不同亚型ZnMT使脱金属碳酸酐酶复活的比较研究

通过荧光探针、酶的活力测定及对金属硫蛋白（ＭＴ）分子中金属与巯基配位键的特征吸收检测,比较研究了锌诱导兔肾不同亚型ＺｎＭＴ使脱金属碳酸酐酶（ＡｐｏＣＡ）复活的能力大小。结果表明,不同亚型ＺｎＭＴ对ＡｐｏＣＡ具有不同的反应活性,ＭＴ１比ＭＴ_２对于ＡｎｏＣＡ的复活具有更强的反应活性,此结论与我们在研究不同亚型ＭＴ清除自由基时的结果相一致。这两种亚型ＭＴ在反应活性上的差异,很可能与其在生物体内功能上的分化密切相关。     

Distinct targeting and recycling properties of two isoforms of the iron transporter DMT1 (NRAMP2, Slc11A2)

The metal transporter DMT1 (Slc11a2) plays a vital role in iron metabolism. Alternative splicing of the 3' exon generates two DMT1 isoforms with different C-terminal protein sequences and a 3' untranslated region harboring (isoform I, +IRE) or not (isoform II, -IRE), an iron-responsive element. Isoform I is expressed at the plasma membrane of certain epithelial cells including the duodenum brush border, where it is essential for the absorption of nutritional iron. Isoform II is expressed in many cells and is essential for the acquisiton of transferrin iron from acidified endosomes. The targeting and trafficking properties of DMT1 isoforms I and II were studied in transfected LLC-PK(1) kidney cells, with respect to isoform-specific differences in function, subcellular localization, endocytosis kinetics, and fate upon internalization. Isoform I showed higher surface expression and was internalized from the plasma membrane with slower kinetics than that of isoform II. As opposed to isoform II, which is efficiently sorted to recycling endosomes upon internalization, isoform I was not efficiently recycled and was targeted to lysosomes. Thus, alternative splicing of DMT1 critically regulates the subcellular localization and site of Fe(2+) transport.     

The role of aldehyde dehydrogenases in the malonic dialdehyde metabolism in the rat liver

The enzymes catalyzing the NAD-dependent oxidation of malonic dialdehyde (MDA) were isolated from rat liver extracts. Upon 5'-AMP-Sepharose chromatography MDA dehydrogenase was separated into two isoforms, I and II. Isoform I was eluted from the affinity carrier with a 0.1 M phosphate buffer pH 8.0. This isoform had a broad substrate specificity towards aliphatic and aromatic aldehydes. Kinetic studies showed that short- and medium-chain aliphatic aldehydes (C2-C6) were characterized by the lowest Km values and the highest Vmax values. The Km' values for MDA and acetaldehyde were 2.8 microM and 0.69 microM, respectively. Isoform II was eluted with a 0.1 M phosphate buffer pH 8.0 containing 0.5 mM NAD, was the most active with medium- and long-chain aliphatic aldehydes (C6-C11) and had Km values for MDA and acetaldehyde equal to 37 microM and 52 microM, respectively. Isoform I was much more sensitive towards disulfiram inhibition than isoform II. Both isoforms had an identical molecular mass (93 kD) upon gel filtration. It is concluded that MDA dehydrogenase isoform I is identical to mitochondrial aldehyde dehydrogenase having a low Km for acetaldehyde, whereas isoform II may be localized in liver cytosol. The role of aldehyde dehydrogenases in the metabolism of aldehydes derived from lipid peroxidation is discussed.     

