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K van Dam

Publications and source records attributed to K van Dam.

At least 37 records · Page 2Linked to original sources
PubMed1994

Respiratory inhibitors affect incorporation of glucose into Saccharomyces cerevisiae cells, but not the activity of glucose transport.

Incubation of starved galactose-grown S. cerevisiae cells with cyanide reduced glucose uptake as measured over a 5-s period. The Vmax for glucose uptake was decreased by over a factor of two but the apparent affinity for glucose doubled. When measured in the sub-second time scale, however, there was no significant inhibition of glucose uptake, by cyanide, up to 200-ms, clearly demonstrating that, in cyanide treated cells, glucose uptake was not linear for the first 5-s. After a 200-ms exposure of untreated cells to radio-labelled glucose, less than 10% of the intracellular label resided in soluble uncharged compounds. In cyanide-treated cells up to 43% of the labelled compounds were uncharged, with a concurrent reduction of intracellular label residing in anionic compounds. The results suggest that, in the presence of 10 mM cyanide when respiration is inhibited, a reduction in the cellular ATP concentration causes a reduction in hexose-kinase activity which results in an accumulation of internal free glucose, which in turn causes a reduction in net glucose transport.

Biological Transport↗
PubMed1994

Rapid kinetics of glucose uptake in Saccharomyces cerevisiae.

In a multiple deletion mutant hxt1deltahxt2deltahxt3delta hxt4deltasnf3delta of Saccharomyces cerevisiae growing on a 2% glucose, high-affinity glucose-uptake (low Km) was exhibited throughout growth on glucose in contrast to the wild-type, which exhibited the usual low-affinity to high-affinity transition as the glucose in the medium was consumed. Elevated levels of invertase activity throughout growth on glucose, in this mutant as compared to the wild-type, indicate that glucose repression may be impaired. However, in a mutant containing only the HXT2 gene (hxt1deltahxt3deltahxt4deltasnf3delta), invertase levels were similar to those in the wild-type. It is likely, therefore, that some of these putative glucose transporters, such as HXT2, also have regulatory roles in cellular metabolism. In triple hexose-kinase mutants, rapid (200-ms) measurements of initial glucose-uptake revealed high-affinity glucose uptake (Km approx. 2 mmol/L) while measurements on the slower 5-s scale clearly demonstrate that uptake is not linear over this longer period. These results suggest that this high-affinity component does not require a functional hexose-kinase.

Biological Transport↗
PubMed1994

Decrease in glycolytic flux in Saccharomyces cerevisiae cdc35-1 cells at restrictive temperature correlates with a decrease in glucose transport.

The glycolytic flux was investigated in the thermosensitive Saccharomyces cerevisiae adenylate cyclase mutant cdc35-1. Directly after a shift to restrictive temperature, the specific CO2 production rate increased from about 250 nmol min-1 (mg protein)-1 to more than 400 nmol min-1 (mg protein)-1, but then the CO2 production gradually fell to about 70 nmol min-1 (mg protein)-1 after 5 h. O2 consumption at restrictive temperature continued at more or less the same rate as at permissive temperature. The temperature shift in the mutant resulted in an increase in the estimated intracellular cAMP concentration from about 1.1 microM to 1.8 microM. This indicates that high cAMP levels are not sufficient for cell cycle progression and high glycolytic activity. The decrease in glycolytic activity at restrictive temperature was not paralleled by a similar decrease in the specific activity of any of the glycolytic enzymes, but correlated with a decrease in hexose transport. A drop in intracellular concentrations of the early metabolites of glycolysis further indicated a defect in transport at restrictive temperature. Our data suggest that glucose transport has a high control on glycolytic flux.

Adenylyl Cyclases↗
PubMed1994

Quantification of the regulation of glycerol and maltose metabolism by IIAGlc of the phosphoenolpyruvate-dependent glucose phosphotransferase system in Salmonella typhimurium.

