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1.
Bacterial chromosomes have genes for transport proteins for inorganic nutrient cations and oxyanions, such as NH 4
+, K +, Mg 2+, Co 2+, Fe 3+, Mn 2+, Zn 2+ and other trace cations, PO 4
3-, SO 4
2- and less abundant oxyanions. Together these account for perhaps a few hundred genes in many bacteria. Bacterial plasmids
encode resistance systems for toxic metal and metalloid ions including Ag + AsO 2
-, AsO 4
3-, Cd 2+, Co 2+, CrO 4
2−, Cu 2+, Hg 2+, Ni 2+, Pb 2+, TeO 3
2−, TI + and Zn 2+. Most resistance systems function by energy-dependent efflux of toxic ions. A few involve enzymatic (mostly redox) transformations.
Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. The Cd 2+-resistance cation pump of Gram-positive bacteria is membrane P-type ATPase, which has been labeled with 32P from [γ- 32P]ATP and drives ATP-dependent Cd 2+ (and Zn 2+) transport by membrane vesicles. The genes defective in the human hereditary diseases of copper metabolism, Menkes syndrome
and Wilson’s disease, encode P-type ATPases that are similar to bacterial cadmium ATPases. The arsenic resistance system transports
arsenite [As(III)], alternatively with the ArsB polypeptide functioning as a chemiosmotic efflux transporter or with two polypeptides,
ArsB and ArsA, functioning as an ATPase. The third protein of the arsenic resistance system is an enzyme that reduces intracellular
arsenate [As(V)] to arsenite [As(III)], the substrate of the efflux system. In Gram-negative cells, a three polypeptide complex
functions as a chemiosmotic cation/protein exchanger to efflux Cd 2+, Zn 2+ and Co 2+. This pump consists of an inner membrane (CzcA), an outer membrane (CzcC) and a membrane-spanning (CzcB) protein that function
together.
Received 08 August 1997/ Accepted in revised form 01 November 1997 相似文献
2.
Essentially all bacteria have genes for toxic metal ion resistances and these include those for Ag+, AsO
−2
, AsO
3−4
, Cd2+, Co2+, CrO
2−4
, Cu2+, Hg2+, Ni2+, Pb2+, TeO
2−3
, Tl+ and Zn2+. The largest group of resistance systems functions by energy-dependent efflux of toxic ions. Fewer involve enzymatic transformations (oxidation, reduction, methylation, and demethylation) or metal-binding proteins (for example, metallothionein SmtA, chaperone CopZ and periplasmic silver binding protein SilE). Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. For example, Cd2+-efflux pumps of bacteria are either inner membrane P-type ATPases or three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump, a periplasmic-bridging protein and an outer membrane channel. In addition to the best studied three-polypeptide chemiosmotic system, Czc (Cd2+, Zn2+, and Co2), others are known that efflux Ag+, Cu+, Ni2+, and Zn2+. Resistance to inorganic mercury, Hg2+ (and to organomercurials, such as CH3Hg+ and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic forms. Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is present in most bacterial genomes and many plasmids, the more recently recognized arr genes for the periplasmic arsenate reductase that functions in anaerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase that functions as an initial electron donor in aerobic resistance to arsenite. 相似文献
3.
A search with the proposed amino acid translation product from the new ‘candidate gene’ for human Menkes disease against protein sequence libraries showed a remarkable similarity to that for the cadmium efflux ATPase from Staphylococcus aureus resistance plasmids. The Menkes sequence appears closer to the CadA Cd 2+ sequence than to P-type ATPases from animal sources. Menkes syndrome is an X-chromosome invariably fatal disease that results from abberant copper metabolism. The gene that is defective in Menkes patients, i.e. the Menkes candidate gene, encodes a P-type ATPase, whose properties satisfactorily explain the phenotype of the disease. P-type ATPases are all cation pumps, either for uptake (e.g. the bacterial Kdp K + ATPase), for efflux (e.g. the muscle sarcoplasmic reticulum Ca 2+ ATPase), or for cation exchange (e.g. the animal cell Na +/K + ATPase). These enzymes have a conserved aspartate residue that is transiently phosphorylated from ATP during the transport cycle, hence the name ‘P-type’ ATPase. The Menkes sequence shares with the staphylococcal CadA ATPase those regions common to all P-type ATPases and also an N-terminal dithiol region that was proposed to be a ‘metal-binding motif’. There are one or two copies of this motif in the available CadA sequences and six copies in the Menkes sequence. 相似文献
4.
