Conformational analysis of biologically active truncated linear analogs of endothelin-1 using NMR and molecular modeling.

Some linear truncated analogs of endothelin-1 display potent agonistic activity at the ET(B) receptor, especially when the side chain of Trp21 is N-formylated. Then, the three-dimensional arrangements of six structurally reduced linear analogs, three formylated and three nonformylated, have been investigated by high resolution NMR spectroscopy and molecular modeling, in order to pinpoint the conformational features related to the biological activity. Two-dimensional double-quantum-filtered correlation spectroscopy (DQFCOSY), total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY) were recorded and analyzed for each molecule. Interspatial distance constraints were derived from the intensity of the NOESY connectivities. The formation of hydrogen bonding was monitored from the temperature dependence of the NH chemical shifts. Molecular models calculated by means of distance geometry, simulated annealing and energy minimization, using the NMR constraints, strongly suggested a global elongated structure for the formylated analogs exhibiting biological activity, and a folded arrangement for the unformylated derivatives. Homology comparisons allowed the identification of a beta-turn-like folding of the C-terminal segment Asp18-Trp21 as a probable key-factor for activity.     

The microheterogeneity of the crystallizable yeast cytoplasmic aspartyl-tRNA synthetase

Yeast aspartyl-tRNA synthetase is a dimeric enzyme (alpha 2, Mr 125,000) which can be crystallized either alone or complexed with tRNAAsp. When analyzed by electrophoretic methods, the pure enzyme presents structural heterogeneities even when recovered from crystals. Up to three enzyme populations could be identified by polyacrylamide gel electrophoresis and more than ten by isoelectric focusing. They have similar molecular masses and mainly differ in their charge. All are fully active. This microheterogeneity is also revealed by ion-exchange chromatography and chromatofocusing. Several levels of heterogeneity have been defined. A first type, which is reversible, is linked to redox effects and/or to conformational states of the protein. A second one, revealed by immunological methods, is generated by partial and differential proteolysis occurring during enzyme purification from yeast cells harvested in growth phase. As demonstrated by end-group analysis, the fragmentation concerns exclusively the N-terminal end of the enzyme. The main cleavage points are Gln-19, Val-20 and Gly-26. Six minor cuts are observed between positions 14 and 33. The present data are discussed in the perspective of the crystallographic studies on aspartyl-tRNA synthetase.     

Identification of the major tRNA(Phe) binding domain in the tetrameric structure of cytoplasmic phenylalanyl-tRNA synthetase from baker's yeast

Native cytoplasmic phenylalanyl-tRNA synthetase from baker's yeast is a tetramer of the alpha 2 beta 2 type. On mild tryptic cleavage it gives rise to a modified alpha 2 beta 2 form that has lost the tRNA(Phe) binding capacity but is still able to activate phenylalanine. In this paper are presented data concerning peptides released by this limited proteolytic conversion as well as those arising from exhaustive tryptic digestion of the truncated beta subunit. Each purified peptide was unambiguously assigned to a unique stretch of the beta subunit amino acid sequence that was recently determined via gene cloning and DNA sequencing. Together with earlier results from affinity labelling studies the present data show that the Lys 172-Ile 173 bond is the unique target of trypsin under mild conditions and that the N-terminal domain of each beta subunit (residues 1-172) contains the major tRNA(Phe) binding sites.     

An auxin-auxotrophic mutant of Nicotiana plumbaginifolia

Summary Auxin (indole-3-acetic acid) is considered to be an important signalling molecule in the regulation of plant growth and development but neither auxin synthesis nor its mode of action is clearly understood. To identify genes involved in these processes, mutations were sought that altered the auxin requirement of plant tissues for growth. For the first time mutant plants were obtained that carry a recessive mutation at a single nuclear locus (auxl) which results in an absolute requirement for exogenous auxin for normal growth. In the absence of auxin treatment, mutant plants undergo premature senescence and die.Abbreviations BAP 6-benzylaminopurine - BUdR 5-bromodeoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - FUdR 5-fluorodeoxyuridine - IAA-EE indole-3-acetic acid ethyl ester - IMS indole-3-methanesulfonic acid     

