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W D Stein

Publications and source records attributed to W D Stein.

At least 73 records · Page 4Linked to original sources
PubMed1987

New permeability pathways induced by the malarial parasite in the membrane of its host erythrocyte: potential routes for targeting of drugs into infected cells.

Malarial parasites propagate asexually inside the erythrocytes of their vertebrate host. Six hours after invasion, the permeability of the host cell membrane to anions and small nonelectrolytes starts to increase and reaches its peak as the parasite matures. This increased permeability differs from the native transport systems of the normal erythrocyte in its solute selectivity pattern, its enthalpy of activation and its susceptibility to inhibitors, suggesting the appearance of new transport pathways. A biophysical analysis of the permeability data indicates that the selectivity barrier discriminates between permeants according to their hydrogen bonding capacity and has solubilization properties compared to those of iso-butanol. The new permeability pathways could result from structural defects caused in the host cell membrane by the insertion of parasite-derived polypeptides. It is suggested that the unique transport properties of the new pathways be used to target drugs into infected cells, to affect the parasite either directly or through the modulation of the intraerythrocytic environment. The feasibility of drug targeting is demonstrated in in vitro cultures of the human malarial parasite Plasmodium falciparum.

Animals↗
PubMed1987

The effect of membrane potential on the mammalian sodium-potassium pump reconstituted into phospholipid vesicles.

1. We have studied effects of electrical diffusion potentials on active Na+-K+ exchange in phospholipid vesicles reconstituted with pig kidney Na+, K+-ATPase. 2. Diffusion potentials, negative inside, were established using outwardly directed K+ gradients plus valinomycin or Li+ gradients plus a Li+ ionophore, AS701. Measurement of fluorescence changes of the carbocyanine dye DiS-C3-(5) showed that the ionophores generated potentials of the expected orientation and of sufficient stability for their effects on active transport to be assessed. Measurement of rates of passive 22Na+ fluxes, over a wide range of diffusion potentials, were consistent with the quantitative predictions of the constant-field flux equation. This result demonstrates that values of diffusion potentials calculated from the Nernst or constant-field equation are accurate. 3. In some conditions, the inside-negative potential (-130 to -180 mV) accelerated the rate of ATP-dependent Na+-K+ exchange on inside-out-oriented pumps, compared to 'control' without the ionophores. Reduction in the size of the diffusion potentials by addition to the medium of Li+ with AS701 or Cs+ with the valinomycin progressively annulled the acceleratory effects, consistent with these being true effects of a change in membrane potentials. 4. At saturating cytoplasmic Na+ and ATP concentrations, the diffusion potential accelerated ATP-dependent Na+-K+ exchange by up to about 30% compared to control but this effect disappeared at rate-limiting ATP concentrations (approximately 1 microM). 5. Using prior knowledge of rate-limiting steps, we interpret this finding to mean that the conformational transition E2(2K)----E12K associated with transport of two K+ ions is voltage insensitive while E1P(3Na)----E2P3Na associated with transport of three Na+ ions is voltage sensitive. The simplest explanation is that the net charge in the transport domain of the protein when no ions, 2K+ or 3Na+ are bound is -2, 0 and +1 respectively. 6. The accelerating effect of the negative-inside diffusion potential on Na+-K+ exchange is greater at limitingly low cytoplasmic Na+ concentrations than at saturating cytoplasmic Na+ concentrations. Cytoplasmic Na+ activation curves show that the diffusion potential increases the apparent cytoplasmic Na+ affinity and reduces the sigmoidicity of cytoplasmic Na+ activation. 7. A kinetic analysis reveals that this effect on apparent affinity is due to an increase in intrinsic Na+ binding and occurs in addition to the effect on a transport rate constant.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1986

Quantitative relationships between the measurable transport resistances of membrane carriers: theory and experiment.

A theoretical analysis is made of the possible quantitative relationships between the transport resistances that characterise membrane carrier systems. It is shown that there exist only five possible patterns in which to rank the four transport resistances. Symbolising these as A, B, C and D, the five possible patterns are (i) A = B = C = D; (ii) A = B much greater than C, D; (iii) A = B much greater than C = D; (iv) A = B = 2C = 2D; (v) A = 2B = 2C much greater than D. A survey of the available experimental data shows that pattern (ii) is the most prevalent, pattern (v) is often found and pattern (iii) has been identified. None of the ten transport systems so far analysed experimentally failed to fit one of the predicted patterns.

