thiol-Sepharose 4B Research Articles

Consensus Substrate Sequence for Protein-tyrosine Phosphatase Receptor Type Z

Protein-tyrosine phosphatase receptor type Z (Ptprz) has multiple substrate proteins, including G protein-coupled receptor kinase-interactor 1 (Git1), membrane-associated guanylate kinase, WW and PDZ domain-containing 1 (Magi1), and GTPase-activating protein for Rho GTPase (p190RhoGAP). We have identified a dephosphorylation site at Tyr-1105 of p190RhoGAP; however, the structural determinants employed for substrate recognition of Ptprz have not been fully defined. In the present study, we revealed that Ptprz selectively dephosphorylates Git1 at Tyr-554, and Magi1 at Tyr-373 and Tyr-858 by in vitro and cell-based assays. Of note, the dephosphorylation of the Magi1 Tyr-858 site required PDZ domain-mediated interaction between Magi1 and Ptprz in the cellular context. Alignment of the primary sequences surrounding the target phosphotyrosine residue in these three substrates showed considerable similarity, suggesting a consensus motif for recognition by Ptprz. We then estimated the contribution of surrounding individual amino acid side chains to the catalytic efficiency by using fluorescent peptides based on the Git1 Tyr-554 sequence in vitro. The typical substrate motif for the catalytic domain of Ptprz was deduced to be Glu/Asp-Glu/Asp-Glu/Asp-Xaa-Ile/Val-Tyr(P)-Xaa (Xaa is not an acidic residue). Intriguingly, a G854D substitution of the Magi1 Tyr-858 site matching better to the motif sequence turned this site to be susceptible to dephosphorylation by Ptprz independent of the PDZ domain-mediated interaction in cells. Furthermore, we found by database screening that the substrate motif is present in several proteins, including paxillin at Tyr-118, its major phosphorylation site. Expectedly, we verified that Ptprz efficiently dephosphorylates paxillin at this site in cells. Our study thus provides key insights into the molecular basis for the substrate recognition of Ptprz.

Large T Antigen Promotes JC Virus Replication in G2-arrested Cells by Inducing ATM- and ATR-mediated G2 Checkpoint Signaling

Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G(2) phase of the cell cycle as a result of the activation of ATM- and ATR-mediated G(2) checkpoint pathways. Transient transfection of cells with a TAg expression vector also induced G(2) checkpoint signaling and G(2) arrest. Analysis of TAg mutants with different subnuclear localizations suggested that the association of TAg with cellular DNA contributes to the induction of G(2) arrest. Abrogation of G(2) arrest by inhibition of ATM and ATR, Chk1, and Wee1 suppressed JCV genome replication. In addition, abrogation of the G(2)-M transition by Cdc2 depletion disabled Wee1 depletion-induced suppression of JCV genome replication, suggesting that JCV replication is facilitated by G(2) arrest resulting from G(2) checkpoint signaling. Moreover, inhibition of ATM and ATR by caffeine suppressed JCV production. The observation that oligodendrocytes productively infected with JCV in vivo also undergo G(2) arrest suggests that G(2) checkpoint inhibitors such as caffeine are potential therapeutic agents for JCV infection.

Appearance of extracellular glutathione peroxidase (eGPx) in the ascite fluid of casein-elicited rats.

Glutathione peroxidase (GPx) activity was detected in the ascite fluid of rats injected intraperitoneally with 2.5% heat-denatured casein solution. Activity in the ascite fluid increased with time after the injection of casein, and reached a maximum at 24 h. The active component was concentrated with successive 35% ammonium sulfate precipitation and Activated Thiol-Sepharose 4B column chromatography from the ascite fluid of rats at 24 h after the injection of casein. No N-terminal amino acid of the protein corresponding to GPx was detected by automatic amino acid sequence analysis following separation with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to a polyvinyl difluoride (PVDF) membrane. Following BrCN treatment of the protein, the N-terminal amino acid sequences of two 14 and 2.6 kDa peptide fragments were found to be S-G-T-I-Y-E-Y-G-A-L and K-I-H-D-I-R-W-N-F-E, respectively. The former and the latter fragments corresponded to sequences beginning at the 37th and 176th amino acid residues of rat extracellular GPx (eGPx), respectively. The exclusive presence of eGPx in the ascite fluid of rats elicited by casein was confirmed immunologically by ELISA, immuno-precipitation and Western blotting assays. No other GPx isozymes such as cytosolic GPx (cGPx), phospholipid hydroperoxide GPx (PHGPx) or intestinal GPx (iGPx) were detected. eGPx activity and protein were also detected in the pleuritic fluid of rats following injection of 2% carrageenan. These findings indicate that eGPx appears at various sites of acute inflammation in rats. This pattern is due to leakage from circulation as a result of the increased capillary permeability at inflammation sites elicited by chemotactic factors.

