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J W Bodley

Publications and source records attributed to J W Bodley.

At least 37 records · Page 2Linked to original sources
PubMed1987

Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu.

Yeast mitochondrial elongation factor Tu (EF-Tu) was purified 200-fold from a mitochondrial extract of Saccharomyces cerevisiae to yield a single polypeptide of Mr = approximately 47,000. The factor was detected by complementation with Escherichia coli elongation factor G and ribosomes in an in vitro phenylalanine polymerization reaction. Mitochondrial EF-Tu, like E. coli EF-Tu, catalyzes the binding of aminoacyl-tRNA to ribosomes and possesses an intrinsic GTP hydrolyzing activity which can be activated either by kirromycin or by ribosomes. Kinetic and binding analyses of the interactions of mitochondrial EF-Tu with guanine nucleotides yielded affinity constants for GTP and GDP of approximately 5 and 25 microM, respectively. The corresponding affinity constants for the E. coli factor are approximately 0.3 and 0.003 microM, respectively. In keeping with these observations, we found that purified mitochondrial EF-Tu, unlike E. coli EF-Tu, does not contain endogenously bound nucleotide and is not stabilized by GDP. In addition, we have been unable to detect a functional counterpart to E. coli EF-Ts in extracts of yeast mitochondria and E. coli EF-Ts did not detectably stimulate amino acid polymerization with mitochondrial EF-Tu or enhance the binding of guanine nucleotides to the factor. We conclude that while yeast mitochondrial EF-Tu is functionally analogous to and interchangeable with E. coli EF-Tu, its affinity for guanine nucleotides and interaction with EF-Ts are quite different from those of E. coli EF-Tu.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Diphtheria toxin-resistant mutants of Saccharomyces cerevisiae.

We developed a selection procedure based on the observation that diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae (Murakami et al., Mol. Cell. Biol. 2:588-592, 1982); this procedure yielded mutants resistant to the in vitro action of the toxin. Spheroplasts of mutagenized S. cerevisiae were transformed in the presence of diphtheria toxin, and the transformed survivors were screened in vitro for toxin-resistant elongation factor 2. Thirty-one haploid ADP ribosylation-negative mutants comprising five complementation groups were obtained by this procedure. The mutants grew normally and were stable to prolonged storage. Heterozygous diploids produced by mating wild-type sensitive cells with the mutants revealed that in each case the resistant phenotype was recessive to the sensitive phenotype. Sporulation of these diploids yielded tetrads in which the resistant phenotype segregated as a single Mendelian character. From these observations, we concluded that these mutants are defective in the enzymatic steps responsible for the posttranslational modification of elongation factor 2 which is necessary for recognition by diphtheria toxin.

Diphtheria Toxin↗
PubMed1984

Anion-exchange chromatography of proteins on AG MP-1 using high-performance liquid chromatography equipment.

That the macroporous anion-exchange resin AG MP-1 can be used with HPLC equipment and common aqueous buffers for the chromatography of proteins is shown. The utility of this system is illustrated by the partial purification and complete resolution of the three protein synthesis elongation factors from each other, starting with a crude extract of Escherichia coli. The factors were purified 10- to 30-fold in a yield of 50 to 90% with a single 60-min chromatographic program of increasing NaCl concentration. Other proteins from various biological sources were purified with similar results. Thus, it appears that AG MP-1 is useful, at least in some applications, for the rapid, reproducible, and economical purification of proteins using HPLC equipment.

Animals↗
PubMed1984

Ribosyl-diphthamide: confirmation of structure by fast atom bombardment mass spectrometry.

Diphtheria toxin inactivates protein synthesis elongation factor 2 by attaching ADP-ribose to an unusual post-translational amino acid derivative, diphthamide, in the factor. Previously, we prepared ribosyl-diphthamide from the ADP-ribosyl-factor and proposed on the basis of NMR spectral analysis that it is 1-alpha-D-ribofuranosyl-2-[3-carboxyamido-3-(trimethylammonio++ +)propyl] histidine [N. J. Oppenheimer, and J.W. Bodley, (1981) J. Biol. Chem. 256, 8579-8581 and op. cit.]. Now, using fast atom bombardment mass spectrometry, the intact cation of ribosyl-diphthamide has been observed in the gas phase. The theoretical mass of the structure proposed for ribosyl-diphthamide uniquely agrees with the observed mass of the inact cation of the compound to within 2 ppm. Collisional activation decomposition mass spectral analysis provided additional structural confirmation. Thus, although the compound has not been synthesized, all available evidence appears uniquely consistent with the structure of ribosyl-diphthamide previously proposed.