The plasmid-determined arsenite and antimonite efflux ATPase of bacteria differs from other membrane transport ATPases, which are classified into several families (such as the F 0F 1-type H +-translocating ATP synthases, the related vacuolar H +-translocating ATPases, the P-type cation-translocating ATPases, and the superfamily which includes the periplasmic binding-protein-dependent systems in Gram-negative bacteria, the human multidrug resistance P-glycoprotein, and the cystic fibrosis transport regulator). The amino acid sequences of the components of the arsenic resistance system are not similar to known ATPase proteins. New findings with the arsenic resistance operons of bacterial plasmids suggest that instead of being an orphan the Ars system will now be the first recognized member of a new class of ATPases. Furthermore, fundamental questions of energy-coupling (ATP-driven or chemiosmotic) have recently been raised and the finding that the arsC gene product is a soluble enzyme that reduces arsenate to arsenite changes the previous picture of the functioning of this widespread bacterial system. 相似文献
5.
Summary Studying metal ion resistances gives us important insights into environmental processes and provides an understanding of basic living processes. This review concentrates on bacterial efflux systems for inorganic metal cations and anions, which have generally been found as resistance systems from bacteria isolated from metal-polluted environments. The protein products of the genes involved are sometimes prototypes of new families of proteins or of important new branches of known families. Sometimes, a group of related proteins (and presumedly the underlying physiological function) has still to be defined. For example, the efflux of the inorganic metal anion arsenite is mediated by a membrane protein which functions alone in Gram-positive bacteria, but which requires an additional ATPase subunit in some Gram-negative bacteria. Resistance to Cd 2+ and Zn 2+ in Gram-positive bacteria is the result of a P-type efflux ATPase which is related to the copper transport P-type ATPases of bacteria and humans (defective in the human hereditary diseases Menkes' syndrome and Wilson's disease). In contrast, resistance to Zn 2+, Ni 2+, Co 2+ and Cd 2+ in Gram-negative bacteria is based on the action of proton-cation antiporters, members of a newly-recognized protein family that has been implicated in diverse functions such as metal resistance/nodulation of legumes/cell division (therefore, the family is called RND). Another new protein family, named CDF for cation diffusion facilitator has as prototype the protein CzcD, which is a regulatory component of a cobalt-zinc-cadmium resistance determinant in the Gram-negative bacterium Alcaligenes eutrophus. A family for the ChrA chromate resistance system in Gram-negative bacteria has still to be defined. 相似文献
6.
Plasmid pl258 carries the cadA gene that confers resistance to cadmium, lead, and zinc. CadA catalyzes ATP-dependent cadmium efflux from cells of Staphylococcus aureus. It is a member of the superfamily of P-type ATPases and belongs to the subfamily of soft metal ion pumps. In this study the membrane topology of this P-type ATPase was determined by constructing fusions with the topological reporter genes phoA or lacZ. A series of 44 C-terminal truncated CadAs were fused with one or the other reporter gene, and the activity of each chimeric protein was determined. In addition, the location of the first transmembrane segment was determined by immunoblot analysis. The results are consistent with the pl258 CadA ATPase having eight transmembrane segments. The first 109 residues is a cytosolic domain that includes the Cys(X) 2Cys motif that distinguishes soft metal ion-translocating P-type ATPases from their hard metal ion-translocating homologues. Another feature of soft metal ion P-type ATPases is the CysProCys motif, which is found in the sixth transmembrane segment of CadA. The phosphorylation site and ATP binding domain conserved in all P-type ATPases are situated within the large cytoplasmic loop between the sixth and seventh transmembrane segments. 相似文献
7.