The amount of IIAGlc, one of the proteins of the phosphoenolpyruvate:glucose phosphotransferase system (PTS), was modulated over a broad range with the help of inducible expression plasmids in Salmonella typhimurium. The in vivo effects of different levels of IIAGlc on glycerol and maltose metabolism were studied. The inhibition of glycerol uptake, by the addition of a PTS sugar, was sigmoidally related to the amount of IIAGlc. For complete inhibition of glycerol uptake, a minimal ratio of about 3.6 mol of IIAGlc to 1 mol of glycerol kinase (tetramer) was required. Varying the level of IIAGlc (from 0 to 1,000% of the wild-type level) did not affect the growth rate on glycerol, the rate of glycerol uptake, or the synthesis of glycerol kinase. In contrast, the growth rate on maltose, the rate of maltose uptake, and the synthesis of the maltose-binding protein increased two- to fivefold with increasing levels of IIAGlc. In the presence of cyclic AMP, the maximal levels were obtained at all IIAGlc concentrations. The synthesis of the MalK protein, the target of IIAGlc, was not affected by varying the levels of IIAGlc. The inhibition of maltose uptake was sigmoidally related to the amount of IIAGlc. For complete inhibition of maltose uptake by a PTS sugar, a ratio of about 18 mol of IIAGlc to 1 mol of MalK protein (taken as a dimer) was required.

ATP-Binding Cassette Transporters↗
PubMed1994

Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose.

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.

Biological Transport↗
PubMed1993

The sum of the control coefficients of all enzymes on the flux through a group-transfer pathway can be as high as two.

In simple metabolic pathways the control exerted by enzyme concentrations on the pathway flux adds up to one when the control is quantified in terms of control coefficients. In this paper we demonstrate that this classical summation theorem has to be modified in pathways where the enzymes participate by transferring a group between each other. We derive the corresponding new control theorem and show how it is consistent with standard metabolic control analysis. In group-transfer pathways lacking enzyme complexes, the sum of the flux control by enzyme concentrations and by the donor and acceptor couples of the pathway, equals two. In group-transfer pathways with enzyme-enzyme interactions the flux control by the dissociation rate constants of the enzyme-enzyme complexes must be added to obtain this sum of two. In all cases, the sum of the controls by all reaction activities remains one. Both by using the new theorem and by numerical simulations, we then demonstrate that, in group-transfer pathways with or without enzyme interactions, the sum of the control of enzymes on the pathway flux is higher than one and can reach a value of two. The total control of all enzymes on the concentration of any intermediate either with or without the transferred group can be equal to one, rather than to the zero found in the classical case. Examples of group-transfer pathways are the bacterial phosphoenolpyruvate:sugar phosphotransferase system, the main pathway for uptake of sugars in Enterobacteriaceae, and the electron-transfer chain in free-energy transducing membranes.

Enzymes↗
PubMed1993

Around the growth phase transition S. cerevisiae's make-up favours sustained oscillations of intracellular metabolites.

Under a limited set of hitherto incompletely defined conditions, inhibition of respiration has been shown to cause transient oscillations in NAD(P)H fluorescence of yeast cells. In this paper, we apply a new method [1992, Anal. Biochem. 204, 118-132] for extraction of intracellular metabolites. This method involves spraying the cells into -40 degrees C methanol; the neutral pH allows extraction of nearly all intracellular metabolites, including NADH. Close to the shift from glucose to ethanol as a growth substrate, the cells acquire a make-up amenable to sustained oscillations in intracellular concentrations of NADH and glycolytic intermediates such as glucose-6-phosphate. NADH was found to oscillate between 200 microM and 400 microM intracellular concentration. The cellular make-up determining the tendency to oscillate is 'remembered' by the cells after three hours of starvation.

Glycolysis↗
PubMed1993

Control and regulation of metabolic fluxes in microbes by substrates and enzymes.

The control of enzymes and substrates on the flux through microbial metabolic pathways can be quantified in terms of flux control coefficients. In pathways involving group transfer, the summation theorem for flux control by the enzymes has to be modified: the sum of control by all enzymes is between 1 and 2. The phosphoenolpyruvate:glucose phosphotransferase system is such a pathway. Experimental determination of the control by the enzymes in this pathway is under way. The control of the enzymes on the glycolytic flux in yeast is low, with the possible exception of the uptake step. In Klebsiella pneumoniae potassium and ammonium ions can simultaneously be 'limiting', (i.e. have significant control on growth) at pH6, but not at pH8. This may be due to the fact that at pH8 the high-affinity potassium uptake system is absent.

Ammonia↗
PubMed1993

Inactivation of the CDC25 gene product in Saccharomyces cerevisiae leads to a decrease in glycolytic activity which is independent of cAMP levels.

