Helga Hinze

Effects of glucose and nitrogen source on the levels of proteinases, peptidases, and proteinase inhibitors in yeast.

In Saccharomyces cerevisiae harvested from early exponential growth on glucose-containing media, the specifc activities of proteinases A and B, carboxypeptidase Y, and the inhibitors IA, IB, IC of these three proteinases, respectively, are found to be 10-30% of the specific activities observed in media without glucose, containing acetate as a carbon source; the activities of two aminopeptidases in glucose-grown cells were 30-50% of those in acetate-grown cells. In contrast to fructose-biphosphatase, phosoenolpyruvate carboxykinase, and cytoplasmic malate dehydrogenase, which are inactivated after the addition of glucose to derepressed cells, the proteinases and inhibitors are not inactivated after glucose addition, but appear to be repressed. Growth of the yeast on poor nitrogen sources or starvation for nitrogen results in 2-3 fold increases in the levels of most proteinases and peptidases, but this effect is not observed with glucose as the carbon source.

Assay of phosphoenolpyruvate car☐ykinase in crude yeast extracts

Abstract The applicability of a spectrophotometric assay of phosphoenolpyruvate car☐ykinase to crude yeast extracts has been studied. The assay measured oxalacetate production by coupling to the malate dehydrogenase reaction (phosphoenolpyruvate + ADP + bicarbonate → oxalacetate + ATP; oxalacetate + NADH → malate + NAD). Disappearance of NADH depended strictly on the presence of phosphoenolpyruvate, bicarbonate, ADP, and Mn2+. Furthermore, the disappearance of NADH was shown to be accompanied by stoichiometric accumulation of malate. Addition of 10 m m quinolinate, which is a known inhibitor of liver phosphoenolpyruvate car☐ykinase, completely prevented phosphoenolpyruvate-dependent NADH disappearance. These observations demonstrated that the assay provides a quantitative measure of phosphoenolpyruvate car☐ykinase activity in crude extracts. The assay could be applied to crude extracts from yeast cells grown under laboratory conditions but not to extracts from commercially produced bakers yeast, because of an extremely high rate of endogeneous oxidation of NADH in the latter extracts. With the spectrophotometric assay, optimal activity was observed at pH 7.0 with both crude extracts and a 15-fold-purified preparation.

Mechanism of sulfite action on the energy metabolism of Saccharomyces cerevisiae

Abstract The pools of ribonucleoside di- and triphosphates decrease within a few min after addition of 5 mM sulfite to a suspension of Saccharomyces cerevisiae at pH 3.6. Levels of the corresponding ribonucleoside monophosphates increase in parallel. The strongest effect was observed with the adenosine phosphate pools. Depletion of ATP by sulfite at pH 3.6 occurs both in the presence and absence of glucose. These findings point to at least two different mechanisms for sulfite action on energy metabolism. Glycolysis is effectively impaired by low sulfite concentrations. The enzymes glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase are inhibited by sulfite in vitro. In addition, formation of adducts between sulfite and aldehydes contributes to the inhibition of enzymatic reactions as shown with alcohol dehydrogenase. Sulfite also causes reduction of oxygen consumption of glucose-starved yeast at pH 3.6 which coincides with ATP depletion. In vitro, the oligomycin-sensitive F1-ATPase of yeast is stimulated 2.8-fold by 1 mM sulfite at pH 5.7. However, this stimulation does not seem to be involved in sulfite-initiated ATP depletion as concluded from experiments with the F1-ATPase-deficient mutant pet 936. At pH 3.6, the intracellular proton concentration of yeast is increased from 3.2−6.3 · 10−8 M to 4.0 · 10−6 M by 1 mM sulfite. In spite of the marked intracellular acidification, stimulation of an ATP-driven proton pump is not the chief cause for the sulfite-initiated ATP decrease. During short exposure of yeast to sulfite the effect on energy metabolism is reversible.

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.

Different selectivities of oxidants during oxidation of methionine residues in the α-1-proteinase inhibitor

Oxidation of the reactive site methionine (Met) in α‐1‐proteinase inhibitor (α‐1‐PI) to methionine sulfoxide (Met(O)) is known to cause depletion of its elastase inhibitory activity. To estimate the selectivity of different oxidants in converting Met to Met(O) in α‐1‐PI, we measured the molar ratio Met(O)/α‐1‐PI at total inactivation. This ratio was determined to be 1.2 for both the myeloperoxidase/H2O2/chloride system and the related compound NH2Cl. With taurine monochloramine, another myeloperoxidase‐related oxidant, 1.05 mol Met(O) were generated per mol α‐1‐PI during inactivation. These oxidants attack preferentially one Met residue in α‐1‐PI, which is identical with Met 358, as concluded from the parallelism of loss of elastase inhibitory activity and oxidation of Met. A similar high specificity for Met oxidation was determined for the xanthine oxidase‐derived oxidants. In contrast, the ratio found for ozone and m‐chloroperoxybenzoic acid was 6.0 and 5.0, respectively, indicating oxidation of additional Met residues besides the reactive site Met in α‐1‐PI, i.e. unselective action of these oxidants. Further studies were performed on the efficiency of oxidants for total depletion of the elastase inhibitory capacity of α‐1‐PI. Ozone and m‐chloroperoxybenzoic acid were 10‐fold less effective and the superoxide anion/hydroxyl radicals were 30–50‐fold less effective to inactivate the elastase inhibitory activity as compared to the myeloperoxidase‐derived oxidants. The myeloperoxidase‐related oxidants are discussed as important regulators of α‐1‐PI activity in vivo.