The Saccharomyces cerevisiae Crs5 Metallothionein metal-binding abilities and its role in the response to zinc overload

Crs5 is a Saccharomyces cerevisiae Metallothionein (MT), non-homologous to the paradigmatic Cu-thionein Cup1. Although considered a secondary copper-resistance agent, we show here that it determines survival under zinc overload in a CUP1-null background. Its overexpression prevents the deleterious effects exhibited by CUP1-CRS5-null cells when exposed to combined Zn/Cu, as it does the mouse MT1 Zn-thionein, but not Cup1. The detailed characterization of Crs5 in vivo and in vitro Zn(II)-, Cd(II)- and Cu(I)-binding abilities fully supports its resemblance to mammalian MTs. Hence, Crs5 exhibits a good divalent metal-binding ability, yielding homometallic, highly chiral and stable Zn and Cd complexes when expressed in media enriched with these metal ions. In Cu-supplemented cultures, heterometallic Zn,Cu complexes are recovered, unless aeration is kept to a minimum. These features define a Crs5 dual metal-binding behaviour that is significantly closer to Zn-thioneins than to Cu-thioneins. Protein sequence similarities fully support these findings. Overall, a Crs5 function in global metal cell homeostasis, based on its Zn-binding features, is glimpsed. The comparative evaluation of Crs5 in the framework of MT functional differentiation and evolution allows its consideration as a representative of the primeval eukaryotic forms that progressively evolved to give rise to the Zn-thionein lineage.     

Isolation, characterization and binding properties of two rat liver fatty acid-binding protein isoforms

Mammalian liver has only one fatty acid-binding protein (L-FABP) while the liver of non-mammalian vertebrates expresses a liver basic FABP (Lb-FABP) in addition to other members of the FABP family. We explore the possibility that L-FABP isoforms accomplish, in the liver of mammals, the metabolic functions corresponding to the different FABPs present in the liver of non-mammalian vertebrates. We have isolated isoforms I and II which have a different residue 105, Asn in the former and Asp in the latter. We made a conformational comparison of the apo-isoforms by intrinsic fluorescence emission and fourth-derivative spectroscopy, native-state proteolysis and unfolding curves. Ligand affinity was studied by measuring cis-parinaric acid displacement by different ligands. They have differences in their molecular conformation, including the environment of the binding site. Isoform II has probably a more open conformation than isoform I, thus allowing the binding of a greater variety of ligands. The affinity of isoform II for lysophospholipids, prostaglandins, retinoids, bilirubin and bile salts is greater than that of isoform I. These characteristics of rat L-FABP isoforms I and II suggest that they may accomplish different functions as happens with those of the different FABP types in non-mammalian species.     

Metal binding of metallothioneins in human astrocytomas (U87 MG,IPDDC-2A)

Astroglia cells structurally and nutritionally support neurons in the central nervous system. They play an important role in guiding the construction of the nervous system and controlling the chemical and ionic environment of neurons. They also represent the major sites for accumulation and immobilisation of toxic metal ions most probably connected with metallothioneins. For this reason astroglia cells possess high cytosolic levels of metallothioneins I, II and III (MT-I,II,III). Our aim was to establish the inducibility and metal binding of MTs in two human astrocytoma cell lines, U87 MG (astrocytoma–glioblastoma, grade IV) and IPDDC-2A (astrocytoma, grade II), on exposure to cadmium chloride (1 μM). MTs were identified by molecular weight (size exclusion chromatography) and their metal content (Cd, Zn and Cu) to follow the interactions between metals. We showed that MTs are constitutively expressed in both human astrocytoma cell lines. In accordance with the higher malignancy grade of U87 MG, the amount of MTs was higher in U87 MG than in IPDDC-2A cells. After 24 hours of exposure to Cd their expression greatly increased in both cell lines and they were capable of immobilising almost all water soluble Cd. Induction of MTs in U87 MG cells was additionally followed up to 48 hours with exposure to different concentrations of CdCl₂ (1, 10 μM). Induction was a time dependent process throughout the period. Isoform III (identified by chromatographic separation of isoform III from I/II) was present at all exposure times, but only in traces with respect to the prevailing amounts of MT-I/II isoforms. So induction can be attributed to isoform I/II only.     