Bovine brain pyroglutamyl aminopeptidase (type-1): Purification and characterisation of a neuropeptide-inactivating peptidase

Pyroglutamyl aminopeptidase type-1 (PAP-I) is reported to be a soluble, broad specificity aminopeptidase, capable of removing the pyroglutamic acid (pGlu) residue from the amino terminus of pGlu-peptides (e.g. TRH, LHRH, neurotensin and bombesin). The central aim of this study was to undertake, for the first time, the complete purification and characterisation of a PAP activity observed within the cytosolic fraction of bovine whole brain and to compare the properties of the enzyme with previous findings. A series of chromatographic steps (DEAE-Sepharose, Sephacryl S-200 and Activated Thiol Sepharose 4B) generated a soluble PAP activity purified to near homogeneity with a total active yield of 6.6%. The enzyme displayed a native molecular mass of approximately 23,700 Da, which compares well with that value obtained under denaturing conditions via SDS-PAGE (24,000 Da), suggesting that the enzyme exists as a monomer. The expression of PAP activity displayed an absolute requirement for the presence of a disulphide bond-reducing agent such as DTT, whilst optimum activity was observed at pH 8.5. Strong inhibition of PAP activity was observed with a number of different agents, including transition metal ions, sulphydryl-blocking agents and 2-pyrrolidone (a pGlu analog). A broad pyroglutamyl substrate specificity, which excludes substrates commencing with the pGlu-Pro bond, was also demonstrated for the bovine brain enzyme. Based on a comparison of these findings with those reported for PAP-I in other mammalian tissues, the soluble PAP activity observed in bovine whole brain can tentatively be classified as a pyroglutamyl aminopeptidase type-1 (EC 3.4.19.3).

Purification and immunoelectron microscopic localization of cellular glutathione peroxidase in rat hepatocytes: quantitative analysis by postembedding method

To measure quantitatively the intracellular distribution of cellular glutathione peroxidase (GPX) in rat hepatocytes, ultrathin sections were stained by a postembedding immunogold technique. GPX had a specific activity of 1670 Units/mg protein, and was purified 2050-fold from rat liver by means of heat denaturation, ammonium sulfate fractionation, and a series of chromatographic procedures including thiol-Sepharose 4B. The purified GPX was shown to be electrophoretically pure, and was a homotetramer of 22 kDa subunits. Monospecific polyclonal antibodies were raised in rabbits by immunization. By immunoblot analysis, both the light mitochondrial the and cytosolic fractions of rat liver homogenate gave a single band with an identical mobility to that of the purified enzyme. Under the light microscope, hepatocytes showed nuclear staining and granular cytoplasmic staining, corresponding to certain intracellular structures. The labeling density (number of gold particles/microns 2) for GPX obtained by immunoelectron microscopy was 11.9 in the nuclei, 19.6 in mitochondria, 3.32 in peroxisomes, 1.95 in lysosomes, and 9.81 in the cytoplasmic matrix. These results suggest that cellular GPX is present in various compartments of rat hepatocytes, and that the GPX occurs in relatively higher amounts in mitochondria.

Assay and Comparative Characterization of the Proteolytic Degradation of Isolated Small Subunit and Holoenzyme of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Chloroplasts from Rye Leaves

