Biomedical subjects

H V Westerhoff

Publications and source records attributed to H V Westerhoff.

At least 127 records · Page 7Linked to original sources

PubMed1994

Rate limitation within a single enzyme is directly related to enzyme intermediate levels.

The extents to which different rate constants limit the steady-state rate of an isolated enzyme can be quantified as the control coefficients of those constants and elemental steps. We have found that the sum of the control coefficients of rate constants characterising unidirectional rates depleting a particular enzyme intermediate is equal to the concentration of that enzyme intermediate as a fraction of the total enzyme concentration. Together with simple measurements this powerful relation may be used (i) to estimate certain enzyme intermediate levels, in particular the free enzyme concentration, and (ii) to estimate the control coefficients of rate constants and steps.

Enzymes↗

PubMed1994

Cooperative P-glycoprotein mediated daunorubicin transport into DNA-loaded plasma membrane vesicles.

Most of the multidrug resistant human tumor cell lines overexpress the MDR1 gene product P-glycoprotein (P-gp) which is believed to function as an energy-dependent drug efflux pump. Here we describe a novel method that allows the kinetic characterization of P-gp-mediated active drug transport. This method is based on the fluorescence quenching of anthracyclines transported into DNA-loaded plasma membrane vesicles. The uptake of daunorubicin (DNR) into the plasma membrane vesicles was saturable in terms of the extravesicular DNR concentration with a Km of 1.5 +/- 0.1 microM. This transport occurred by a cooperative process with a Hill coefficient close to 2 for DNR. A model is discussed in which P-gp pumps two molecules of drug per turnover.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Magainin oligomers reversibly dissipate delta microH+ in cytochrome oxidase liposomes.

Magainin peptides present in the skin of Xenopus laevis and identified as antimicrobial agents are shown to decrease the membrane potential in cytochrome oxidase liposomes. They also released respiratory control with a third or higher order concentration dependence. Respiratory control was restored by proteolytic digestion of the added magainin. The amount of magainin required for half-maximal stimulation of respiration was proportional to lipid concentration. At appreciably higher concentrations magainins inhibited uncoupled respiration. The results are discussed in terms of a model in which most of the added magainin adsorbs as a monomer to the membranes but equilibrates with a multimeric pore that causes rather general permeability of membranes. The ensuing ion permeation dissipates membrane potential and stimulates respiration.

Animals↗

PubMed1994

The multidrug-resistance-reverser verapamil interferes with cellular P-glycoprotein-mediated pumping of daunorubicin as a non-competing substrate.

We examined P-glycoprotein-mediated verapamil transport, using two drug-sensitive and multi-drug resistant cell-line couples, i.e. A2780, 2780AD and SW-1573, SW-1573/1R500. The interaction of 3H-labeled verapamil with cells was measured using a flow-through system. The verapamil-containing medium was pumped over the cells and monitored on-line for radioactivity. In the P-glycoprotein-expressing cells, verapamil accumulation was increased by vinblastine and some known multidrug resistant (MDR) modifiers. Subsequent removal of these modifiers caused release of verapamil into the medium against a verapamil concentration gradient. In this manner, we obtained evidence that verapamil is actively transported by the MDR-related P-glycoprotein. Using the flow-through system, we also exposed the cells to flowing culture medium containing daunorubicin, and measured the inhibition of daunorubicin efflux by verapamil. We found that, although the active efflux of daunorubicin was maximally blocked by verapamil short-term, longer-term active efflux of daunorubicin resumed. At a daunorubicin concentration in the flowing medium of 5 microM, increasing the verapamil concentration resulted in the same short-term effects, but in a significantly longer period of a maximal inhibition of daunorubicin efflux from the cells. At a daunorubicin concentration of 20 microM, increasing the verapamil concentration affected neither the short-term nor the long-term effects. These and other observations are in agreement with a model in which daunorubicin and verapamil are non-competing substrates for P-glycoprotein. In conclusion, we obtained evidence that verapamil is actively transported by the MDR-related P-glycoprotein and that verapamil and daunorubicin are non-competing substrates for P-glycoprotein. Consequently, the effectiveness of verapamil as an MDR antagonist may be compromised because it is extruded by P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Yeast cells with a specific cellular make-up and an environment that removes acetaldehyde are prone to sustained glycolytic oscillations.

