Interactions of gold with cytosolic selenium-containing proteins in rat kidney and liver

Rats injected with aurothioglucose (ATG) for 5 days were subsequently injected with [75Se]selenious acid and killed after 3 days. Kidney and liver cytosols were chromatographed on Sephadex G-150. 75Se in kidney was associated with high molecular weight (HMW), 85,000 Mr, 26,000 Mr, and 10,000 Mr proteins and with a nonprotein fraction. The elution profile of liver cytosol was similar to that of kidney, but without a 26,000 Mr protein. ATG injection increased the association of 75Se with all fractions of kidney cytosol except the 85,000 Mr fractions, which contained Se-glutathione peroxidase (SeGSHPx) activity; 75Se in liver was increased only in HMW fractions. Unfractionated kidney cytosolic SeGSHPx activity was decreased 14% by ATG injection, but liver enzyme activity was not changed. However, Sephadex G-150 chromatography showed that total and specific activities, respectively, were decreased 28 and 23% in kidney and 25 and 16% in liver. Au coeluted with HMW and 10,000 Mr 73Se-containing kidney proteins; the latter contained 50% of the Au eluted from the column. DEAE Sephacel chromatography of the 10,000 Mr kidney protein showed that both Au and 75Se were tightly associated with metallothionein-like proteins. This study demonstrates the interaction of Au with rat liver and kidney 75Se-containing proteins.     

Calcitonin-induced phosphorylation of rat liver cytosolic proteins

Calcitonin (CT) stimulated phosphorylation of two liver cytosolic proteins whose molecular weights are 67,000 and 93,000. Stimulation of 67,000-Mr protein phosphorylation began shortly after subcutaneous injection of CT, reaching a maximum at 5 min and decreasing to below the control level at 30 min. The reaction was independent of cyclic AMP or Ca2+, and was not influenced by a calmodulin antagonist, W7. Stimulation of 93,000-Mr protein phosphorylation became evident by 30 min. This reaction was also stimulated by administration of vasopressin or epinephrine, which is known to cause increased phosphorylation of glycogen phosphorylase having the same molecular weight. The phosphorylation of 93,000-Mr protein, stimulated by CT, was dependent on Ca2+ but not on cyclic AMP, and appeared to be inhibited by W7. In addition, CT did not influence the phosphorylation of 61,000-Mr protein, a major protein phosphorylated in a cyclic AMP-dependent manner. These results suggest that CT may exert its effect on liver cells through protein phosphorylation, most probably in a cyclic AMP-independent manner.     

Interactions of rab5 with cytosolic proteins.

Rab proteins, one of the subfamilies of ras-like small GTP-binding proteins, are attached to cellular compartments or transport vesicles and may determine the specificity of fusion between these compartments and vesicles. It has been proposed that they alternate between a membrane-bound and a cytosolic state during their functional cycle. We have used a photo-crosslinking approach to identify their cytosolic interaction partners. In vitro synthesized rab5 was cross-linked in the presence of ATP mainly to three cytosolic proteins of 52, 65, and 85 kDa. Sucrose density gradient centrifugation of the cross-linked products suggested that they were part of a 10-14 S complex. Furthermore, rab5 was cross-linked to these and additional cytosolic proteins of 42, 48, and 160 kDa in the absence of ATP. Unexpectedly, upon ATP depletion of the cytosol cross-linked and noncross-linked rab5 was found in a sedimentable high molecular weight structure. Other members of the rab subfamily, but not N-ras, also sedimented under these conditions. Electrophoretic and electron microscopic analysis of the pelleted material revealed that it contained actin filament bundles and intermediate filaments. Our data suggest that cytosolic rab proteins interact with several proteins in a 10-14 S complex, and that the rab proteins may interact directly or indirectly via this complex with the cytoskeleton.     

Interactions of cadmium and selenium in rat plasma in vivo and in vitro.

75Se and 109Cd tracers were used to study the binding of Se and Cd to plasma proteins at various SeO32- doses and times upt to 24 h after the simultaneous subcutaneous administration of SeO32- markedly increased both Se and Cd plasma levels over that in control animals. Gel permeation chromatography of plasma indicated that at all times up to 24 h Cd and Se were bound in an atomic ratio of approx. 1 : 1 in 330 000 and 130 000 dalton fractions. From 4 to 24 h, Cd and Se appeared in the 420 000 dalton fraction, also with an atomic ratio of approx. 1 : 1. The 330 000 dalton molecules appeared to have a maximal binding capacity for the Cd-Se complex at a concentration of approx. 30 mumol/ml of plasma, while the 130 000 and 420 000 dalton molecules show a higher binding capacity. Studies in vitro revealed that SeO32- does not interact directly with Cd and plasma proteins. It is metabolized by erythrocytes to a form that interacts in an atomic ratio of 1 : 1 with Cd to form a protein-bound complex of 130 000 daltons.     