Summary The cleavage of purified and fluorescent or radioactive labeled ribulose-1,5-bisphosphate carboxylase/oxygenase and its isolated small subunit by proteolytic activities of purified chloroplasts from rye leaves ( Secale cereale L.) were compared. Degradation of the small subunit was mainly catalysed by the soluble supernatant fraction of chloroplasts or ribosome-deficient plastids, with highest activity at pH 6. Proteolytic cleavage of the ribulose-1,5-bisphosphate carboxylase holoenzyme was found in the soluble supernatant, but also to some extent associated with the membrane fraction of chloroplasts, having its maximal activity at pH 8. The K m for cleavage of the holoenzyme was 1.2 μM; the K m for cleavage of the small subunit was 52.7 μM. Proteolytic breakdown of the small subunit was enhanced by the addition of SDS, particularly in the presence of dithioerythritol. The activities of the proteolytic degradation of labeled ribulose-1,5-bisphosphate carboxylase/oxygenase and its small subunit were not specifically affected by various protease inhibitors that were representative for the different mechanistic classes of proteases. Proteolytic activities were enriched to higher specific activities by affinity chromatography with activated Thiol-Sepharose 4B. A function of a small-subunit-specific protease in chloroplast biogenesis for the control of the subunit stoichiometry of the ribulose-1,5-bisphosphate carboxylase/oxygenase is discussed.

Characterization of the covalent chromatography of thymidylate synthase on thiopropyl-Sepharose 6B

We have examined the covalent chromatography of Lactobacillus casei thymidylate synthase on thiopropyl-Sepharose 6B resin. This enzyme is a dimer of identical subunits, each of which contains one active site that features a catalytic sulfhydryl group (Cys-198). Reversible coupling was achieved via the attack of one of the enzyme's two catalytic cysteine residues, causing displacement of 2-thiopyridone, the reactive moiety of the resin. To establish the usefulness of this matrix for immobilization and the conditions required for chromatography, model studies were conducted with 2,2′-dithiodipyridine. The chemical modification of thymidylate synthase with 2,2′-dithiodipyridine was shown to be specific for the catalytic sulfhydryl groups of the native dimer, titrating 1.51 sulfhydryl groups, while 2.93 cysteines were modified in the GnHCl-denatured protein. The former reaction, which resulted in total loss of enzyme activity, was reversible with complete recovery of control activity within 30 min after addition of 100 mM 2-mercaptoethanol. Characterization of the protein pools generated in the covalent chromatography procedure indicated that the enzyme fraction washing through the column without attachment had substantially lower catalytic and ligand binding activities than the original protein stock; conversely, the enzyme fraction eluted from the column by 2-mercaptoethanol exhibited higher levels of these activities. Gel electrophoresis studies further illustrated that the unique application of the covalent chromatography technique described herein fractionated homogeneous thymidylate synthase protein into enzyme pools exhibiting distinct biochemical properties. As immobilization reaction times were increased beyond 6 h, the coupling of thymidylate synthase was demonstrated to occur through more than one enzymic sulfhydryl group. Interestingly, no covalent coupling was detected in attempts using activated thiol-Sepharose 4B, a result underlining the importance of the structure of the resin linker arm in enzyme immobilization.

Purification and properties of aminopeptidase H from chicken skeletal muscle.

Aminopeptidase H was purified from fresh chicken breast muscle by ammonium sulfate fractionation and successive chromatographies on DEAE-cellulose, Ultrogel AcA 34, activated thiol-Sepharose 4B, phenyl-Sepharose CL-4B and DEAE-cellulose again. The purified enzyme migrated as a single band on SDS/PAGE. Aminopeptidase H exhibits activity against both L-leucine beta-naphthylamide and alpha-N-benzoyl-DL-arginine beta-naphthylamide. The molecular mass of this enzyme was found to be 52 kDa on SDS/PAGE and 400 kDa on Sepharose 6B column chromatography. The optimum pH for the hydrolysis of both substrates was 8.0 and this activity was remarkably enhanced by reducing agents. The enzyme was strongly inhibited by monoiodoacetate and leupeptin, but not affected by EDTA, phenylmethylsulfonyl fluoride, pepstatin, bestatin or puromycin. Aminopeptidase H has been shown to hydrolyze di-, tri- and tetrapeptides in the manner of an aminopeptidase, as well as the beta-naphthylamide derivatives of amino acids. However, the enzyme has not been shown to hydrolyze proteins such as hemoglobin, bovine serum albumin, myofibrillar proteins or sarcoplasmic proteins.

Affinity Chromatography of Ovine Casein

Sheep milk casein was separated into two fractions: one containing αs1-plus β- and the other αs2 plus κ-caseins by affinity chromatography on activated thiol-Sepharose 4B. Milk samples were from the Leccese breed with the most common electrophoretic pattern. Electrophoresis of the chromatographic fractions on SDS-PAGE and on starch urea gel at pH 8.6 and 1.7 clarified the electrophoretic pattern of whole casein. Acidic pH electrophoresis of the two fractions obtained by affinity chromatography may be useful for investigations on the polymorphism of the casein fractions.

