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W D Stein

Publications and source records attributed to W D Stein.

At least 19 recordsLinked to original sources
PubMed1992

Chemical modification of Glu-953 of the alpha chain of Na+,K(+)-ATPase associated with inactivation of cation occlusion.

We have investigated the role, number, and identity of glutamate (or aspartate) residues involved in cation occlusion on Na+, K(+)-ATPase, using the carboxyl reagent N,N'-dicyclohexylcarbodiimide (DCCD). Extensive use is made of selectively trypsinized Na+,K(+)-ATPase--the so-called "19-kDa membranes"--containing a 19-kDa COOH-terminal, smaller (8-11 kDa) membrane-embedded fragments of the alpha chain, and a largely intact beta chain; these membranes have normal Rb+ and Na+ occlusion capacities. The 19-kDa peptide and a smaller (approximately 9 kDa) unidentified peptide(s) are labeled by [14C]DCCD in a Rb(+)-protectable fashion. Rb(+)-protected [14C]DCCD incorporation into the "19 kDa membranes" and into native Na+,K(+)-ATPase is linearly correlated with inactivation of Rb+ occlusion. Similar linear correlations are observed when Rb(+)-protected [14C]DCCD incorporation is measured by examination of labeling of 19-kDa peptide purified from "19-kDa membranes" or of alpha chain purified from native enzyme. Stoichiometries, estimated by extrapolation, are as follows: (for "19-kDa membranes") close to one DCCD per Rb+ site and one DCCD per 19-kDa peptide; and (for native enzyme) close to two DCCD per phosphoenzyme and two DCCD per alpha chain. We suggest that each of two K+ (or Na+) sites contains a carboxyl group, one located in the 19-kDa peptide and one elsewhere in the alpha chain. After cyanogen bromide digestion of purified, labeled alpha chain, or of 19-kDa peptide, a labeled fragment of apparent M(r) approximately 4 kDa was detected and was identified as that with NH2-terminal Lys-943. Rb(+)-protected [14C]DCCD incorporation was associated almost exclusively with Glu-953. We suggest that the cation occlusion "cage" consists of ligating groups donated by different trans-membrane segments and includes two carboxyl groups such as Glu-953 (and perhaps Glu-327) as well as neutral groups, in two K+ (or Na+) sites, but only neutral groups in the third Na+ site.

Amino Acid Sequence↗
PubMed1992

Modulation of bone calcium-binding sites regulates plasma calcium: an hypothesis.

A new model of calcium (Ca) homeostasis is proposed. It is based on the kinetics of restoration of the plasma Ca level following positive or negative Ca loads in animals of different endocrine status. As others, we can account for the kinetics of plasma Ca restoration as being the result of a very rapid dilution of Ca into extracellular water (t1/2 less than 1 minute) and an uptake or release by bone (t1/2 = 14-80 minutes) that occurs as the fraction of cardiac output directed to bone is partially cleared of or repleted with Ca. In this model, bone surfaces have Ca-binding sites that demonstrate a range of affinities and whose average Km determines the plasma Ca level. Acute regulation is brought about by controlling access to subpopulations of Ca binding sites in bone, comprising the extremes of high and low affinity. Osteoblasts, when active and extended, block the low affinity sites, and osteoclasts, when active and extended, block the high affinity sites. Exposure of low- or high-affinity sites is brought about when these cells respond to hormonal signals by contraction, parathyroid hormone (PTH), and vitamin D leading to osteoblast, and calcitonin to osteoclast, contraction. These reciprocal cell shape changes are the first in a cascade of metabolic events that lead to bone formation and resorption, as well as changes in the number or affinity of the binding sites. The model also accounts for the prolongation of the response time to Ca loads in animals deprived of PTH, calcitonin, or vitamin D.

Animals↗
PubMed1992

Facilitated diffusion of calcium across the rat intestinal epithelial cell.

