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Biomedical subjects

K P Wheeler

Publications and source records attributed to K P Wheeler.

At least 19 recordsLinked to original sources
PubMed1992

Modulation by betaine of cellular responses to osmotic stress.

Various solutes were tested to see if they could modify the responses of SV-3T3 cells to hyperosmotic (0.5 osM) conditions, which cause an inhibition of general cell protein synthesis and of the rate of cell proliferation, coupled with an induction of amino acid transport activity. The added solutes were glycerol, proline, taurine, betaine, dimethylglycine and sarcosine. Of these, betaine produced the most dramatic and consistent effects. Addition of 10-25 mM-betaine to the hyperosmotic medium largely prevented the 90% inhibition of cell proliferation that occurred in its absence. Whether it was added initially or after the cells were exposed to hyperosmotic medium, 25 mM-betaine also converted a 50% recovery of the rate of protein synthesis into 100%. Similarly, the same concentrations of betaine prevented a 30% decrease in cell volume and decreased the induction of amino acid transport via system A by 73%. Lower concentrations of betaine produced smaller but still significant changes in these functional responses. With chick-embryo fibroblasts, under identical hyperosmotic conditions, 25 mM-betaine completely counteracted a 75% inhibition of the rate of protein synthesis. At present it is not clear how betaine modulates these effects of hyperosmolarity on cell functions.

3T3 Cells↗
PubMed1990

Induction of amino acid transport activity in chick embryo fibroblasts by replacement of extracellular sodium chloride with disaccharide.

The activity of amino acid transport System A in avian fibroblasts was increased following incubation of the cells in a medium in which most of the NaCl normally present had been isoosmotically replaced by sucrose. This increase was detectable after 2 h of incubation, reached a maximum at about 4 h, and remained constant thereafter. Transfer of treated cells back to a normal medium resulted in decay of the induced transport activity, with a half-life of less than 2 h. Kinetic analysis revealed that the increase in transport activity arose from an increase in Vmax, with little change in Km. This induction of System A activity did not occur if an inhibitor of either RNA or protein synthesis was present in the modified medium. The use of various different solutes as replacements for NaCl in the incubation medium showed that, although each replacement caused a decrease in both cellular Na+ content and protein synthesis, only disaccharides produced the increase in amino acid transport activity. In addition, estimates of cell volume indicated that, even under iso-osmotic conditions, incubation in the sucrose-containing medium caused initial cell shrinkage, followed by swelling. It is concluded that this induction of System A activity is associated with a volume regulatory process and that this process probably accounts for the parallel responses previously observed when cells were incubated in hyperosmolar media. Induction of amino acid transport activity by this process is distinct from adaptive regulation, caused by amino acid starvation; but the two processes are not strictly additive, and so appear to converge at some step.

Animals↗
PubMed1987

Multicomponent analysis of amino acid transport System L in normal and virus-transformed fibroblasts.

Amino acid transport System L in both normal Balb/c 3T3 cells and in those transformed with simian virus 40 (SV 3T3) was analysed kinetically under two different experimental conditions. Under 'zero-trans' conditions the results for both types of cell could be interpreted satisfactorily in terms of System L consisting of two components (L1 and L2) characterized by different Km values. This conclusion is in agreement with previous reports. However, under 'infinite-trans' conditions, the experimental data could not be accounted for in terms of only two components; the introduction of a third component (L3) was necessary to provide a satisfactory fit. Viral transformation affects only the L1 component, either by modification or by replacement, giving it a higher 'affinity' (lower Km) but a lower 'capacity' (lower Vmax).

Amino Acids↗
PubMed1987

Sodium ions protect a membrane transport protein from proteolysis.