Human adipocyte lipid-binding protein: purification of the protein and cloning of its complementary DNA

Human adipocyte lipid-binding protein (H-ALBP) was purified from normal subcutaneous adipose tissue to greater than 98% homogeneity, utilizing a combination of acid fractionation, gel filtration, covalent chromatography on activated thiol-Sepharose 4B, and anion-exchange chromatography. Human ALBP comprised about 1% of total cytosolic protein in human adipose tissue, had a relative molecular mass of about 15 kDa, and existed as a monomer in solution. The amino terminus of H-ALBP was blocked to sequencing. When a liposome ligand delivery assay was used, H-ALBP saturably bound oleic acid with about 1 mol of ligand bound per mole of protein. Additionally, H-ALBP saturably bound retinoic acid as determined by the quenching of intrinsic tryptophan fluorescence. A full-length H-ALBP cDNA has been cloned; the sequence predicts a 649-base mRNA comprised of a 62-base 5'-noncoding region containing an 18S ribosome-binding site, a single 396-base open-reading frame, and a 191-base 3'-noncoding region. Comparative sequence analysis indicated that the 132 amino acid H-ALBP is a member of a multigene family of intracellular lipid-binding proteins and contains the consensus substrate phosphorylation sequence for tyrosyl kinases.

Immobilized phenylalanine hydroxylase through the SH groups

Purified rat liver phenylalanine hydroxylase [L-phenylalanine:tetrahydropteridine:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1] was immobilized with activated thiol-Sepharose 4B via disulfide bond formation, which is expected to immobilize the enzyme in its activated form through the SH modification. This immobilized enzyme was more stable against thermal denaturation than the free enzyme. When tetrahydrobiopterin was used as the natural cofactor, the K(m) value for phenylalanine was decreased and that for the cofactor was increased. Constant conversion from phenylalanine to tyrosine was demonstrated continuously for over 8 h at 25 degrees C.

Covalent chromatography by thiol-disulfide interchange of the highly-purified non-transformed rat liver glucocorticoid-receptor

Highly-purified non-transformed rat liver [ 3H]triamcinolone acetonide-receptor complex was shown to be covalently adsorbed on activated tbiol sepharose 4B, a reactive sulfhydryl matrice. Elution by mercaptoethanol in excess and inhibition of binding by previous treatment of the complex with N-ethylmaleimide clearly demonstrated the specificity of the binding by tbiol disulfide interchange. The transformed [ 3H]triamcinolone acetonide-receptor complex, partially purified by DNA-cellulose chromatography, was also retained on activated thiol sepharose 4B. The physicochemical characteristics of both the transformed and non-transformed glucocorticoid receptor complexes eluted from the covalent chromatography column were studied by HP size exclusion chromatography on a TSK G 3000 SW column and were found to be identical to those of the starting complexes. These results provide direct evidence for accessible sulfhydryl groups on the glucocorticoid receptor complex surface, probably distinct from the steroid binding essential sulfhydryl group.

Studies on toxin of Aspergillus fumigatus. XXII. Fashion of binding of Asp-hemolysin to human erythrocytes and Asp-hemolysin-binding proteins of erythrocyte membranes.

The fashion of binding of Asp-hemolysin to human erythrocytes and the isolation of Asp-hemolysin-binding proteins from erythrocyte membranes were investigated by the immunocytochemical technique and affinity chromatography. Asp-hemolysin bound best at a pH range from 5 to 7. The erythrocytes treated with Asp-hemolysin showed diffuse, ring-like or cap-like staining by the peroxidase-labeled antibody method under the light microscope. The distribution of Asp-hemolysin on the erythrocyte surface was clearly observed as patches or caps in the scanning electron microscope. The erythrocyte ghosts were extracted with 1% sodium deoxycholate-0.1 M Tris-HC1 buffer (pH 7.5) containing 0.2 M NaCl and 1 mM EDTA, and the extract was chromatographed on an affinity column consisting of Asp-hemolysin attached to activated thiol-Sepharose 4B. Four proteins present in the membrane extract were retained by activated thiol-Sepharose 4B and eluted with 50 mM cysteine as toxin-membrane components. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the polypeptides correspond to band 2.1, one protein of the 2 region, band 3 and band 7 in the Steck nomenclature system.