Biological Transport↗
PubMed1986

Intrinsic, apparent, and effective affinities of co- and countertransport systems.

Solutions to kinetic schemes for the simple carrier, the countertransporter (antiport, exchanger), and the rapid equilibrium cases of the cotransporter (symport) and co-chemiporter (cation-dependent ATPase) are listed. A distinction is made between the intrinsic, apparent, and effective affinities of the transporters for their substrates. Effective pumping requires that the active transporter binds the pumped substrate, at high affinity, realized at the "whence side" (from which pumping takes place) and, at low affinity, at the "whither side" (to which pumping takes place). It is demonstrated how effective pumping might be achieved by appropriate design of the transporter or chemiporter in terms of the energies of the intrinsic binding sites, the energies of the conformation changes that the pump protein undergoes, the dissociation constant of the chemical reaction that drives the co-chemiport, and the order of binding of the cosubstrates, appropriate at different prevailing levels of the driving substrate.

Adenosine Triphosphatases↗
PubMed1986

An analysis of intestinal calcium transport across the rat intestine.

Kinetic analysis of transmural calcium transport, as evaluated by in situ intestinal loops, has confirmed the existence of two transport processes, a saturable, transcellular one that is regulated by vitamin D and predominates in the proximal intestine and a nonsaturable process similar in intensity throughout the intestine. Transport data obtained from everted sac experiments are kinetically consistent with events in the in situ loop. Analysis of the three component steps making up the saturable process, i.e., entry across the brush-border membrane, intracellular diffusion, and extrusion across the basolateral membrane, indicates that intracellular diffusion is likely to be the limiting step. Active calcium transport varies directly and proportionately with the content of calcium-binding protein (CaBP), a specific molecular expression of the action of vitamin D. Since CaBP is a cytosolic protein, it may act to facilitate calcium diffusion, a proposition advanced by Kretsinger, Mann, and Simmons and supported here quantitatively. We calculate that the rate of intracellular calcium diffusion in the absence of CaBP is only approximately 1/70 of what is found in the vitamin D-replete cell. Similar considerations have led to the proposal that calcium moved by the nonsaturable process travels largely via the paracellular route. The kinetic parameters derived here, i.e., Vm = 22 mumol X h-1 X g (wt wt-1, Km = 3.9 mM, and a nonsaturable rate of 0.16/h, can be used to predict calcium absorption data as determined in previously published balance experiments.

Aging↗
PubMed1985

Cation activation of the pig kidney sodium pump: transmembrane allosteric effects of sodium.

We have studied activation by Na or Rb ions of different transport modes of the Na-K pump, using phospholipid vesicles reconstituted with pig kidney Na-K-ATPase. The shape of the activation curves, sigmoid or quasi-hyperbolic, depends on the nature of the cation at the opposite surface and not on the specific mode of transport. ATP-dependent Na uptake into K-containing vesicles (Na-K exchange) is activated by cytoplasmic Na along a highly sigmoid curve in the absence of extracellular Na (Hill number, nH = 1.9). Activation displays progressively less-sigmoid curves as extracellular Na is raised to 150 mM (nH = 1.2). The maximal rate of the Na-K exchange is not affected. Na is not transported from the extracellular face by the pump in the presence of excess extracellular K, and the transmembrane effects of the extracellular Na are therefore 'allosteric' in nature. ATP-dependent Na-Na exchange (Lee & Blostein, 1980) and classical ATP-plus-ADP-dependent Na-Na exchange are activated by cytoplasmic Na along hyperbolic curves. ATP-dependent Na uptake into Tris-containing vesicles is activated by cytoplasmic Na along a somewhat sigmoidal curve. (ATP + Pi)-dependent Rb-Rb exchange is activated by cytoplasmic and extracellular Rb along strictly hyperbolic curves. The same applies for Rb-Rb exchange in the presence or absence of ATP or Pi alone. The presence of a high concentration of extracellular Na together with extracellular Rb induces a sigmoidal activation by cytoplasmic Rb of (ATP + Pi)-dependent Rb-Rb exchange (nH = 1.45) but does not affect the maximal rate of exchange. Slow passive Rb fluxes through the pump observed in the absence of other pump ligands (see Karlish & Stein, 1982 alpha) are activated by cytoplasmic Rb along a strictly hyperbolic curve with extracellular Rb, nH = 1.0 (Rb-Rb exchange), along a strongly sigmoid curve with extracellular Na, nH = 1.5 (Rb-Na exchange), and along less-sigmoid curves with extracellular Tris, nH = 1.24 (net Rb flux) or extracellular Li, nH = 1.2 (Rb-Li exchange). Activation of the passive Rb fluxes by extracellular Rb is hyperbolic in the presence of cytoplasmic Rb, Li or Tris but is sigmoid in the presence of cytoplasmic Na (nH = 1.36). Inhibition by cytoplasmic Na of passive Rb fluxes from the cytoplasmic to the extracellular face of the pump depends on the nature of the cation at the extracellular surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1984

