Sara H. Goldemberg

Biosynthesis of glycogen from uridine diphosphate glucose

Abstract An enzyme which leads to the formation of glycogen according to the equation: UDPG + primer → UDP + glucosyl α (1 → 4) primer has been studied. The optimal conditions for activity were determined with a partially purified preparation from rat muscle. The reaction requires the presence of a polysaccharide as primer and is strongly activated by hexose 6-phosphates. Using UDPG labeled in the glucose moiety, it was found that the radioactivity was transferred to the glycogen from which it could be removed as maltose with β-amylase or as G-1-P with phosphorylase. Thus it seems that the glucose residue becomes linked α (1 → 4) to the polysaccharide. Several inhibitors were tested as well as the occurrence of the enzyme in different organs.

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.

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.

Translation of natural mRNA in cell-free systems from a polyamine-requiring mutant of Escherichia coli.

Abstract Cell-free extracts of polyamine-deficient and supplemented bacteria were used to study the translation of MS 2 bacteriophage RNA. The decreased synthetic activity observed with preparations of starved cells depends on a deficient initiation of polypeptide synthesis, as indicated by a lower capacity for the binding of formyl-methionyl-tRNA to ribosomes, induced by the trinucleotide AUG. The analysis of polypeptides formed showed that extracts from polyamine-depleted and supplemented cells are both able to synthesize complete protein chains.

Initiation, elongation and termination of polypeptide synthesis in cell-free systems from polyamine-deficient bacteria

Abstract Protein synthesis in a polyamine-auxotrophic mutant of E. coli cultivated in the absence and presence of putrescine has been studied. The ratio of in vivo polypeptide synthesis under both conditions was the same as that obtained with cell-free extracts derived from polyamine-depleted and supplemented bacteria. The markedly reduced activity in systems from polyamine starved cells is most probably due to a poorly efficient initiation step. The estimation of nascent peptide chains and their release from tRNA support the idea that polypeptide elongation and termination processes do not have decreased rates in systems from polyamine-depleted bacteria.

Altered heat-shock response in polyamine-depleted bacteria

A polyamine‐auxotrophic mutant of E. coli was cultivated in the presence or absence of putrescine and submitted to heat shock over 3 different ranges of temperature. In all cases, protein synthetic capacity measured in comparison to that of cultures at the preshift temperature was much higher in polyamine‐depleted bacteria under thermic stress. Addition of putrescine only before the shift‐up was able to restore gradually normal control of the relative protein synthetic capacity.

Glucan biosynthesis in Bacillus stearothermophilus: I. Properties of the polysaccharide

Abstract A glucan which contains α-1,4 and presumably α-1,6 bonds has been isolated from a spontaneous variant of Bacillus stearothermophilus 1503-4R by trichloroacetic acid extraction and precipitation with ethanol. It resembles glycogen in solubility, characteristics of the iodine complex and internal chain length. It has longer average and external chains and also different electrophoretic mobility on glass fiber paper. The product synthesized in vitro in the presence of acceptor and ADP-glucose, by cell-free extracts containing glucan synthetase, has elongated external chains. The newly added glucosyl residues are α-1,4 linked. The glucan accumulates in stationary phase cells. No polysaccharide nor synthetic activity were detected in wild-type cells.