Israel D. Algranati

Defective 30S ribosomal particles in a polyamine auxotroph of Escherichia coli

Abstract Polypeptide synthesis directed by poly(U) or MS 2 phage RNA is several fold more active in cell-free systems prepared from polyamine supplemented bacteria than in extracts of polyamine depleted cells. This effect depends on the presence of defective 30S ribosomal subunits in the starved bacteria. It is concluded that polyamines play a role in the normal biosynthesis, maturation and/or assembly of the small ribosomal subparticles.

Lack of Arginine Decarboxylase in Trypanosoma cruzi Epimastigotes

Abstract The presence of arginine decarboxylase (ADC) enzymatic activity in Trypanosoma cruzi epimastigotes is still a matter of controversy due to conflicting results published during the last few years. We have investigated whether arginine might indeed be a precursor of putrescine via agmatine in these parasites. We have shown that wild-type T. cruzi epimastigotes cultivated in a medium almost free of polyamines stopped their growth after several repeated passages of cultures in the same medium, and that neither arginine nor ornithine were able to support or reinitiate parasite multiplication. In contrast, normal growth was quickly resumed after adding exogenous putrescine or spermidine. The in vivo labelling of parasites with radioactive arginine showed no conversion of this amino acid into agmatine, and attempts to detect ADC activity measured by the release of CO2 under different conditions in T. cruzi extracts gave negligible values for all strains assayed. The described data clearly indicate that wild-type T. cruzi epimastigotes lack ADC enzymatic activity.

Protein synthesis and ribosomal distribution in a polyamine auxotroph of Escherichiacoli: Studies in cell-free systems

Summary Phenylalanine incorporation into polypeptides induced by poly (U) is markedly reduced in extracts obtained from polyamine depleted bacteria, and this effect, totally independent of RNA synthesis, is related to some deficiency at the level of the ribosomal particles and/or the factors bound to them. The ribosomal profiles corresponding to polyamine starved cells change when cultures are supplemented with putrescine.

The enzymic synthesis of yeast mannan

Abstract When guanosine diphosphate mannose (GDP-mannose) was first isolated from yeast, it was proposed that this compound might be the precursor of mannan ( Cabib and Leloir, 1954 ). Evidence supporting this hypothesis will be outlined in the present communication.

Differential regulation of putrescine uptake in Trypanosoma cruzi and other trypanosomatids.

Putrescine uptake in Trypanosoma cruzi epimastigotes is 10 to 50-fold higher than in Leishmania mexicana or Crithidia fasciculata. Polyamine transport in all these trypanosomatids is an energy-dependent process strongly inhibited by the presence of 2,4-dinitrophenol or KCN. Putrescine uptake in T. cruzi and L. mexicana was markedly decreased by the proton ionophore carbonylcyanide m-chlorophenylhydrazone but it was not affected by ouabain, a Na(+)-K+ pump inhibitor. The depletion of intracellular polyamines by treatment of parasite cultures with alpha-difluoromethylornithine elicited a marked induction of putrescine uptake in L. mexicana and C. fasciculata by increasing considerably the Vmax of this process. Conversely, the uptake of putrescine in T. cruzi was essentially unchanged by the same treatment. The differential regulation of putrescine transport in T. cruzi might be related to some distinctive features of polyamine metabolism in this parasite.

Spermidine is essential for normal proliferation of trypanosomatid protozoa

Trypanosomatid parasites containing a metabolically unstable ornithine decarboxylase (ODC) are naturally resistant to high levels of α‐difluoromethylornithine (DFMO) because this ODC inhibitor, though causing a drastic reduction of intracellular putrescine, elicits only a moderate decrease of the spermidine endogenous pool. In this study we have used a combination of DFMO with cyclohexylamine (CHA; bis‐cyclohexylammonium sulfate), an inhibitor of spermidine synthase, to reach a more complete depletion of spermidine. Under these conditions we have observed the arrest of proliferation not only in trypanosomatids with stable ODC but also in parasites with an enzyme of high turnover rate. In all cases the reinitiation of proliferation occurred only after the addition of exogenous spermidine, and neither putrescine nor spermine were able to induce the same effect.

