The actvity of K+-activated yeast aldehyde dehydrogenase following rapid changes in cation environment

Springham, Mary G.; Betts, Graham F.

doi:10.1016/0005-2744(73)90335-5

Cited by 5 publications

(5 citation statements)

References 13 publications

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“…Yeast ALDH was first purified by Steinman and Jakoby (49), who found that the enzyme was activated by K+ ions, and later by others (48,54). Though some detailed kinetic studies were performed (5,8,12,50), neither the amino acid sequence nor the subcellular localization of the enzyme has been reported.…”

Section: Discussionmentioning

confidence: 99%

Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation

et al. 1991

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Mutants of Saccharomyces cerevisiae deficient in mitochondrial aldehyde dehydrogenase (ALDH) activity were isolated by chemical mutagenesis with ethyl methanesulfonate. The mutants were selected by their inability to grow on ethanol as the sole carbon source. The ALDH mutants were distinguished from alcohol dehydrogenase mutants by an aldehyde indicator plate test and by immunoscreening. The ALDH gene was isolated from a yeast genomic DNA library on a 5.7-kb insert of a recombinant DNA plasmid by functional complementation of the aldh mutation in S.cerevisiae. An open reading frame which specifies 533 codons was found within the 2.0-kb BamHI-BstEII fragment in the 5.7-kb genomic insert which can encode a protein with a molecular weight of 58,630. The N-terminal portion of the protein contains many positively charged residues which may serve as a signal sequence that targets the protein to the mitochondria. The amino acid sequence of the proposed mature yeast enzyme shows 30% identity to each of the known ALDH sequences from eukaryotes or prokaryotes. The amino acid residues corresponding to mammalian cysteine 302 and glutamates 268 and 487, implicated to be involved at the active site, were conserved. S. cerevisiae ALDH was found to be localized in the mitochondria as a tetrameric enzyme. Thius, that organelle is responsible for acetaldehyde oxidation, as was found in mammalian liver.Saccharomyces cerevisiae, unlike higher eukaryotes, can metabolize as well as produce ethanol. During fermentation, the consumption of sugars results in the accumulation of ethanol. When glucose or any other fermentable sugar is absent from the culture medium, S. cerevisiae can utilize ethanol as the carbon source aerobically. The metabolism of ethanol is well understood in mammals. In the liver, cytosolic alcohol dehydrogenase (ADH) oxidizes ethanol to acetaldehyde and then mitochondrial aldehyde dehydrogenase (ALDH) oxidizes the intermediate to acetate (14). In S. cerevisiae, it appears that a mitochondrial alcohol dehydrogenase (ADH3) is responsible for the initial oxidation (65,67). Little is known about the role of ALDHs in the next step.Various groups have purified and characterized yeast ALDH (12,49,54). It was reported to be a tetrameric enzyme with a subunit molecular mass of 60 kDa and a low Km for acetaldehyde. Unlike the liver enzymes, the yeast enzyme is activated by K+ ions (4, 32). Whereas the mammalian enzymes have been sequenced at the cDNA (16,19,29,34) as well as the protein (24, 33, 60) level, no sequence work has been reported for the yeast enzyme.One of our interests was to study the subcellular localization of acetaldehyde metabolism in S. cerevisiae. In the mammalian liver tissue, this was studied by selectively inhibiting the cytosolic or the mitochondrial isozyme of ALDH (11,52). An alternative approach would be to introduce the enzyme into a cell deficient in ALDH activity. S. cerevisiae would be a suitable model system, as it can metabolize ethanol and could serve as a host for the expression of foreign g...

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Section: Discussionmentioning

confidence: 99%

Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation

et al. 1991

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“…This is much slower than the likely rate of dissociation of T1 ÷ from the enzyme [ 12]. Such behaviour has previously been demonstrated when K* instead of T1 ÷ was the activating cation [ 13], and has been interpreted in terms of a conformation change induced by activating cation. Fig.5 demonstrates the stability of aldehyde dehydrogenase in various levels of T1 NO;.…”

Section: Methodsmentioning

confidence: 55%

Thallium activation and inhibition of yeast aldehyde dehydrogenase

et al. 1975

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“…Additional information on the inhibitory effect of Tl+ at high concentrations was obtained by rapid mixing and stopped-flow experiments. It has been shown that removal of activating K+ by rapid dilution and addition of Li+, which is a competitive inhibitor with respect to activator, gives transiently the high rate of activity expected in the environment existing before the unfavourable change of cation status (Springham & Betts, 1973;Bostian et al, 1975). Activity takes several seconds to reflect the new unfavourable environment.…”

Section: Ks(1+k +[Tl+1 (1+k-mentioning

confidence: 99%

“…It has been shown that the activity of yeast aldehyde dehydrogenase is rapidly lost on removal of activating K+ (Black, 1951). Provided the activator cation environment is quickly restored enzyme activity rapidly recovers (Springham & Betts, 1973;Betts et aL, 1979). However, continued incubation of enzyme in the absence of activator results in the accumulation of an enzyme form attaining maximal activity only after a 10min recovery in the presence of K+ .…”

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confidence: 99%

Multiple binding of thallium and rubidium to potassium-activated yeast aldehyde dehydrogenase. Influences on tertiary structure, stability and catalytic activity

Bostian¹,

Betts²,

Man³

et al. 1982

Biochemical Journal

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Univalent cation activators of aldehyde dehydrogenase have dual effects, both interpreted as cation-induced or -stabilized conformation changes. These two processes are differentiated by the time scales of their associated changes in activity. Using Tl+ as an activator, under certain conditions, the slower change in activity saturates at a Tl+ concentration which is only 0.1 Ks for the faster change. This, together with evidence for cation-induced rather than cation-stabilized conformation changes, is used to propose separate binding sites for cations responsible for the two activation processes. Equilibrium dialysis indicates 4 binding sites per active site for Rb+ or 6 sites for Tl+. At least one of the additional sites for Tl+ is an inhibitory site which has been differentiated from the activator sites on the basis of steady-state and pre-steady-state kinetic data.

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The actvity of K+-activated yeast aldehyde dehydrogenase following rapid changes in cation environment

Cited by 5 publications

References 13 publications

Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation

Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation

Thallium activation and inhibition of yeast aldehyde dehydrogenase

Multiple binding of thallium and rubidium to potassium-activated yeast aldehyde dehydrogenase. Influences on tertiary structure, stability and catalytic activity

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