Large scale isolation, purification, and partial characterization of the intermediate filament-specific, Ca 2+-activated proteinase from porcine kidney and Ehrlich ascites tumor cells: A comparative study

The Ca 2+-activated, neutral thiol proteinase specific for intermediate filament subunit proteins was isolated at large scale from the postribosomal supernatant of a low-ionic-strength extract of porcine kidney and Ehrlich ascites tumor (EAT) cells, respectively. The purification procedure encompassed DEAE-Sephacel ion exchange chromatography of the material precipitating between 23 and 55% (NH 4) 2SO 4 saturation, followed by hyroxylapatite chromatography and activated thiol Sepharose 4B covalent chromatography. On the average, 25 mg of 62% pure enzyme was obtained from 500 g frozen kidney and 55 mg of 51% pure enzyme from 500 g EAT cells within a week. Both enzyme preparations were free of Ca 2+-independent proteolytic activities and indistinguishable with respect to their physicochemical and functional properties; their catalytic properties were indistinguishable from those of enzyme purified to homogeneity on arginine methylester Sepharose 4B. Because of this identity, porcine kidney proves to be an inexpensive source for the Ca 2+-activated proteinase which had previously been isolated and purified at small scale from EAT cells (W. J. Nelson and P. Traub, (1983) J. Biol. Chem. 257, 5544–5553). Despite a 38% protein contamination, the partially purified enzyme from porcine kidney is useful for the in vitro study of structure-function relationships of intermediate filaments and their subunit proteins. During affinity chromatography of the partially purified proteinase from EAT cells on arginine methylester Sepharose 4B, a 100-kDa protein was purified which has a high affinity for arginine residues. It also occurs in porcine kidney, although at a considerably lower concentration. Its cellular localization and function remain to be determined.

Purification of human platelet calcium-activated protease. Effect on platelet and endothelial function.

Calcium-activated protease (CAP) was purified from the cytosol fraction of homogenized human platelet concentrates using a combination of gel filtration chromatography and affinity chromatography on antipain aminohexyl-Sepharose and activated thiol-Sepharose 4B. Purified CAP is composed of two different polypeptides of Mr = 80,000 and 27,000. Half-maximal protease activity was observed at 0.52 mM Ca2+, and all activity was inhibited by antipain, leupeptin, and N-ethylmaleimide. Activated CAP showed a time-dependent inactivation in the presence of 1 mM Ca2+ with only 5% of the control protease activity remaining after a 1-h exposure to calcium. Preincubation of washed platelets with varying amounts of CAP (0.2-0.4 units) significantly interfered with thrombin-induced platelet aggregation. In addition, ristocetin-induced platelet agglutination in the presence of von Willebrand factor was completely inhibited by 0.4 units of CAP. Concomitant with these protease-induced changes in platelet function, a decrease was observed in a major glycoprotein band of Mr = 150,000 present in platelet membranes and presumed to be glycoprotein Ib. In addition to these effects on platelets, CAP inhibited thrombin-induced production of prostacyclin by cultured human endothelial cells in a dose-dependent manner when the cells were pretreated with CAP. Thus platelet CAP can modulate membrane functions in both platelets and endothelial cells and may thus contribute to the regulation of hemostasis.

Affinity chromatography of brain cyclic nucleotide phosphodiesterase using 3-(2-pyridyldithio)propionyl-substituted calmodulin linked to thiol-sepharose.

[3-(2-Pyridylthio)propionyl]calmodulin (PDP-CaM), an activated thiol derivative of calmodulin (CaM), was synthesized. Preparations of this derivative containing an average of 2.8 mol of substituent/mol of protein activated purified cyclic nucleotide phosphodiesterase in a manner indistinguishable from that of native CaM. PDP-CaM was covalently coupled to free thiol-Sepharose 4B through formation of a stable mixed disulfide bond for use in affinity chromatography. The binding capacity of the disulfide-linked CaM-Sepharose for phosphodiesterase activity was proportional to substituent level up to 4 mg of CaM/mL of gel; the total capacity of the gel for binding phosphodiesterase was 4 times that of CNBr-coupled CaM-Sepharose. Quantitative recovery was achieved by desorption of both ligand and bound proteins with a reducing agent. The thiolated CaM derivative was then separated from phosphodiesterase by rapid gel filtration; the overall recovery of phosphodiesterase activity was greater than 70%. Preparations of homogeneous enzyme in good yield were obtained after a second chromatography step on CaM-Sepharose. Binding and recovery of phosphodiesterase activity were entirely reproducible, since each preparation of affinity gel was used only once. As it permits separation of interacting species in free solution, this general method may be useful with other ligands for increasing yields from affinity chromatography, particularly when dissociation of molecules in their matrix-bound conformation may be difficult to achieve.