High-performance hydrophobic interaction chromatography of estrogen receptors and magnesium dependent protein kinase(s): detection of two molecular forms of estrogen receptors in the presence and absence of sodium molybdate

The separation characteristics of estrogen receptors (ER) from human breast cancer were evaluated based on their hydrophobic properties. Results show that (1) two distinct hydrophobic isoforms of ER exist either in the presence of sodium molybdate (peaks MI and MII with retention times of 15-17 min and 24-26 min) or in its absence (peaks I and II with retention times of 25-27 min and 34-36 min respectively); (2) this is observed whether molybdate (MoO2-4) is added to prepared cytosol or to the buffer prior to homogenization; (3) isoform MII and I separated with similar retention times suggesting they are the same ER species; and (4) isoform MI (Rt = 15-17 min) is a distinct ER species from either MII/I (Rt = 25-28 min) or II (Rt = 34-36 min). The latter isoform represents a highly hydrophobic species seen only in the absence of MoO2-4. Finally, (5) MoO2-4 ions appear to interconvert the most hydrophobic species (II) into the least hydrophobic isoform (MI) with virtually no change in the quantity of isoform(s) MII/I. However, it cannot be ascertained if the II----MI interconversion proceeds via isoform MII/I. Isoform II may result from the interaction with the stationary phase via its DNA binding site since MoO2-4, which is suggested to directly interact with this site, selectively interacts with peak II. These results imply the usefulness of inclusion of receptor stabilizing reagents in the mobile phase for preserving receptor integrity and in elucidating the interrelationships of ER isoforms and associated macromolecules.     

Cadmium removal from human plasma by Cibacron Blue F3GA and thionein incorporated into polymeric microspheres

Poly(2-hydroxyethylmethacrylate–ethyleneglycoldimethacrylate) [poly(HEMA–EGDMA)] microspheres carrying Cibacron Blue F3GA and/or thionein were prepared and used for the removal of cadmium ions Cd(II) from human plasma. The poly(HEMA–EGDMA) microspheres, in the size range of 150–200 μm in diameter, were produced by a modified suspension copolymerization of HEMA and EGDMA. The reactive triazinyl dye-ligand Cibacron Blue F3GA was then covalently incorporated into the microspheres. The maximum dye incorporation was 16.5 μmol/g. Then, thionein was bound onto the Cibacron Blue F3GA-incorporated microspheres under different conditions. The maximum amount of thionein bound was 14.3 mg/g. The maximum amounts of Cd(II) ions removed from human plasma by poly(HEMA–EGDMA)–Cibacron Blue F3GA and poly(HEMA–EGDMA)–Cibacron Blue F3GA–thionein were of 17.5 mg/g and 38.0 mg/g, respectively. Cd(II) ions could be repeatedly adsorbed and desorbed with both types of microspheres without significant loss in their adsorption capacity.     

Formation, circular dichroism and x-ray photoelectron spectroscopy of hepatic Zn-thionein.

The formation of the powerful Zn binding protein called Zn-thionein was examined using male albino rats and [14C]cysteine, as cystein is known to be the most abundant constituent of this metal protein. 65% of the hepatic [14C]cysteine was incorporated into the protein portion of freshly prepared Zn-thionein. The protein was isolated by a combination of ethanol/chloroform treatment and various chromatographic steps, including ion exchange and gel filtration. 4.7 mol of Zn, 0.02 mol of Cd and less than 0.001 mol of either Cu or Hg were found per 12 000 g of portein. It was presumed that considerable amounts of Zn were lost during these isolation procedures, with the consequence of disulphide gridge formation. Indeed, the presence of R-S-S-R was deduced from circular dichroism and X-ray photoelectron spectroscopy. Due to the clearly detectable disulphide chromophore in the circular dichroism spectrum, it was possible to assign the shoulder at S 2p1/2,3/2 = 162.7 eV of the X-ray photoelectron spectrum of native Zn-thionein to R-S-S-R and not to strongly polarized sulphur. Upon reducing R-S-S-R-containing native Zn-thionein with dithiothreitol, all oxidised thiolate moieties of the thionein molecule could be restored. The addition of ZnCl2 with the subsequent desalting of extraneously bound Zn2 yielded a homogeneous Zn-thionein with 9.6 mol Zn2 per mol protein. A stoichiometry of ZnRS 1:3 was seen, which confirmed earlier reports of the existence of the mixed Cd,Zn-thionein. The conversion of mixed Cd,Zn-thionein into homogeneous Zn-, Cd-, Hg- and Cu-thionein by the gel filtration technique proved successful. From chiroptical measurements, the extraordinary contribution of the metal chromophores to the circular dichroism was seen. Due to the differences in the geometry of complexes formed by the respective metal ions, dramatic changes in the protein portion were expected. Polyacrylamide disc electrophoresis of purified native untreated Zn-thionein resulted in the appearance of two or more bands. This phenomenon was attributed to the different migration rates of cystine-thionein and thiolate-rich Zn-thionein, and was consistent with the spectral properties of the above Zn-protein species. By contrast, only one single band was monitored when a homogeneous metal-thionein was electrophoresed.     

