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W D Stein

Publications and source records attributed to W D Stein.

At least 91 records · Page 5Linked to original sources
PubMed1982

Effects of atp or phosphate on passive rubidium fluxes mediated by Na-K-ATPase reconstituted into phospholipid vesicles.

1. The passive Rb fluxes mediated by the Na-K pump in reconstituted vesicles, described by Karlish & Stein (1982), are affected by ATP or by phosphate acting separately.2. Rb-Rb exchange through inside-out pumps is stimulated by ATP at low concentrations and is inhibited at high concentrations. There are mutual effects of Rb at cytoplasmic sites and ATP. The higher is the Rb concentration, the greater is the degree of stimulation and the less is the inhibition of exchange by ATP, and the higher are the concentrations of ATP required to produce effects. ATP stimulates Rb-Rb exchange maximally by about 5-fold.3. There are similar effects of ATP on zero-trans net Rb uptake through inside-out pumps. However, much lower degrees of stimulation and greater inhibition of the net flux by ATP are observed, and much lower concentrations of ATP are required for these effects, by comparison with those on Rb-Rb exchange.4. Rb uptake on inside-out pumps in Na-loaded vesicles shows only inhibition by ATP.5. Phosphate effects require the presence of Mg(0) ions. At low Mg(0) concentrations (up to 100 muM) phosphate moderately stimulates Rb uptake into Rb-free or Rb-loaded vesicles (about 50%), but has no effect on Rb uptake into Na-loaded vesicles. At millimolar concentrations of Mg(0) ions, phosphate strongly inhibits the Rb uptake into Rb-free or Na-loaded vesicles but has no effect on Rb uptake into Rb-loaded vesicles.6. The separate effects of ATP and of phosphate are explained in terms of the model proposed by Karlish & Stein (1982), modified to take into account stimulation of the conformational transition E(2)(Rb)(occ) --> E(1) Rb by ATP, and stimulation of the conformational transition E(2)(Rb)(occ) --> E(2) Rb by phosphate due to phosphorylation of the protein.

Adenosine Triphosphate↗
PubMed1982

Combined effects of ATP and phosphate on rubidium exchange mediated by Na-K-ATPase reconstituted into phospholipid vesicles.

1. Phospholipid vesicles reconstituted with Na-K-ATPase show an (ATP+phosphate)-stimulated Rb-Rb exchange, with properties similar to the K-K exchange of human red cells. This includes a rate 15-20% of the rate of active ATP-dependent Na-K exchange.2. We have studied activation of this Rb-Rb exchange by ATP at fixed phosphate concentrations and by phosphate at fixed ATP concentrations. It is found for both ATP and phosphate that with low concentrations of the fixed ligand an increase in concentration of the complementary ligand produces first stimulation and then inhibition of Rb-Rb exchange. At high concentrations of the fixed ligand the complementary ligand shows only saturation behaviour.3. The pattern of activation and of inhibition by ATP and by phosphate is affected by the Rb(0) concentration in the exterior medium, in that higher concentrations of Rb(0) counteract inhibitory effects of high concentrations of ATP and phosphate.4. (ATP+phosphate)-stimulated Rb-Rb exchange is activated by Rb(0) in the exterior medium along a sigmoid curve. An increase of Rb(i) within the vesicles, which raises the maximal velocity of Rb-Rb exchange, is accompanied by a smaller increase in the Rb(0) concentration required for half-maximal stimulation of the Rb-Rb exchange.5. The data are interpreted in terms of a model similar to those proposed by Karlish & Stein (1982a,b), but extended to include simultaneous effects of ATP and phosphate. Inhibitions by high concentration of ATP or phosphate arise as a result of stabilization of E(1) ATP or E(2)-P forms respectively, in the presence of low concentrations of the complementary ligand. With high concentrations of the fixed ligand, saturation behaviour of the varying ligand is observed because the occluded Rb forms become the dominant transport intermediates. The occluded Rb forms bind both ATP and phosphate weakly and independently. The effects of ATP together with phosphate are accounted for by a simple combination of their separate effects on the Rb-Rb exchange.6. We suggest that the functional role of the occluded Rb form E(2) (Rb)(occ) in active transport is to minimize passive cation leaks through the system and allow control of the direction of cation movements by binding of physiological ligands such as ATP or phosphate.

Adenosine Triphosphate↗
PubMed1979

The kinetic dissection of transport from metabolic trapping during substrate uptake by intact cells. Uridine uptake by quiescent and serum-activated Nil 8 hamster cells and their murine sarcoma virus-transformed counterparts.

1. We present a theoretical analysis of the tandem processes of transport and metabolic trapping which together constitute uptake of a substrate by intact cells. 2. Transport is assumed to occur by means of a simple carrier here analysed in its general form. Trapping is assumed to occur by a simple enzymic reaction. 3. We show how to obtain the separate parameters of the steps by analysing uptake data over a range of uptake times and substrate concentrations. 4. We present uptake data for uridine and cytosine-beta-D-arabinoside entering Nil 8 hamster fibroblasts, normal and murine sarcoma virus transformed, in the quiescent condition and after stimulation by added serum. We analyse the data in terms of the theory for tandem processes. 5. Transport is characterised by a system having a high Km and a high V for entry. The data for cytosine-beta-D-arabinoside suggest that the cytosine-beta-D-arabinoside system is not far from a symmetric one. The data for uridine transport do not differ when quiescent and serum-activated cells are compared. Transformed cells transport uridine at half the maximum velocity of normal cells, with or without added serum. 6. Trapping of cytosine-beta-D-arabinoside is insignificant. Trapping of uridine occurs by a system with both V and Km at least an order of magnitude smaller than are these parameters for transport. Trapping of uridine by non-transformed cells activated by serum, has twice the V of such cells in the quiescent state. 7. In the virus-transformed cells, the control of uridine trapping by added serum is lost, along with control of growth by this stimulant.