Immunochemical investigations of Rh0(D) activity detected in Band 3 of red cell membrane.

Stroma from D positive red cell was solubilized with 1% deoxycholic acid (DOC) and was applied onto Sepharose 4B and 6B, followed by fractionation with activated thiol Sepharose 4B. By this method Band 3 containing no carbohydrate was prepared. The Band 3 was incubated with human anti-D, then immune complex soluble in DOC solution was produced. This complex was not prepared from red cell components other than Band 3, nor from Band 3 from D negative red cell. The complex agglutinated papainized D positive red cells. Molecular weight of the complex was estimated to be 500,000 to 700,000 by the gel filtration method. The molecular weight increased according to the time of incubation. The complex gave a precipitation line in counter immunoelectrophoresis (IEP), a single peak in crossed IEP against anti-Band 3, and a line at gamma-globulin region in IEP against anti-human serum. SDS-polyacrylamide gel disc electrophoresis of the complex showed the pattern consisting of fragments of immunoglobulin G and Band 3 which was not stained with periodic acid Schiff reagent. These support our results that Rh-Hr blood type activities are located in Band 3 almost free of carbohydrate.

Isolation and purification of Rh0(D) antigen of human erythrocyte membrane and its serological property.

To elucidate immunochemical properties of Rh-Hr antigens, influence of various solubilizers on the activity, and isolation of the antigens were investigated. As a very sensitive method to detect Rh-Hr antigens, the agglutination inhibition test was recommended in which diluted antisera (1:400-1:1,600), papainized red cells and microtitrate plates were used. Extraction of D antigen with Brij 35 and Triton X-100 was effective, but SDS was not successful. However, D activity of SDS solubilized stroma was recovered by adding reductants. The activity was slightly demonstrated at the preparation from D negative red cells. The influence of the reductants was discussed. SDS solubilized stroma was fractionated with gel filtrations and activated thiol Sepharose 4B. The final preparation with D activity was proved to correspond with Band 3 which was not stained with PAS reagent. Deoxycholic acid solubilized stroma was fractionated with gel filtrations and affinity chromatography, resulting in the preparation corresponding to Band 3 with potent Rh-Hr blood type activities. The activities were detected without reductants and the preparation from D negative red cells had no D activity. Rh-Hr blood type activities other than D were observed in the same fraction.

Affinity chromatography purification of cytochrome c binding enzymes.

An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.

Induction of Kidney Metallothionein and Metallothionein Messenger RNA by Zinc and Cadmium

The influence of a 7.7-µmole (0.5-mg) dose of parenteral zinc on the synthesis of metallothionein in rat kidney was examined. The amount of zinc bound to metallothionein was maximal 9–15 hours after zinc administration. Pulse labeling with [35S]cystine showed the rate of renal metallothionein was stimulated to a maximum 6 hours after zinc administration and declined thereafter. Total RNA was extracted by the guanidine thiocyanate procedure from kidneys that had been flash frozen in liquid N2. Polyadenylated RNA (mRNA) was isolated by oligo (dT)-cellulose chromatography. The mRNA was translated in a wheat germ system and newly synthesized metallothionein was isolated by activated thiol-Sepharose 4B chromatography. Metallothionein mRNA activity nearly doubled after zinc administration and was closely correlated with the enhancement in synthetic rate of this protein in kidney. Actinomycin D administered prior to either zinc or cadmium completely blocked both the stimulation of metallothionein synthesis and mRNA activity found in kidney in response to these metals. The results suggest renal metallothionein is induced by zinc or cadmium through a mechanism that requires altered expression of the metallothionein gene(s).kidney metallothionein induction mRNA zinc cadmium protein synthesis