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.     

Zur Mauser spanischer Weiden- und Haussperlinge (Passer hispaniolensis unddomesticus)

Zusammenfassung Die Mauserperiode westspanischer Weidensperlinge(Passer hispaniolensis) und Haussperlinge(P. domesticus) reicht von Ende Juli bis Ende September/Anfang Oktober. Beim Weidensperling endet der Federwechsel im Durchschnitt etwa fünf Tage früher als beim Haussperling. Es gibt keine Geschlechtsunterschiede in der Chronologie der Mauser beim Weidensperling. Ad. beider Arten mausern schneller und synchronisierter als juv., die ihr Gefieder um so rascher erneuern, je später sie mit der Mauser begonnen haben. Die Handschwingenmauser dauert etwa 66 Tage beim Weidensperling und 69 Tage beim Haussperling. Beide Arten brauchen ca. 3 weitere Tage für die Verhornung der 5. und 6. Armschwingen. Die ad. beider Arten und die juv. Weidensperlinge beginnen die Mauser im Durchschnitt am selben Tag (24. Juli), die juv. Haussperlinge später (29. Juli). Der Mauserverlauf und die Beziehungen zwischen den verschiedenen Federreihen sind bei beiden Arten identisch. Die Synchronisation der Mauser ist beim Weidensperling höher. Brut und Mauserperiode überschneiden sich beim Haussperling; beim Weidensperling, bei dem noch kurze Wanderungen gleich nach der Fortpflanzungsperiode und vor der Mauser erfolgen, nicht. Das frühere und höher synchronisierte Mauserende beim Weidensperling scheint eine Anpassung an die stärkere Zugtendenz zu sein.

---

On the moult of Spanish Sparrows(Passer hispaniolensis) and House Sparrows(Passer domesticus) in Iberia

Summary The moulting period of Spanish sparrows(Passer hispaniolensis) and House Sparrows(Passer domesticus) in Western Spain extends from late July to late September/early October. House Sparrows finish moulting on average some five days later than Spanish Sparrows. There are no sexual differences in the moulting chronology of adult Spanish Sparrows. Ad. of both species moult faster and better synchronized. The speed of moulting is also higher in later moulting juveniles. The estimated durations of wing feather replacement were 66 days for the Spanish Sparrow and 69 days for the House Sparrow. Some three more days are needed to complete the growth of the 5th and 6th secondary remiges in both species. Adults of both species and juvenile Spanish Sparrows start moulting on average on the same date: 24th July; juvenile House Sparrows start moulting on 29th July. The sequence of moult and the relations between different feather tracts are identical in both species. The synchronization of the moult is higher in the Spanish Sparrow. Breeding and moulting seasons slightly overlap in the House Sparrow, but not in the Spanish Sparrow. In this species the time lapse between both periods allows the birds to wander to suitable areas, where they moult. The earlier ending and higher synchronization of the moult in the Spanish Sparrow is related to its higher migratory tendency.

     

Restriction cleavage maps of the DNAs of Streptococcus pneumoniae bacteriophages containing protein covalently bound to their 5'' ends

Summary Several pneumococcal bacteriophages showing a morphology similar to that previously described for Cp-1 (Ronda et al. 1981) have been isolated and purified from throat samples taken from healthy children. Three of these phages (Cp-5, Cp-7 and Cp-9) have been studied in detail and compared to Cp-1. The four phages differed in several respects, e.g. size, structural polypeptides, restriction enzyme cleavage patterns, etc. The DNA of Cp-5, Cp-7 and Cp-9 showed protease-sensitive transfecting activity. This, together with the results obtained by electrophoretic analyses as well as by isotopic labelling of these DNAs with [-³²P] ATP and polynucleotide kinase indicated that all these new phages have a protein covalently linked to the 5 ends of their DNAs as in the case of Cp-1 (García et al. 1983). Restriction enzyme cleavage maps of Cp-1, Cp-5, Cp-7 and Cp-9 have been constructed.     

Biochemical properties of alcohol dehydrogenase from Drosophila lebanonensis.