Glycolytic oscillations can be induced by adding glucose to starved Saccharomyces cerevisiae cells and, after a steady state has been established, cyanide. Transient oscillations or limit-cycle oscillations can be induced depending on the growth phase in which the cells are harvested. To find what causes these differences in the dynamic behaviour, we analyzed glycolytic enzyme activities at different growth phases. The hexokinase activity increased by a factor of three after growth substrate transition from glucose to ethanol; the other measured activities remained constant. Cyanide was found not only to block respiration, but also to trap acetaldehyde. Both cyanide actions appear necessary for the occurrence of sustained glycolytic oscillations.

Acetaldehyde↗

PubMed1994

Getting to the inside of cells using metabolic control analysis.

Metabolic control analysis can relate control properties of an intact system to kinetic properties (elasticity coefficients) of the enzymes within that system. The method formulating the former as matrix inverse of the latter is elaborated here for the general case and founded in standard metabolic control theory. Then a method is developed that accomplishes the reverse: it is shown that a matrix containing all elasticity coefficients and information concerning the pathway structure equals the inverse of a matrix containing flux and concentration control coefficients. As a consequence, by measuring the control properties of an intact system, one is able to deduce its in situ pathway structure and enzyme kinetic properties: This solves the ever-present question of whether the kinetic properties of enzymes in their isolated state differ from those under the conditions prevailing in the cell.

Cells↗

PubMed1994

How to determine control of growth rate in a chemostat. Using metabolic control analysis to resolve the paradox.

The chemostat makes it possible to study microbial physiology at steady state. However, because growth rate in a chemostat is set by the experimenter, it seems impossible to employ the chemostat to study the control of microbial growth by processes within the microorganism. In this paper we show how, paradoxically, one can determine control of growth rate, of growth yield and of other fluxes in a chemostat. We develop metabolic control analysis for the chemostat. This analysis does not depend on the particular way in which specific growth rate varies with the concentration of the growth limiting substrate.

Bacteria↗

PubMed1993

Dramatic changes in control properties that accompany channelling and metabolite sequestration.

A simple summation theorem describes the control of fluxes in 'ideal' metabolic pathways. This paper shows how this theorem and the control properties of a pathway change when direct transfer of intermediates and/or sequestration of metabolites involved in moiety conservations (by enzymes present at high concentrations) take place. The derived generalized summation theorem quantifies the extent to which metabolite sequestration decreases and direct metabolite transfer can increase the control exerted by enzymes on the flux. The implications of metabolite channelling for the control of fluxes are discussed quantitatively.

Enzymes↗

PubMed1993

A plasma membrane 'vacuum cleaner' for daunorubicin in non-P-glycoprotein multidrug-resistant SW-1573 human non-small cell lung carcinoma cells. A study using fluorescence resonance energy transfer.

A multidrug resistant (MDR) human non-small cell lung carcinoma cell line, SW-1573/2R120 (2R120), not containing the drug-efflux pump P-glycoprotein (PgP), has been studied for the transport of daunorubicin (DN) across the cellular plasma membrane. Earlier, reduced initial DN-uptake rates and lower cellular DN steady-state concentrations were found for this cell line, when it was compared to the SW-1573 wild-type cell line. This finding was an indication for the presence of another cellular drug-efflux pump. However, we found similar DN-efflux rates in drug-free medium for the two cell lines, while for Pgp-containing MDR SW-1573/2R160 (2R160) cells the efflux rate was increased compared to wild-type cells. In order to elucidate differences in DN transport across the cellular plasma membrane, the association of DN with plasma membranes of intact cells was investigated, using fluorescence-resonance-energy transfer. For this purpose, the plasma-membrane probe 1-(4-trimethyl-ammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was chosen since, because of the overlap between the emission spectrum of TMA-DPH and the excitation spectrum of DN, transfer of energy can be achieved from TMA-DPH to DN. Cells were loaded with TMA-DPH and, after addition of 10 microM DN, the TMA-DPH fluorescence was quenched. Rapid initial quenching proved to be similar in the MDR 2R160 (Pgp-containing) cells and in the SW-1573 wild-type cells (21 +/- 1% and 20 +/- 2%, respectively), but was less in the MDR 2R120 cells not containing Pgp (14 +/- 1%). This finding correlated with a lowered amount of DN dissolved in the plasma membrane of 2R120 cells. We interpret these data to be the result of a 'vacuum-cleaner' pumping system other than Pgp which removes DN from a plasma membrane compartment and equilibrates relatively slowly with the interior of the cell.