Kinetics of in vitro ageing of mouse embryo fibroblasts.

The kinetics of ageing of normal mouse embryo fibroblast cells in culture have been determined. The growth pattern during every passage was established. It was observed that the growth pattern was not exponential, but that the growth rate declined progressively both within and with every passage. We also estimated the cell cycle parameters using the Fraction of Labelled Mitoses method at every passage. We found that the cell cycle duration was constant throughout the lifespan of this cell strain; the median value of the cell cycle duration was found to be 15.5 +/- 0.5 h (S.D., n = 8). From these two sets of observations we infer that the fraction of dividing cells declines smoothly from the beginning of the culture. Our data exclude quite positively any description of ageing of the fibroblast population in terms of a catastrophe or any abrupt change in the population. Our data are also inconsistent with a linear decline in growth fraction. On the contrary, we observed that there was a gradual and smooth decline in the growth rate of the strain, due to a smoothly declining growth fraction. This smooth change in the growth behaviour of this cell strain is accurately described by the mortalization theory of Shall & Stein in which the single parameter gamma (gamma), describes the change in reproductive potential over the entire lifespan. The parameter gamma describes the rate at which the doubling time of the culture increases. It is the number of generations at which half of the newborn cells are themselves reproductively sterile. Our present data provided an estimate of gamma for this cell strain, which was consistent during the entire lifespan of the strain; the best estimate of gamma for this cell strain was 20.3 +/- 0.6 generations (S.D., n = 19).

Animals↗
PubMed1983

Multiple controls on the intracellular trapping of uridine.

Uridine uptake by mouse or hamster cells grown in conditions which support good growth is very sensitive to inhibition by cyanide and azide, at concentrations which only slightly reduce overall cellular ATP levels. Iodoacetate, when present alone, reduces uridine uptake only insofar as it reduces cellular ATP levels. At concentrations which by themselves do not affect uridine uptake, iodoacetate greatly reduces the sensitivity of uridine uptake to cyanide or azide. The effect of cyanide is on intracellular trapping of uridine and not on its transport into the cell. The specific effect of cyanide is confined to uridine and not found for the uptake of adenine, thymidine or 2-deoxyglucose. The effect is of rapid onset (within 2 min) and is rapidly reversible (also within 2 min). Phosphorylation of uridine in homogenised cells or in Triton X-100-permeabilised cells is unaffected by cyanide. The data are interpreted in terms of a model in which intracellular trapping of uridine is subject to multiple controls, including one regulated by some factor requiring intact functioning of the mitochondrion. These multiple control systems interact synergistically to affect trapping of uridine by the intact cell.

Adenosine Triphosphate↗
PubMed1983

Kinetic tests of models for sugar transport in human erythrocytes and a comparison of fresh and cold-stored cells.

We studied the time course of the entry of galactose into human erythrocytes from an external concentration of 500 mM, and analyzed the data by an integrated rate equation treatment. We found evidence for only a single, high-affinity site for sugar at the inner face of the membrane. We studied the effect of pre-loading cells with galactose at various concentrations on the entrance of galactose into the cell from 128 mM, and compared the result we found with a previous report of a similar experiment from 500 mM external sugar. We found no evidence of other than a high affinity for sugar at the inner face of the membrane. The data reject a model in which sugar transport occurs on two asymmetric, oppositely directed carriers. We studied exchange of glucose into and out of the cells as a function of sugar concentration, taking care to minimize metabolism of sugar. We found no evidence for other than a single component for glucose exchange. Our data reject the 'allosteric pore' model for sugar transport. The explanation of the high-affinity site for sugar at the inner membrane face thus remains enigmatic. We find a very significant difference in the kinetics of glucose exchange when we compare freshly drawn and long cold-stored blood. The Km for exchange was almost twice as large for cold-stored as for fresh blood.

