Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.     

Incorporation of [3H]thymidine into DNA and of [35S]sulfate into sulfatides of oligodendroglial cells during development: Effect of malnutrition

Incorporation of [³H]thymidine into DNA and of [³⁵S]sulfate into sulfatides of oligodendroglial cells isolated from brain slices incubated with the radioactive precursor was studied in normal and malnourished rats at different ages. The pattern and the values of incorporation of [³H]thymidine into DNA were similar in both groups of animals. The maximum value of incorporation was observed at 7 days of age decreasing rapidly thereafter and leveling off between 18–21 days. In both groups of animals labeling of sulfatides attained a maximum at 18 days of age, showing similar values of incorporation up to that age. However, at 21 days of age; the values corresponding to malnourished rats were found to be 40% lower in comparison to controls. The results suggest that (a) proliferation of oligodendroglial cells stops at similar ages in normal and malnourished rats, (b) expression of sulfatide synthesis by oligodendroglial cells is similar in both groups of animals up to 18 days, and (c) the starved rats seem to be unable to maintain normal synthesis of these galactolipids throughout the entire period of active myelinogenesis.     

Neonatal malnutrition in the rat affects the delivery of sulfatides from microsomes and their entry into myelin

Brain slices from 18 day old normal and malnourished rats were incubated in the presence of [³⁵S]sulfate to explore its incorporation into sulfatides of a total brain homogenate and the appearance of labeled sulfatides in different subcellular fractions. While the incorporation of label into sulfatides of the total homogenate was similar in both groups of animals, in subcellular fractions separated on a linear sucrose density gradient, labeling of sulfatides in malnourished animals was relatively higher in the region corresponding to the microsomal fraction. Time course incorporation and pulse-chase experiments were carried out to explore the kinetics of labeling of microsomal and myelin sulfatides. In pulse-chase experiments, normal controls showed a decrease in the specific radioactivity of sulfatides in the microsomal fraction after the chase, which was not observed in malnourished animals, while the appearance of labeled sulfatides in the myelin fraction of the latter group of animals was found to be lower than in normals. These results suggest that in neonatal malnutrition there is a defect in the transport of de novo synthesized sulfatides towards myelin or/and a problem in the assembly of these lipids into the myelin membrane.     

Variants within the yeast Ty sequence family encode a class of structurally conserved proteins.

The Ty transposable elements of Saccharomyces cerevisiae form a heterogeneous family within which two broad structural classes (I and II) exist. The two classes differ by two large substitutions and many restriction sites. We show that, like class I elements a class II element, Tyl-17, also appears to contain at least two major protein coding regions, designated TYA and TYB, and the organisational relationship of these regions has been conserved. The TYA genes of both classes encode proteins, designated p1 proteins, with an approximate molecular weight of 50 Kd and, despite considerable variation between the TYA regions at the DNA level, the structures of these proteins are remarkably similar. These observations strongly suggest that the p1 proteins of Ty elements are functionally significant and that they have been subject to selection.     

3H-uridine labelling patterns and chromosomal polymorphism inDrosophila subobscura: J and U chromosomes

The³H-uridine labelling patterns in J and U polytene chromosomes ofDrosophila subobscura were determined. The analysis was carried out in two developmental stages and in two strains proceeding from the same geographical origin whose genotypes were: J_st/J_st; U₁₊₂/U₁₊₂ and J₁/J₁; U₁₊₂₊₈/U₁₊₂₊₈ respectively. It was observed that the labelling pattern coincided very approximately with the puffing pattern in the same stages and chromosomal arrangements. Comparison of the³H-Uridine incorporation patterns between chromosomal arrangements showed light quantitative differences. These results are discussed in relation to the inversion effect.     

The thermodynamics of complex formation of cyclic tetra-aza-tetracetic acids

Enthalpy changes for the complexation of alkaline-earth and transition metals with three cyclic tetra-aza-tetracetic acids (cDOTA, cTRITA and cTETA) were obtained by continuous titration calorimetry. From these values and free energy data, the entropy changes for the same reactions were derived. The results show that these complexes are stabilised by both favourable enthalpy and entropy changes, except those of Mg²⁺ and those of Sr²⁺ and Ba²⁺ with cTETA. Generally, the entropy changes for the reactions of the alkaline-earth metals are higher than for the reactions of the non cyclic polyaminocarboxylic acids, but for the reactions of the transition metals the entropy changes are comparable for the cyclic and non cyclic ligands. These results are discussed in terms of a model of ‘cage’ coordination of the metals.The enthalpy changes decrease with the increase in size of the tetra-aza ring (except in the case of Cu²⁺) but no specific cavity size effect is noticeable. Consideration of the temperature-dependent and temperature-independent contributions to ΔH supports the idea that the number of coordinated nitrogen atoms and carboxylate groups vary along the series.     

High density of transmembrane glycoproteins on the flagellar surface of boar sperm cells

Membrane halves of boar sperm flagella were produced by freeze-fracture and labeled in situ with concanavalin A and wheat germ agglutinin; the lectins were visualized with protein-gold complexes. Concanavalin A and wheat germ agglutinin binding sites partition with both protoplasmic and exoplasmic halves of the membrane. A high density of lectin marking was found on protoplasmic membrane halves; we conclude that the label corresponds to transmembrane glycoproteins that, on freeze-fracture, are dragged across the outer (exoplasmic) half of the phospholipid bilayer. Our demonstration of numerous transmembrane proteins in sperm flagella offers the structural setting for previous models on flagellar surface motility that postulate accessibility of motile membrane components to the submembranous cytoskeleton.