By using a modified technique to measure glucose uptake in Saccharomyces cerevisiae, potential uncertainties have been identified in previous determinations. These previous determinations had led to the proposal that S. cerevisiae contained a constitutive low-affinity glucose transporter and a glucose-repressible high-affinity transporter. We show that, upon transition from glucose-repressed to -derepressed conditions, the maximum rate of glucose transport is constant and only the affinity for glucose changes. We conclude that the transporter or group of transporters is constitutive and that regulation of glucose transport occurs via a factor that modifies the affinity of the transporters and not via the synthesis of different kinetically independent transporters. Such a mechanism could, for instance, be accommodated by the binding of kinases causing a change in affinity for glucose.Until very recently, glucose uptake in Saccharomyces cerevisiae was thought to be mediated by two kinetically distinct mechanisms. A constitutive low-affinity transport system, consisting of a nonconcentrative facilitated diffusion process with a Km of approximately 20 mM (50 mM for fructose), and a kinase-dependent, glucose-repressible high-affinity transport system, also consisting of a facilitated diffusion process with a Km of approximately 1 mM (5 mM for fructose), have been described (3). The diagnostic technique used in the elucidation of these two systems was the biphasic nature of Eadie-Hofstee plots obtained from short-time-scale (5-s) uptake experiments using radiolabelled glucose. Analysis of such plots, however, is complex and requires computer-assisted nonlinear regression. Simple linearization of the two slopes in the biphasic plot can produce poor estimations of the kinetic parameters or, in severe cases, fail to detect all of the systems present (24, 26).The widespread use of these experimental and analytical techniques has led to the description of high-and low-affinity glucose transporters in several Saccharomyces species (7) as well as other yeasts such as Kluyveromyces lactis (23), Kluyveromyces marxianus (9), and Candida wickerhamii (20).More recent genetic studies have identified a number of genes involved in glucose transport in S. cerevisiae. SNF3 (sucrose nonfermenting), a glucose-repressible component of high-affinity glucose transport (4) and HXT1 and HXT2 (hexose transport) are proposed components of high-affinity glucose transport (14,16). Further studies have revealed that HXT3, HXT4, and an unidentified component are likewise involved in high-affinity glucose transport while SNF3 may be involved only in the regulation of high-affinity transport (13). While at least six components have been identified as being involved in the high-affinity uptake, the nature of the lowaffinity component has remained elusive, and this has led to questions regarding the nature of this component. Several groups have suggested that low-affinity uptake may be a consequence of passive diffusion (8,10,11 Strains and growth con...

Infrared (IR) spectra in combination with chemical analyses have recently shown that the active Ni-Fe site of the soluble NAD(+)-reducing [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanide groups and one carbon monoxide as ligands. Experiments presented here confirm this result, but show that a variable percentage of enzyme molecules loses one or two of the cyanide ligands from the active site during routine purification. For this reason the redox conditions during the purification have been optimized yielding hexameric enzyme preparations (HoxFUYHI(2)) with aerobic specific H(2)-NAD(+) activities of 150-185 mumol/min/mg of protein (up to 200% of the highest activity previously reported in the literature). The preparations were highly homogeneous in terms of the active site composition and showed superior IR spectra. IR spectro-electrochemical studies were consistent with the hypothesis that only reoxidation of the reduced enzyme with dioxygen leads to the inactive state, where it is believed that a peroxide group is bound to nickel. Electron paramagnetic resonance experiments showed that the radical signal from the NADH-reduced enzyme derives from the semiquinone form of the flavin (FMN-a) in the hydrogenase module (HoxYH dimer), but not of the flavin (FMN-b) in the NADH-dehydrogenase module (HoxFU dimer). It is further demonstrated that the hexameric enzyme remains active in the presence of NADPH and air, whereas NADH and air lead to rapid destruction of enzyme activity. It is proposed that the presence of NADPH in cells keeps the enzyme in the active state.

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The kinetics of glucose transport in a number of different mutants of Saccharomyces cerevisiae with multiple deletions in the glucose transporter gene family were determined. The deletions led to differences in maximal rate and affinity for glucose uptake by the cells, dependent on the growth conditions. At the same time, there were changes in glucose repression, as determined by expression of invertase activity. Only in the strain with genes HXT1-4 and SNF3 deleted but carrying HXT6/7 were glucose uptake kinetics and invertase activity independent of the presence or concentration of glucose in the growth medium. Some degree of glucose sensitivity was recovered if the SNF3 or HXT2 gene was present in the multiple-deletion background. It is hypothesized that during growth on glucose, both modulation of the kinetics of glucose uptake and derepression of invertase activity require the presence of more than one active gene of the glucose transporter family.

Activity changes of a number of enzymes involved in carbohydrate metabolism were determined in cell extracts of fractionated exponential-phase populations of Saccharomyces cerevisiae grown under excess glucose. Cell-size fractionation was achieved by an improved centrifugal elutriation procedure. Evidence that the yeast populations had been fractionated according to age in the cell cycle was obtained by examining the various cell fractions for their volume distribution and their microscopic appearance and by flow cytometric analysis of the distribution patterns of cellular DNA and protein contents. Trehalase, hexokinase, pyruvate kinase, phosphofructokinase 1, and fructose-1,6-diphosphatase showed changes in specific activities throughout the cell cycle, whereas the specific activities of alcohol dehydrogenase and glucose-6-phosphate dehydrogenase remained constant. The basal trehalase activity increased substantially (about 20-fold) with bud emergence and decreased again in binucleated cells. However, when the enzyme was activated by pretreatment of the cell extracts with cyclic AMP-dependent protein kinase, no significant fluctuations in activity were seen. These observations strongly favor posttranslational modification through phosphorylation-dephosphorylation as the mechanism underlying the periodic changes in trehalase activity during the cell cycle. As observed for trehalase, the specific activities of hexokinase and phosphofructokinase 1 rose from the beginning of bud formation onward, finally leading to more than eightfold higher values at the end of the S phase. Subsequently, the enzyme activities dropped markedly at later stages of the cycle. Pyruvate kinase activity was relatively low during the Gl phase and the S phase, but increased dramatically (more than 50-fold) during G2. In contrast to the three glycolytic enzymes investigated, the highest specific activity of the gluconeogenic enzyme fructose-1,6-diphosphatase 1 was found in fractions enriched in either unbudded cells with a single nucleus or binucleated cells. The observed changes in enzyme activities most likely underlie pronounced alterations in carbohydrate metabolism during the cell cycle.Biochemical conversions which occur in bioreactors are the combined contributions of cells of different ages, i.e., cells at different stages of the cell cycle. Knowledge of possible variations in enzyme activities during the cell cycle is necessary to understand fully the metabolic behavior of the entire microbial culture. This requires methods to obtain subpopulations at defined stages of the cell cycle.In the past, the activity patterns of a variety of enzymes have been determined in synchronously dividing yeast cultures (10,27,28). However, as discussed by other investigators, many of these earlier results may have been flawed by technical artifacts, since the synchronization techniques that were used could cause serious perturbations in enzyme activity (6, 7,10,29). Creanor and Mitchison (7) developed a method of producing synchronous cultures of S...