Analysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite

After addition of 5 mM sulfite or nitrite to glucose-metabolizing cells of Saccharomyces cerevisiae a rapid decrease of the ATP content and an inversely proportional increase in the level of inorganic phosphate was observed. The concentration of ADP shows only small and transient changes. Cells of the yeast mutant pet 936, lacking mitochondrial F₁ATPase, after addition of 5 mM sulfite or nitrite exhibit changes in ATP, ADP and inorganic phosphate very similar to those observed in wild type cells. They key enzyme of glucose degradation, glyceraldehyde-3-phosphate dehydrogenase was previously shown to be the most sulfiteor nitrite-sensitive enzyme of the glycolytic pathway. This enzyme shows the same sensitivity to sulfite or nitrite in cells of the mutant pet 936 as in wild type cells. It is concluded that the effects of sulfite or nitrite on ATP, ADP and inorganic phosphate are the result of inhibition of glyceraldehyde-3-phosphate dehydrogenase and not of inhibition of phosphorylation processes in the mitochondria. Levels of GTP, UTP and CTP show parallel changes to ATP. This is explained by the presence of very active nucleoside monophosphate kinases which cause a rapid exchange between the nucleoside phosphates. The effects of the sudden inhibition of glucose degradation by sulfite or nitrite on levels of ATP, ADP and inorganic phosphate are discussed in terms of the theory of Lynen (1942) on compensating phosphorylation and dephosphorylation in steady state glucose metabolizing yeast.Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - P_i inorganic orthophosphate Dedicated to Prof. Dr. Hans Grisebach on the occasion of his sixtieth birthday     

Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90°C

A novel type of bacterium has been isolated from various geothermally heated locales on the sea floor. The organisms are strictly anaerobic, rod-shaped, fermentative, extremely thermophilic and grow between 55 and 90°C with an optimum of around 80°C. Cells show a unique sheath-like structure and monotrichous flagellation. By 16S rRNA sequencing they clearly belong to the eubacteria, although no close relationship to any known group could be detected. The majority of their lipids appear to be unique in structure among the eubacteria. Isolate MSB8 is described as Thermotoga maritima, representing the new genus Thermotoga.Dedicated to Otto Kandler on the occasion of his 65th birthday Present addresses: University of South Dakota, Vermillion, USA; University of Illinois, Urbana, USA; Universität für Bodenkultur, Wien, Austria     

Cytochrome c oxidase. Time dependence of optical and EPR spectral changes related to the ‘oxygen-pulsed’ form

Time-dependent changes in the optical spectrum (450–920 nm) of cytochrome c oxidase, following oxidation with oxygen of the stoichiometrically reduced form, have been investigated and where possible, attempts have been made to correlate our observations with variations in the EPR spectrum over a parallel time course at 2°C. In this regard, particular emphasis has been placed on establishing absorption features related to the presence of EPR resonances at g 5, 1.78 and 1.69, which have been tentatively assigned to a spin-coupled state involving cytochrome a₃ and ‘EPR-undetectable Cu’ (Beinert, H., Shaw, R.W., Dunham, R.W. and Sands, R.H. (1982) in Oxidases and Related Redox Systems (King, T.E., Mason, H.S. and Morrison, M., eds.), Pergamon Press, Oxford, in the press). For optical studies we have used a versatile rapid-scanning spectrophotometer to obtain well resolved spectra down to 2 ms reaction time. Concomitant with the appearance (within 10 ms) of EPR signals at g 5, 1.78 and 1.69 is the presence of an enhanced absorption (Δε = 0.25 mM (heme a)^?1·cm^?1) at 660 nm, with a trough (relative to following spectra) at 580 nm. In our hands, this feature disappears in a first-order process with a half-life of 46 s at pH 7.2 and 2°C. The effect of this spectral transformation is to decrease considerably the acuteness of the 655 nm absorption band, previously suggested as representing a state of the enzyme in which ferric cytochrome a₃ is coupled to oxidised EPR-undetectable Cu (Beinert, H., Hansen, R.E. and Hartzell, C.R. (1976) Biochim. Biophys. Acta 423, 339–355). This observation can be correlated satisfactorily with a small field shift of the high-field resonances at g 1.78 and 1.69 and a broadening at g 1.78. Support for this and further correlative assignments arises from parallel experiments using cytochrome c oxidase purified via an alternative procedure, which displays different kinetic behavior. Further transformations of the oxidized enzyme are evident through an approx. 10% decrease in absorbance at 600 nm together with small changes centered at 640 and 665 nm (which serve to restore the sharpness of the 655 nm band). The kinetics, as analyzed by the Guggenheim procedure using the absorbance at 597 nm, indicate approx. 50% first-order linearity (half-life 40 min) with additional species contributing at longer times, while over a parallel time course (0–3 h) the EPR resonances at g 5, 1.78 and 1.69 virtually disappear. These novel signals can also be seen at a lower intensity in samples of cytochrome c oxidase anaerobically reoxidized by porphyrexide and frozen after a 6 min incubation period at 4°C. This observation, along with the establishment of similar optical changes over the time course of 1 min to 3 h, suggests that aerobic and anaerobic reoxidation produce common forms of the enzyme. Comparison of the g 1.78 and 1.69 resonances between samples rapidly aerobically reoxidized in the presence of H₂¹⁶O and H₂¹⁷O yielded no evidence for the presence of any labile oxygen ligand (including OH^?, H₂O) in the coordination sphere of the species involved.     