Essentially all bacteria have genes for toxic metal ion resistances and these include those for Ag+, AsO2-, AsO4(3-), Cd2+ Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, TeO3(2-), Tl+ and Zn2+. The largest group of resistance systems functions by energy-dependent efflux of toxic ions. Fewer involve enzymatic transformations (oxidation, reduction, methylation, and demethylation) or metal-binding proteins (for example, metallothionein SmtA, chaperone CopZ and periplasmic silver binding protein SilE). Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. For example, Cd2+-efflux pumps of bacteria are either inner membrane P-type ATPases or three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump, a periplasmic-bridging protein and an outer membrane channel. In addition to the best studied three-polypeptide chemiosmotic system, Czc (Cd2+, Zn2+, and Co2), others are known that efflux Ag+, Cu+, Ni2+, and Zn2+. Resistance to inorganic mercury, Hg2+ (and to organomercurials, such as CH3Hg+ and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic forms. Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is present in most bacterial genomes and many plasmids, the more recently recognized arr genes for the periplasmic arsenate reductase that functions in anaerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase that functions as an initial electron donor in aerobic resistance to arsenite. 相似文献
8.
Bacterial plasmids have genes that confer highly specific resistances to As, Bi, Cd, Cu, Cr, Hg, Pb, Te, Zn, and other toxic heavy metals. For each toxic cation or anion, generally a different resistance system exists, and these systems may be “linked” together on multiple resistance plasmids. For Cd 2+, AsO 2 ?, AsO 4 3?, Hg 2+, and organomercurials, DNA sequence analysis has supplemented direct physiological and biochemical experiments to produce sophisticated understanding. The cadA ATPase of S.aureus plasmids is a 727 amino acid membrane ATPase that pumps Cd 2+ from the cells as rapidly as it is accumulated. This polypeptide is related by sequence to other cation translocating ATPases, including the membrane K + ATPases of Escherichia coli and Streptococcus faecalis, the H + ATPases of yeast and Neurospora, the Na +/K + ATPases of vertebrate animals, and the Ca 2+ ATPases of rabbit muscle. The conserved residues include the aspartyl residue that is phosphorylated, the lysine involved in ATP binding, and the proline within a membrane translocating region. The arsenate and arsenite translocating ATPase consists of 3 polypeptides (from DNA sequence analysis), including a recognizable ATP binding protein ( arsA), an integral membrane protein ( arsB gene), and a substrate specificity subunit ( arsC gene). Inorganic mercury and organomercurial degradation is carried out by a series of about 6 polypeptides, including 2 soluble intracellular enzymes (organomercurial lyase and mercuric reductase). The latter is related by sequence and function to glutathione reductase and lipoamide dehydrogenase of prokaryotes and eukaryotes. These enzymes are dimeric, FAD-containing, NAD(P)H-dependent oxidoreductases. Other recognizable polypeptides in the mer system include a DNA-binding regulatory protein from the merR gene and a Hg 2+ transport system consisting of a periplasmic Hg 2+-binding protein ( merP gene) and a membrane protein ( merT gene) in gram negative systems. 相似文献
9.
P-type ATPases catalyze the selective active transport of ions like H +, Na +, K +, Ca 2+, Zn 2+, and Cu 2+ across diverse biological membrane systems. Many members of the P-type ATPase protein family, such as the Na +,K +-, H +,K +-, Ca 2+-, and H +-ATPases, are involved in the development of pathophysiological conditions or provide critical function to pathogens. Therefore, they seem to be promising targets for future drugs and novel antifungal agents and herbicides. Here, we review the current knowledge about P-type ATPase inhibitors and their present use as tools in science, medicine, and biotechnology. Recent structural information on a variety of P-type ATPase family members signifies that all P-type ATPases can be expected to share a similar basic structure and a similar basic machinery of ion transport. The ion transport pathway crossing the membrane lipid bilayer is constructed of two access channels leading from either side of the membrane to the ion binding sites at a central cavity. The selective opening and closure of the access channels allows vectorial access/release of ions from the binding sites. Recent structural information along with new homology modeling of diverse P-type ATPases in complex with known ligands demonstrate that the most proficient way for the development of efficient and selective drugs is to target their ion transport pathway. 相似文献
10.