3-O-Methylglucose↗
PubMed1983

A quantitative analysis of the aging of human glial cells in culture.

The kinetics of aging of normal human diploid brain cells in culture have been determined using the miniclone technique in which cells are cloned in the presence of a large number of other cells. The miniclone technique records the behaviour of every viable cell in the sample, not merely those cells capable of forming visible clones. This technique permits the direct measurement of the reproductive potential of individual cells growing in bulk culture and of the dispersion of the sizes of colonies generated by dividing cells. The fraction of cells that are able to divide declines smoothly and continuously from the beginning of in vitro cultures of human glial cells. There is a broad distribution of colony sizes; even at the earliest passages there are significant numbers of small colonies. With increasing age of the culture there is a shift in the distribution, so that fewer large colonies and more small colonies occur. The distribution of intermitotic times is almost identical in young and middle-aged cultures. Our data seem to exclude quite positively any description in terms of a catastrophe or any abrupt change in the population. On the contrary, the decline in reproductive potential may be described adequately either as a linear change with time, or as predicted by the mortality theory of Shall and Stein (1979), in which the single constant, gamma, describes the change in reproductive potential over the entire lifetime.

Adult↗
PubMed1982

Passive rubidium fluxes mediated by Na-K-ATPase reconstituted into phospholipid vesicles when ATP- and phosphate-free.

1. Phospholipid vesicles reconstituted with Na-K-ATPase from pig kidney, show slow passive pump-mediated (86)Rb fluxes in the complete absence of ATP and phosphate.2. The Rb fluxes are inhibited in vesicles prepared from enzyme pre-treated with either ouabain or vanadate ions. Rb fluxes through Na-K pumps oriented inside-out or right-side out by comparison with the normal cellular orientation can be distinguished by effects of vanadate on one or both sides of the vesicle.3. (86)Rb uptake into Rb-loaded vesicles represents a (86)Rb-Rb exchange. The maximal rate of exchange through inside-out and right-side out oriented pumps is equal, suggesting a random arrangement of the pumps across the vesicle membrane. This Rb-Rb exchange is half-saturated on inside-out and right-side out pumps at about 0.6 and 0.2 mM-external Rb respectively.4. (86)Rb uptake into Rb-free vesicles represents a net Rb flux. The Rb uptake through inside-out pumps has a maximal rate about equal to the Rb-Rb exchange, half-saturates at an external Rb concentration of roughly 0.5 mM, and shows evidence for co-operativity. Net Rb uptake through right-side out pumps is very slow, and half-saturates at roughly 0.1 mM external Rb.5. K ions at low concentrations in the exterior medium stimulate (86)Rb uptake, but at high concentrations, inhibit. Na ions in the exterior medium always inhibit (86)Rb uptake. The result suggests that K ions are transported in co-operative fashion together with Rb ions, while Na ions block the Rb fluxes.6. The presence of Rb congeners at the vesicle interior raises the (86)Rb uptake through inside-out pumps with the decreasing order of effectiveness: Li > Na > Cs > K > Rb. Stimulation by Na ions involves a Rb-Na exchange.7. Turnover numbers were estimated from parallel measurement of Na/K pump mediated fluxes and amount of covalent phosphoenzyme. In units of moles of ion per mole of phosphoenzyme per second at 20 degrees C the following values were obtained: ATP-dependent Na-Rb exchange, 43; (ATP+phosphate)-stimulated Rb-Rb exchange, 7. For (ATP+phosphate)-independent fluxes: Rb-Rb exchange 0.25; net Rb uptake 0.15 and Rb-Na exchange 0.65.8. Mg ions in the exterior medium inhibited both net and exchange Rb fluxes through inside-out pumps in a manner antagonistic with respect to Rb. Mg and vanadate ions inhibit the Rb fluxes in a synergistic fashion.9. The results are interpreted in terms of a model in which net and exchange (86)Rb fluxes occur via conformational transitions between form E(1) which binds Rb at the cytoplasmic face of the protein, the form E(2) (Rb)(occ) containing occluded Rb ions and a form E(2) which binds Rb at the extracellular face of the protein. A kinetic analysis allows us to identify rate-limiting steps of the transport cycle by making use of our transport data in combination with values of rate-constants for conformational transitions observed directly in isolated Na-K-ATPase.

Animals↗