Bernard D. Davis

Polysomes Extracted from Escherichia coli by Freeze-Thaw-Lysozyme Lysis

Polysomes can be extracted from Escherichia coli by freezing and thawing in the presence of lysozyme, followed by treatment with sodium deoxycholate. The method is simple and convenient; the yields consistently high.

On the importance of being ionized

Abstract A survey of metabolic reactions discloses that all the known lowmolecular weight and water-soluble biosynthetic intermediates possess groups that are essentially completely ionized at neutral pH. In some sequences (e.g., glycolysis, the pentose phosphate pathway, most of the histidine pathway, and purine biosynthesis) the components are all phosphorylated and usually not carboxylated. In many other sequences (e.g., the tricarboxylic acid cycle pyrimidine biosynthesis, most amino acid pathways) the components are carboxylated and usually not phosphorylated. In a few instances (histidinol, spermine) there is an ionizable basic but not an acidic group. Uncharged metabolites are found only as excretory and fermentation products, and occasionally as intermediates in purely degradative reactions (e.g., acetaldehyde in alcoholic fermentation). This generalization should have value in guiding investigation of undisclosed biosynthetic reactions. More important is the problem of determining the principle of cell physiology that lends such evolutionary advantage to ionized biosynthetic intermediates. One possibility is the more efficient retention of ionized compounds within the cell or its organelles.

Significance of the free 70 s ribosomes in Escherichia coli extracts.

Abstract We have confirmed the report (Mangiarotti & Schlessinger, 1966) that a new method of lysing Escherichia coli cells yields polysomes and ribosomal subunits but no 70 s ribosomes. However, two features of that method are shown to promote ribosome dissociation: growth in hypertonic medium, and the use of an excess of deoxycholate (which precipitates Mg 2+ ). When the cells are lysed by a freeze-thaw-lysozyme method, with a lower deoxycholate concentration, a substantial 70 s peak is seen along with polysomes and subunits. Moreover, when the products of polysome run-off are caused to accumulate in the cells by treatment with actinomycin D or with puromycin, or by starvation for the carbon source or for a required amino acid, they appear only as 70 s particles. Finally, treatment of complexed 70 s particles (fragmented polysomes) with puromycin in vitro does not cause dissociation into subunits, though the nascent polypeptide is removed. We conclude that (a) polysomes release their run-off ribosomes as stable, free 70 s units, and (b) the dissociation of these units in the cell is subject to a physiological control which maintains an essentially constant pool of subunits. These findings, together with recent work on initiation factors, suggest that dissociation of ribosomes depends on complexing with a factor that is present in the cell in limited supply.

Inhibition of Protein Synthesis by Spectinomycin

Spectinomycin selectively inhibits protein synthesis in cells and in extracts of Escherichia coli. Mutations to high-level resistance to this antibiotic map close to the streptomycin locus, and the site of action of spectinomycin, like that of streptomycin, is the 30S ribosomal subunit, as shown by experiments with reconstituted 70S ribosomes containing subunits from sensitive and from resistant ribosomes. In contrast to streptomycin, however, spectinomycin is not bactericidal and causes no detectable misreading of polyribonucleotides.

Magnesium ion dependence of free and polysomal ribosomes from Escherichia coli

Abstract This paper presents a simple means of distinguishing two kinds of 70 s ribosomes from Escherichia coli: intact complexes with mRNA and peptidyl-tRNA, obtained by ribonuclease treatment of polysomes; and free ribosomes, relieved of the polypeptide (and presumably of tRNA and mRNA) by treatment with puromycin in vitro. Free ribosomes are completely dissociated into subunits at 1 to 2 m m -Mg2+, while preparations containing polysomal ribosomes are only partly dissociated. Evidence obtained by the application of this criterion supports the conclusion, presented in the preceding paper, that the cell normally contains undissociated free ribosomes, and that their concentration increases when the products of polysomes runoff are caused to accumulate.

The sequence of some effects of streptomycin in Escherichia coli

The effects of streptomycin on a number of biochemical parameters in growing cultures of Escherichia coli have been studied. Using low concentrations of drug and radioactive tracer techniques we have timed these effects relative to each other and to the killing action of the drug. The earlier effects noted were: first, an immediate (primary) uptake of streptomycin; next, an acceleration of K efflux and a transient stimulation of net RNA synthesis; and then, inhibition of protein synthesis and loss of viability. Later effects were: a further (secondary) uptake of streptomycin, increased permeability to a β-galactoside, impairment of respiration, inhibition of RNA and DNA synthesis and RNA breakdown and nucleotide excretion. The present findings cannot be integrated into a definitive theory on the action of streptomycin, but they do emphasize the importance of effects on membrane integrity and on the synthesis of RNA and protein.