Biological Transport↗

PubMed1993

Movement of fatty acids, fatty acid analogues, and bile acids across phospholipid bilayers.

How lipophilic acids move across membranes, either model or biological, is the subject of controversy. We describe experiments which better define the mechanism and rates in protein-free phospholipid bilayers. The transbilayer movement of lipophilic acids [fatty acids (FA), covalently-labeled FA, bile acids, and retinoic acid] was monitored by entrapping pyranin, a water-soluble, pH-sensitive fluorescent molecule to measure pH inside unilamellar vesicles [Kamp, F., & Hamilton, J.A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11367-11370]. Equations for the pseudo-unimolecular rate constants for transbilayer movement of un-ionized (kappa FAH) and ionized (kappa FA-) acids are derived. All FA studied (octanoic, lauric, myristic, palmitic, stearic, oleic, elaidic, linoleic, linolelaidic, and arachidonic) and retinoic acid exhibited rapid transbilayer movement (t 1/2 < 1 s) via the un-ionized form across small unilamellar egg phosphatidylcholine (PC) vesicles. FA produced by phospholipase A2 in the outer leaflet of PC vesicles equilibrated rapidly to the inner leaflet. Ionized FA showed enhanced transbilayer movement (kappa FA- = 0.029 s-1) in the presence of equimolar valinomycin. The three FA analogues [12-(9-anthroyloxy)stearic acid, 5-doxylstearic acid, and 1-pyrenenonanoic acid] moved across PC bilayers via the un-ionized form; except for the anthroyloxy FA (kappa FAH = 4.8 x 10(-3) s-1), the rates were too fast to measure (t 1/2 < 1 s). The rate for cholic acid (CA) transbilayer movement was slow (kappa CAH = 0.056 s-1) compared to that of the more hydrophobic bile acids, deoxy- and chenodeoxycholic acid (t 1/2 < 1 s). The taurine conjugates of the three bile acids did not cross the bilayer (t 1/2 > 1 h). A further application of the pyranin method was to measure the partitioning of FA and bile acids among water, albumin, and PC vesicles. Our results show that the ability of lipophilic acids to permeate a PC bilayer rapidly is dependent on the presence of the un-ionized acid in the membrane interface. Considering the fast unfacilitated movement of FA across protein-free phospholipid bilayers, it is unlikely that there is a universal need for a transport protein to enhance movement of FA across membrane bilayers. Physiological implications of proton movement accompanying fast movement of un-ionized lipophilic acids (and the consequent generation of a pH gradient) are discussed.

Bile Acids and Salts↗

PubMed1993

Control analysis of the dependence of Escherichia coli physiology on the H(+)-ATPase.

The H(+)-ATPase plays a central role in Escherichia coli free-energy transduction and hence in E. coli physiology. We here investigate the extent to which this enzyme also controls the growth rate, growth yield, and respiratory rate of E. coli. We modulate the expression of the atp operon and determine the effect on said properties. When quantified in terms of control coefficients, we find that, in the wild-type cell growing on glucose in minimal medium, this key enzyme (H(+)-ATPase) exerts virtually no control on growth rate (magnitude of C < 0.01), a minor positive control on growth yield (C = 0.15), and a small but negative control on respiration rate (C = -0.25). The control the enzyme exerts on the consumption rate of the carbon and free-energy substrate is negative (C = -0.15). We also studied how the control coefficients themselves vary with the expression of the atp operon. As the level of expression of the atp operon was reduced, the control exerted by the H(+)-ATPase on growth rate and growth yield increased slightly; the control on growth rate passed through a maximum (C = 0.1) and disappeared when the atp operon was not expressed at all, reflecting that with this substrate there are alternative routes for ATP synthesis. At elevated levels of the H(+)-ATPase compared to the wild type, the control exerted by the enzyme on growth rate became negative. The evolutionary context of the absence of control by the atp operon on growth rate is discussed.

Cell Division↗

PubMed1993

Electric potentiation, cooperativity, and synergism of magainin peptides in protein-free liposomes.

