Biomedical subjects

K van Dam

Publications and source records attributed to K van Dam.

At least 55 records · Page 3Linked to original sources

PubMed1990

Changes in the incorporation of carbon derived from glucose into cellular pools during the cell cycle of Saccharomyces cerevisiae.

The rate of incorporation of 14C derived from [U-14C]glucose into cells of Saccharomyces cerevisiae X2180(1B) was investigated as a function of the cell cycle. After pulse-labelling of exponentially growing populations, centrifugal elutriation was used to isolate various cell fractions of increasing cell size, representing successive stages of the cell cycle. The total amount of 14C incorporated per cell was found to increase continuously during the cell cycle along with cellular protein content and Coulter counter cell volume. This pattern supports the model of exponential cell growth. In order to evaluate changes in intracellular carbon flow during the cell cycle, chemical extraction procedures were used to obtain four cellular fractions enriched in either low-molecular-mass components, lipid material, polysaccharides or proteins. The distribution of 14C among these cellular fractions varied during successive stages of the cell cycle, indicating cell-cycle-dependent fluctuations in intracellular carbon flow. During the G1 phase the flow of 14C into the low-molecular-mass pool increased markedly; concurrently, the rate of incorporation into the polysaccharide-enriched pool decreased.

Carbon↗

PubMed1990

Adaptation of Salmonella typhimurium mutants containing uncoupled enzyme IIGlc to glucose-limited conditions.

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. As a result of the ptsG mutation, the apparent Km of the system for glucose transport is increased about 1,000-fold (approximately 18 mM) compared with wild-type PTS-mediated glucose transport. An S. typhimurium mutant containing uncoupled enzyme IIGlc as the sole system for glucose uptake was grown in glucose-limited chemostat cultures. Selective pressure during growth in the chemostat resulted in adaptation to the glucose-limiting conditions in two different ways. At first, mutations appeared that led to a decrease in Km value of uncoupled enzyme IIGlc. These results suggested that uncoupled enzyme IIGlc had significant control on the growth rate under glucose-limiting conditions. More efficient glucose uptake enabled a mutant to outgrow its parent and caused a decrease in the steady-state glucose concentration in the chemostat. At very low glucose concentrations (10 microM), mutants arose that contained a constitutively synthesized methyl-beta-galactoside permease. Apparently, further changes in the uncoupled enzyme IIGlc did not lead to a substantial increase in growth rate at very low glucose concentrations.

Biological Transport↗

PubMed1989

Effect of concentration of substrates and products on the growth of Klebsiella pneumoniae in chemostat cultures.

Non-equilibrium thermodynamics (NET) can be used to describe microbial growth. In this description, the concentrations of products contribute to the driving forces of the metabolic processes (anabolism and catabolism). Thus, in contrast to the model of bacterial growth of Monod (Recherches sur la Croissance les Cultures Bactériennes (1942) Herman et Cie, Paris), it is predicted that the growth rate of a bacterial chemostat culture is, in principle, dependent on the concentration of the catabolic product (for instance HCO3-) during catabolite limitation and on the concentration of the anabolic product (for instance biomass) during anabolite limitation. In order to test this prediction, Klebsiella pneumoniae was grown in aerobic citrate-limited, glucose-limited or ammonia-limited chemostat cultures. Ammonia-limited cultures were considered to be essentially anabolite-limited, whereas citrate limitation was used as a representative for catabolite limitation. In ammonia-limited or in glucose-limited cultures it was found that the growth rate was independent of the biomass concentration present. In the NET description this means that the 'back' reaction (i.e., in the direction from biomass to substrates) is saturated with respect to biomass. On the other hand, in citrate-limited cultures, the steady-state concentration of citrate increased with the concentration of the catabolic product HCO3-. At relatively low concentrations of HCO3-, 'thermodynamic back-pressure' of growth (i.e., increase in product concentration was compensated by an increase in substrate concentration so that the driving force for growth remained almost constant) was demonstrated as predicted by the NET model. At concentrations above 40 mM, a kinetic (allosteric) effect of HCO3- was detected. This was concluded from a reduced growth yield on citrate, and from a significant decrease in the maximal growth rate and the maximal oxygen consumption rate after relief of the citrate limitation.