The effect of heavy metal cations on the mitochondrial ornithine/citrulline transporter was tested in proteoliposomes reconstituted
with the protein purified from rat liver. The transport activity was measured as [ 3H]ornithine uptake in proteoliposomes containing internal ornithine (ornithine/ornithine antiport mode) or as [ 3H]ornithine efflux in the absence of external substrate (ornithine/H + transport mode). 0.1 mM Cu 2+, Pb 2+, Hg 2+, Cd 2+ and Zn 2+ strongly inhibited (more than 85%) the antiport; whereas Mn 2+, Co 2+ and Ni 2+ inhibited less efficiently (25, 47 and 69%, respectively). The IC 50 values of the transporter for the different metal ions ranged from 0.71 to 350 μM. Co 2+ and Ni 2+ also inhibited the [ 3H]ornithine efflux whereas Cu 2+, Pb 2+, Hg 2+, Cd 2+ and Zn 2+ stimulated the [ 3H]ornithine efflux. The stimulation of the [ 3H]ornithine efflux by Cu 2+ and Cd 2+ (as well as by Pb 2+, Hg 2+ and Zn 2+) was not prevented by NEM and was reversed by DTE. These features indicated that the inhibition of the antiport was due to
the interaction of the Cu 2+, Pb 2+, Hg 2+, Cd 2+ and Zn 2+ with a population of SH groups, of the transporter, responsible for the inhibition of the physiological function; whereas
the stimulation of [ 3H]ornithine efflux was due to the induction of a pore-like function of the transporter caused by interaction of cations with
a different population of SH groups. Differently, the inhibition of the ornithine transporter by Ni 2+, Co 2+ or Mn 2+ was caused by interaction with the substrate binding site, as indicated by the competitive or mixed inhibition. 相似文献
11.
CadA is a membrane protein of the P-type ATPase family which is the major determinant of the resistance to Cd2+ in Listeria monocytogenes. During its catalytic cycle, CadA undergoes auto-phosphorylation from ATP at Asp398, which allows Cd2+ translocation across the membrane. In the reverse mode, Asp398 is phosphorylated from Pi. From the data obtained so far, the CadA catalytic mechanism is similar to that proposed for the sarcoplasmic reticulum Ca2+-ATPase, the model of the P-type ATPase family. We show here that CadA is sensitive to two different ranges of Cd2+ concentration. The 0.1-10 microM range of added CdCl2 corresponds to Cd2+ binding at the transport site of unphosphorylated CadA which induces the reaction of the enzyme with ATP and impairs its reaction with Pi. The 0.1-1 mM range of added CdCl2 could correspond to Cd2+ binding to the transport site accessible from the extracellular medium. In addition, although it is widely accepted that the actual substrate of P-type ATPases is the MgATP complex, we show here that CadA can also perform its cycle in the absence of Mg2+, using CdATP in the place of MgATP at the catalytic site. 相似文献
12.
Powered by ATP hydrolysis, P IB‐ATPases drive the energetically uphill transport of transition metals. These high affinity pumps are essential for heavy metal detoxification and delivery of metal cofactors to specific cellular compartments. Amino acid sequence alignment of the trans‐membrane (TM) helices of P IB‐ATPases reveals a high degree of conservation, with ~60–70 fully conserved positions. Of these conserved positions, 6–7 were previously identified to be important for transport. However, the functional importance of the majority of the conserved TM residues remains unclear. To investigate the role of conserved TM residues of P IB‐ATPases we conducted an extensive mutagenesis study of a Zn 2+/Cd 2+ P IB‐ATPase from Rhizobium radiobacter (rrZntA) and seven other P IB‐ATPases. Of the 38 conserved positions tested, 24 had small effects on metal tolerance. Fourteen mutations compromised in vivo metal tolerance and in vitro metal‐stimulated ATPase activity. Based on structural modelling, the functionally important residues line a constricted ‘channel’, tightly surrounded by the residues that were found to be inconsequential for function. We tentatively propose that the distribution of the mutable and immutable residues marks a possible trans‐membrane metal translocation pathway. In addition, by substituting six trans‐membrane amino acids of rrZntA we changed the in vivo metal specificity of this pump from Zn 2+/Cd 2+ to Ag +. 相似文献
13.