Similarities and differences between the models for facilitated diffusion of substrates across cytoplasm and across cell membranes are discussed. It is valuable to consider that calbindin acts to increase the partitioning of calcium within the cytoplasm. Calculations on the basis of a simple model for this facilitated diffusion show that the rate of transcellular calcium movement depends in a complex way on KCAB, the dissociation constant for calcium and calbindin. At each value of the transcellular concentration gradient of free calcium, there is an optimal value of KCAB that gives the maximal augmentation of calcium flow. For low values of the ratio of free calcium concentrations at the opposite poles of the cell, the optimal value of KCAB is close to the prevailing concentration of free calcium. An analysis of possible effects of pH on binding of calcium to calbindin shows that it may be useful to consider cotransport of calcium and protons within the cell. Small values of a transcellular pH gradient can have large effects on enhancing transcellular calcium flow, provided the hydrogen ion concentration affects the binding of calcium to calbindin or the rate of diffusion of the calcium/calbindin complex.

Animals↗
PubMed1992

Identification of the cation binding domain of Na/K-ATPase.

This paper summarises results and conclusions from experiments with renal Na/K-ATPase, utilising proteolytic digestion to define minimal peptide structures involved in cation occlusion and chemical modification with dicyclohexylcarbodiimide (DCCD) to investigate the role of carboxyl groups and location of K (Rb) and/or Na binding residues. Extensive digestion with trypsin or non-selective proteases in the presence of Na or Rb and absence of divalent cations reveals an essential C-terminal 19Kd fragment of the alpha chain (N-terminal asn 830) and indicates that occlusion sites of Na or K ions must reside within transmembrane segments. The bulk of the beta chain is not involved. Kinetics of inactivation of Rb or Na occlusion and covalent labelling with DCCD indicate that each of two Rb(K) or Na sites contains a carboxyl group. The third Na site may contain only neutral ligating groups. One carboxyl group is located on the 19Kd fragment and the other on tryptic fragment of about 9Kd. When cyanogen bromide was used to digest labelled alpha chain, glu 953 was found to be labelled in a Rb-protectable fashion. In tryptic "19Kd-membranes", fragments containing all putative transmembrane segments of the alpha chain have been identified (i.e. 19, 10.9, 8.7 and 8.0 Kda respectively). The cation occlusion "cage" is apparently composed of ligating groups from different trans-membrane segments, including segments of the 19Kd fragment. Construction of models is hampered by the fact that the number of the transmembrane segments is still uncertain, particularly in the crucial C-terminal domain. Alternative ways of arranging the tryptic fragments across the membrane are discussed.

Animals↗
PubMed1991

Rate-limiting steps in carcinogenesis and their bearing on the incidence/age data for cutaneous melanoma and hepatocellular carcinoma.

We analyze published data on cancer incidence as a function of age for (i) cutaneous melanoma for cohorts of males in Connecticut and (ii) hepatocellular cancer in Mozambique, China and Romania, on the basis of multi-hit models for carcinogenesis. The simple multi-hit model suggests an unlikely scenario in which, as the cohorts advance or the intensity of mutagenesis increases, fewer mutations are required to bring about the appearance of cancer. We present a new version of the multi-hit model in which two sets of mutatable loci are present, of differing sensitivities to mutagens. This provides a more acceptable description in which the number of rate-limiting steps for the appearance of cancer drops as the frequency of mutation rises, so that some of these steps are no longer rate-limiting.

Adolescent↗
PubMed1991

Kinetic modelling of chloroquine uptake by malaria-infected erythrocytes. Assessment of the factors that may determine drug resistance.

The antimalarial chloroquine, by virtue of its weak base properties, concentrates in the acidic compartment(s) of the intraerythrocytic parasite. Drug accumulation is essential for it to exert its pharmacological activity. Drug resistance has been thought to result from insufficient acidification of drug-accumulating organelle(s), (due to weakened proton pump activity and/or proton leak) or to result from the action of the recently suggested active efflux drug pump. In this work we have devised a kinetic model which takes into account the various processes that have been postulated to account for acidification and drug fluxes. Using this model to analyse the time-course of chloroquine uptake and the steady-state levels of drug accumulation, in strains of Plasmodium falciparum which display variable drug resistance, we demonstrate that drug resistance is compatible with the existence of a weakened proton pump in the resistant parasite strains. Consistent with recent molecular studies that show no correlation between the presence of the multidrug efflux pump gene and the phenotypic expression of chloroquine resistance, our analysis fails to detect any such pump activity. We also show that analysis of drug efflux kinetics cannot distinguish between the possible modes of drug resistance.

Animals↗
PubMed1990

Energetics and the design principles of the Na/K-ATPase.