Chemical Phenomena↗
PubMed1983

Biosynthetic labeling of diphthamide in Saccharomyces cerevisiae.

Diphthamide, the post-translational amino acid derivative in the diphtheria toxin-modification site of protein synthesis elongation factor 2 (EF-2), has the proposed structure 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine (Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Biol. Chem. 255, 10710-10716). The identification of the biosynthetic precursors of diphthamide would provide a means of evaluating its proposed structure and determining if the amino acid occurs in proteins other than EF-2. Toward this end, yeast were grown on potential radiolabeled precursors and the resulting radiolabeled protein was hydrolyzed in acid. The acid hydrolysates were subjected to amino acid analysis with a program optimized to resolve diphthine, the acid hydrolysis product of diphthamide, from the common amino acids. Radiolabel from [beta-3H]histidine, [alpha-3H]methionine, and [Me-3H] methionine was found to be incorporated into diphthine in a molar ratio of 1:1:3 while that of [35S]methionine was not incorporated. These results are in accord with the proposed structure of diphthamide and suggest that in its biosynthesis the backbone and 3 methyl groups of methionine are added to a histidine residue in the peptide chain of EF-2. These labeling experiments show that diphthine (diphthamide) constitutes approximately 6 X 10(-6) mol fraction of the total amino acids in yeast protein hydrolysates. Estimates of the amount of diphthamide present in the diphtheria toxin-modification site of EF-2 indicate that it constitutes from 4.5 to 9 X 10(-6) mol fraction of the total amino acids in yeast protein. The present evidence suggests that diphthamide occurs only in EF-2.

Amino Acids↗
PubMed1983

Occurrence of diphthamide in archaebacteria.

We examined the nature of the diphtheria toxin fragment A recognition site in the protein synthesis translocating factor present in cell-free preparations from the archaebacteria Thermoplasma acidophilum and Halobacterium halobium. In agreement with earlier work (M. Kessel and F. Klink, Nature (London) 287:250-251, 1980), we found that extracts from these organisms contain a protein factor which is a substrate for the ADP-ribosylation reaction catalyzed by diphtheria toxin fragment A. However, the rate of the reaction was approximately 1,000 times slower than that typically observed with eucaryotic elongation factor 2. We also demonstrated the presence of diphthine (the deamidated form of diphthamide, i.e., 2-[3-carboxyamide-3-(trimethylammonio)propyl]histidine) in acid hydrolysates of H. halobium protein in amounts comparable to those found in hydrolysates of similar preparations from eucaryotic cells (Saccharomyces cerevisiae and HeLa). Diphthine could not be detected in hydrolysates of protein from the eubacterium Escherichia coli. Whereas both archaebacterial and eucaryotic elongation factors contain diphthamide, they differ importantly in other respects.

Adenosine Diphosphate Ribose↗
PubMed1982

Saccharomyces cerevisiae spheroplasts are sensitive to the action of diphtheria toxin.

Diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae but not the intact yeast cells. After 2 h of exposure to ca. 10(-7) M toxin, less than 1% of spheroplasts were able to regenerate into intact cells. The same high levels of toxin inhibited the rate of protein synthesis by more than 90% within 1 h, whereas RNA and DNA synthesis were not inhibited until 4 h or exposure. Both killing and protein synthesis inhibition were dependent on toxin concentration. The nature of the toxin-cell interaction was also studied by using fragments of intact toxin and mutant toxin proteins. Neither toxin fragment A nor CRM45 nor CRM197 affected spheroplasts, but CRM197 and ATP prevented the inhibitory action of intact toxin. These results suggest that toxin acts on S. cerevisiae spheroplasts in much the same manner as it acts on sensitive mammalian cells.

Cell Division↗
PubMed1981

Diphtheria toxin. Site and configuration of ADP-ribosylation of diphthamide in elongation factor 2.

Diphtheria toxin inactivates protein synthesis elongation factor 2 by catalyzing the ADP-ribosylation of a novel derivative of histidine, diphthamide, in the protein (Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Biol. Chem. 255, 10710-10716). In this report, we describe experiments involving nuclear Overhauser enhancement NMR spectroscopy which were undertaken to elucidate the site of ADP-ribosylation of diphthamide and the configuration of the glycosidic bond formed by the toxin. The essential result of these experiments is that, in ribosyl-diphthamide obtained by enzymatic digestion of ADP-ribosyl-elongation factor-2, the H-5 imidazole proton is near the R-4 proton of ribose. This result and others are consistent with the interpretation that diphtheria toxin covalently attaches ADP-ribose to the imidazole N-1 of diphthamide via an alpha-glycosidic linkage.

