Spiralin is defined as the major membrane protein of the helical mollicute Spiroplasma citri. According to the S. citri strain used, spiralin shows polymorphism in its electrophoretic mobility. The spiralin gene sequences of eight S. citri strains were determined by direct sequencing of the PCR-amplified genes. All spiralins were found to be 241 amino acids long, except for the spiralin of strain Palmyre, which is 242 amino acids long. The molecular masses calculated from these sequences did not explain the differences observed in the electrophoretic mobilities. In all of the spiralins examined, the first 24 N-terminal amino acids were conserved, including a cysteine at position 24, and had the features of typical signal peptides of procaryotic lipoproteins. When S. citri strains were grown in the presence of [ ]palmitic acid labeled, and it had almost the same electrophoretic mobility irrespective of the spiralins used. Samples of the unlabeled peptide fragments from the four representative strains had slightly different electrophoretic mobilities (⌬DaХ800 Da); however, these were much smaller than those of the whole spiralins before formic acid hydrolysis (⌬DaХ8,000 Da). These results suggest that spiralin polymorphism in S. citri is not due to differences in posttranslational modification by palmitic acid and is certainly a structural property of the whole protein or could result from an unidentified posttranslational modification of spiralin.Among the mollicutes (i.e., wall-less eubacteria with low GϩC contents and small genomes), spiroplasmas are characterized by their helical morphology and motility. Spiroplasma citri, the first spiroplasma to have been cultured, is the causal agent of ''Stubborn'' disease of citrus (24) and affects many other plant species. S. citri also multiplies in various leafhoppers, which act as insect vectors. The spiroplasmas belong to the recently established order Entomoplasmatales, which also contains Entomoplasma and Mesoplasma species (31). All of these mollicutes are associated with arthropods, mainly insects. The spiroplasmas, like the mycoplasmas, ureaplasmas, mesoplasmas, and entomoplasmas, but unlike the acholeplasmas, use the triplet UGA, usually a stop codon, as a tryptophan codon (8,21).Spiralin is the most abundant membrane protein in S. citri (36). Its amphiphilic and antigenic properties (34, 38) and its membrane location and arrangement (30, 35) have been extensively studied. The spiralin gene has been identified, cloned, and expressed in Escherichia coli cells (19). Several forms of spiralin were produced in E. coli, including a major form with a slightly larger molecular mass than that of spiralin in S. citri. S. citri spiralin is free of tryptophan, and its gene has no UGA codon (6). This feature explains why the gene could be efficiently translated in E. coli at a time when it was not yet known that in spiroplasmas, UGA is not read as a stop codon but instead is read as a tryptophan codon. However, the function of spiralin in the spiroplasma cell remains undet...

The aminoacylation reaction catalyzed by the dimeric tryptophanyl-tRNA synthetase from beef pancreas was studied under pre-steady-state conditions by the quenched-flow method. The transfer of tryptophan to tRNATrp was monitored by using preformed enzyme-bis(tryptophanyl adenylate) complex. Combinations of either unlabeled or L-[14C]tryptophan-labeled tryptophanyl adenylate and of aminoacylation incubation mixtures containing either unlabeled tryptophan or L-[14C]tryptophan were used. We measured either the formation of a single labeled aminoacyl-tRNATrp per enzyme subunit or the turnover of labeled aminoacyl-tRNATrp synthesis. Four models were proposed to analyze the experimental data: (A) two independent and nonequivalent subunits; (B) a single active subunit (subunits presenting absolute "half-of-the-sites reactivity"); (C) alternate functioning of the subunits (flip-flop mechanism); (D) random functioning of the subunits with half-of-the-sites reactivity. The equations corresponding to the formation of labeled tryptophanyl-tRNATrp under each labeling condition were derived for each model. By use of least-squares criteria, the experimental curves were fitted with the four models, and it was possible to disregard models B and C as likely mechanisms. Complementary experiments, in which there was no significant excess of ATP-Mg over the enzyme-adenylate complex, emphasized an activator effect of free L-tryptophan on the rate of aminoacylation. This result disfavored model A. Model D was in agreement with all data. The analyses showed that the transfer step was not the major limiting reaction in the overall aminoacylation process.

Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [32P]PO43− from [γ‐32P]ATP into protein constituents. Among these, P0 glycoprotein is the major phosphorylated species. To identify the phosphorylated sites, P0 protein was in vitro phosphorylated, purified, and cleaved by CNBr. Two 32P‐phosphopeptides were isolated by HPLC. The exact localization of the sequences around the phosphorylated sites was determined. The comparison with rat P0 sequence revealed, besides a Lys172 to Arg substitution, that in the first peptide, two serine residues (Ser176 and Ser181) were phosphorylated, Ser176 appearing to be modified subsequently to Ser181. In the second peptide, Ser197, Ser199, and Ser204 were phosphorylated. All these serines are clustered in the C‐terminal region of P0 protein. This in vitro study served as the basis for the identification of the in vivo phosphorylation sites of the C terminal region of P0. We found that, in vivo, Ser181 and Ser176 are not phosphorylated, whereas Ser197, Ser199, Ser204, Ser208, and Ser214 are modified to various extents. Our results strongly suggest that the phosphorylation of these serine residues alters the secondary structure of this domain. Such a structural perturbation could play an important role in myelin compaction at the dense line level.

The kinetics of formation of tryptophanyl adenylate by tryptophanyl-tRNA synthetase from beef pancreas has been followed by stopped-flow, using the quenching of fluorescence of the enzyme linked to the amino acid activation reaction. Both subunits of this LYZ enzyme catalyze the adenylate formation. At saturation with substrates the rate constant of the activation reaction is the same for both subunits. The same behaviour is observed for the pyrophosphorolysis reaction. Both subunits exhibit the same affinity for ATP-Mg in the forward reaction and the same affinity for magnesium pyrophosphate in the backward reaction. On the contrary the formation of tryptophanyl adenylate follows biphasic kinetics when tryptophan concentration is much below saturation. This is independent of ATP-Mg concentration and is the consequence of different affinities of the two subunits for tryptophan as already observed by Graves et al. (1979, Eur. J. Biochem. 96, 509-518) in equilibrium dialysis experiments. A monoadenylate-enzyme complex on one subunit has been prepared. This complex made possible the study of the formation of the second adenylate on the other subunit. The formation of this second adenylate followed first-order kinetics at all ATP-Mg and tryptophan concentrations. The tryptophan concentration dependence of the rate of formation of this second adenylate leads to a Michaelis constant close to the dissociation constant of the low affinity tryptophan site of the enzyme. No isomerization step could be evidenced. The experiments were carried out under two conditions corresponding to those used by Merault et al. ( 3 978, Eur. J. Biochem. 87, 541 -550) in the steady state of the tRNATrp aminoacylation reaction (10 mM total magnesium in 100 mM KCI and 1 mM free magnesium ions, both at pH 8.0, 25 "C). No great difference either in the mechanism or in the dissociation and rate constants was observed but an inhibitory effect of KCl. It is concluded that the enzyme is symmetrical as far as the ATP-Mg and the magnesium pyrophosphate sites are concerned and that the rate of the activation reaction reflects the anticooperative occupancy of the tryptophan sites carried by the two subunits.