Following on the pioneering analysis by Pickart and Jencks of the energetics of the calcium pump, the present mini review attempts a similar analysis of the somewhat more complicated sodium--and potassium--activated ATPase pump-enzyme. The analysis is based on the measurements of the rate-constants of the individual steps in the enzymic reaction which brings about pumping and uses data assembled by Stürmer et al., in the laboratory of Peter Läuger in Konstanz. The aim of such an analysis is to calculate the overall free energy released on ATP hydrolysis and to apportion this energy among the successive steps of the pump-enzyme reaction. We calculate the so-called basic free energy changes that take into account the prevailing ligand and ion concentrations, rather than the standard-state free energies that refer to 1 M concentrations of these ions and ligands. Using appropriate values of the ion and ligand concentrations in cardiac muscle, the available free energy which can be released from the hydrolysis of ATP (at 20 degrees C) comes out at 575 mV. Following a complete cycle of pumping, 371 mV of this free energy are found stored in the sodium and potassium ion gradients. The remaining 204 mV from the free energy of hydrolysis of ATP are lost to the ATP system. This part of the energy, that had been transduced into the concentration gradient of sodium, has presumably been used in the living cell to drive the co- and counter-transport (symport and antiport) of ions and metabolites in secondary transports. The free energy changes are pretty evenly apportioned along the various steps in the pumping cycle. The steps that one might naively have thought to be "powered", such as the step in which covalently bound phosphate is transformed from a high-energy to a low-energy state, or the step in which sodium is released into the phase containing a high concentration of sodium, show some of the lowest drops of free energy, 61 mV and 27 mV, respectively. The most surprising step in the overall reaction of ATP hydrolysis and synthesis is the phosphorylation of the protein from inorganic phosphate with formation of the acylphosphate bond. The stabilization of the acylphosphate bond presumably arises from ionic interactions between the covalently bound phosphate itself and appropriate groupings on the enzyme. ATP formation on the F0F1 ATPase (the F-type ATPase) is in an analogous way stabilized in the first place by phospho-ligand/enzyme interactions.

Animals↗
PubMed1990

Testing and characterizing the two-stage model of carcinogenesis for a wide range of human cancers.

The age dependence of incidence for 45 cancer types in three populations is analyzed on a two-stage kinetic model containing three determinable parameters: (i) the fraction of population at risk for a cancer; (ii) the product of the frequencies of cancer-producing mutations, and (iii) the growth rate of the transformed clone from which a cancer ultimately evolves. The model, simplifying that proposed by others, fits many cancers. Data are easily handled in terms of the derived parameters, providing the basis for epidemiological analysis, here applied in detail to liver, cervix and testis cancers for nearly 50 world-wide populations. We identify 12 cancers for which only a limited fraction of the population is at risk. We argue that the appearance of most cancers requires at least three mutational events. For child, youth, or early adult cancers, one mutation may be congenital. We arrange the various cancers in a descending scale, defining six groups that differ in the derived mutation frequencies. A cancer appearing later in life, for which the whole population can be at risk, shows a low mutation frequency, consistent with background spontaneous mutation. The other cancers require increases in mutation frequency, arising from an increased rate of cell division and/or mutation rate.

Age Factors↗
PubMed1990

Kinetic properties of F0F1-ATPases. Theoretical predictions from alternating-site models.

We present an analysis of models based on current structural concepts of the F0F1 synthases, accounting for coupling between proton transport and ATP synthesis. It is assumed that each of the three alpha beta-subunits of the synthase can exist in three different conformational states E, Eo and E*. Proton translocation is coupled to cyclic interconversion of the conformations of the alpha beta-subunits. The conformational changes of these subunits are assumed to be coordinated so that all three interconvert simultaneously, in a rate-limiting transition. Binding and release of the ligands ATP, ADP, Pi, and protons are assumed to be equilibrium steps. In one family of models, interconversion of the alpha beta-subunits of F1 is coupled to the translocation event in F0 acting as a proton carrier. In a second family of models, protons combine with F0F1 and are translocated during the interconversion step in a chemiport. Kinetic tests involving the mutual effects of [ATP], [ADP], H+', and H+" are described, allowing us to make a distinction between the different models and submodels.