Zur gegenwärtigen problematik der evolutionsforschung

Reflections on the actual problematics of the palaeontological evolution research are observed under epistemological aspects. When discussing new or only pretended new beginnings, sometimes with claim to neutral-point elucidations, success and merit of the traditional methods in palaeontology should not be forgotten. Gradualism and punctualism reduce the multilateral reality of nature.     

Amino acid sequence of the active site of human pseudocholinesterase

The usualE ₁ ^u and atypicalE ₁ ^a human pseudocholinesterases (acylocholine acylhydrolase, EC 3.1.1.8) were purified to homogeneity. The active-site serine residue was conjugated with diisopropyl fluorophosphate and digested with trypsin. The tryptic peptide containing the active site was isolated by gel filtration followed by two-dimensional paper chromatography and electrophoresis. The amino acid sequence of the active site peptide obtained from the usualE ₁ ^u enzyme was found to be Gly-Glu-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu. A remarkable structural homology exists between the human and the horse enzymes in their active sites. From the difference in electrophoretic mobility of the active-site peptides obtained from the usual and atypical enzymes, the probable structure of the atypical human enzyme was deduced as Gly-His-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu.     

Elektron-Spin-Resonanz-Untersuchungen der Reaktionskinetik strahleninduzierter freier Radikale des Cholesterins

Zusammenfassung Die Reaktionskinetik strahleninduzierter freier Radikale des Cholesterins wurde in flüssiger Phase bei Raumtemperatur mittels ESR-Spektroskopie untersucht. Mit Hilfe eines geeigneten photochemischen Initiationssystems ließen sich in Cyclohexanlösung unter UV-Bestrahlung (235 nm265 nm) genau dieselben freien Radikale des Cholesterins darstellen, die schon früher [9, 7] in röntgenbestrahltem Cholesterinpulver beobachtet worden waren. Bei ausreichendem O₂-Partialdruck (3·10⁴Torr) über der Probenlösung trat das ESR-Spektrum eines Peroxyradikals auf, das mittels der Analyse seiner Reaktionsprodukte (7-Hydroxy-Cholesterin und 7-Keto-Cholesterin) mit dem Cholesteryl-7-peroxyradikal identifiziert wurde. Die Kinetik sowohl der Bildung als auch des Zerfalls des Radikals entsprachen einer Reaktion von 2. Ordnung. Die Geschwindigkeitskonstante für den bimolekularen Zerfall, eine Disproportionierung in Alkohol und Keton unter Abgabe eines Moleküls O₂, wurde bei Raumtemperatur zuk ₂=(1,8 _–0,6 ^+0,9 )·10⁶ sec^–1M^–1·l bestimmt. Ferner wurde gezeigt, daß das Cholesteryl-7-peroxyradikal aus dem freien Radikal Cholesteryl-7 durch Anlagerung eines Moleküls O₂ entsteht. Für die Geschwindigkeitskonstante dieser Reaktion ergab sich eine untere Schranke vonk ₁=0,40·10¹⁰ sec^–1M^–1·l.