Aromatic biosynthesis: XII. Conversion of 5-dehydroquinic acid to 5-dehydroshikimic acid by 5-dehydroquinase☆

5-Dehydroquinase, an enzyme that catalyzes the conversion of 5-dehydroquinic acid to 5-dehydroshikimic acid through removal of a molecule of water, has been extracted from E. coli and partially purified. The reaction apparently involves a single enzyme and has no demonstrable cofactor requirement. The activity of the enzyme is readily measured by virtue of the absorption peak of 5-dehydroshikimic acid at 234 mμ. The reaction is reversible, with an equilibrium constant of 15. Certain properties of the enzyme are described.

Aromatic biosynthesis. XIII. Conversion of quinic acid to 5-dehydroquinic acid by quinic dehydrogenase.

Abstract Quinic dehydrogenase, which catalyzes the conversion of quinic acid to 5-dehydroquinic acid, has been extracted from cells of an Aerobacter mutant and partially purified. The enzyme is DPN-specific. With initial extracts TPN also promotes the reactin, but its activity has been shown to be due to coupling with DPN by pyridine nucleotide transhydrogenase. Quinic dehydrogenase activity is conveniently measured by spectrophotometric determination of DPNH (using cyanide to inactivate DPNH oxidase). However, initial extracts also contain the necessary enzymes for further converting 5-dehydroquinic acid to 5-dehydroshikimic acid, and for reducing the latter compound (with TPNH) to shikimic acid. The coupling of this last reaction (via transhydrogenase) reoxidizes the DPNH formed by quinic dehydrogenase, and hence interferes with the spectrophotometric assay. This interference can be eliminated by removing TPN through pretreatment of the extract with charcoal, or by conventional methods of enzyme purification. Alternatively, enzyme activity can be determined by a bioassay method which is not affected by the coupled reaction. Certain properties of quinic dehydrogenase are described. No cofactor requirement (other than DPN) could be detected. In the series of coupled reactions, the overall equilibrium constant for shikimic/quinic is approximately i.o. The limited distribution of quinic dehydrogenase, compared with the broader distribution of known enzymes of aromatic biosynthesis, supports the conclusion that quinic acid is probably not an intermediate in this biosynthetic sequence.

Synergism between Streptomycin and Penicillin: a Proposed Mechanism.

Brief treatment of growing Escherichia coli with penicillin hastened subsequent killing of these cells by streptomycin. It also hastened the secondary uptake of streptomycin, which represents an increase in the number of freely accessible binding sites. In contrast, brief treatment with streptomycin failed to affect subsequent killing by penicillin. These findings suggest that the synergism of penicillin with streptomycin depends on the damaging effect of penicillin on the cell membrane, which promotes further damage by streptomycin and increases its subsequent access to intracellular sites. Observations on a streptomycin-resistant mutant are also reported.

Effect of spectinomycin on polypeptide synthesis in extracts of Escherichia coli

Abstract Spcotinomycin, a dibasic aminoglycoside antibiotic, inhibits protein, synthesis reversibly on Escherichia coli ribosomes. Its effect depends strikingly on the nucleotide composition of the messenger: inhibition is completely absent with poly U or poly A; it becomes increasingly pronounced with increasing C or G content in mixed polymers; and it is almost complete with poly I. The degree of inhibition becomes maximal within a relatively narrow range of increasing Spc§ concentration. Moreover, one-step mutants with a highly resistant 30 s ribosome subunit are readily isolated. These properties suggest that the drug acts on only a single site on this subunit. In contrast to streptomycin, however, which also acts on the 30 s subunit, Spc does not cause detectable misreading in translation, and its action in vitro is independent of Mg2+ concentration over a wide range. Spc does not appear to influence directly the initiation, recognition, peptideforming, or termination steps in polypeptide synthesis. Moreover, when ribosomes are preloaded with peptidyl-tRNA Spc does not affect amino acid transfer for the first few minutes after its addition; and evidence is presented that this lag depends on having the ribosomes reach the susceptible state, rather than on a slow interaction of Spc with some component of the system. These findings suggest that Spc blocks some aspect of the complex translocation process in the cycle of amino acid transfer.