Quantitative immunolocalization of four immunodominant antigens of Toxoplasma gondii

Ultrastructural localization of four immunodominant antigens of Toxoplasma gondii was investigated quantitatively on thin sections and replicas by an immunogold technique using four monoclonal antibodies (Mab). On immunoblot Mab IV47, GII9, II38 and IE10 identified proteins of 28, 30, 45 and 66-70 kDa, respectively. Use of digital image analyzer and a semi-automatic procedure developed by us, the patterns of label distribution were compared in three cell structures: cell surface, submembrane area and rhoptries. On the whole cell surface, protein P28 and P30 were 2.5 and 4 times more abundant than P66-70 respectively. The protein P28 was essentially concentrated in the submembrane area with a labeling of 195.4 +/- 46.7 gold particles/microns 2 that follows a decreasing gradient from this area to the cell centre. In the rhoptries, all four antigens were detected, P45 and P66-70 being major with a labeling of 97.1 +/- 31.1 gold particles/microns 2 and 155.1 +/- 39.3 gold particles/microns 2 respectively. The results support the hypothesis that rhoptries are the essential site for antigen storage.     

Cytoplasmic methionyl-tRNA synthetase from Bakers' yeast. A monomer with a post-translationally modified N terminus

Methionyl-tRNA synthetase has been purified from a yeast strain carrying the MES1 structural gene on a high copy number plasmid (pFL1). The purified enzyme is a monomer of Mr = 85,000 in contrast to its counterpart from Escherichia coli which is a dimer made up of identical subunits (Mr = 76,000; Dardel, F., Fayat, G., and Blanquet, S. (1984) J. Bacteriol. 160, 1115-1122). The yeast enzyme was not amenable to Edman's degradation indicating a blocked NH2 terminus. Its primary structure as derived from the DNA sequence (Walter, P., Gangloff, J., Bonnet, J., Boulanger, Y., Ebel, J.P., and Fasiolo, F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2437-2441) has been confirmed using the fast atom bombardment-mass spectrometric method. This method was applied to tryptic digests of the carboxymethylated enzyme and the corresponding data provided extensive coverage of the translated DNA sequence, thus confirming its correctness. The ambiguity concerning which of the three NH2-terminally located methionine codons is the initiation codon was easily resolved from peptides identified in this region. It was possible to show that the first methionine had been removed and that the new NH2 terminus, serine, had been acetylated. A comparison between the yeast and E. coli sequences shows that the former has an N-terminal extension of about 200 residues as compared to the latter. It also lacks the C-terminal domain which is responsible for the dimerization of the E. coli methionyl-tRNA synthetase.     

Purification and characterization of a toxin inhibiting protein synthesis from Croton mongue, a Madagascar Euphorbiaceae

Monguine, a thermostable toxic protein was extracted from the seeds of Croton mongue (Euphorbiaceae) and purified by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-25 and G-15. Polyacrylamide gel electrophoresis of purified monguine in the presence of sodium dodecyl sulfate and after treatment with 2-mercaptoethanol showed one band corresponding to a molecular weight of 9000. The same molecular weight was determined by analytical centrifugation. Amino acid analysis revealed a high content in both aspartic and glutamic acids (or the corresponding amides). The LD50 (24 h) is 12 mg/kg of mouse body weight. Monguine inhibits protein synthesis in hepatoma tissue culture cells and globin synthesis in a rabbit reticulocyte lysate.     

Improved Method for Virus Structural Polypeptide Analysis on Dissociating Acylamide Gel

A method is described in which polypeptides can be separated, with a high band resolution, by electrophoresis through "pore gradient" acrylamide gel (15 cm in length) in a sodium dodecyl sulfate-urea dissociating system. The applicability of this technique to the analysis of virus structural components was examined with the adenovirus type 2 model system.