Inhibition of rat liver and kidney arginase by cadmium ion

Cadmium ion activates arginase from many species of organisms but is an inhibitor of arginase from many other species. The purpose of this study was to investigate the inhibition of rat liver and kidney arginase by cadmium ion. Rat kidney arginase was inhibited by much lower concentrations of cadmium ion than rat liver arginase. Cadmium ion was a mixed noncompetitive inhibitor of both rat liver and kidney arginase. Cadmium ion enhanced the substrate activation of rat kidney arginase while still inhibiting the enzyme. Cadmium ion prevented the substrate inhibition of rat kidney arginase by fluoride while still inhibiting the enzyme. Cadmium ion also inhibited rat kidney arginase in the presence of manganese ion.     

Inhibition of rat liver and kidney arginase by cadmium ion

Cadmium ion activates arginase from many species of organisms but is an inhibitor of arginase from many other species. The purpose of this study was to investigate the inhibition of rat liver and kidney arginase by cadmium ion. Rat kidney arginase was inhibited by much lower concentrations of cadmium ion than rat liver arginase. Cadmium ion was a mixed noncompetitive inhibitor of both rat liver and kidney arginase. Cadmium ion enhanced the substrate activation of rat kidney arginase while still inhibiting the enzyme. Cadmium ion prevented the substrate inhibition of rat kidney arginase by fluoride while still inhibiting the enzyme. Cadmium ion also inhibited rat kidney arginase in the presence of manganese ion.     

Selenium-containing proteins of rat kidney and liver microsomes

Selenium (Se)-containing proteins in microsomal fractions of rat kidney and liver were investigated after isotopic labeling of rats with [75Se]selenite. More than 85% of the 75Se in the solubilized microsomal extracts precipitated with protein after trichloroacetic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), used to separate the labeled protein subunits in the solubilized microsomal extracts, revealed several 75Se-containing proteins in addition to glutathione peroxidase. 75Se-labeled subunits with molecular weights of 55, 30, 26, 22, 19, and 17 kDa were present in microsomal fractions of kidney and liver. The 75Se-labeled tryptic peptide of the 55 kDa subunit had the same Rf value on a 17% SDS-PAGE gel as the peptide from plasma selenoprotein P. A time-course study of the labeling of individual protein subunits in kidney and liver microsomes from Se-supplemented and Se-deficient rats showed that most of the 75Se was associated with the 55 kDa subunit 3 hr after injection. The amount of 75Se associated with this protein subunit decreased by 12 hr, with a concurrent increase in the labeling of lower molecular-weight subunits. The results support the hypothesis that there is a mechanism for transfer of Se from the 55 kDa subunit to other Se-containing proteins.     

Interactions between different selenium compounds and zinc, cadmium and mercury

In this paper, we present a polarographic study of systems containing different inorganic and organic selenium compounds (sodium selenite, sodium selenate, seleno-methionine and seleno-urea) and metal ions (Zn2+, Cd2+ Hg2+) of the 12th group of elements in the periodic table. While zinc is a trace element known to be essential for plants and animals, cadmium and mercury are exogenous elements and are harmful pollutants that accumulate during aging; selenium is also recognized as an important micronutrient and is sometimes added to the diet. Experiments investigating the interactions were carried out using polarographic techniques in unbuffered systems. The three metal cations originated complexes with different strength and solubility in the presence of selenite anions; in the presence of selenate, polarography was not able to detect formation of complexes with these metal ions, at least under the experimental conditions used: a decrease of Hg2+ ion concentration was observed. Seleno-methionine did not react with Cd2+; in the presence of Zn2+, a soluble complex with a co-ordination number 1 was formed, while, again, the concentration of Hg2+ decreased in the presence of increasing concentrations of the selenium derivative. Seleno-urea did not react with Zn2+, but formed a complex with Cd2+ with limited solubility. Finally, this ligand could not be studied with Hg2+ because of the overlapping of the reduction potentials of both the ligand and the metal cation. Overall equilibrium constants for complex formation (Kf) and the solubility product (Ksp) for poorly soluble species are also reported.     

Effect of gold thioglucose on subcellular selenium distribution in rat liver and kidney

Biological Trace Element Research - Reported are the subcellular distributions of selenium (Se), gold, glutathione peroxidase, and enzyme markers for nuclei, mitochondria, lysosomes, and soluble...     

