Nucleotide pools of growing,synchronized and stressed cultures of Saccharomyces cerevisiae

High pressure liquid chromatography has been used to study the acid soluble nucleotide pool of Saccharomyces cerevisiae under different conditions of growth. ATP, ADP, AMP, NAD, GTP, UTP, UDP, CTP, CDP, and UDP-sugars plus UMP could be separated and were found in concentrations higher than 0.1 mumol per g yeast cell dry weight (= detection limit). During glucose-limited continuous culture the levels of individual nucleotides depended on the growth rate, which was most pronounced with pyrimidine (uridine, cytidine) nucleotides. The energy charge (E.C.) remained high (0.9) at all growth rates (0.07-0.3 h-1). During synchronized growth at a constant growth rate (0.11 h-1) almost all nucleotide levels and the E.C. remained at constant values with the only exception of UDP-sugars and UMP of which increased levels were found during the phase of budding. Under conditions of metabolic stress (addition of antimycin A, deoxyglucose plus iodoacetate) pronounced changes in the levels of purine (adenine and guanine) nucleotides and the E.C. were observed. All other nucleotides were less influenced by these conditions. Only under these conditions IMP accumulation was observed. The results strongly argue against the significance of purine nucleotide or E.C. measurements under viable conditions. In contrast, changes in the levels of pyrimidine nucleotides seem to be indicative of changes in the flux through the metabolic pathways where they act as coenzymes.     

Electron-paramagnetic-resonance studies on cobalt(II) carbonic anhydrase–sulphonamide complexes

Sulphonamide adducts of three Co(II) carbonic anhydrases were investigated by e.p.r. (electron paramagnetic resonance) at helium temperatures. The highly anisotropic 9 GHz spectra exhibited only three distinct features, with g values between 6.3 and 1.5. Such spectra arise from an electronic state with effective spin S'=(1/2), indicating that the high-spin (S=3/2) ground level is split into two spin doublets differing in energy by an amount large compared with the microwave quantum, but small in relation to thermal energies at ambient temperature. This situation would occur in a tetrahedral system suffering a large rhombic distortion. Calculations based on this model accounted for apparent discrepancies in integrated spectral intensities, and yielded magnetic moments in good agreement with independent measurements, especially in the case of certain small Co(II) complexes resembling the enzyme adducts in their e.p.r. signals. Precise sets of g values, reflecting a particular co-ordination geometry, were found to be representative of each enzyme variant and the type of sulphonamide inhibitor, whether benzocyclic or heterocyclic. A series of substituted benzene sulphonamides bound to the same enzyme gave rise to closely similar spectra despite a wide range of pK(i) values. Thus benzocyclic and heterocyclic sulphonamides were evidently held in the active-site cleft in characteristic orientations irrespective of side chains that might considerably influence the total binding strength. Visible absorption spectra of various sulphonamide adducts at room temperature showed a similar pattern of inhibitor dependence to the e.p.r. spectra, suggesting a correspondence between the co-ordination structures in liquid and frozen solution. E.p.r. spectra of the sulphonamide complexes were remarkable not only for their range of g values, but also for their variations in line-width and spin-lattice relaxation behaviour. Addition of glycerol to the medium produced marked enhancement in resolution, owing to the creation of a more homogeneous frozen matrix. The non-uniform spin relaxation was probably a consequence of the large anisotropy in effective g tensor.     

Effects of potassium,lanthanum and black widow spider venom on miniature synaptic potentials in theTorpedo electroplax

Summary Using a method of focal drug application it is demonstrated that high potassium concentration, lanthanum, and black widow spider venom accelerate spontaneous transmitter release inTorpedo electric tissue.This investigation was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 138). Thanks are due to Dr. R. Martin and the staff of the Stazione Zoologica, Naples, for supplyingTorpedo, and Dr. N. Frontali, Rome, for a gift of frozen black widow spiders.     

The effect of energization on the apparent Michaelis–Menten constant for oxyge in nmitochondrial respiration

Lineweaver-Burk plots of 1/v against 1/[O(2)] for rat liver mitochondrial respiration with succinate or ascorbate+NNN'N'-tetramethyl-p-phenylenediamine as substrates are non-linear. In state 3u (uncoupled by trifluoromethoxycarbonyl cyanide phenylhydrazone) such plots tend to be concave upward, whereas in state 4 (energized) the plots were concave downward. The apparent K(m) for oxygen is larger in state 4 than in state 3u, despite the higher turnover in the latter system. It is postulated that at least one reversible reaction occurs between cytochrome c and cytochrome c oxidase, whose rate is increased on energization (reversed electron transfer); a model including such a reaction is proposed which accounts semiquantitatively for the observations.     

Effect of arterial perfusion on net water flux and active sodium transport across the isolated skin ofBufo bufo bufo (L.)

Summary A method has been developed which allows perfusion of the blood vessels in isolated pelvic skins ofB. bufo. The effect of various doses of vasotocin (AVT) on net water flux (inside medium 220 mOsM, outside medium 11 mOsM) and active sodium transport were compared in perfused and unperfused skins. The unstimulated water flux (Fig. 3) and the active sodium transport (Fig. 6) were unaffected by perfusion. The threshold for stimulation of water flux was between 0.01 and 0.1 nM vasotocin in perfused skins and between 0.1 and 1 nM in unperfused skins. The threshold for stimulation of active sodium transport was between 0.005 and 0.05 nM vasotocin in perfused skins and between 0.05 and 0.5 nM in unperfused skins. The maximal water flux through perfused skins, 4 l/cm²min, was obtained at 1 nM vasotocin. At 10 nM vasotocin the water flux was only 0.7 l/cm²min in unperfused skins. The maximal active sodium transport was approximately of the same magnitude in perfused and unperfused skins, at 0.5 mM and at 50 nM, respectively.     

Synthese der Hämolymphproteine und die Aufnahme der Dotterfraktion in die Oocyte unter Actinomycin-Einflu\ (Untersuchungen anMusca domestica)

Zusammenfassung Mit Hilfe von autoradiographischen und elektrophoretischen Methoden wurde die Dottereinlagerung in den wachsenden Oocyten vonMusca domestica untersucht. Sie beginnt nach 30 min im Autoradiogramm sichtbar zu werden. Durch ihre Färbbarkeit und Markierung konnte die Dotterfraktion im Pherogramm von Ovar und Hämolymphe eines mittleren Wachstumsstadiums (Stadium 3) nachgewiesen werden. Nach Abschlu\ der Vitellogenese tritt sie in der Hämolymphe nicht mehr auf. Die Einlagerung der Dotterproteine wird durch Actinomycin gestört, dagegen läuft ihre Synthese nahezu unbeeinflu\t weiter. Die Transporthemmung kann als bisher unbekannter Nebeneffekt des Actinomycins gedeutet werden.

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Synthesis of haemolymph proteine and the uptake of the yolk fraction in the oocyte during Actinomycin-treatment. (Studies onMusca domestica)

Summary By means of radioautographic and electrophoretic techniques yolk protein uptake in the growing oocytes ofMusca domestica was investigated. After 30 min yolk protein becomes visible in the radioautograms. By stainability and labeling the yolk fraction could be demonstrated in the pherogram of ovary and haemolymph in an intermediate developmental stage (stage 3). After the end of vitellogenesis it does not appear in the haemolymph. The yolk protein uptake is inhibited by Actinomycin, but the synthesis goes on nearly as normal. This inhibition can be interpretated as a new accessory effect of Actinomycin.