Purified Drosophila lebanonensis alcohol dehydrogenase (Adh) revealed one enzymically active zone in starch gel electrophoresis at pH 8.5. This zone was located on the cathode side of the origin. Incubation of D. lebanonensis Adh with NAD+ and acetone altered the electrophoretic pattern to more anodal migrating zones. D. lebanonensis Adh has an Mr of 56,000, a subunit of Mr of 28 000 and is a dimer with two active sites per enzyme molecule. This agrees with a polypeptide chain of 247 residues. Metal analysis by plasma emission spectroscopy indicated that this insect alcohol dehydrogenase is not a metalloenzyme. In studies of the substrate specificity and stereospecificity, D. lebanonensis Adh was more active with secondary than with primary alcohols. Both alkyl groups in the secondary alcohols interacted hydrophobically with the alcohol binding region of the active site. The catalytic centre activity for propan-2-ol was 7.4 s-1 and the maximum velocity of most secondary alcohols was approximately the same and indicative of rate-limiting enzyme-coenzyme dissociation. For primary alcohols the maximum velocity varied and was much lower than for secondary alcohols. The catalytic centre activity for ethanol was 2.4 s-1. With [2H6]ethanol a primary kinetic 2H isotope effect of 2.8 indicated that the interconversion of the ternary complexes was rate-limiting. Pyrazole was an ethanol-competitive inhibitor of the enzyme. The difference spectra of the enzyme-NAD+-pyrazole complex gave an absorption peak at 305 nm with epsilon 305 14.5 X 10(3) M-1 X cm-1. Concentrations and amounts of active enzyme can thus be determined. A kinetic rate assay to determine the concentration of enzyme active sites is also presented. This has been developed from active site concentrations established by titration at 305 nm of the enzyme and pyrazole with NAD+. In contrast with the amino acid composition, which indicated that D. lebanonensis Adh and the D. melanogaster alleloenzymes were not closely related, the enzymological studies showed that their active sites were similar although differing markedly from those of zinc alcohol dehydrogenases.     

In vitro modification of Candida albicans invasiveness

Candida albicans produces germ-tubes (GT) when it is incubated in animal or human serum. This dimorphism is responsible for its invasive ability.The purpose of the present paper is (1) to evaluate the ability of rat peritoneal macrophages to inhibit GT production of ingested Candida albicans, obtained from immunized rats and then activated in vitro with Candida-induced lymphokines; (2) to determinate any possible alteration of phagocytic and candidacidal activities.The phagocytes were obtained from rats immunized with viable C. albicans. Some of them were exposed to Candida-induced lymphokines in order to activate the macrophages in vitro. The monolayers of activated, immune and normal macrophages were infected with a C. albicans suspension during 4 hr.Activated macrophages presented not only the highest phagocytic and candidacidal activities but a noticeable inhibition of GT formation and incremented candidacidal activity.     

Sensory projections to the nucleus basalis prosencephali of the pigeon

Summary The afferent pathways to the nucleus basalis prosencephali of the pigeon were studied by use of the horseradish peroxidase (HRP) technique. It was confirmed that this nucleus receives a direct pathway from the nucleus sensorius principalis nervi trigemini and that, as in the starling, it receives a direct input from the nucleus lemnisci lateralis, pars ventralis, an auditory relay. Totally novel is the finding that the nucleus basalis prosencephali is the target of a direct pathway originating in the medullary nucleus vestibularis superior. All three pathways bypass the thalamus. From within the telencephalon the nucleus basalis prosencephali also receives fibres from the tuberculum olfactorium and the peri-ectostriatal belt, suggestive of olfactory and visual input. Marked cell bodies were also found in the neostriatum frontolaterale. It is assumed that these arose from HRP uptake by axons of the tractus fronto-archistriatalis that course through the nucleus basalis prosencephali to the anterodorsal archistriatum. Marked fibres and bouton-like formations were observed in the latter structure. The afferents to the nucleus basalis prosencephali are discussed in conjunction with the probable role of the nucleus as a sensorimotor coordinator of the pecking/feeding behaviour of the pigeon.     

Monoclonal antibodies to neurospora adenylate cyclase

A monoclonal antibody against $\text{Neurospora}$ soluble adenylate cyclase was obtained. The antibody inhibits cyclase activities from several lower eucaryotic organisms but not activities associated to testicular cytosol or turkey erythrocyte membranes.     

NADPH/NADP+ ratio: regulatory implications in yeast glyoxylic acid cycle

Summary The utilization by yeast of two carbon sources is carried out through the operation of the glyoxylic acid cycle. Kinetic data from the isocitrate transforming enzymes suggest that the flow of isocitrate through the glyoxylic acid cycle depends upon the inhibition of the isocitrate decarboxylating enzymes. Both isocitrate dehydrogenases are inhibited by a mixture of glyoxylate + oxaloacetate, but for the reasons described in the text we consider that this inhibition is of no physiological significance. On the other hand, we have found that NADPH is a competitive inhibitor of NADP-isocitrate dehydrogenase with respect to NADP⁺, with a K_I similar to its K_M. It also produces an additive effect on the NADH-produced inhibition of NAD-isocitrate dehydrogenase. We propose NADPH as the compound that channels the utilization of isocitrate into the glyoxylic acid cycle. This is supported by the finding of an increased NADPH/NADP⁺ ratio in acetate grown yeast with respect to glucose grown cells.