Substitution of the methionine residues of calmodulin with the unnatural amino acid analogs ethionine and norleucine: biochemical and spectroscopic studies.
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Calmodulin (CaM) is a 148-residue regulatory calcium-binding protein that activates a wide range of target proteins and enzymes. Calcium-saturated CaM has a bilobal structure, and each domain has an exposed hydrophobic surface region where target proteins are bound. These two "active sites" of calmodulin are remarkably rich in Met residues. Here we have biosynthetically substituted (up to 90% incorporation) the unnatural amino acids ethionine (Eth) and norleucine (Nle) for the nine Met residues of CaM. The substituted proteins bind in a calcium-dependent manner to hydrophobic matrices and a synthetic peptide, encompassing the CaM-binding domain of myosin light-chain kinase (MLCK). Infrared and circular dichroism spectroscopy show that there are essentially no changes in the secondary structure of these proteins compared to wild-type CaM (WT-CaM). One- and two-dimensional NMR studies of the Eth-CaM and Nle-CaM proteins reveal that, while the core of the proteins is relatively unaffected by the substitutions, the two hydrophobic interaction surfaces adjust to accommodate the Eth and Nle residues. Enzyme activation studies with MLCK show that Eth-CaM and Nle-CaM activate the enzyme to 90% of its maximal activity, with little changes in dissociation constant. For calcineurin only 50% activation was obtained, and the K(D) for Nle-CaM also increased 3.5-fold compared with WT-CaM. These data show that the "active site" Met residues of CaM play a distinct role in the activation of different target enzymes, in agreement with site-directed mutagenesis studies of the Met residues of CaM. (+info)
DL-ethionine treatment of adult pancreatic donors. Amelioration of diabetes in multiple recipients withe tissue from a single donor.
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Transplantation of adult rat pancreatic islet tissue as a free graft requires the separation of islet from exocrine tissue to avoid host injury or graft destruction by digestive enzymes. The poor yield from islet isolation techniques currently necessitates the use of multiple donors to ameliorate diabetes in a single recipient. DL-ethionine (DLE) is an agent selectively toxic to the exocrine pancreas. We examined the effect of DLE administration on pancreatic digestive enzyme content and islet mass in adult Lewis rats and the ability of such pancreatic tissue dispersed by collagenase digestion without specific islet isolation to ameliorate diabetes when transplanted to the portal vein of syngeneic rats with streptozotocin induced diabetes. Rats fed normal chow supplemented with 0.5% DLE for 14-20 days showed a logarithmic loss of pancreatic mass. Total pancreatic amylase content declined to 0.3 + 0.1 mg, less than 3% of control values (14.3 +/- 1.0 mg). Total insulin content in DLE treated rats was 87 +/- 8 microg, not significantly different from control rats (101 +/- 7 microg). Histological examination confirmed the selective atrophy of exocrine tissue in DLE treated rats. Fresh pancreatic tissue prepared from a single DLE treated donor ameliorated diabetes 75% of the time when transplanted to one or two recipients and 65% of the time when divided between three of four recipients. Tissue prepared from a single DLE treated donor and stored for 24-48 hours ameliorated diabetes 91% of the time when divided between one or two recipients. Only four of 31 diabetic rats transplanted with fresh pancreatic tissue from untreated adult donors became normoglycemic. Pretreatment of adult rats with DLE induces selective exocrine atrophy, permits dispersed pancreatic tissue from a single donor to ameliorate experimental diabetes in up to four recipients, and allows tissue to be preserved by culture for up to 48 hours without specific islet isolation. (+info)
Tryptophan fluorescence of calmodulin binding domain peptides interacting with calmodulin containing unnatural methionine analogues.
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The interactions between the abundant methionine residues of the calcium regulatory protein calmodulin (CaM) and several of its binding targets were probed using fluorescence spectroscopy. Tryptophan steady-state fluorescence from peptides encompassing the CaM-binding domains of the target proteins myosin light chain kinase (MLCK), cyclic nucleotide phosphodiesterase (PDE) and caldesmon site A and B (CaD A, CaD B), and the model peptide melittin showed Ca(2+)-dependent blue-shifts in their maximum emission wavelength when complexed with wild-type CaM. Blue-shifts were also observed for complexes in which the CaM methionine residues were replaced by selenomethionine, norleucine and ethionine, and when a quadruple methionine to leucine C-terminal mutant of CaM was studied. Quenching of the tryptophan fluorescence intensity was observed with selenomethionine, but not with norleucine or ethionine substituted protein. Fluorescence quenching studies with added potassium iodide (KI) demonstrate that the non-native proteins limit the solvent accessibility of the Trp in the MLCK peptide to levels close to that of the wild-type CaM-MLCK interaction. Our results show that the methionine residues from CaM are highly sensitive to the target peptide in question, confirming the importance of their role in binding interactions. In addition, we provide evidence that the nature of binding in the CaM-CaD B complex is unique compared with the other complexes studied, as the Trp residue of this peptide remains partially solvent exposed upon binding to CaM. (+info)
Methionine depletion enhances the antitumoral efficacy of cytotoxic agents in drug-resistant human tumor xenografts.