Ammonia↗

PubMed1989

Thermodynamic efficiency of bacterial growth calculated from growth yield of Pseudomonas oxalaticus OX1 in the chemostat.

In order to determine the thermodynamic efficiency of bacterial growth, Pseudomonas oxalaticus OX1 was grown in carbon-limited continuous cultures. 11 different carbon sources, ranging from oxalate (most oxidised component) to ethanol (most reduced component), were used as limiting substrate in these experiments. From the experimental yield values (expressed as C-mol dry weight produced per C-mol carbon substrate consumed) the thermodynamic efficiencies were calculated. On substrates more reduced than biomass (such as ethanol and glycerol) the thermodynamic efficiency of growth of P. oxalaticus was negative but it reached a maximum of 23 +/- 3% with substrates with a degree of reduction of 3 (citrate) and lower. The actual concentrations of the components involved were incorporated into the calculations but this affected the overall thermodynamic efficiency only to a small extent. This result strengthens the conclusion of Westerhoff et al. (Westerhoff, H.V., Hellingwerf, K.J. and Van Dam, K. (1983) Proc. Natl. Acad. Sci. 80, 305-309) that bacteria have been optimised towards a theoretical thermodynamic efficiency of 24%, corresponding with maximisation of growth rate at optimal efficiency, with highly oxidised substrates.

Bicarbonates↗

PubMed1989

On the evaluation of data from flow-dialysis experiments.

Flow dialysis can be used to measure (i) ligand binding to macromolecules and (ii) the size of transmembrane ion gradients. Generally an approximate method is used to calculate the binding or gradient parameters from the raw data. Here we present a simple but exact method and evaluate the errors that may arise when the approximate method is used to calculate the magnitude of ion gradients. In addition, equations are presented that allow for a correction for sampling from or additions to the upper compartment of a flow-dialysis vessel during the measurements. Setty and Hendler [(1982) J. Biochem. Biophys. Methods 7, 35-46] have reported artifacts in the measurement of ion-gradients caused by the addition of electron donors to the upper compartment of a flow-dialysis cell. Here we extend their observations and suggest additional methods to prevent such artifacts.

Biopolymers↗

PubMed1988

Effect of macromolecular composition of microorganisms on the thermodynamic description of their growth.

Most models describing bacterial growth, including the original mosaic non-equilibrium thermodynamic (MNET) description, do not take into account that the macromolecular composition of the cells varies with growth rate. The MNET description of bacterial growth is extended to account for such a variation in macromolecular composition of the cells in order to make the MNET description more generally applicable. Klebsiella aerogenes NCTC 418 was cultured in a chemostat under glucose- or ammonia-limited conditions to determine the macromolecular composition at varying growth rate. The dilution rate has a strong influence on the macromolecular composition of the cells. Under glucose-limited conditions an increase of the RNA content of the cells was observed with increasing growth rate. The RNA content of the cells was much lower under ammonia-limited conditions of the cells than under glucose-limited conditions but also showed an increase with increasing growth rates. Under ammonia-limited conditions, the polysaccharide content strongly decreased with increasing growth rate. The other cellular components changed relatively less with changing growth rate. It is shown that the slope of the line relating catabolism to anabolism varies very little due to variation of the macromolecular cell composition with growth rate, at least under the tested conditions.

Ammonia↗

PubMed1988

Uncoupler-inhibitor titrations of ATP-driven reverse electron transfer in isolated rat-liver mitochondria.

Uncoupler-inhibitor titrations of ATP-driven reverse electron transfer across the first site of the respiratory chain were performed in isolated rat-liver mitochondria, and the experimental results were compared with the predictions of a simple delocalized chemiosmotic mechanism. The rates of ATP hydrolysis (Jp) and reverse electron transfer (-J0) were measured at different uncoupler (S-13) concentrations, either in the absence or in the presence of rotenone. When the rates -J0 and Jp measured at different uncoupler concentrations were expressed as percentages of the activity at zero uncoupler concentration, it was found that the efficiency of S-13 to uncouple the reverse electron transfer and to stimulate ATP hydrolysis was not significantly changed upon partial inhibition with rotenone. These results are in contrast with data from a study of uncoupler-inhibitor titrations in submitochondrial particles published previously, in which a higher effectiveness of several uncouplers to inhibit ATP-driven reverse electron transfer was observed in the presence of rotenone.