Na+-dependent alanine transport activity in vesicles prepared from pigeon erythrocyte membranes was examined after exposure of the vesicles to some proteinases under various conditions. The presence of sodium ions during proteolysis affords considerable protection of alanine transport activity from the inhibitory action of the proteinases. The concentration of sodium ions required for half-maximum protection is greater than that needed for half-maximum activation of alanine uptake. The site of protective action could be at either or both surfaces of the membrane because the vesicles are very permeable to sodium ions. Neither measurement of residual protein content nor analysis by polyacrylamide gel electrophoresis revealed any differences in the extent of protein degradation occurring in the presence and absence of sodium ions, suggesting that the transporter constitutes only a minor membrane component. We conclude that sodium ions probably induce a conformation change in the transporter.

Alanine↗
PubMed1985

Sodium-induced conformation changes in membrane transport proteins.

In the presence of KCl, tryptic digestion of vesicles derived from pigeon erythrocyte membranes inactivates sodium-dependent uptake of alanine by the vesicles, whereas digestion in the presence of NaCl does not. Extensive degradation of vesicle proteins occurs under both conditions. Similarly, the extent of inhibition by N-ethylmaleimide of the sodium-dependent influxes of both glycine and alanine into human erythrocytes is greater when the cells are exposed to the thiol reagent in the presence of KCl than when NaCl is used. These observations are interpreted as providing evidence for sodium-induced conformation changes in these transport proteins.

Alanine↗
PubMed1985

Metabolic disposition study of chlorinated hydrocarbons in rats and mice.

Chlorinated hydrocarbons found in a bioassay to be carcinogenic to both B6C3F1 mice and Osborne-Mendel rats (1,2-dichloroethane), carcinogenic only to mice (1,1,2-trichloroethane, 1,1,2,2-tetrachloroethane, hexachloroethane, trichloroethylene, and tetrachloroethylene), and noncarcinogenic to either species (1,1-dichloroethane and 1,1,1-trichloroethane) were used to investigate the biochemical bases for tumorigenesis. Studies were conducted after chronic oral dosing of adult mice and rats with the MTD and 1/4 MTD of each compound. The extent to which the compounds were metabolized in 48 hr, hepatic protein binding, and urinary metabolite patterns were examined. Metabolism of the compounds (mmoles per kg body weight) was 1.7 to 10 times greater in mice than in rats. Hepatic protein binding (nanomole equivalents bound to 1 mg of liver protein) was 1.2 to 8.3 times higher in mice than in rats except for 1,2-dichloroethane and 1,1,1-trichloroethane. The noncarcinogens 1,1-dichloroethane and 1,1,1-trichloroethane exhibited 2 to 18 times more binding in mice than did the carcinogens 1,2-dichloroethane and 1,1,2-trichloroethane. Urinary metabolite patterns of the compounds were similar in both species. The biochemical parameters measured provided no clue to differentiate the carcinogens from the noncarcinogens.

Administration, Oral↗
PubMed1979

Enhanced transport of natural amino acids after activation of pig lymphocytes.

Na+-dependent uptake of the amino acids L-proline and L-methionine was greatly accelerated when pig lymphocytes were activated with phytohaemagglutinin or other mitogens. The increased influx was apparent after incubation with phytohaemagglutinin for 1 h, and reached a maximum after 24 h. The lymphocytes appear to possess at least three different transport systems for neutral amino acids with properties similar to, but not identical with, those described for other cells. The activity of a system resembling the A system of other cells was increased most dramatically after activation, its activity in unstimulated lymphocytes being extremely low or absent. A second Na+-dependent system, which has properties similar to those of the ASC system in other cells, but with a broader specificity for amino acids, was more active in unstimulated lymphocytes, and uptake by this system was also accelerated after incubation with phytohaemagglutinin. The activity of a third system, very similar to the L system in other cells, was increased to a much smaller extent after lymphocyte activation.

Amino Acids↗
PubMed1979

Requirement for negatively charged dispersions of phospholipids for interaction with lipid-depleted adenosine triphosphatase.