Adenosine Diphosphate Ribose↗
PubMed1980

ADP-ribosylation of elongation factor 2 by diphtheria toxin. Isolation and properties of the novel ribosyl-amino acid and its hydrolysis products.

We have isolated the novel amino acid, amino acid X, in elongation factor 2 (EF-2) which is ADP-ribosylated by diphtheria toxin (Robinson, E. A., Hendriksen, O., and Maxwell, E. S. (1974) J. Biol. Chem. 249, 5088-5093). The pure ribosyl-amino acid was obtained in milligram quantities following sequential enzymatic degradation of yeast ADP-ribosyl-EF-2 (Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1978) J. Biol. Chem. 253, 8687-8690) with an overall yield of 30 to 60%. In the course of acid hydrolysis, the ribosyl-amino acid was converted first to an intermediate compound which did not contain ribose. Further acid hydrolysis converted the intermediate to amino acid X and ammonia. Alkaline hydrolysis of the ribosyl-amino acid produced a different intermediate which contained ribose. We have isolated the two hydrolysis intermediates and on the basis of the properties of all four compounds we conclude that the ribosyl-amino acid contains an amido function which is not involved in a glycosidic linkage and a net positive charge of 2 at acid pH. We believe that the amino acid amide occurs in EF-2 and for this compound we propose the trivial name diphthamide. Acid hydrolysis of diphthamide yields amino acid X for which we propose the trivial name diphthine.

Adenosine Diphosphate Ribose↗
PubMed1980

ADP-ribosylation of elongation factor 2 by diphtheria toxin. NMR spectra and proposed structures of ribosyl-diphthamide and its hydrolysis products.

NMR spectral analysis of the novel amino acid, diphthamide, in elongation factor 2 which is ADP-ribosylated by diphtheria toxin suggests that it is 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine. Ribosyl-diphthamide was prepared by enzymatic hydrolysis of ADP-ribosyl-elongation factor 2 and three compounds were produced by its chemical hydrolysis (Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Biol. Chem. 255, 10717-10720). Proton NMR spectroscopy in 2H2O of diphthine demonstrated the elements of histidine minus the carbon 2 proton plus 14 additional nonexchangeable protons. These protons were attributed to an extensive modification at carbon 2 of the imidazole ring. Proton NMR spectroscopy in 2H2O of ribosyl-diphthamide showed only those protons seen in diphthine plus those expected of a ribofuranosylmoiety. Chemical shift dependence on pH was demonstrated for the histidine-derived protons as well as several protons of the modifying side chain. Natural abundance carbon 13 NMR spectroscopy of ribosyldiphthamide also showed the elements of histidine and ribose plus 7 additional carbon atoms attributed to the modification of the imidazole ring. Based on the NMR spectral properties of the anomeric proton of ribosyldiphthamide we propose that the ribose is linked to one of the nitrogens of the histidine imidazole ring.

Adenosine Diphosphate Ribose↗
PubMed1978

Isolation and properties of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2.

We have developed a method for the purification in micromole amounts of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2 (EF-2). EF-2 was partially purified (15 to 20% purity) by ammonium sulfate precipitation and DEAE-Sephadex chromatography. After [3H]ADP-ribosylation by [3H]nad+ and diphtheria toxin, EF-2 was digested with trypsin and a homogeneous [3H]ADP-ribosyl peptide was isolated by chromatography on DEAE-Sephadex and dihydroxyboryl-substituted cellulose. Based on the amount of ADP-ribose acceptor activity in the crude extract, the overall yield of the peptide was 35%. The yeast peptide contains an unusual amino acid (X) which is the site of ADP ribosylation and is apparently identical to the amino acid reported from rat liver EF-2 by Robinson et al. (Robinson, E. A., Hendriksen, O., and Maxwell, E.S. (1974) J. Biol. Chem. 249, 5088-5093). The sequence of the 15-residue yeast peptide was determined to be: Val-Asn-Ile-Leu-Asp-Val-Thr-Leu-His-Ala-Asp-Ala-Ile-X-Arg. The 11 COOH-terminal residues of this peptide and of the homologous 15-residue peptide reported by Maxwell and co-workers from rat liver EF-2 are identical.

Adenosine Diphosphate Sugars↗