Acetoacetates↗

PubMed1988

Unique relationships between the rates of oxidation and phosphorylation and the protonmotive force in rat-liver mitochondria.

The rate of ATP synthesis (JP) in isolated rat-liver mitochondria was strongly dependent on the magnitude of the protonmotive force (delta mu H+) across the mitochondrial inner membrane. Addition of different concentrations of various uncouplers or malonate to mitochondrial incubations in State 3 led to a depression of delta mu H+ and a concomitant decrease in JP. A unique relationship between JP and delta mu H+ was obtained, which was independent of the way in which delta mu H+ was varied. This unique relationship was observed when K+ (in the presence of valinomycin) was used as a probe for delta psi. Different relationships between JP and delta mu H+ were observed when K+ was used as a probe for delta psi and when K+ was measured after separation of the mitochondria by centrifugation without silicone oil. This led to a serious underestimation of delta psi, specifically when uncouplers were present, and non-unique flow-force relationships were thus obtained. Anomalous relationships between JP and delta mu H+ were also found when TPMP+ was used as a probe for delta psi. However, in uncoupler incubations the presence of TBP- strongly affected the TPMP+ accumulation ratio without any effect on the K+ accumulation or on JP and in the presence of TBP- unique relationships between JP and delta mu H+ were again obtained. This indicates that the accumulation of TPMP+ inside the mitochondria is not a straightforward function of delta psi but also depends on conditions like the presence of TBP- or uncouplers. We conclude that there is a unique relationship between the rate of phosphorylation and the protonmotive force in mitochondria and that under some conditions the behaviour of TPMP+ is anomalous.

Adenosine Triphosphate↗

PubMed1988

Energetic consequences of two mutations in Escherichia coli K+ uptake systems for growth under potassium-limited conditions in the chemostat.

The energetics of growth of two Escherichia coli strains (TK 2240 and TK 2242) differing in Km of the high-affinity potassium uptake system and lacking the low-affinity system were studied in the chemostat under potassium-limited conditions. The results were compared with the results obtained previously (Mulder, M.M., Teixeira de Mattos, M.J., Postma, P.W. and Van Dam, K. (1986) Biochim. Biophys. Acta 851, 223-228) with the wild-type FRAG-1, having two potassium uptake systems, and FRAG-5, a mutant which lacks the high-affinity potassium uptake system. We postulated that the high-affinity potassium uptake system was able to generate such a steep gradient across the membrane that the low-affinity system would act in reverse, thus creating a futile cycle of potassium ions at the cost of energy. As a result, FRAG-1 would show a higher ATP turnover at all growth rates tested than the mutant FRAG-5, in which strain the proposed futile cycle is interrupted because of the lack of the high-affinity system. It is shown here that the results obtained with TK 2240 and TK 2242 are in line with our hypothesis of futile potassium cycling. Under our experimental conditions, the yield on potassium was not dependent on the kinetic parameters of the uptake systems. The (thermodynamic) energy demand of the uptake systems determined the carbon substrate conversion required to achieve this yield.

Adenosine Triphosphate↗

PubMed1987

ATP-driven transhydrogenase provides an example of delocalized chemiosmotic coupling in reconstituted vesicles and in submitochondrial particles.