Isolation of copper thionein from rat liver

The inducible Cu-binding protein from adult rat liver previously referred to as Cu-chelatin has been purified and shown to be Cu-thionein. The Cu-protein was purified to homogeneity by gel filtration and thiopropyl-Sepharose chromatography. The Cu-thionein exhibited an amino acid composition similar but not identical to that of the two forms of rat liver Cd,Zn-thionein. The polypeptide-chain molecular weight of Cu-thionein was indistinguishable from that of Cd,Zn-thionein. The identification of the Cu-protein as metallothionein was substantiated by the complete immunological cross-reactivity with antisera prepared against purified rat liver Cd,Zn-thionein. Purified Cu-thionein bound 9–11 g atoms of Cu per mole of protein in an electron paramagnetic resonance nondetectable form. The $\text{Cu}\text{Zn}$ ratio of the protein is about 100. Ion-exchange chromatography resolved the Cu-protein into three polymorphic forms which differed from the polymorphism of Cd,Zn-thionein.     

Beta-hexosaminidase,an enzyme from ripening bell capsicum (Capsicum annuum var variata)

beta-Hexosaminidase activity increased significantly during fruit development and ripening of bell capsicum (Capsicuum annuum var. variata). Three isoforms of beta-hexosaminidase from bell capsicum could be resolved upon ion exchange chromatography with step wise gradient (0.10, 0.15 and 0.20 M NaCl) having an abundance of 38, 47 and 15% for isoforms I, II and III respectively. Isoforms I and II were further purified on gel permeation chromatography. The pH optimum for these two isoforms was around 5. Isoform II exhibited higher thermal stability. Hg(2+) and Zn(2+) inhibited both, but isoform I showed a much higher inhibition by Cu(2+) also. The K(m) for isoforms I and II with pnp-beta-D-N-acetyl glucosamine pyranoside was 3.00 and 1.75 mM, respectively. Isoform II on SDS-PAGE was found to be a monomer with a relative molecular mass of 85 kD. This isoform (the most major) appeared to be electrophoretically homogeneous. beta-Hexosaminidase is novel in the context of fruit ripening. This enzyme has not been reported from fruits and studied hitherto.     

The purification and characterisation of the two forms of soluble starch synthase from developing pea embryos

Soluble starch synthase was purified 10000-fold from developing embryos of pea (Pisum sativum L.). The activity was resolved into two forms which together account for most if not all of the soluble starchsynthase activity in the embryo. The two isoforms differ in their molecular weights but are similar in many other respects. Their kinetic properties are similar, neither isoform is active in the absence of primer, and both are unstable at high temperatures, the activity being abolished by a 20-min incubation at 45° C. Both isoforms are recognised by antibodies raised to the granule-bound starch synthase of pea. Isoform II, which has the same molecular weight (77 kDa) as the granulebound enzyme, is recognised more strongly than Isoform I.     

Molecular biology of copper. A circular dichroism study on copper complexes of thionein and penicillamine.