Previous work in our laboratory led to the isolation of a cadmium (Cd)-resistant variant (Cd r2C10) of the line CHO Chinese Hamster cell having a 10-fold greater resistance to the cytotoxic action of Cd 2+ compared with the CHO cell. This resistance was attributed to an increased capacity of the Cd 2+-resistant Cd r2C10 subline to induce synthesis of the Cd 2+- and Zn 2+-binding protein(s), metallothionein(s) (MT). Evidence that Cd 2+ behaves as an analog of the essential trace metal, Zn 2+, especially as an inducer of MT synthesis, suggested that the Cd r and CHO cell types could be employed to investigate cellular Zn 2+ metabolism. In the present study, measurements were made to compare CHO and Cd r cell types for (a) growth as a function of the level of ZnCl 2 added to the culture medium, (b) uptake and subcellular distribution of Zn 2+, and (c) capacity to induce MT synthesis. The results of these measurements indicated that (a) both CHO and Cd r cell types grew normally ( T
d≊16–18 h) during exposures to Zn 2+ at levels up to 100 μ M added to the growth medium, but displayed abrupt growth inhibition at higher Zn 2+ levels, (b) Cd r cells incorporate fourfold more Zn 2+ during a 24-h exposure to the maximal subtoxic level of Zn 2+ and (c) the CHO cell lacks the capacity to induce MT synethesis while the Cd r cell is proficient in this response during exposure to the maximal subtoxic Zn 2+ level. These findings suggest that (a) the CHO and Cd r cell systems will be useful in further studies of cellular Zn 2+ metabolism, especially in comparisons of Zn 2+ metabolism in the presence and absence of induction of the Zn 2+-sequestering MT and (b) a relationship exists between cellular capacity to induce MT synthesis and capacity for cellular
Zn 2+ uptake. 相似文献
14.
The mechanism of Zn resistance in multiple metal-resistant Pseudomonas putida strain S4 is based on inducible efflux. An ATPase in the strain S4 mediated active extrusion of Zn 2+, which occurred during the exponential phase of growth. The ATPase activity was inhibited by micromolar concentrations (50 M) of vanadate, suggesting the involvement of a P-type ATPase. The effluxed Zn 2+ were not ejected out of the cell but stored in the outer membrane and periplasm, which provided the required binding sites. The strain S4, thus, employs a dual strategy of efflux and binding to bring about a proper management of essential ions like Zn. 相似文献
15.
According to in silico analysis, the genome of Pseudomonas putida KT2440 encodes at least four Zn/Cd/Pb efflux transporters-two P-type ATPases (CadA1 and CadA2) and two czc chemiosmotic transporters (CzcCBA1 and CzcCBA2). In this study we showed that all these transporters are functional, but under laboratory conditions only two of them were involved in the mediation of heavy metal resistance in P. putida KT2440. CadA2 conferred Cd(2+) and Pb(2+) resistance, whereas CzcCBA1 was involved in export of Zn(2+), Cd(2+), and possibly Pb(2+). CadA1, although nonfunctional in P. putida, improved Zn(2+) resistance and slightly improved Cd(2+) resistance when it was expressed in Escherichia coli. CzcCBA2 contributed to Zn resistance of a czcA1-defective P. putida strain or when the CzcA2 subunit was overexpressed in a transporter-deficient strain. It seemed that CzcA2 could complex with CzcC1 and CzcB1 subunits and therefore complement the loss of CzcA1. The CzcCBA2 transporter itself, however, did not function. Expression of cadA1, cadA2, and czcCBA1 was induced by heavy metals, and the expression levels were dependent on the growth medium and growth phase. Expression of cadA2 and czcCBA1 was nonspecific; both genes were induced by Zn(2+), Cd(2+), Pb(2+), Ni(2+), Co(2+), and Hg(2+). On the other hand, remarkably, expression of cadA1 was induced only by Zn(2+). Possible roles of distinct but simultaneously functioning transporters are discussed. 相似文献
16.