The mechanism of coupling between mitochondrial ATPase (EC 3.6.1.3) and nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) was studied in reconstituted liposomes containing both purified enzymes and compared with their behavior in submitochondrial particles. In order to investigate the mode of coupling between the transhydrogenase and the ATPase by the double-inhibitor and inhibitor-uncoupler methods, suitable inhibitors of transhydrogenase and ATPase were selected. Phenylarsine oxide and A3'-O-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)-NAD+ were used as transhydrogenase inhibitors, whereas of the various ATPase inhibitors tested aurovertin was found to be the most convenient. The inhibition of the ATP-driven transhydrogenase activity was proportional to the inhibition of both the ATPase and the transhydrogenase. Inhibitor-uncoupler titrations showed an increased sensitivity of the coupled reaction towards carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)--an uncoupler that preferentially uncouples localized interactions, according to Herweijer et al. (Biochim. Biophys. Acta 849 (1986) 276-287)--when the primary pump was partially inhibited. However, when the secondary pump was partially inhibited the sensitivity towards FCCP remained unchanged. Similar results were obtained with submitochondrial particles. These results are in contrast to those obtained previously with the ATP-driven reverse electron flow. In addition, the amount of uncoupler required for uncoupling of the ATP-driven transhydrogenase was found to be similar to that required for the stimulation of the ATPase activity, both in reconstituted vesicles and in submitochondrial particles. Uncoupling of reversed electron flow to NAD+ required much less uncoupler. On the basis of these results, it is proposed that, in agreement with the chemiosmotic model, the interaction between ATPase and transhydrogenase in reconstituted vesicles as well as in submitochondrial particles occurs through the delta mu H+. In contrast, the energy transfer between ATPase and NADH-ubiquinone oxidoreductase appears to occur via a more direct interaction, according to the above-mentioned results by Herweijer et al.

Adenosine Triphosphatases↗

PubMed1987

On the origin of the limited control of mitochondrial respiration by the adenine nucleotide translocator.

A thermodynamic control theory previously developed has been applied to mitochondrial oxidative phosphorylation with emphasis on the role of delta microH and coupling and within the paradigm of delocalized chemiosmotic coupling. The basis for the observed distribution of flux control over the participating enzymes is shown to lie in the relative magnitudes of so-called delta microH elasticity coefficients, i.e., the delta microH dependencies of the different mitochondrial processes. In particular the relatively strong delta microH dependence of mitochondrial respiration is responsible for the significant role of the adenine nucleotide translocator in the control of oxidative phosphorylation. Uncoupling decreases the control exerted by this translocator on respiration but increases that exerted on phosphorylation.

Adenosine Diphosphate↗

PubMed1987

Energetics of glucose uptake in Salmonella typhimurium.

We have studied the energetics of glucose uptake in Salmonella typhimurium. Strain PP418 transports glucose via the phosphoenolpyruvate: glucose phosphotransferase system, while strain PP1705 lacks this system and can only use the galactose permease for glucose uptake. These two strains were cultured anaerobically in glucose-limited chemostats. Both strains produced ethanol and acetate in equimolar amounts but a significant difference was observed in the molar growth yield on glucose (YGlc). It is suggested that this difference is due to a difference in the energetics of the glucose uptake systems in the two strains. Assuming an equal YATP for both strains, we could calculate that uptake of 1 mole of glucose via the galactose permease consumes the equivalent of 0.5 mole of ATP. With the additional assumption that one proton is transported in symport with one glucose molecule, these results imply a stoichiometry of two protons per ATP hydrolysed.

Adenosine Triphosphate↗

PubMed1986

Energetic consequences of multiple K+ uptake systems in Escherichia coli.

The energetics of growth of Escherichia coli FRAG 1 under potassium-limited growth conditions and with glucose as sole carbon and energy source were studied in the chemostat and compared with those of a mutant, FRAG 5, defective in the high-affinity potassium uptake system. The steady-state concentration of biomass decreased with increasing growth rate and was the same in both parent and mutant. For each growth rate, the rate of production of ATP was higher in the parent than the mutant strain. Under potassium-limited conditions, FRAG 1 has at least two potassium uptake systems, an inducible high-affinity uptake system and a constitutive low-affinity uptake system (Rhoads, D.B., Waters, F.B. and Epstein, W. (1976) J. Gen. Physiol. 67, 325-341). Apparently, the presence of the high-affinity uptake system in the parent leads to an energy drain. We suggest that this energy drain is due to futile cycling of potassium ions. On the basis of a mosaic non-equilibrium thermodynamic description of bacterial growth, it is concluded that the growth behaviour under potassium limitation corresponds to that expected for a catabolite limitation.

Adenine Nucleotides↗

PubMed1986

Flow-force relationships in mitochondrial oxidative phosphorylation.