Chicken liver Cd, Zn-thionein (metallothionein) was isolated from Cd-pretreated chickens weighing 1 500 g. The native Cd, Zn-thionein contained 9 g-atoms of metals per 12 000 g of protein. Upon the addition of Cu(CH3CN)4ClO4, all Cd2 and Zn2 were successfully replaced. 15 g-atoms of Cu from the acetonitrile perchlorate complex were bound to the protein. Due to the absence of aromatic amino acid residues, thionein has unique ultraviolet and circular dichroism properties. The shoulder of the ultraviolet spectrum at 250 nm (A250 X A280(-1) = 23.9) was shifted to 275 nm (A250 X A280(-1) = 1.6). No significant absorption was detected in the visible region. Th conformational changes of the protein moiety were much more visible in the circular dichroism spectra. The titration with Cu(CH3CH)2 caused the appearence of three new Cotton effects: 257.5 nm (+), 350 nm (+) and 301 nm (-). The negative Cotton effect at 239 nm of the original metallothionein was completely levelled off. The binding strength of copper with thionein is extraordinarily high: it survives proton treatment up to pH 1.9. Displacement of the Cd2 by Cu employing Cd-thionein which was formed at pH 2.2 resulted in the same circular dichroism properties as observed for Cu-thionein. D-Penicillamine proved a suitable model for the metal-free thionein, since redox reactions and polymerization of the sterically hindered thiol residue are known to be slow. The correlation of the circular dichroism properties of either copper complex using thionein or D-penicillamine was surprisingly high. Circular dichroism measurements of Cu(I)-D-penicillamine revealed Cotton effects at 255 nm (+), 280 nm (+) and 355 nm (-). Upon examining the red-violet mixed Cu(-i)-cu(II)-D-penicillamine complex, Cotton bands in the visible region at 425 nm (-) and 495 nm (+) were seen. In many blue copper enzymes, the copper is assumed to be in the neighborhood of both cysteine and aromatic amino acid residues, which are known to play an important role in the electron transfer. This is not the case in the Cu-thionein, which would explain many different properties of this copper protein. It is very attractive to conclude that the sterically hindered SH-group of D-penicillamine reacts with excess copper in a specific way, similar to the Cu-thionein. This phenomenon could explain the considerable success of D-penicillamine in the treatment of Wilson's disease.     

Transgenic analysis of the physiological functions of Mahogunin ring finger‐1 isoforms

Mahogunin Ring Finger‐1 (Mgrn1) null mutant mice have a pleiotropic phenotype that includes the absence of yellow hair pigment, abnormal head shape, reduced viability, and adult‐onset spongiform neurodegeneration. Mgrn1 encodes a highly conserved E3 ubiquitin ligase with four different isoforms which are differentially expressed and predicted to localize to different subcellular compartments. To test whether loss of specific isoforms causes different aspects of the mutant phenotype, we generated transgenes for each isoform and bred them onto the null mutant background. Mice expressing only isoform I or III appeared completely normal. Isoform II rescued or partially rescued the mutant phenotypes, whereas isoform IV had little or no effect. Our data show that different Mgrn1 isoforms are not functionally equivalent in vivo and that the presence of only isoform I or III is sufficient for normal development, pigmentation, and neuronal integrity. genesis 47:524–534, 2009. © 2009 Wiley‐Liss, Inc.     

Nonoxidative cadmium-dependent dimerization of Cd7-metallothionein from rabbit liver.

The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concentration-dependent dimerization of this protein was observed by gel filtration chromatographic studies. The dimeric MT form, isolated by gel filtration, contains approximately two additional and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissociation of dimers to monomers. The results of circular dichroism (CD) and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chemical shift positions similar to those observed for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.     

Synthesis and inhibition potency of novel ureido benzenesulfonamides incorporating GABA as tumor-associated carbonic anhydrase IX and XII inhibitors

New ureido benzenesulfonamides incorporating a GABA moiety as a linker between the ureido and the sulfonamide functionalities were synthesized and their inhibition potency determined against both the predominant cytosolic (hCA I and II) and the transmembrane tumor-associated (hCA IX and XII) isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The majority of these compounds were medium potency inhibitors of the cytosolic isoform hCA I and effective hCA II inhibitors, whereas they showed strong inhibition of the two transmembrane tumor-associated isoforms hCA IX and XII, with K_Is in nanomolar range. Only one derivative had a good selectivity for inhibition of the tumor-associated hCA IX target isoform over the cytosolic and physiologically dominant off-target hCA I and II, being thus a potential tool to develop new anticancer agents.     

