James W. Bodley

Formation of the ribosome-G factor-GDP complex in the presence of fusidic acid

Abstract When ribosomes are incubated with G factor and GTP, a ribosome-G factor-GDP complex is formed. Fusidic acid does not inhibit the formation of this complex; in fact, more complex is isolated in the presence of the antibiotic (10−3 M) than in its absence. No complex containing GTP is detectable in the presence or absence of the antibiotic.

The activity and stability of alkaline phosphatase in solutions of water and the fused salt ethylammonium nitrate

The fused salt ethylamonium nitrate has several properties which resemble those of the biologically important solvent water. In order to shed light on the role of solvent in determining protein structure we have examined the influence of ethylammonium nitrate on the activity and stability of the enzyme alkaline phosphatase. Significantly, although reduced enzymatic activity was observed in ethylammonium nitrate solutions up to 60% (v/v) in water, the enzyme was stable to brief exposure to solutions as high as 80% (v/v) in the fused salt.

Primary structure at the site in beef and wheat elongation factor 2 of ADP-ribosylation by diphtheria toxin

Elongation factor 2 (W-2) is the eukaryotic protein which catalyzes the translocation of the nascent peptide chain on the ribosome during the elongation phase ofyrotcin synthesis. W-2 is selectively inactivated by covalent ADFribosylation catalyzed by diphtheria toxin [ 1.2 3_ In vitro studies of this reaction show that EF-2 from all eukaryotic sources tested is specifically modified-by the toxin while other proteins, including the prokaryotic analog of W-2, elongation factor G, are 1101: [3 1. The site of ADP-ribosylation in EF-2 from rat liver [4] and yeast [5] has been shown to be a new amino acid, amino acid X, which presumably resuitr from a post-translational modification of a standard amino acid. The exact chemical structure of the modified residue or the functional nature of the inactivation has yet to be determined_ 2.1_ Purijk23. I of the pc.pti&s The assay &Cd partial purification of EF-2 and the

Studies on translocation VI: Thiostrepton prevents the formation of a ribosome-G factor-guanine nucleotide complex☆

Abstract Prior treatment of ribosomes with an approximate molar equivalence of the antibiotic inhibitor of translocation, thiostrepton, destroys their ability to participate in the formation of a ternary complex involving the ribosome, G factor, and either GTP or GDP.

A volatile liquid chromatography system for nucleotides

Abstract A liquid chromatography system useful for the rapid purification of radioactively labeled and nonradioactive ribonucleoside mono-, di-, and triphosphates has been developed. The system employs the strong anion-exchange resin AG MP-1 and low concentrations of the volatile eluting agent trifluoroacetic acid, and is suitable for use with high-performance liquid chromatography systems in either isocratic or gradient elution modes. Most common nucleotides can be completely resolved and nearly quantitatively recovered following solvent volatilization.

Vitellogenin synthesis in the lady beetle Coccinella septempunctata

Abstract To elucidate the hormonal mechanisms which regulate reproduction in a beneficial insect, we have begun to investigate the process of vitellogenesis in Coccinella septempunctata , the seven-spotted lady beetle. Vitellin (Vn) constitutes 60–70% of the total protein in C. septempunctata eggs and is composed of four polypeptides with molecular weights determined by electrophoresis in denaturing gels of 133,000, 130,000, 46,000 and 43,000. In the egg these polypeptides occur in a ratio of approx. 1:1:1:2. The two larger Vn polypeptides yielded similar peptide fragments upon partial proteolytic digestion which suggests that they are structurally related. The two smaller Vn polypeptides appear structurally distinct because they yielded unique proteolytic fragments. The Vn precursor, vitellogenin (Vg), was observed in the haemolymph of mature females, but was not detected in the haemolymph of immature females or males. The electrophoretic mobilities, antigenicity, and proteolytic fragmentation patterns of the Vg polypeptides were indistinguishable from those of their Vn counterparts. Thus the major processing of the Vn polypeptides appears to occur prior to their secretion into the haemolymph. The synthesis of Vg was examined in whole animals and in organ cultures. Vg synthesis was observed in the fat body and to a smaller extent in the ovaries of mature females. The newly synthesized Vg was rapidly secreted. Vg synthesis was not detectable in brain or thoracic muscle of mature females or in the fat body of males or immature females. Very little vitellogenin synthesis occurred in female insects reared on artificial diets. The topical application of a juvenile hormone analogue induced Vg synthesis in non-fecund females but not in males.

Saccharomyces cerevisiae elongation factor 2 is phosphorylated by an endorenous kinase

Mammalian cells contain a Ca2+/calmodulin‐dependent protein kinase that specifically phosphorylates and inactivates elongation factor 2 (EF‐2) in response to hormones and other agents which increase intracellular Ca2+ concentrations. Therefore, it has been proposed that the rate of translation in mammals is regulated by EF‐2 phosphorylation. In the present study, EF‐2 purified from the yeast Saccharomyces cerevisiae is shown to be a substrate for the mammalian EF‐2 kinase. Furthermore, evidence was obtained using two‐dimensional gel electrophoresis and peptide mapping which suggests that yeast EF‐2 is a substrate for an endogenous kinase which phosphorylates the same site as the mammalian EF‐2 kinase. Based on these findings, we propose that in yeast as in higher eukaryotes, the protein synthesis elongation cycle is regulated by phosphorylation of EF‐2.

The ribosomes of Aspergillus giganteus are sensitive to the cytotoxic action of α‐sarcin

Ribosomes in lysates prepared from the mycelia of Aspergillus giganteus MDH 18894, which are actively secreting α‐sarcin, do not contain the α‐sarcin lesion. However, the addition of exogenous α‐sarcin to these same lysates results in cleavage of the 26 S rRNA of the 60 S ribosomal subunit, characteristic of the cytotoxic action of α‐sarcin. We conclude that A. giganteus ribosomes are not inherently resistant to the action of α‐sarcin but are protected in vivo by producing α‐sarcin in an inactive form and/or by the efficient cotranslational secretion of the toxin.

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.

U.V. induced covalent crosslinking of E. coli ribosomal RNA to specific proteins.

Abstract E. coli 70S ribosomes uniformly labeled in vivo with 32PO4 were subjected to varying doses of u.v. radiation and then to the combined action of the RNases A and T1. Following these treatments the ribosomal proteins were separated by trichloroacetic acid precipitation from the noncovalently attached RNA degradation fragments. Subsequent two-dimensional gel electrophoresis and autoradiography of these proteins revealed that significant 32PO4 was associated with unique ribosomal proteins, L2 was among these.