The biosynthesis of metallothionein in rat liver and kidney after administration of cadmium

The biosynthesis of the cadmium-binding protein, metallothionein, was studied in rat liver and kidney after injection of cadmium chloride. A simplified procedure for the isolation of metallothionein from liver and kidney tissues was devised. It was found that the concentration of a subcutaneously injected dose of 30 μmoles of ¹⁰⁹CdCl₂/kg in the liver reached the maximum within 36 h. Thereafter, a slow decrease in the concentration of the isotope was noted during the 3 week period. In the kidney, the isotope was taken up in two phases. During the first phase the uptake was faster and lasted for about 4 days. The second phase of ¹⁰⁹Cd accumulation showed a slower increase in the concentration of the isotope. In both liver and kidney tissues 75–80% of the ¹⁰⁹Cd was associated with metallothionein. Amino acid incorporation studies revealed that active biosynthesis of metallothionein took place in the kidney as well as in the liver of cadmium-exposed rats. The turnover of ³⁵S-labeled metallothionein was also investigated and the half-lives of the hepatic and the renal metallothionein were found to be 2.8 and 5 days, respectively.     

Interactions between selenium and methylmercury in rat brain

The interaction of selenium with methylmercury was investigated in brain of animals labeled with ⁷⁵SeO₃^2? and CH₃²⁰³Hg⁺. Brains were fractionated into subcellular components and the cytosol was further fractionated by chromatography on Sephadex G-150 and G-200. The main result of these studies was evidence suggesting a shift of ⁷⁵Se from the cytosol to the mitochondrial fraction in brain when CH₃Hg⁺ was given. Concurrent equimolar (10 μmoles/kg) selenite injections increased the uptake of Hg but did not alter ²⁰³Hg distribution in brain. Changing the dose of CH₃Hg⁺ from 1 to 38 μmoles/kg had little effect on Hg uptake (% of dose per g). Gel filtrations on Sephadex G-150 and G-200 revealed that ²⁰³Hg in cytosol followed a pattern more closely related to protein (A₂₈₀) than to ⁷⁵Se, although a considerable portion of both isotopes eluted with proteins in the void volume. Assays of whole brain homogenates revealed a slight reduction in glutathione peroxidase activity in CH₃Hg⁺-treated rats which was not seen when equimolar selenite was injected with the CH₃Hg⁺.     

Differential regulation of expression of cytosolic and mitochondrial thioredoxin reductase in rat liver and kidney

Adequate supply of selenium (Se) is critical for synthesis of selenoproteins through selenocysteine insertion mechanism. To explore this process we investigated the expression of the cytosolic and mitochondrial isoenzymes of thioredoxin reductase (TrxR1 and TrxR2) in response to altered Se supply. Rats were fed diets containing different quantities of selenium and the levels of TrxR1 and TrxR2 protein and their corresponding mRNAs were determined in liver and kidney. Expression of the two isoenzymes was differentially affected, with TrxR1 being more sensitive to Se depletion than TrxR2 and greater changes in liver than kidney. In order to determine if the selenocysteine incorporation sequence (SECIS) element was critical in this response liver and kidney cell lines (H4 and NRK-52E) were transfected with reporter constructs in which expression of luciferase required read-through at a UGA codon and which contained either the TrxR1 or TrxR2 3'UTR, or a combination of the TrxR1 5' and 3'UTRs. Cell lines expressing constructs with the TrxR1 3'UTR demonstrated no response to restricted Se supply. In comparison the Se-deficient cells expressing constructs with the TrxR2 3'UTR showed considerably less luciferase activity than the Se-adequate cells. No disparity of response to Se supply was observed in the constructs containing the different TrxR1 5'UTR variants. The data show that there is a prioritisation of TrxR2 over TrxR1 during Se deficiency such that TrxR1 expression is more sensitive to Se supply than TrxR2 but this sensitivity of TrxR1 was not fully accounted for by TrxR1 5' or 3'UTR sequences when assessed using luciferase reporter constructs.     

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells

Summary In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, _DBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID_50,DBDS,MCD (0.5±0.1 m) for the H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is in agreement with the ID_50,Cl ^–,_MCD (0.94±0.07 m) for H₂-DIDS inhibition of MCD cell Cl^– flux, thus relating _DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl^– exchange by a factor of two, from _Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO₃). The ID_50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 m, so that binding is about an order of magnitude tighter than that for inhibition of rbc K⁺ flux (K _I,K ⁺,_rbc=0.017 m). These experiments indicate that the Na⁺,K^–-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID_50,DBDS,MCD (0.076±0.005 m); 2 m CE also more than doubles the Cl^– exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO₃). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.     