P 1B-type ATPases transport a variety of metals (Cd 2+, Zn 2+, Pb 2+, Co 2+, Cu 2+, Ag +, Cu +) across biomembranes. Characteristic sequences CP[C/H/S] in transmembrane fragment H6 were observed in the putative transporting metal site of the founding members of this subfamily (initially named CPx-ATPases). In spite of their importance for metal homeostasis and biotolerance, their mechanisms of ion selectivity are not understood. Studies of better-characterized P II-type ATPases (Ca-ATPase and Na,K-ATPase) have identified three transmembrane segments that participate in ion binding and transport. Testing the hypothesis that metal specificity is determined by conserved amino acids located in the equivalent transmembrane segments of P 1B-type ATPases (H6, H7, and H8), 234 P 1B-ATPase protein sequences were analyzed. This showed that although H6 contains characteristic CPX or XPC sequences, conserved amino acids in H7 and H8 provide signature sequences that predict the metal selectivity in each of five P 1B-ATPase subgroups identified. These invariant amino acids contain diverse side chains (thiol, hydroxyl, carbonyl, amide, imidazolium) that can participate in transient metal coordination during transport and consequently determine the particular metal selectivity of each enzyme. Each subgroup shares additional structural characteristics such as the presence (or absence) of particular amino-terminal metal-binding domains and the number of putative transmembrane segments. These differences suggest unique functional characteristics for each subgroup in addition to their particular metal specificity. 相似文献
17.
The amino acid sequences of 47 P-type ATPases from several eukaryotic and bacterial kingdoms were divided into three structural segments based on individual hydropathy profiles. Each homologous segment was (1) multiply aligned and functionally evaluated, (2) statistically analyzed to determine the degrees of sequence similarity, and (3) used for the construction of parsimonious phylogenetic trees. The results show that all of the P-type ATPases analyzed comprise a single family with four major clusters correlating with their cation specificities and biological sources as follows: cluster 1: Ca 2+-transporting ATPases; cluster 2: Na +- and gastric H +-ATPases; cluster 3: plasma membrane H +-translocating ATPases of plants, fungi, and lower eukaryotes; and cluster 4: all but one of the bacterial P-type ATPases (specific for K +, Cd 2+, Cu 2+ and an unknown cation). The one bacterial exception to this general pattern was the Mg 2+-ATPase of Salmonella typhimurium, which clustered with the eukaryotic sequences. Although exceptions were noted, the similarities of the phylogenetic trees derived from the three segments analyzed led to the probability that the N-terminal segments 1 and the centrally localized segments 2 evolved from a single primordial ATPase which existed prior to the divergence of eukaryotes from prokaryotes. By contrast, the C-terminal segments 3 appear to be eukaryotic specific, are not found in similar form in any of the prokaryotic enzymes, and are not all demonstrably homologous among the eukaryotic enzymes. These C-terminal domains may therefore have either arisen after the divergence of eukaryotes from prokaryotes or exhibited more rapid sequence divergence than either segment 1 or 2, thus masking their common origin. The relative rates of evolutionary divergence for the three segments were determined to be segment 2 < segment 1 < segment 3. Correlative functional analyses of the most conserved regions of these ATPases, based on published site-specific mutagenesis data, provided preliminary evidence for their functional roles in the transport mechanism. Our studies define the structural and evolutionary relationships among the P-type ATPases. They should provide a guide for the design of future studies of structure-function relationships employing molecular genetic, biochemical, and biophysical techniques.
