interaction between cadmium and metallothionein isoforms by capillary zone electrophoresis.

The effects of cadmium on the peak area of metallothionein (MT) protein were studied by capillary zone electrophoresis with a polyacrylamide-coated tube at neutral pH. When cadmium was added to a commercial standard MT-1 isoform prepared from rabbit liver, the peak are of the MT-1 isoform decreased in a time- and dose-dependent manner. The MT-2 isoform also decreased with time, but the rate of decrease was lower than that of the MT-1 isoform. When ethylene glyco-bis-(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA) was added to a solution containing cadmium and MT-1, the peak area recovered with increasing concentration of EGTA. Zinc caused a slight decrease in the peak areas of MT-1 and MT-2 compared with cadmium, while addition of sodium did not decrease the areas. Furthermore, the peak areas of MT isoforms of the crude extract prepared from zinc-treated mice also decreased with increasing concentration of cadmium. These results indicate that cadmium may changed the charge of MT, which may account for the observed differences in electrophoretic behavior.

Constitutive Expression of Metallothionein-III (MT-III), but Not MT-I, Inhibits Growth When Cells Become Zinc Deficient

BHK cells were stably transformed with plasmid constructs that allowed constitutive expression of either mouse metallothionein-I (MT-I) or MT-III to determine whether these isoforms have different physiological properties. Cells expressing equivalent amounts of MT-I or MT-III could grow in 60-fold more cadmium than nontransfected cells and they were 2- to 3-fold more resistant to zinc, copper, and cobalt. The results suggest that the two MT isoforms detoxify these metals similarly. MT-III reduced the amount of zinc available to activate a zinc-sensitive reporter gene; MT-I actually increased the expression of the reporter gene at low concentrations of zinc. Cells expressing MT-I or MT-III also responded differently to zinc deficiency. When cells expressing MT-I were deprived of zinc, the amount of MT-I protein declined to undetectable levels, even though MT-I mRNA was still abundant, and cell proliferation was unaffected. In contrast, when cells expressing the MT-Ill gene were deprived of zinc, cell proliferation was arrested and MT-III protein persisted. These results suggest that MT-I does not compete with essential zinc-requiring proteins and is degraded, whereas MT-III competes for zinc and exacerbates zinc deficiency.

In Vivo Expression of Metallothionein in Rat Alveolar Macrophages and Type II Epithelial Cells Following Repeated Cadmium Aerosol Exposures

This study examined the ability of alveolar macrophages and alveolar type II epithelial cells to accumulate cadmium (Cd) and to express metallothionein (MT). Lung cells were isolated from Lewis rats repeatedly exposed to a Cd aerosol (1.6 mg Cd/m3). Intracellular Cd concentration rose following Cd exposure and showed an increasing rend as a function of exposure number. Alveolar macrophages accrued approximately four times more Cd than type II epithelial cells similarly exposed. Macrophages and type II cells responded to the presence of intracellular Cd by increasing MT protein levels. MT concentration was highly correlated with intracellular Cd. Immunocytochemical studies revealed that not all macrophages and type II cells from Cd-exposed animals were immunopositive for MT and that the intensity of immunostaining varied within each cell population. Although a greater percentage of macrophages were immunopositive for MT than type II cells, a greater proportion of type II cells showed moderate and dark MT staining patterns. Oligonucleotide probes, shown to distinguish between MT-1 and MT-2 mRNA isoforms, were used to test for cell-specific differences in MT isoform gene expression. The basal level of MT-1 mRNA was greater in macrophages than in type II cells. Following Cd administration, the level of MT-1 mRNA and MT-2 mRNA increased in each cell class but the response to Cd was three times greater in alveolar macrophages. Neither macrophages nor type II cells expressed MT mRNA isoforms in equal proportions. Macrophages expressed more MT-1 mRNA when exposed to air and more MT-2 mRNA in response to Cd exposure. Type II cells, on the other hand, expressed more MT-2 mRNA than MT-1 mRNA regardless of whether the cells were exposed to air or Cd. In conclusion, the present study has demonstrated that (1) alveolar macrophages and type II cells respond to in vivo Cd exposure by increasing MT protein and mRNA levels; (2) MT expression is greater in macrophages than in type II cells and correlates well with intracellular Cd concentration; and (3) the MT-2 mRNA to MT-1 mRNA ratio is cell and treatment specific.

The use of solid phase concentrators for on-line preconcentration of metallothionein prior to isoform separation by capillary zone electrophoresis.

UV absorbance is a convenient detection method for monitoring a wide variety of different components separated by capillary electrophoresis (CE). However, a significant disadvantage of UV detection is its low sensitivity and this is a major factor limiting the application of CE as an analytical technique. In order to improve sensitivity, several methods of on-line sample preconcentration by electrofocusing, including "stacking" and isotachophoresis, have been investigated and the potential of on-line solid phase preconcentrators for this purpose has also been demonstrated. We have developed methods for the separation of isoforms of metallothionein (MT), a low M(r) metal-binding protein which functions as a detoxification system and cellular buffer for heavy metals, primarily in liver and kidney. While a variety of modifications to the capillary surface and electrolyte chemistry have been used to resolve many MT isoforms in liver samples from a variety of species, there are few available alternatives to UV detection and thus sensitivity limits the application of these methods to samples with low MT levels, such as urine or plasma. In this study we have investigated the use of an inexpensive and easy to assemble C18 concentrator which was fitted to the capillary inlet to extend the detection limits for MT isoform analysis. Samples were pressure loaded onto the concentrator for up to 5 min, eluted using 33% acetonitrile and then subjected to electrophoresis in borate or phosphate buffer. The purified isoforms MT-1 and MT-2 from rabbit liver were best resolved using an acidic acetonitrile eluent and a phosphate buffer electrolyte at pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)