In the budding yeast Saccharomyces cerevisiae cyclic AMP (cAMP) can influence the activity of key enzymes in carbohydrate metabolism through modulation of the activity of cAMP-dependent protein kinase. One of the components involved in cAMP production is the CDC25 gene product, which can activate the RAS/adenylate cyclase pathway by promoting the exchange of guanine nucleotides bound to RAS. In two yeast strains carrying different thermosensitive alleles of the CDC25 gene, cAMP levels respond differently to an increase in growth temperature from 23 degrees C (permissive) to 36 degrees C (restrictive). In strain OL86 (cdc25-5) the estimated intracellular concentration of cAMP dropped after transfer to restrictive temperature whereas in strain ts321 (cdc25-1) the cAMP level rose under the same conditions. Despite the differences in cAMP levels the glycolytic flux in the two mutants responded in a very similar way to the shift from permissive to restrictive temperature; after the increase in the incubation temperature, the specific glycolytic flux in both cdc25-1 and cdc25-5 initially increased from about 300 nmol min-1 (mg protein)-1 to about 500 nmol min-1 (mg protein)-1 (presumably mainly as a consequence of the increase in temperature), but then gradually fell to 100-200 nmol min-1 (mg protein)-1. A similar pattern of CO2 production to that found in the two cdc25 mutants was also observed for several other thermosensitive mutants displaying a Start-II type of G1 arrest. In contrast, in a wild-type strain and in strains giving a Start-I type of G1 arrest, CO2 production did not drop after a temperature shift. The specific activities of glycolytic enzymes in the two cdc25 mutants did not show much change after the temperature shift, indicating that the decrease in glycolytic flux was not caused by a decrease in the activity of any of the glycolytic enzymes. Our data show that, at least in long-term regulation, the cAMP levels per se are not likely to be a prime factor controlling glycolytic flux.

Alleles↗
PubMed1992

A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH.

Glucose metabolism in yeast can be stopped within 0.1 s by spraying the cells in 60% methanol at -40 degrees C. With this procedure the integrity of the cells is not damaged. Using stopped-flow equipment for the incubation with glucose, major changes within a second are shown to occur in intracellular glucose-6-phosphate whereas the fructose-1,6-bisphosphate concentration remains constant. After quenching, the cells can be separated from the medium, washed with cold methanol when required, and extracted using chloroform at -40 degrees C at neutral pH, ensuring minimal degradation of labile metabolites. With partly automated enzymatic methods, a large variety of metabolites, including all glycolytic intermediates, can be determined in the neutral extracts. During the first second after addition of glucose, a significant increase in free intracellular glucose is found.

Chloroform↗
PubMed1992

Analysis of mutations that uncouple transport from phosphorylation in enzyme IIGlc of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.

Mutations that uncouple glucose transport from phosphorylation were isolated in plasmid-encoded Escherichia coli enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The uncoupled enzymes IIGlc were able to transport glucose in the absence of the general phosphoryl-carrying proteins of the PTS, enzyme I and HPr, although with relatively low affinity. Km values of the uncoupled enzymes IIGlc for glucose ranged from 0.5 to 2.5 mM, 2 orders of magnitude higher than the value of normal IIGlc. Most of the mutant proteins were still able to phosphorylate glucose and methyl alpha-glucoside (a non-metabolizable glucose analog specific for IIGlc), indicating that transport and phosphorylation are separable functions of the enzyme. Some of the uncoupled enzymes IIGlc transported glucose with a higher rate and lower apparent Km in a pts+ strain than in a delta ptsHI strain lacking the general proteins enzyme I and HPr. Since the properties of these uncoupled enzymes IIGlc in the presence of PTS-mediated phosphoryl transfer resembled those of wild-type IIGlc, these mutants appeared to be conditionally uncoupled. Sequencing of the mutated ptsG genes revealed that all amino acid substitutions occurred in a hydrophilic segment within the hydrophobic N-terminal part of IIGlc. These results suggest that this hydrophilic loop is involved in binding and translocation of the sugar substrate.

Base Sequence↗
PubMed1991

Energetics of glucose uptake in a Salmonella typhimurium mutant containing uncoupled enzyme IIGlc.

Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. We have studied the energetics of glucose uptake catalyzed by this uncoupled enzyme IIGlc. The molar growth yields on glucose of two strains cultured anaerobically in glucose-limited chemostat- and batch cultures were compared. Strain PP799 transported and phosphorylated glucose via an intact PTS, while strain PP952 took up glucose exclusively via uncoupled enzyme IIGlc, followed by ATP-dependent phosphorylation by glucokinase. Thus the strains were isogenic except for the mode of uptake and phosphorylation of the growth substrate. PP799 and PP952 exhibited similar YGlc values. Assuming equal YATP values for both strains this result indicated that there were no energetic demands for glucose uptake via uncoupled enzyme IIGlc.