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Electron spin resonance investigations on radiation-induced free radicals of cholesterol in liquid phase

Summary The reaction kinetics of radiation-induced free radicals of cholesterol was studied in liquid phase at room temperature by means of e.s.r. spectroscopy on a solution of cholesterol in cyclohexane. Using a convenient photochemical initiation system, just those free radicals of cholesterol could be generated by the filtered u.v. radiation from a Xe high pressure lamp (235 nm265 nm) as were observed already a decade ago by Gordy [9] and by Ehrenberg, Löfroth [7] in X-irradiated cholesterol powder. At sufficiently high O₂-pressures (3·10^–4 Torr) over the sample solution a peroxy radical e.s.r. spectrum arose during u.v. irradiation which was identified by product analysis (7-hydroxy-cholesterol and 7-keto-cholesterol) to be dueto a cholesteryl-7-peroxyradical. The radical'sgeneration and decay kinetics was governed by a second order reaction. The velocity constant for bimolecular decay of the cholesteryl-7-peroxyradical was found to be k₂=(1.8 _–0,6 ^+0,9 )·10⁶sec^–1M^–1·l at room temperature. Furthermore it could be shown that the cholesteryl-7-peroxyradical was built up by the addition of one molecule of O₂ to a cholesteryl-7 free radical. For this reaction a value ofk ₁=0.4·10¹⁰ sec^–1 M^–1·l was estimated as a lower limit of the velocity constant.

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Die Arbeit stellt einen Auszug aus einer Dissertation an der Technischen Hochschule München dar.     

Book reviews

Ohne Zusammenfassung

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Book reviews

     

Bukettförmige Strukturen im Ependym der Regio hypothalamica des III. Ventrikels beim Kaninchen

Zusammenfassung Die Wand des III. Ventrikels (Regio hypothalamica) vom erwachsenen Kaninchen wurde elektronenmikroskopisch untersucht. — Unterschiedliche keulenförmige portsätze, deren Perikaryen in nahen und entfernten Regionen liegen, dringen in den Ventrikel ein. Sie heben eine Ependymzelle ab und bilden eine bukettförmige Struktur. Einige Fortsätze enthalten eine spärliche feingranulierte Masse, andere Bläschen (600–1500 Å) oder Bläschen mit dichtem Kern (450–1200 Å). Inmitten des Buketts kann eine Nervenzelle liegen, die einen dicken Fortsatz gegen das Hypendym schickt. Unterhalb des Buketts findet man eine andere Nervenzelle, die Fortsätze ins Bukett sendet. Zahlreiche in das Bukett eintretende Fortsätze stammen aus entfernteren Kerngebieten. Die funktionelle Bedeutung der Strukturen (Neurosekretion ? Resorption ? Rezeptor ?) wird diskutiert.

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Bouquet-like ependymal structures in the hypothalamic region of the 3rd Ventricle (rabbit)The question of neurosecretion or receptor function

Summary The wall of the III. ventricle (Regio hypothalamica) of the adult rabbit has been investigated electron microscopically. —Various bulblike processes of nerve cells, having their perikarya in close or distant regions, penetrate into the ventricle. They lift an ependymal cell and form a bouquet-like structure. Some of the processes contain a sparse granulated material and others include vesicles (600–1500 Å) or dense-cored vesicles (450–1200 Å). In the middle of the bouquet a perikaryon may be seen extending a thick process towards the hypendyma. Below the bouquet another perikaryon may be found extending several processes into the bouquet. The functional significance of these structures (neurosecretion ? resorption? receptor?) is discussed.

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Die Untersuchung wurde mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft durchgeführt.