The basis of the requirement for a net negative charge on phospholipid dispersions able to re-activate lipid-depleted (Na++K+)-dependent adenosine triphosphatase was studied. The origin and density of the charge in phospholipid dispersions were varied before interaction with the adenosine triphosphatase protein, and the charge density on restored phospholipid-adenosine triphosphatase complexes was changed after interaction. The results indicated that: (a) re-activation requires a lamellar arrangement of the lipid molecules with sufficient density of negative charge, but not necessarily negatively charged phospholipid molecules; (b) the net charge appears to be necessary for the correct interaction between the enzyme protein and the phospholipids, although the amount of phospholipid that binds to the protein is also a function of the nature of the acyl chains; (c) it is not possible on the basis of these findings and those in the literature to decide unequivocally if the charge is also required for the enzyme reaction itself. The possible relevance of the findings to the situation in vivo is discussed in terms of the charge being concerned only with lipid-protein interaction.

Bile Acids and Salts↗
PubMed1979

Relative effects of different surfactants on intestinal absorption and the release of proteins and phospholipids from the tissue.

The actions of anionic, cationic and non-ionic surfactants on the absorptive capability of rat jejunal tissue in vivo were compared with their effects on the amounts of protein and phospholipid released from the mucosal surface under the same conditions. Release of a comparatively small amount of protein was accompanied by large increases in the absorption rates of both L-valine and salicylate, whereas much larger quantities of phospholipid were released before any increase in absorption were observed. Much of the released material appeared to be derived from mucus which was partly degraded after exposure to the higher concentrations of surfactants. The liberation of cholesterol by high concentrations of anionic surfactants suggested that some disruption of the mucosal membrane occurred under those conditions. The relative potency of the surfactants in stimulating both absorption of the solutes and the release of polypeptides and lipids followed the order: anionic greater than non-ionic greater than cationic. The possible pharmaceutical relevance of these findings is discussed.

Animals↗
PubMed1978

Protein and lipid components of the pigeon erythrocyte membrane.

The plasma membrane of the nucleated pigeon erythrocyte was isolated by a method that is simple, reproducible and minimally disruptive, the final preparation consisting of whole cell 'ghosts', recovered at over 40% yield. Alternative methods, which yield membrane fragments, were also tested and some of their possible disadvantages demonstrated. Analysis of the protein components of the isolated membranes by gel elctrophoresis in the presence of sodium dodecyl sulphate revealed that their composition is very similar to that of the proteins of human erythrocyte membranes. However, two major proteins are unique to the nucleated cell membrane; these have apparent mol.wts. of 97000 and 57000. Also, the bands designated 4.2 (74500 mol.wt.) and 6 (35000 mol wt.) by Steck [(1974) J. Cell Biol. 62, 1-19] for the human cell membrane are absent from pigon cell membrane. Glycosylated membrane proteins could not be detected in gels stained with the periodate-Schiff-base procedure. Analysis of membrane phospholipids revealed the same components known to be present in mammalian erythrocytes, though in different proportions. These findings are discussed in the light of known physiological and biochemical differences between avian and mature mammalian erythrocytes.

Animals↗
PubMed1978

A simple and rapid method for the reversible removal of lipids from a membrane-bound enzyme.

A simple, rapid and reproducible method for the reversible removal of lipids from a membrane-bound enzyme is described. Essentially, a membrane preparation containing (Na+ + K+)-dependent adenosine triphosphatase was extracted with the non-ionic detergent Lubrol WX in the presence of glycerol, and partial separation of protein from lipid was achieved with the use of only two centrifugations. About 74% of the endogenous phospholipid and 79% of the cholesterol were removed, concomitant with a virtually complete loss of ouabain-sensitive adenosine triphosphatase activity, but with retention of 60-100% of the K+-dependent phosphatase activity. The addition of pure phosphatidylserine re-activated the enzyme to more than 80% of the initial activity, and up to 30% of the protein was recovered. Excess of phosphatidylserine could be washed off the enzyme to give a stable 'reconstituted' preparation. The effects of variation in the experimental conditions were examined, and the results are discussed with respect to the possibility of adapting the method to the study of other lipid-dependent enzymes bound to membranes.