Effect of ozone on ATP, cytosolic enzymes and permeability of Saccharomyces cerevisiae

Treatment of a yeast suspension with ozone inactivates a number of cytosolic enzymes. Among 15 studied, the most drastic inactivation was found for glyceraldehyde-3-phosphate dehydrogenase and to lesser extents: NAD-glutamate dehydrogenase, pyruvate decarboxylase, phosphofructokinase-1 and NAD-alcohol dehydrogenase. Ozone treatment also effects the quantity of ATP and of other nucleoside triphosphates, reducing to about 50% of the initial value. The ATP missing in the cells appears in the medium. NAD and protein also accumulate in the medium suggesting that the yeast cells have been permeabilized. Permeabilization of the yeast cells by treatment with ozone preceeds the inactivation of glyceraldehyde-3-phosphate dehydrogenase and other cytosolic enzymes.

Accumulation of nitrite and sulfite in yeast cells and synergistic depletion of the intracellular ATP content

ZusammenfassungWenn Hefezellen mit Nitrit oder Sulft bei pH-Werten unter 5,0 inkubiert werden, beobachtet man eine starke intracelluläre Akkumulation von Nitrit, bzw. Sulfit. Es ist anzunehmen, daß Nitrit und Sulfit in ihrer undissoziierten Form als salpetrige Säure (pK=3,3) bzw. schweflige Säure (pK=1,8) penetrieren und dann in den Zellen durch Neutralisation zu den anionischen Formen, die die Zellmembran nicht mehr permeieren können, abgefangen werden. Ähnlich dem früher beschriebenen raschen Abfall des ATP-Gehaltes nach Zusatz von Sulfit in Hefe [Schimz, KL und Holzer H (1979) Arch Microbiol 121:225–229] und in Bakterien [Hinze H, Maier K, Holzer H (1981) Z Lebensm Unters Forsch 172:389-392] verursacht auch Nitrit einen raschen Abfall des ATP-Gehaltes in Hefe auf weniger als 10 % des Anfangswertes. Werden Nitrit und Sulfit in Kombination verabreicht, so beobachtet man einen wesentlich stärkeren Abfall des ATP-Gehaltes als er aus der Summe der Einzeleffekte von Nitrit, bzw. Sulfit zu erwarten wäre („Synergistischer Effekt”).SummaryWhen nitrite or sulfite are applied to yeast cells below pH 5.0, an enormous intracellular accumulation occurs. It is assumed that nitrite and sulfite penetrate the cell membrane in their undissociated forms as nitrous acid (pK = 3.3) or sulfurous acid (pK =1.8), respectively. Due to the neutral intracellular pH they are trapped inside the cell in their anionic forms, which are impermeable to the cell membrane. It has previously been shown that sulfite causes a rapid depletion of the ATP content of yeast cells [Schimz, K. L. and Holzer, H. (1979) resp. Hinze et al. as above]. Similarly, millimolar concentrations of nitrite decrease the ATP level to less than 10% of the initial value. Nitrite and sulfite in combination deplete the ATP content of yeast cells much stronger than expected for the sum of the separate effects of these compounds (“synergistic effect”).

Effect of sulfite or nitrite on the ATP content and the carbohydrate metabolism in yeast