The rates of oxidation and phosphorylation in isolated rat-liver mitochondria have a steep dependence on the protonmotive force (delta mu H+) across the membrane. These experimentally observed relationships proved to be independent of the way in which delta mu H+ was varied. These results were obtained when the membrane potential (delta psi) was calculated from the distribution of K+ (in the presence of valinomycin). When triphenylmethylphosphonium (TPMP+) was used as a probe for delta psi, slightly different flow-force relationships were obtained. We conclude that unique relationships exist between delta mu H+ and the rates of oxidation and phosphorylation, and that under some conditions the behaviour of the probe TPMP+ is anomalous.

Animals↗

PubMed1985

Transport-associated phosphorylation of 2-deoxyglucose in rat adipocytes.

The relationship between ATP levels and 2-deoxyglucose uptake was investigated. When the concentration in the medium lies between 1 and 10 mM 2-deoxyglucose uptake causes a marked decrease in ATP level. This could partly be explained by an inhibiting effect of 2-deoxyglucose and 2-deoxyglucose 6-phosphate on ATP synthesis in the mitochondria. A good correlation between the various ATP levels induced by 2,4-dinitrophenol and the rate of uptake of 5 microM and 0.5 mM (but not 5 mM) 2-deoxyglucose was observed. The addition of glucose and 2-deoxyglucose to cells incubated in the presence of trace amounts of 2-deoxy-[1-14C]glucose induced marked changes in the uptake of the tracer that were associated with a rapid decline in ATP level. It appeared that the phosphorylation of 2-deoxyglucose is an important step in the uptake of the sugar. It is hypothesized that the processes of transport and phosphorylation of 2-deoxyglucose are coupled in rat adipocytes.

2,4-Dinitrophenol↗

PubMed1985

The phosphate potential maintained by mitochondria in State 4 is proportional to the proton-motive force.

Evidence is presented for a proportional relationship between the extramitochondrial phosphate potential (delta Gexp) and the proton-motive force (delta mu H+) across the mitochondrial membrane in rat-liver mitochondria oxidising succinate in State 4, when delta mu H+ is varied by addition of uncouplers or malonate. This relationship was found when precautions were taken to minimise interference with the determination of delta Gexp and delta mu H+ by intramitochondrial nucleotides, adenylate kinase activity, the quenching method, and delta mu H+-dependent changes in matrix volume. A non-proportional delta Gexp/delta mu H+ relationship was obtained when these precautions were omitted. Our results do not support mosaic protonic coupling, but are not necessarily in conflict with other localised coupling schemes.

Adenine Nucleotides↗

PubMed1984

The effects of 1-methyl-3-isobutylxanthine on insulin-sensitive 2-deoxyglucose transport.

The effect of 1-methyl-3-isobutylxanthine on insulin-sensitive 2-deoxyglucose uptake in rat adipocytes was studied. 1-Methyl-3-isobutylxanthine inhibited 2-deoxyglucose uptake rate substantially in both the absence and presence of insulin. The lag-time for the effect of insulin on 2-deoxyglucose uptake was prolonged. At the same time 1-methyl-3-isobutylxanthine caused a decrease in ATP levels. From experiments with isolated rat liver mitochondria it appeared that 1-methyl-3-isobutylxanthine inhibits glutamate plus malate oxidation in State 3.

1-Methyl-3-isobutylxanthine↗

PubMed1983

Progressive poliodystrophy (Alpers' disease) with a defect in cytochrome aa3 in muscle: a report of two unrelated patients.

We present two unrelated patients, a boy and a girl, with a progressive neurologic disorder, characterized by psychomotor retardation, seizures and paresis, the illness being exacerbated during stressful periods. Lactate levels in serum and cerebrospinal fluid were elevated in both patients. Histopathologic studies of muscle tissue revealed mitochondrial abnormalities in the boy; in the girl, slight neuronal degeneration was observed. A cerebral biopsy in the girl showed abnormalities compatible with progressive poliodystrophy. Autopsy in the boy demonstrated progressive poliodystrophy. Biochemical studies in muscle tissue showed a defect of cytochrome aa3 in both patients, connected with a defect of cytochrome b in the girl. The association of defective pyruvate metabolism and progressive poliodystrophy is discussed.

Atrophy↗