Adenosine Triphosphatases↗
PubMed1978

Ouabain binding to phospholipid-dependent adenosine triphosphatase.

The role of phospholipid in the binding of ouabain to the (Na+ + K+)-dependent adenosine triphosphatase was studied. Enzyme preparations obtained from rabbit kidney were treated with Lubrol WX to remove the phospholipid component essential for ATPase activity. Reconstituted enzyme samples were prepared by the addition of phosphatidylserine and sedimentation of an enzymically active lipid-protein complex. The binding of ouabain to both kinds of preparations was measured under equilibrium conditions with the use of 3H-labelled ouabain and initial ouabain concentrations in the range 0.01-1 micrometer. The main findings were: (i) (Mg2+ + Pi) promoted binding of significant quantities of ouabain only to the reconstituted enzyme; (ii) the absence of added Na+, (Mg2+ + ATP) similarly promoted binding only to the reconstituted samples; (iii) the addition of Na+ in the presence of (Mg2+ + ATP) increased the amount of ouabain bound to the reconstituted enzyme when the ouabain concentration was below about 0.1 micrometer, but it had no effect when the ouabain concentration was about 1 micrometer; (iv) (Mg2+ + ATP) induced ouabain binding to the depleted enzyme only when Na+ was also added; (v) the amount of ouabain bound to both depleted and reconstituted enzymes was the same in the presence of (Mg2+ + ATP + Na+); (vi) the reconstituted enzyme appeared to have a greater affinity for Na+ than did the depleted enzyme.

Adenosine Triphosphatases↗
PubMed1975

Polar head-group and acyl side-chain requirements for phospholipid-dependent (Na-+ plus K-+)-ATPase.

The abilities of different phospholipids to reactivate a lipid-depleted (Na-+ plus K-+)-ATPase (EC 3.6.1.3) have been compared. The phospholipids contained either the same group of hydrocarbon chains withhh different polar groups, or different hydrocarbon groups wit the same polar group; they were prepared by enzymic modification of the polar group of phosphatidylserine isolated from bovine brain and of phosphatidylcholine extracted from egg. Only the acidic phospholipids reactivated the ATPase but the amount of reactivation depended on the nature of the hydrocarbon chains as well as the polar group. These findings are discussed in relations to the compositions of the two different groups of fatty acyl chains constituting the hydrocarbon portions of the phospholipids.

Adenosine Triphosphatases↗
PubMed1975

Lipid requirement of the membrane sodium-plus-potassium ion-dependent adenosine triphosphatase system.

The dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) on lipid has been examined in a number of different ways, with the use of various preparations from kidney tissue. The main findings were as follows. (1) The ATPase activities of the preparations examined were closely correlated with their total phospholipid content. (2) Extraction of the ATPase with deoxycholate or Lubrol W, combined with suitable salt-fractionation and washing procedures, removed phospholipid, cholesterol and enzymic activity in parallel; but activity was completely lost before all lipid had been removed. (3) The loss of activity could not be attributed to inhibition by residual detergent. (4) No selective removal of any particular phospholipid class by detergent could be detected. (5) Consistent reactivation of the Lubrol-extracted enzymes was obtained by adding dispersions of exogenous phospholipid, but only some, bearing a net negative charge, such as phosphatidylserine and phosphatidylglycerol, were effective. (6) The degree of reactivation was correlated with the amount of residual activity remaining after lipid depletion. (7) Partial purification of the ATPase, giving a 50-fold increase in specific activity, was not accompanied by selective enhancement of any particular class of phospholipid. We conclude that although the ATPase is dependent on phospholipid, only the reactivation results provide evidence for specificity.

Adenosine Triphosphatases↗