Animals↗
PubMed1978

Increase in the affinity of the uridine phosphorylation system for ATP after serum or insulin activation of 3T3 fibroblasts.

The stimulation of uridine uptake, brought about by the addition of serum or insulin to quiescent 3T3 fibroblasts, is associated in the half-saturation concentration of the uridine phosphorylating system for the substrate ATP, with relatively little change in the maximum uptake or in the affinity for uridine. In stimulated cells the Km towards ATP fell in the range 0.053--0.187 mM, while V max was 34 to 52 pmoles/10(6) cells/min. In quiescent cells these values were 2.89--4.22 mM and 74.5--126 pmoles/10(6) cells/min, respectively. No difference was found, however, between the Km's for ATP when phosphorylation of uridine was determined using cell-free extracts prepared from either quiescent cells or from stimulated cells.

Adenosine Triphosphate↗
PubMed1978

Diffusion within egg lecithin bilayers resembles that within soft polymers.

An analysis is presented of how the permeability coefficient/octanol:water partition coefficient ratio for 33 different chemical substances crossing egg lecithin bilayers depends on the molecular volume of the substances. From this analysis we conclude that bilayers made from egg lecithin behave as soft polymers in their discrimination between permeants of different sizes and shapes.

Diffusion↗
PubMed1977

How the kinetic parameters of the simple carrier are affected by an applied voltage.

The kinetic parameters (K, R00, R12, R21 and Ree) of the simple carrier model are analysed as a function of applied voltage for various cases of charged substrates and carriers. If certain parameters are invariant with voltage, strong conclusions can be made as to the charge on the free carrier. In one particular case (where the parameter R00 is invariant while R12 and R21 display a non-linear dependence on the exponential of the voltage) it may be possible to identify the isomerisation of the carrier-substrate complex.

Biological Transport, Active↗
PubMed1977

Models for the active transport of cations...the steady-state analysis.

We summarise the progress that has been made in the analysis of active transport models, at the steady-state level. The two general classes of such model, counter-and co-transport, can be treated by a kinetic analysis which makes no assumptions as to the symmetry or asymmetry of the systems nor as to the presence of any particular rate-limiting steps. Precisely the same formalism is obeyed for primary active transport as for secondary active transport. Both are merely a generalisation of facilitated diffusion, in that they follow directly from accepted properties of carrier models. How affinities of such carriers for their substrates affect the efficiency of active transport is discussed and it is shown that in a number of cases, the affinity changes that the carrier demonstrates arise from inherent properties of the free carrier and not from any "high energy" properties of the chemical reactants. Methods of obtaining the kinetic parameters of the system from experimental data are reviewed, together with methods for testing and characterising the different transport models.

Binding Sites↗
PubMed1977

Regulatin of uridine uptake by serum and insulin in density-inhibited 3T3 cells.

Stimulation of nucleoside uptake in quiescent 3T3 cells by insulin and serum is preceded by a substantial lag phase. Our findings point to the length of the lag phase as a major target for regulation. The length of such phase varies markedly with the concentration of insulin (10(-9)-10(-6) M) or serum (0.5-10%) but it is not eliminated by high saturating levels of the activating agents. Further, variations in the temperature at which the stimulation process occurs (24-39 degrees C), addition of compounds like prostaglandin E1 (1-5 mug/ml) or theophylline (0.4 mM) and differences in the age of the cultures primarily affect the length of the lag time while the final uptake rates achieved are remarkably constant. Analysis of the temporal order of the events in the lag phase reveals that there is a discrete temperature-sensitive period located in the early and middle part of the lag, while the prostaglandin E1-sensitive step(s) appear to be toward the end of the lag. The transition from the basal to the stimulated rate of uptake is abrupt. Indeed, the kinetics of activation does not fit a sample exponential law but a high power of an exponential, suggesting that the switching mechanism involves cooperative steps. Since the transition is abrupt, the nucleoside uptake system exists largely in two alternative states either switched off or on. The regulation of the lag period is by the control of the time at which this switch occurs. On the basis of the data presented here, we propose a working hypothesis of uptake stimulation.

Biological Transport, Active↗
PubMed1976

Testing the simple carrier using irreversible inhibitors.

1. We analyse the kinetics of irreversible inhibition of the simple carrier. We consider how the rate of inactivation is influenced by the concentrations of permeant on the two sides of the membrane. 2. We consider various kinetic schemes for the simple carrier and show that these are all indistinguishable kinetically, using steady-state transport or inactivation data. We point out the advantages of using the simplest kinetic scheme and the possible pitfalls of using more complicated schemes, including the conventional carrier model. 3. We show that in the absence of information on the transport properties of the system, irreversible inhibition data are ambiguous. Taken together with transport data, however, inactivation data provide new tests for the applicability of the simple carrier. 4. The new tests show that for the simple carrier model to be applicable, the substrate dependencies of transport and of inactivation must be identical in comparable experimental situations. Further, the maximal rates of transport and of stimulation (or inhibition) of inactivation must obey a simple relationship, which we derive. We illustrate the use of these tests with published data on the glucose and choline transport systems of the human red blood cell.

Biological Transport↗