Effect of low temperature on ethanolic fermentation in rice seedlings.

Rice seedlings (Oryza sativa L.) were incubated at 5-30 degrees C for 48 h and the effect of temperature on ethanolic fermentation in the seedlings was investigated in terms of low-temperature adaptation. Activities of alcohol dehydrogenase (ADH, EC 1.1.1.1) and pyruvate decarboxylase (PDC, EC 4.1.1.1) in roots and shoots of the seedlings were low at temperatures of 20-30 degrees C, whereas temperatures of 5, 7.5 and 10 degrees C significantly increased ADH and PDC activities in the roots and shoots. Temperatures of 5-10 degrees C also increased ethanol concentrations in the roots and shoots. The ethanol concentrations in the roots and shoots at 7.5 degrees C were 16- and 12-times greater than those in the roots and shoots at 25 degrees C, respectively. These results indicate that low temperatures (5-10 degrees C) induced ethanolic fermentation in the roots and shoots of the seedlings. Ethanol is known to prevent lipid degradation in plant membrane, and increased membrane-lipid fluidization. In addition, an ADH inhibitor, 4-methylpyrazole, decreased low-temperature tolerance in roots and shoots of rice seedlings and this reduction in the tolerance was recovered by exogenous applied ethanol. Therefore, production of ethanol by ethanolic fermentation may lead to low-temperature adaptation in rice plants by altering the physical properties of membrane lipids.

Modification of thiamine pyrophosphate dependent enzyme activity by oxythiamine in Saccharomyces cerevisiae cells.

Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phosphate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS-PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger inhibition of mitochondrial pyruvate dehydrogenase than of cytosolic enzymes.

Pyruvate decarboxylase, the target for omeprazole in metronidazole-resistant and iron-restricted Tritrichomonas foetus.

The substituted benzimidazole omeprazole, used for the treatment of human peptic ulcer disease, inhibits the growth of the metronidazole-resistant bovine pathogen Tritrichomonas foetus in vitro (MIC at which the growth of parasite cultures is inhibited by 50%, 22 microg/ml [63 microM]). The antitrichomonad activity appears to be due to the inhibition of pyruvate decarboxylase (PDC), which is the key enzyme responsible for ethanol production and which is strongly upregulated in metronidazole-resistant trichomonads. PDC was purified to homogeneity from the cytosol of metronidazole-resistant strain. The tetrameric enzyme of 60-kDa subunits is inhibited by omeprazole (50% inhibitory concentration, 16 microg/ml). Metronidazole-susceptible T. foetus, which expresses very little PDC, is only slightly affected. Omeprazole has the same inhibitory effect on T. foetus cells grown under iron-limited conditions. Similarly to metronidazole-resistant cells, T. foetus cells grown under iron-limited conditions have nonfunctional hydrogenosomal metabolism and rely on cytosolic PDC-mediated ethanol fermentation.

The transduction of the signal for grape bud dormancy breaking induced by hydrogen cyanamide may involve the SNF-like protein kinase GDBRPK.

Alterations in gene expression during early stages of dormancy release in grapevine buds were analyzed to facilitate the identification of gene products that may mediate the signal transduction of a dormancy-release signal, or derepression of meristematic activity. In the present report we describe the identification of GDBRPK, a transcript for an SNF-like protein kinase that is up-regulated upon chemical induction of dormancy release by hydrogen cyanamide (HC). Since SNF and SNF-like protein kinases are known as sensors of stress signals, we hypothesize that GDBRPK may be involved in the perception of a stress signal induced by HC. We also describe a simultaneous and remarkable induction of both PDC and ADH transcripts that was observed shortly after HC application, and was of a transient nature. These data may imply that HC application leads to a transient respiratory stress, which likely results in a temporary increase in the AMP/ATP ratio. Since AMP is known as a stress signal that is sensed by SNF-like kinases, we suggest that the SNF-like GDBRPK could serve as the sensor of this signal.

[Inactivation of yeast pyruvate decarboxylase in the presence of substrate and quinone electron acceptors]. / Inaktivatsiia drozhzhevoi piruvatdekarboksilazy v prisutstvii substrata i khinonovykh aktseptorov élektronov.

The rate constants of paracatalytic inactivation of pyruvate decarboxylase in the presence of 1,4-naphthoquinones and 1,4-benzoquinones are determined by redox potentials of the oxidant. The logarithm of k2 depends hyperbolically on the redox potential of quinone E0(Q/Q-.) with the coefficient of proportionality which approximates 8.4, The absence of considerable deviations in this correlation for the oxidants of different structures with closed values Eo(Q/Q-.) indicates that the enzyme produces no additional steric barrier.

Correlation of cofactor binding and the quaternary structure of pyruvate decarboxylase as revealed by 31P NMR spectroscopy.

The pH dependence of the quaternary structure of pyruvate decarboxylase (EC 4.1.1.1) has recently been discovered [(1990) FEBS Lett. 266, 17-20; (1992) Biochemistry (in press)]. In the present study we have investigated the change in quaternary structure by observing the binding of the cofactor, thiamine pyrophosphate, using 31P NMR spectroscopy. The dissociation of the native tetramers into dimers when increasing the pH coincides with a weaker binding of the cofactor and loss of enzyme activity. The results provide further evidence that thiamine pyrophosphate is bound primarily via the beta-phosphate moiety. In addition, a phosphoserine has been discovered in two of the four subunits.

[Pyruvate decarboxylase inactivation by interaction with substrate and molecular oxygen]. / Inaktivatsiia piruvatdekarboksilazy pri vzaimodeistvii s substratom i molekuliarnym kislorodom.

Pyruvate may promote the yeast pyruvate decarboxylase inactivation when affected by molecular oxygen. In the presence of pyruvate and O2 inactivation of enzyme increases with the initial substrate concentration increasing. pH-dependence of pyruvate decarboxylase inactivation under joint action of substrate and O2 has maximum in the region 6.9-7.5. It is suggested that the influence of pyruvate and molecular oxygen is connected with the coenzyme-substrate complex oxidation in an active site of yeast pyruvate decarboxylase on the steps preceding the release of free acetaldehyde.