Correspondence to: M.H. Saier, Jr. 相似文献
18.
Leadmium Green is a commercially available, small molecule, fluorescent probe advertised as a detector of free intracellular cadmium (Cd 2+) and lead (Pb 2+). Leadmium Green has been used in various paradigms, such as tracking Cd 2+ sequestration in plant cells, heavy metal export in protozoa, and Pb 2+ absorption by vascular endothelial cells. However very little information is available regarding its affinity and selectivity for Cd 2+, Pb 2+, and other metals. We evaluated the in vitro selectivity of Leadmium Green using spectrofluorimetry. Consistent with manufacturer’s claims, Leadmium Green was sensitive to Cd 2+ (K D ~600 nM) and also Pb 2+ (K D ~9.0 nM) in a concentration-dependent manner, and furthermore proved insensitive to Ca 2+, Co 2+, Mn 2+ and Ni 2+. Leadmium Green also responded to Zn 2+ with a K D of ~82 nM. Using fluorescence microscopy, we evaluated Leadmium Green in live mouse hippocampal HT22 cells. We demonstrated that Leadmium Green detected ionophore-mediated acute elevations of Cd 2+ or Zn 2+ in a concentration-dependent manner. However, the maximum fluorescence produced by ionophore-delivered Zn 2+ was much less than that produced by Cd 2+. When tested in a model of oxidant-induced liberation of endogenous Zn 2+, Leadmium Green responded weakly. We conclude that Leadmium Green is an effective probe for monitoring intracellular Cd 2+, particularly in models where Cd 2+ accumulates rapidly, and when concomitant fluctuations of intracellular Zn 2+ are minimal. 相似文献
19.
Cultures of Aspergillus parasiticus produce the polyketide versicolorin A in response to elevation of the Zn 2+ content of the growth medium. With suboptimal Zn 2+ (0.8 μ M) mycelial growth is about half maximal, and versicolorin synthesis is essentially zero. Inclusion of Cd 2+ (1–100 μ M) in the Zn 2+-limiting growth medium allows optimal growth and stimulates full versicolorin synthesis. Cd 2+, like Zn 2+, will stimulate versicolorin sysnthesis only when added within the first 30 h after conidial inoculation. The transport system for Cd 2+ uptake may be the same as that for Zn 2+, as judged by in vivo competition studies. Cd 2+ is a competitive inhibitor of Zn 2+ uptake, with Ki = 20 μM. 相似文献
20.
In addition to the previously studied Zn 2+, low concentrations (about 0.5 mM) of Be 2+, Ba 2+, Cd 2+, Ni 2+, Cu 2+, Pt 4+ and, outstandingly, 0.5 µ M of UO 2
2+, potentiate the twitch of frog sartorius and toe muscles by prolonging the active state of contraction. The degree of potentiation is a roughly S-shaped function of p(metal 2+), suggesting that each metal binds to a ligand of the muscle fiber, representative apparent affinity constants being: UO 2
2+, 5 x 10 6; Zn 2+, 2.8 x 10 5; and Cd 2+, 2 x 10 4. UO 2
2+ potentiation effects are rapidly reversed by PO 4, and Zn 2+ and Cd 2+ effects by EDTA, PO 4, and cysteine. The rapidity of these reversals by the nonpenetrating EDTA and PO 4, and the fact that heavy metal ions evidently potentiate by prolonging the action potential, indicate that the metal potentiators exert their primary action at readily accessible (i.e. plasma and T tubular) membrane sites. The relatively slow kinetics of development of potentiation, and the even slower reversal of it in pure Ringer''s solution, indicate that the metal ions are bound to connective tissue, as well as to muscle fibers. The binding effects at the readily accessible membrane sites evidently impairs delayed rectification and thus modifies the action potential and excitation-contraction coupling so as to cause potentiation. SH is excluded, and PO 4 and imidazole are possibilities, as the membrane ligand binding the potentiating metal ions. 相似文献
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