Interaction with deoxycholate of rat liver peroxisomal and cytosolic catalase

Rat liver catalase was found to interact with deoxycholate (DOC). When purified, the peroxisomal catalase was precipitated at pH 6 in the presence of DOC, whereas in the peroxisomal extract (with DOC) it was unsedimentable at pH 6. The membrane fraction in the extract interacted with the catalase instead of DOC, and prevented the precipitation of catalase with DOC at pH 6. The peroxisomal catalase seemed to be easily modified by lysosomal protease during manipulation, and this proteolytic cleavage rendered the molecule able to interact with the membrane. On the other hand, the cytosolic catalase, both in the cytosol fraction and in the purified preparation, sedimented at pH 6 in the presence of DOC. The cytosolic catalase was far more resistant to proteolytic modification than the peroxisomal catalase. The molecule of peroxisomal catalase is assumed to have a site for recognizing the membrane, whereas such a structure may be absent in the cytosolic catalase or may not be easily exposed by proteolytic cleavage.     

Interactions of cadmium and zinc in cultured rat embryos

Rat embryos were cultured in serum taken from animals dosed with cadmium, or serum with cadmium added $\text{in}$$\text{vitro}$ in the presence or absence of additional zinc. Embryos explanted at day ten and grown in serum taken from animals sooner than 4 h after dosing had a reduced DNA content after 24 h culture. In one-hour serum, the yolk sac had become thick and brittle. Zinc ameliorated the effects but had no stimulatory effect on post eight-hour serum when serum zinc levels were at their lowest. The hypothesis that cadmium induces a maternal zinc deficiency sufficient to cause teratogenic changes could not be sustained. Embryos explanted at nine days were much more susceptible to cadmium added $\text{in}$$\text{vitro}$ than ten-day embryos. The principal anomaly, apart from a reduced DNA content, was a thickening of the yolk sac similar to that seen in embryos grown in serum taken from animals one hour after cadmium dosing. Addition of zinc to the medium prevented both of these effects. The suggestion is made that the cadmium-induced dysgenesis of the yolk sac precludes appropriate embryonic nutrition.     

Interactions of bacteria with cadmium

Cadmium pollution arises mainly from contamination of minerals used in agriculture and from industrial processes. The usual situation is of large volumes of soil and water that are contaminated with low — but significant — concentrations of cadmium. Therefore, detoxification of the polluted water and soil involves the concentration of the metal, or binding it in a way that makes it biologically inert.Cadmium is one of the more toxic metals, that is also carcinogenic and teratogenic. Its effects are short term, even acute (diseases like Itai-itai), or long term. The long term effects are intensified due to the fact that cadmium accumulates in the body.This paper describes a study involving several hundred cadmium-resistant bacterial isolates. These bacteria could be divided into three groups—the largest group consisted of bacteria resistant to cadmium by effluxing it from the cells. The bacteria of the other two groups were capable of binding cadmium or of detoxifying it. We concentrated on one strain that could bind cadmium very efficiently, depending on the bacterial biomass and on the pH. This strain could effectively remove cadmium from contaminated water and soil.     

Interaction of platinum with metallothionein-like ligands in the rat kidney after administration of cis-dichlorodiammine platinum II

After the administration of the anticancer drug cis-dichlorodiammine platinum II (cisplatin) to male rats, the Pt in the soluble fraction of the kidney is isolated, by gel filtration, in association with a high molecular weight component and a low molecular weight fraction. At 24 h, Pt is also recovered in a metallothionein-like fraction which elutes from Sephadex G-50 with a lower apparent molecular weight than endogenous (Cu, Zn)-thionein or Cd-thionein isolated from the kidneys of Cd2+-treated rats. None of these low molecular weight metal-binding fractions binds to Octyl Sepharose CL-4B. On DE-52 ion exchange chromatography, Cd-thionein is resolved into two isometallothioneins whereas the low molecular weight Pt-binding fraction is only partially purified and contains at least six components which elute at higher gradient concentrations than metallothionein. Pretreatment with Cd2+ which stimulates the synthesis of renal and hepatic metallothionein has no effect on the uptake and subcellular distribution of Pt in the liver and kidneys. Cisplatin treatment reduces the concentration of Cu and Zn in the renal metallothionein and other soluble protein fractions in the kidney. When administered to Cd2+-pretreated rats, cisplatin promotes the loss of Zn from the soluble protein fractions but causes the redistribution of Cd from the metallothionein to the high molecular weight fraction and fails to inhibit the Cd2+-induced accumulation of Cu in the kidneys and the binding of Cu to the soluble protein fractions. It is suggested that metallothionein probably does not have a significant role in the renal metabolism of Pt following the administration of cisplatin to rats.