Magainins, positively charged peptides present in the skin of Xenopus laevis, are known to permeabilize free-energy transducing membranes. Structural studies in otherwise protein-free model systems show alpha-helical magainins parallel to the membrane water interface. However, functional studies in biological membranes suggest that magainins operate as oligomeric complexes. Here we investigate whether magainins function as oligomers in protein-free liposomes also. We report that they do exhibit strong positive heterocooperativity. The magainins, magainin 2 and PGLa, act synergistically. Both activity and cooperativity are enhanced by net negative charge of the liposomal membranes. A transmembrane electric potential, negative inside, enhanced the activity of the peptides. We propose a model in which (i) binding to the surface of the membrane, mainly guided by electrostatic interactions, occurs and (ii) the bound form is in equilibrium with an n-meric complex of magainins spanning the membrane.

Amino Acid Sequence↗

PubMed1993

Metabolic channelling and control of the flux.

Metabolic control theory is extended to include channelled metabolism in general. A simple relationship between the flux control by the enzymes and the degree of metabolite channelling is derived. This relationship suggests experiments in which modulation of gene expression allows one to quantify channelling.

Enzymes↗

PubMed1993

'Channelled' pathways can be more sensitive to specific regulatory signals.

In 'simple' metabolic pathways the response to an external signal is readily described in terms of the effect of the signal on its receptor enzyme and the control exerted by that enzyme. We show here that in the response of 'channelled' pathways to such a signal, additional terms appear that reflect the direct enzyme-enzyme interactions. They tend to enhance the responsiveness of the pathway. The normalized value of the response is called the signal transduction coefficient. We show that in channelled pathways these coefficients are usually larger than in corresponding non-channelled (simple) pathways.

Enzymes↗

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

The use of lac-type promoters in control analysis.

For control analysis, it is necessary to modulate the activity of an enzyme around its normal level and measure the changes in steady-state fluxes or concentrations. We describe an improved method for effecting the modulation, as elaborated for Escherichia coli. The chromosomal gene, encoding the enzyme of interest, is put under the control of a lacUV5 or a tacI promoter. The alternative use of the two promoters leads to an expression range which should make it suitable for the use in control analysis of many enzymes. The lacUV5 promoter should be used when the wild-type expression level is low, the tacI promoter when the latter is high. The endogenous lac operon is placed under the control of a second copy of the lacUV5 promoter and a lacY7am mutation (eliminating lactose permease, the transport system for the inducer isopropyl-thio-beta-D- galactoside) is introduced. The method was demonstrated experimentally by constructing E. coli strains, in which the chromosomal atp operon is transcribed from the lacUV5 and the tacI promoter. We measured the concentration of the c subunit of H(+)-ATPase, and found that the expression of this enzyme could be modulated between non-detectable levels and up to five times the wild-type level. Thus, in the absence of inducer, no expression of atp genes could be detected when the atp operon was controlled by the lacUV5 promoter, and we estimate that the expression was less than 0.0025 times the wild-type level. We show that the introduction of a lacY mutation facilitated the attainment of steady induction levels of partially induced cells. The mutation also reduced positive cooperativity in the dependence of expression on the concentration of isopropyl-thio-beta-D-galactoside (the inducer) and shifted the concentration of inducer needed for half maximum induction to higher values. These properties should facilitate the experimental modulation of the enzyme activity by varying the concentration of the inducer.

Cloning, Molecular↗

PubMed1993

Excess capacity of H(+)-ATPase and inverse respiratory control in Escherichia coli.

With succinate as free-energy source, Escherichia coli generating virtually all ATP by oxidative phosphorylation might be expected heavily to tax its ATP generating capacity. To examine this the H(+)-ATPase (ATP synthase) was modulated over a 30-fold range. Decreasing the amount of H(+)-ATPase reduced the growth rate much less than proportionally; the H(+)-ATPase controlled growth rate by < 10%. This lack of control reflected excess capacity: the rate of ATP synthesis per H(+)-ATPase (the turnover number) increased by 60% when the number of enzymes was decreased by 40%. At 15% H(+)-ATPase, the enzyme became limiting and its turnover was increased even further, due to an increased driving force caused by a reduction in the total flux through the enzymes. At smaller reductions of [H(+)-ATPase] the total flux was not reduced, revealing a second cause for increased turnover number through increased membrane potential: respiration was increased, showing that in E.coli, respiration and ATP synthesis are, in part, inversely coupled. Indeed, growth yield per O2 decreased, suggesting significant leakage or slip at the high respiration rates and membrane potential found at low H(+)-ATPase concentrations, and explaining that growth yield may be increased by activating the H(+)-ATPase.

Adenine Nucleotides↗