Ornithine decarboxylase from Crithidia fasciculata is metabolically unstable and resistant to polyamine down-regulation

Ornithine decarboxylase (ODC) of Crithidia fasciculata extracts shows maximal activity during exponential growth of the parasite and decreases markedly in the stationary phase. The inhibition of protein synthesis by cycloheximide evoked a rapid loss of enzyme activity with a half‐life of about 30 min. Upon removal of DFMO from Crithidia cultures treated with the drug for 24 h, the ODC activity increased at the same rate as total protein synthesis. The addition of putrescine at high concentrations to parasites cultivated in a synthetic medium showed that Crithidia CDC levels were not reduced by polyamines.

Putrescine distribution in Escherichia coli studied in vivo by 13C nuclear magnetic resonance

In order to study the intracellular polyamine distribution in Escherichia coli, 13C-NMR spectra of [1,4-13C]putrescine were obtained after addition of the latter to intact bacteria. The 13C-enriched methylene signal underwent line broadening. When the cells were centrifuged after 90 min the cell-bound putrescine peak had a linewidth of 23 Hz, while the supernatant liquid showed an unbound putrescine signal with a linewidth smaller than 1 Hz. By using 13C-enriched internal standards it could be shown that the linewidening was not due to the heterogeneity of the medium or to an in vivo paramagnetic effect. Cell-bound putrescine was liberated by addition of trichloroacetic acid and was therefore non-covalently linked to macromolecular cell structures. Cell-bound [13C]putrescine could be displaced by addition of an excess of [12C]putrescine. When samples of membranes, soluble protein, DNA, tRNA and ribosomes from E. coli were incubated with [1,4-13C]putrescine, strong binding was detected only in the ribosomal and membrane fractions. The ribosome-putrescine complex showed properties similar to those determined with the intact cells. By measuring the nuclear Overhauser enhancements eta, it was possible to estimate that only about 50% of the polyamine was linked to the macromolecules. Determination of the T1 values of free and ribosomal-bound putrescine allowed the calculation of a correlation time, tau c = 4 X 10(-7) s for the latter. T1 and tau c values found for the ribosome-putrescine complex were those expected for a motional regime of slowly tumbling molecules.

Effect of GTP on the dissociation of 70 S ribosomes.

Zamecnik’s discovery of the GTP requirement for protein synthesis [l] was followed by a series of investigations on the role of this nucleotide both in microbial and mammalian systems. In bacterial extracts GTP is needed in several steps of the protein synthesis process: (a) The binding of N-formyhnethionyl-tRNA to the ribosomes [2,3]. (b) An interaction with the soluble transfer factors T [4,5] and subsequent binding of the aminoacyl-tRNA to the ribosomes [6-lo] . Apparently GTP is not hydrolyzed in these reactions, although there are some conflicting results on this point [6,7,9,10]. (c) The formation of a complex containing ribosomes and transfer factor G [ 1 l] , and the translocation of peptidyl-tRNA [ 121 or N-formylmethionyl-tRNA [ 131 from the aminoacyl (A) to the peptidyl (P) site on the ribosome with concomitant hydrolysis of the nucleotide into GDP and orthophosphate. This communication reports a new effect of GTP: an enhancement of the in vitro dissociation of 70 S ribosomes. The properties and physiological significance of dissociating factors from Bacillus stearothermophilus and Escherichia coli were recently investigated [ 141. The ribosomal dissociation by extracts from E. coli was previously studied by Subramanian et al. [ 151 who did not mention any effect of nucleotides on the reaction.

Polyamines and protein synthesis: Studies in various polyamine-requiring mutants ofEscherichia coli

SummaryDifferentEscherichia coli mutants auxotrophic for polyamines were studied in order to investigate the relationships among polypeptide synthesis in cell-free systems, ribosomal distribution profiles and endogenous polyamine pools. Thein vitro protein synthetic activity and the polyribosomal content were reduced in extracts from putrescine-starved cells of the double mutants MA 255 and MA 261, but not in the arginine-conditional auxotroph DK 6. Putrescine addition to the cultures of all these strains previously starved for polyamines, provoked a shift towards monomers in the equilibrium involving ribosomal particles. Concomitant changes in the intracellular levels of polyamines were observed: putrescine and spermidine increased markedly, and cadaverine disappeared.