Binding Sites↗
PubMed1990

A 19-kDa C-terminal tryptic fragment of the alpha chain of Na/K-ATPase is essential for occlusion and transport of cations.

Tryptic digestion of pig renal Na/K-ATPase in the presence of Rb and absence of Ca ions removes about half of the protein but leaves a stable 19-kDa membrane-embedded fragment derived from the alpha chain, a largely intact beta chain, and essentially normal Rb- and Na-occlusion capacity. Subsequent digestion with trypsin in the presence of Ca or absence of Rb ions leads to rapid loss of the 19-kDa fragment and a parallel loss of Rb occlusion, demonstrating that the fragment is essential for occlusion. The N-terminal sequence of the 19-kDa fragment is Asn-Pro-Lys-Thr-Asp-Lys-Leu-Val-Asn-Glu-Arg-Leu-Ile-Ser-Met-Ala, beginning at residue 830 and extending toward the C terminus. Membranes containing the 19-kDa fragment have the following functional properties. (i) ATP-dependent functions are absent. (ii) The apparent affinity for occluding Rb is unchanged, the affinity for Na is lower than in the control enzyme, and activation is now strongly sigmoidal rather than hyperbolic. (iii) Membranes containing the 19-kDa fragment can be reconstituted into phospholipid vesicles and sustain slow Rb-Rb exchange. Thus the transport pathway is retained. We conclude that cation occlusion sites and the transport pathway within transmembrane segments are quite separate from the ATP binding site, located on the cytoplasmic domain of the alpha chain. Interactions between cation and ATP sites, the heart of active transport, must be indirect--mediated, presumably, by conformational changes of the protein.

Adenosine Triphosphate↗
PubMed1989

Acute plasma calcium regulation in rats: effect of vitamin D deficiency.

Vitamin D-replete (+D) and vitamin D-deficient (-D) rats received large doses of calcium (2-18 mg) by intraperitoneal injection and their responses to the calcium load was analysed in terms of the instantaneous and time-dependent responses of the plasma calcium concentration, [Cas]. Following an initial expansion, [Cas] returned to the preinjection value in a strictly exponential manner, with t1/2 = 22.5 +/- 2.0 (SE) min in +D and 51 +/- 5.2 min in -D animals. In both groups of animals, these rates were independent of the calcium load. Extraprolation of [Cas] to t = 0, i.e., the time just after administration of the calcium, revealed that the amount of calcium circulating at that moment was only about one-fifth of the amount that would have been found if all of the injected calcium had remained in the plasma. Calculations suggest that in all animals about four-fifths of the injected calcium load became distributed virtually instantaneously in the extracellular water. In both +D and -D groups the fraction of the injected load that left the plasma instantaneously was independent of the calcium load, of [Cas] at t = 0 or of the animals' plasma volume. The ability of rats to disperse some 80% of the load to outside the plasma would seem to constitute a major mechanism of acute plasma calcium regulation. Dilution was insufficient, however, totally to reduce [Cas] to the preinjection level. That occurred exponentially, with most of the calcium presumed to enter the skeleton. This exponential rate was markedly and significantly slower in the vitamin D-deficient animals than in their controls.

Animals↗
PubMed1988

CaBPr facilitates intracellular diffusion for Ca pumping in distal convoluted tubule.

The system of renal Ca transport in the rat is modeled in terms of two classes of processes: a nonsaturable flux that predominates in the proximal tubule, and an active, vitamin D-dependent flux with major expression in the distal convoluted tubule. There transport is against an electrochemical gradient, with much of the efflux probably mediated by the Ca/Mg-ATPase. Calculations of the rate of free Ca diffusion in tubular cells indicate that an unaided flux would be only one-seventy-seventh of that found experimentally. It is suggested that the vitamin D-induced renal calcium binding protein, CaBPr, Mr approximately 28,000, in raising total cellular calcium by three orders of magnitude, increases the transcellular Ca flux and thus the free intracellular Ca ion concentration at the basolateral pole, allowing the Ca/Mg-ATPase to function near its maximum. Analysis of the rate of nonsaturable Ca flux throughout the kidney tubule suggests a paracellular pathway via bulk flow, following water that is driven osmotically. Evaluation of whole animal data in terms of these two classes of calcium fluxes indicates that our model is consistent with experimental observations and assigns a functional role to active calcium transport.

Animals↗