Anaerobiosis↗
PubMed1991

Changes in the activities of key enzymes of glycolysis during the cell cycle in yeast: a rectification.

Activities of glycolytic enzymes were determined in elutriation fractionated cultures of Saccharomyces cerevisiae grown on different carbon sources. Almost pure fractions of single cells at the G1 state of cell division were obtained for some of the growth conditions tested, whereas other stages were enriched in particular fractions. Specific activities of glucose-6-phosphate dehydrogenase and alcohol dehydrogenase were found to be constant during the cell cycle, as reported by van Doorn et al. (1988a), Journal of Bacteriology 170, 4808-4815, and (1988b), Journal of General Microbiology 134, 785-790. In contrast to the earlier reports, the activities of hexokinase, phosphofructokinase, pyruvate kinase and trehalase were also constant in different states of the cell cycle. For hexokinase and phosphofructokinase it was shown that the apparent specific activity in a cell-free extract strongly diminished when extracts contained less that 0.5-1 mg protein ml-1. In the experiments of van Doorn et al. (1988a) the protein content of the outer fractions was up to 20 times lower than that of the central fractions, suggesting an alternative explanation for the observed changes in enzyme activities during the cell cycle. Therefore, we want to rectify the observations presented by van Doorn et al. (1988a), and conclude that the activities of the glycolytic enzymes do not vary greatly during the cell cycle of S. cervisiae.

Cell Cycle↗
PubMed1991

Control of glucose metabolism by enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli.

The quantitative effects of variations in the amount of enzyme IIGlc of the phosphoenolpyruvate:glucose phosphotransferase system (PTS) on glucose metabolism in Escherichia coli were studied. The level of enzyme IIGlc could be adjusted in vivo to between 20 and 600% of the wild-type chromosomal level by using the expression vector pTSG11. On this plasmid, expression of the structural gene for enzyme IIGlc, ptsG, is controlled by the tac promoter. As expected, the control coefficient (i.e., the relative increase in pathway flux, divided by the relative increase in amount of enzyme) of enzyme IIGlc decreased in magnitude if a more extensive pathway was considered. Thus, at the wild-type level of enzyme IIGlc activity, the control coefficient of this enzyme on the growth rate on glucose and on the rate of glucose oxidation was low, while the control coefficient on uptake and phosphorylation of methyl alpha-glucopyranoside (an enzyme IIGlc-specific, nonmetabolizable glucose analog) was relatively high (0.55 to 0.65). The implications of our findings for PTS-mediated regulation, i.e., inhibition of growth on non-PTS compounds by glucose, are discussed.

Enzyme Induction↗
PubMed1990

Differentiation between leaks and slips in oxidative phosphorylation.

We have measured the thermodynamic efficiency of oxidative phosphorylation of isolated rat-liver mitochondria during oxidation of succinate. Furthermore, we have calculated what the effect of proton leak or slip in the redox pumps should be on the efficiency of energy transduction in oxidative phosphorylation. These calculations were compared with experiments in which the efficiency was determined in the presence of induced proton leak or redox slip. The results of these experiments are in agreement with the predictions. It is concluded that it is possible to distinguish experimentally between effects of proton leak and redox slip on energy transduction.

Animals↗
PubMed1990

Quantification of multiple-substrate controlled growth--simultaneous ammonium and glucose limitation in chemostat cultures of Klebsiella pneumoniae.

In chemostat cultures of Klebsiella pneumoniae (K. aerogenes) NCTC 418 we measured the concentrations of glucose and ammonium and we varied the ratio of the (limiting) concentrations of glucose and ammonium in the feed medium. By doing this at different dilution rates we found a range where growth rate varies with either concentration in the culture when the other concentration in the culture is held constant. This proves that within this range, dual-substrate controlled growth occurs. Dual substrate-controlled growth was accompanied by yield coefficients for glucose and for ammonium that were intermediate between the yield coefficients obtained for single glucose or single ammonium limitation. We quantified the control by either substrate in terms of the flux control coefficient with respect to that substrate, where flux refers to growth rate. Dual-substrate controlled growth is reflected by the finding that both flux control coefficients exceed zero, simultaneously. In the transition of glucose to ammonium limitation, the control gradually shifts from glucose to ammonium.

Ammonium Chloride↗