ZusammenfassungKonzentrationen von Sulfit oder Nitrit um 0.5 mmol bewirken bei der Inkubation mit Hefe bei pH 3,6 in wenigen Minuten einen starken Abfall der ATP-Konzentration in den Zellen. Unter diesen Bedingungen wird die alkoholische Gärung durch Sulfit oder Nitrit gehemmt. Der Alkoholabbau unter aeroben Bedingungen ist empfindlicher gegen Nitrit als gegen Sulfit. Dies deutet auf eine höhere Empfindlichkeit der Atmungskette gegenüber Nitrit als gegenüber Sulfit. Unter 15 verschiedenen Enzymen, deren Aktivität nach der Inkubation von Hefezellen mit Sulfit oder Nitrit in Extrakten bestimmt wurde, erwies sich Glycerinaldehyd-3-phosphat-dehydrogenase als am empfindlichsten. Die Analyse stationärer Konzentrationen von Zwischenprodukten der alkoholischen Gärung in intakten Hefezellen unter der Einwirkung von Sulft oder Nitrit deutet auf Blokkade bei der Glycerinaldehyd-3-phosphat-dehydrogenase-Reaktion. Außerdem erhöht Sulfit die intrazelluläre stationäre Konzentration von Glycerinaldehyd-3-phosphat 10 bis 100 fach gegenüber den Konzentrationen bei Abwesenheit von Sulfit. In-vitro-Versuche über die Beeinflussung des von Triosephosphat-Isomerase oder Aldolase katalysierten Gleichgewichts ergaben bei Gegenwart von Sulfit eine Verschiebung dieser Gleichgewichte in Richtung Glycerinaldehyd-3-phosphat durch Komplexbildung dieser Substanz mit Sulfit.SummaryLow concentrations of sulfite or nitrite (about 0.5 mmol) when applied at pH 3.6, caused a rapid and drastic decrease of the concentration of ATP in yeast cells. Under these conditions, alcoholic fermentation was inhibited by sulfite and to a lesser extent by nitrite. Ethanol consumption under aerobic conditions was shown to be more sensitive to nitrite than to sulfite. This indicates a higher sensitivity of respiratory processes to nitrite than to sulfite. Among 15 enzyme activities assayed in extracts from yeast cells after incubation with sulfite or nitrite, glyceraldehyde-3-phosphate dehydrogenase was shown to be the most sensitive. Analysis of the steady-state concentrations of intermediates of alcoholic fermentation in intact yeast cells also implies inhibition by sulfite or nitrite of the glyceraldehyde-3-phosphate dehydrogenase step of fermentation. In contrast to nitrite, sulfite had an additional effect by accumulating the intracellular steady state concentration of glyceraldehyde-3-phosphate 10 to 100-fold over the concentration in the absence of sulfite.In vitro studies on the equilibrium catalyzed by triosephosphate isomerase or aldolase confirmed the postulated shift of equilibrium concentrations by a formation of complex of glyceraldehyde-3-phosphate with sulfite.

Inactivation of enzymes and an enzyme inhibitor by oxidative modification with chlorinated amines and metal-catalyzed oxidation systems.

Oxidative inactivation of various key enzymes and alpha-1-proteinase inhibitor (alpha-1-PI) was studied by treatment with N-chloramines and the metal-catalyzed oxidation (MCO)-systems ascorbate/Fe(III) and ascorbate/Cu(II). Chlorinated amines completely inhibited alpha-1-PI, fructose-1,6-bis phosphatase (Fru-P2ase) and glyceraldehyde phosphate dehydrogenase (GAPD) at a low molar excess, and glucose-6-phosphate dehydrogenase (G6PD) at a high molar excess, but did not impair beta-N-acetylglucosaminidase (beta-NAG), alkaline phosphatase (AP) or lactate dehydrogenase (LDH). MCO-systems affected the activities of Fru-P2ase, GAPD, AP, LDH and G6PD, but not those of beta-NAG or alpha-1-PI. EDTA prevented inactivation of Fru-P2ase, G6PD and LDH by ascorbate/Cu(II) and of Fru-P2ase by ascorbate/Fe(III) suggesting a site-specific oxidation catalyzed by a protein-bound metal ion. In conclusion, N-chloramines and MCO-systems exhibited different properties with regard to oxidative inactivation, sulfhydryl-enzymes were susceptible to both systems, but other enzymes were only susceptible to one or neither system.

Metal-catalyzed inactivation of bovine glucose-6-phosphate dehydrogenase — role of thiols

The role of thiols as oxidant scavengers during inactivation of bovine glucose‐6‐phosphate dehydrogenase by metal‐catalyzed oxidation systems has been studied in vitro. Partial inactivation of the enzyme was achieved by the metal‐catalyzed oxidation systems Fe(II)/H2O2/EDTA or Fe(II)/ H2O2/ADP under specific conditions. When EDTA as chelator was present in the oxidation system, both cysteine and N‐acetylcysteine at low concentrations (0.1 – 1 mM) drastically enhanced inactivation, while cysteinyl‐glycine and glutathione did not. The thiol‐mediated inactivation was inhibitable by superoxide dismutase. Depletion of enzyme activity by cysteine was paralleled by an increase of the carbonyl content, which indicates oxidative injury. However, when EDTA as chelator was replaced by the natural chelator ADP, all thiols studied acted as antioxidants. It is therefore concluded that the nature of the chelator as a constituent of the metal‐catalyzed oxidation systems determines whether the antioxidative function of some thiols is shifted to a prooxidative function against glucose‐6‐phosphate dehydrogenase.