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Efficacy of chemotherapy is limited in numerous tumors by specific cellular mechanisms that inactivate cytotoxic antitumoral drugs, such as ATP-dependent drug efflux and/or drug detoxification by glutathione. In reducing ATP pools and/or glutathione synthesis, it might be possible to enhance the efficacy of drugs affected by such resistance mechanisms. Reduction of the ATP pool and glutathione content is achievable in cancer cells by depleting the exogenous methionine (Met) supply and ethionine. Thus, the rationale for the present study was to use Met depletion to decrease the ATP and glutathione pools so as to sensitize tumors refractory to cytotoxic anticancer drugs. Met depletion was achieved by feeding mice a methionine-free diet supplemented with homocysteine. The effects of Met depletion combined with ethionine and/or chemotherapeutic agents were studied using human solid cancers xenografted into nude mice. TC71-MA (a colon cancer) SCLC6 (a small cell lung cancer), and SNB19 (a glioma) were found to be refractory to cisplatin, doxorubicin, and carmustine, respectively. These three drugs are used to treat such tumors and are dependent for their activity on the lack of cellular ATP- or glutathione-dependent mechanisms of resistance. TC71-MA, SCLC6, and SNB19 were Met dependent because their proliferation in vitro and growth in vivo were reduced by Met depletion. Cisplatin was inactive in the treatment of TC71-MA colon cancer, whereas a methionine-free diet, alone or in combination with ethionine, prolonged the survival of mice by 2-fold and 2.8-fold, respectively. When all three approaches were combined, survival was prolonged by 3.3-fold. Doxorubicin did not affect the growth of SCLC6, a MDR1-MRP-expressing tumor. A Met-deprived diet and ethionine slightly decreased SCLC6 growth and, in combination with doxorubicin, an inhibition of 51% was obtained, with survival prolonged by 1.7-fold. Combined treatment produced greater tumor growth inhibition (74%) in SCLC6-Dox, a SCLC6 tumor pretreated with doxorubicin. Growth of SNB19 glioma was not inhibited by carmustine, but when it was combined with Met depletion, survival duration was prolonged by 2-fold, with a growth inhibition of 80%. These results indicate the potential of Met depletion to enhance the antitumoral effects of chemotherapeutic agents on drug-refractory tumors. (+info)
Methyl-deficient mammalian transfer RNA: II. Homologous methylation in vitro of liver tRNA from normal and ethionine-fed rats: ethionine effect on 5-methyl-cytidine synthesis in vivo.
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Following hydroxyapatite chromatography, rat liver tRNA methylase activity was assayed with liver tRNA from normal rats and with methyl-deficient liver tRNA from ethionine-fed rats. The difference in homologous methylation between normal and methyl-deficient tRNA was maximal in certain fractions in presence of cadaverine, and much less in presence of Mg(++) or Mg(++) plus cadaverine. These methylase fractions, which contained endogenous tRNA, were used for preparative homologous methylation of added normal and methyl-deficient tRNA in presence of 30 mM cadaverine. The (14)C-methylated tRNA was digested with RNase T(2) and the resulting methylated mononucleotides were characterized and quantitated after twodimensional thinlayer chromatography and autoradiography. The major products of homologous tRNA methylation were m(5)C and m(1)A. However, the methylase fraction used here did not catalyze the formation of m(6) (2)A with m(6) (2)A-deficient tRNA as substrate.- In addition to the previously described, analytically detectable m(6) (2)A-deficiency, a partial m(5)C-deficiency was demonstrated in liver tRNA from ethionine-fed rats by measuring the methylacceptance in vitro. In presence of cadaverine, with the methylase fraction used here, methyl-deficient tRNA from ethionine-fed rats was a twofold more efficient methyl-acceptor in vitro than normal liver tRNA, while endogenous tRNA isolated from the methylase fraction was a threefold more efficient methyl-acceptor than normal liver tRNA. Homologous methylation of normal tRNA, as observed here, has not been described before. (+info)
Biochemical and regulatory effects of methionine analogues in Saccharomyces cerevisiae.
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The effect of three methionine analogues, ethionine, selenomethionine, and trifluoromethionine, on the biosynthesis of methionine in Saccharomyces cerevisiae has been investigated. We have found the following to be true. (i) A sharp decrease in the endogenous methionine concentration occurs after the addition of any one of these analogues to growing cells. (ii) All of them can be transferred to methionine transfer ribonucleic acid in vitro as well as in vivo with, as a consequence, their incorporation into proteins. In the absence of radioactive trifluoromethionine, this conclusion results from experiments of an indirect nature and must be taken as an indication rather than a direct demonstration. (iii) Ethionine and selenomethionine can be activated as homologues of S-adenosylmethionine, whereas trifluoromethionine cannot. (iv) All of them can act as repressors of the methionine biosynthetic pathway. This has been shown by measuring the de novo rate of synthesis of methionine in a culture grown in the presence of any one of the three analogues. (+info)
Acute hemorrhagic pancreatitis (massive necrosis) with fat necrosis induced in mice by DL-ethionine fed with a choline-deficient diet.
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Female, albino mice were fed a choline-deficient diet containing 0.5% DL-ethionine. All animals died within 5 days due to the development of an acute hemorrhagic pancreatis with fat necrosis throughout the peritoneal cavity. The apancreatitis was characterized by a massive necrosis of the exocrine parenchyma with intense hemorrhage and inflammatory reaction of the stroma. The sequence of histologic and ultrastructural alterations occurring in the acinar cells of the pancreas were studied in mice fed the diet for 1, 2, and 3 days. Major findings consited of accumulation of zymogen granules, vacuolation due to foci of cytoplasmic degradation, and alterations in the morphology of the zymogen granules. The pancreatitis appears to be due to intraparenchymal activation of zymogens, resulting from a synergistic action of choline deficiency with the basic toxicity of ethionine toward the acinar cells of the pancreas. The experimental model simulates closely the acute hemorrhagic pancreatitis with fat necrosis occurring in humans and may prove useful for exploring the pathogenesis of this condition. (+info)
Genetic characterization of the metK locus in Escherichia coli K-12.
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Three independently isolated metK mutants have been shown to have leisions lying between speB and glc near 57 min on the Escherichia coli chromosome. Two deletions result in a lack of the metC gene product but neither extends into the metK glc region. The three metK mutations are recessive to the wild-type allele carried on the KLF16 episome. (+info)