Carbonic anhydrase inhibitors. Phenacetyl-, pyridylacetyl- and thienylacetyl-substituted aromatic sulfonamides act as potent and selective isoform VII inhibitors

A series of aromatic/heterocyclic sulfonamides incorporating phenyl(alkyl), halogenosubstituted-phenyl- or 1,3,4-thiadiazole-sulfonamide moieties and thienylacetamido; phenacetamido- and pyridinylacetamido tails were prepared and assayed as inhibitors of cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I, II and VII. The new compounds showed moderate inhibition of the two ubiquitous isoforms I and II (K_Is of 50–390 nM) and excellent inhibitory activity against the brain associated hCA VII (K_Is in the range of 4.7–8.5 nM). Isoform VII highly selective inhibitors are being detected for the first time, with selectivity ratios for inhibiting CA VII over CA II of 11–75, and for inhibiting CA VII over CA I of 10–49, which may be useful for understanding the role of CA VII in epileptogenesis and other physiologic processes.     

Chromone containing sulfonamides as potent carbonic anhydrase inhibitors

A series of sulfonamide derivatives incorporating substituted 3-formylchromone moieties were investigated for the inhibition of three human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, hCA I, II, and VI. All these compounds, together with the clinically used sulfonamide acetazolamide, were investigated as inhibitors of the physiologically relevant isozymes I, II (cytosolic), and VI (secreted isoform). These sulfonamides showed effective inhibition against all these isoforms with K_I’s in the range of 0.228 to 118 µM. Such molecules can be used as leads for discovery of novel effective CA inhibitors against other isoforms with medicinal chemistry applications.     

A novel alliinase from onion roots. Biochemical characterization and cDNA cloning

We have purified a novel alliinase (EC 4.4.1.4) from roots of onion (Allium cepa L.). Two isoforms with alliinase activity (I and II) were separated by concanavalin A-Sepharose and had molecular masses of 52.7 (I) and 50.5 (II) kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 51 (I) and 57.5 (II) kD by gel filtration fast-protein liquid chromatography. Isoform I had an isoelectric point of 9.3, while isoform II had isoelectric points of 7.6, 7.9, 8.1, and 8.3. The isoforms differed in their glycosylation. Both contained xylose/fucose containing complex-type N-linked glycans, and isoform II also contained terminal mannose structures. Both isoforms had activity with S-alk(en)yl-L-cysteine sulfoxides. Unlike other allium alliinases, A. cepa root isoforms had cystine lyase activity. We cloned a gene from A. cepa root cDNA and show that it codes for A. cepa root alliinase protein. Homology to other reported allium alliinase genes is 50%. The gene coded for a protein of mass 51.2 kD, with two regions of deduced amino acid sequence identical to a 25- and a 40-amino acid region, as determined experimentally. The A. cepa root alliinase cDNA was expressed mainly in A. cepa roots. The structure and function of the alliinase gene family is discussed.     

A novel isoform of ATPase c subunit from pearl millet that is differentially regulated in response to salinity and calcium

Vacuolar ATPases help in maintaining the pH of the vacuoles and thereby play a crucial role in the functioning of vacuolar sodium-proton antiporter. Though the various subunits that make V₁ and V₀ sector have been reported in plants their regulation is not understood completely. We have cloned three different isoforms of vacuolar ATPase subunit c (VHA-c) from Pennisetum glaucum with homologies among themselves varying from 38% to ∼73% at the nucleic acid level. Using real-time PCR approach we have shown that the three isoforms are regulated in a tissue-specific manner under salinity stress. While isoform III is constitutively expressed in roots and shoots and does not respond to stress, isoform I is upregulated under stress. Isoform II is expressed mainly in roots; however, under salinity stress its expression is downregulated in roots and upregulated in shoots. Tissue specific expression under salinity stress of isoform II was also seen after exogenous application of calcium. This study for the first time shows the presence of three isoforms of PgVHA-c and their differential regulation during plant development, and also under abiotic stress.