Efecto Pasteur

Biochem. J. (1983) 212, 749-754 Printed in Great Britain

749

Glycolysis and respiration in yeasts
The Pasteur effect studied by mass spectrometry
David LLOYD,* Bodil KRISTENSEN and Hans DEGN Institute of Biochemistry, Odense University, Campusvej 55, DK-5230 Odense M, Denmark

(Received 20 December 1982/Accepted 10 March 1983)
1. Simultaneous and continuous measurements of changes inCO2 and 02 concentrations in glucose-metabolizing yeast suspensions by mass spectrometry enabled a study of the Pasteur effect (aerobic inhibition of glycolysis) in Saccharomyces uvarum and Schizosaccharomyces pombe. 2. A different control mechanism operates in Candida utilis to give a damped oscillation after the anaerobic-aerobic transition. 3. The apparent Km values for respiration of the threeyeasts were in the range 1.3-1.8 pM-02. 4. The apparent Km values for 02 of the Pasteur effect were 5 and 13 pM for catabolite-repressed and derepressed S. uvarum respectively and 7 UM for Sch. pombe. 5. These results are discussed with respect to currently accepted mechanisms for the control of glycolysis. The inhibition of carbohydrate utilization by has been observed in many cell-types (Krebs,1972), and since the original discovery of this effect by Pasteur (1861) different mechanisms have been proposed. Energy demand in many yeasts may be satisfied either by highly efficient mitochondrial energy conservation, or by the much less efficient glycolytic generation of ATP. Transition from the anaerobic to the aerobic state leads to a suppression of glycolytic flux, observable as adecreased rate of CO2 evolution; the more efficient ATP-generating system suppresses the less efficient one. The effect is reversible, so that an increased rate of production of CO2 is observed going from aerobiosis to the anaerobic state. The Pasteur effect in yeast involves the integration of a series of feedback mechanisms (Sols, 1967), including allosteric activation of NAD+-dependent isocitratedehydrogenase by AMP (Hathaway & Atkinson, 1963), inactivation of phosphofructokinase by ATP and citrate (or activation by AMP or P,) (Ramaiah et al., 1964; Salas et al., 1965), and inactivation of glucose transport (Sols, 1967). Despite an extensive literature, there is just one previous report of the 02-concentration dependency of the Pasteur effect (Laser, 1937). In the present
02

work we havetaken advantage of a novel method to do this; simultaneous continuous measurements of °2 and CO2 concentrations in a suspension of yeasts using a quadrupole mass spectrometer with a membrane-covered inlet. We show that the Pasteur effect is more easily seen in catabolite-derepressed (but highly fermentative) yeasts, e.g. Saccharomyces uvarum and Schizosaccharomyces pombe, than in the more aerobicspecies Candida utilis, and also that the apparent Km for °2 of the Pasteur effect is higher than that for respiration.

Experimental Maintenance, growth and harvesting of the
organisms

Abbreviation used: CCCP, m-chlorocarbonylcyanide phenylhydrazone. * Permanent address: Department of Microbiology, University College, Newport Road, Cardiff CF2 ITA, Wales, U.K.

C. utilis N.C.Y.C. 193 wasmaintained and grown defined medium containing 1% (w/v) glucose as described previously (Kader & Lloyd, 1979). S. uvarum (formerly S. carlsbergensis) N.C.Y.C. 74S and Sch. pombe 972h- were maintained on 10% (w/v) glucose/malt extract/agar slopes and grown on defined medium containing 1% (w/v) glucose, as described by Mitchison (1970), but with 0.5% (w/v) (NH4)2S04 as nitrogen source. All threeyeasts were grown at 300C with rotary shaking (100rev./min) in 200ml cultures in 500ml conical flasks. Organisms were counted in a Thoma haemocytometer slide (Hawkesley, Lancing, Sussex, U.K.) after suitable dilution. They were harvested by centrifugation at 2000g for 2min at 40C, washed twice in 20vol. of
on

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15 mM-NaCl and finally resuspended in 5 vol. of this solution.

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