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R M Schiller

Publications and source records attributed to R M Schiller.

8 recordsLinked to original sources
PubMed1979

Catabolic control of the enhanced alanine-preferring system for amino acid transport in glucose-starved hamster cells requires protein synthesis.

In cultured hamster cells starved for glucose for 24 hr there is an enhancement of the rate of alpha-aminoisobutyric acid transport ("shiftup"). When the starved cells are re-fed with glucose, the rate of transport shifts back down to the low, "regulated" rate typical of cells continuously fed with medium containing glucose ("shiftdown"). The high, deregulated rate of transport is maintained, however, when cycloheximide is present for 24 hr during the re-feeding with glucose. Maintenance of the high transport rate is evident only when the cells are incubated in amino acid-free medium just prior to the transport assay or when the assays are conducted with isolated membrane vesicles. A premature, pseudoshiftdown was observed in intact cells within as little as 2 hr after re-feeding when care was not taken to deplete the amino acid pool prior to the transport assay. In addition, a cycloheximide-insensitive increase in transport was observed when cultures were re-fed for 2 hr with amino acid-free medium containing fresh serum. These results emphasize the often overlooked precautions that should be taken to guard against artifacts that could mislead interpretations of amino acid transport data. More important, however, is the finding that Na(+)-dependent amino acid transport in cultured animal cells is regulated in part by a factor (or factors) that becomes inactivated when the cells are maintained under nonglycolytic culture conditions. In order to reactivate the control mechanism, starved cells that have been re-fed with glucose must resynthesize the regulatory factor(s). Thus, in at least cultured hamster cells, Na(+)-dependent amino acid transport regulation is much like the hexose transport regulation in that catabolic control (shiftdown) requires protein synthesis.

Alanine↗
PubMed1978

Sodium-dependent amino acid transport by cultured hamster cells: membrane vesicles retain transport changes due to glucose starvation and cycloheximide.

Enhanced alpha-aminoisobutyric acid transport by hamster cells cultured in the absence of D-glucose has been demonstrated in isolated membrane vesicles. The observed enhancement was seen in the presence but not in the absence of Na+. Kinetic analysis of transport using both the intact cells and the membrane vesicles showed that the overall enhancement was associated with an increase in Vmax. Decreases in transport activity by intact cells resulting from extended exposure of the cells to inhibitors of protein synthesis, such as cycloheximide, were also evident in membrane vesicles. The use of metabolically inactive membrane vesicles demonstrated that amino acid uptake by intact cells is a transport property of the plasma membrane. In addition, this study shows that membrane vesicle preparations can be exploited for the purpose of studying the regulation of amino acid transport. Taken together, the data suggest that carrier turnover is involved in the regulation of amino acid transport in animal cells.

Amino Acids↗
PubMed1978

Role of Na+ in alpha-aminoisobutyric acid uptake by membrane vesicles from mouse fibroblasts transformed by simian virus 40.

The uptake of alpha-amino[(3)H]isobutyric acid (AIB) was studied in membrane vesicles from mouse fibroblasts transformed by simian virus 40 to examine the features of the Na(+)-stimulated and Na(+)-dependent AIB transport process. The simultaneous addition of NaCl and AIB to these vesicles produced a transient accumulation, or "overshoot," of amino acid 3-4 times the equilibrium value. Both the initial rate of uptake and the rate of fall of intravesicular AIB after maximal accumulation were sensitive to the temperature of incubation. The overshoot of AIB uptake was enhanced with Na(+) salts of highly permeant lipophilic anions, such as SCN(-) and NO(3) (-), and was decreased by the addition of SO(4) (2-), a relatively impermeant ion. Gramicidin D, which enhances the membrane conductance of Na(+) electrogenically, decreased the overshoot, while a potassium diffusion potential, induced by valinomycin (in K(+)-preloaded membrane vesicles), produced a Na(+)-dependent overshoot of AIB uptake. When vesicles were preincubated with both Na(+) and AIB, followed by the generation of an interior negative membrane potential (by the addition of SCN(-)), an overshoot of AIB uptake resulted. However, this did not occur in the absence of Na(+). It is concluded that, apart from its role in the generation of a transmembrane electrochemical potential, Na(+) is essential for the overshoot of AIB uptake.

Aminoisobutyric Acids↗
PubMed1978

Sodium-stimulated active transport of aminoisobutyric acid by reconstituted vesicles from partially purified plasma membranes of mouse fibroblasts transformed by simian virus 40.

Plasma membrane fractions isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40 were partially purified by treatment with dimethylmaleic anhydride followed by extraction with 2% cholate. The extracted proteins were combined with exogenous phospholipids and eluted through a Sephadex G50 column. Reconstituted vesicles thus obtained were shown to possess the ability of Na+-stimulated transport of alpha-aminoisobutyric acid. The simultaneous addition of NaSCN and alpha-aminoisobutyric acid to these vesicles produced a transient accumulation above the equilibrium level (overshoot, active transport). The Na+-stimulated transport of alpha-aminoisobutyric acid was sensitive to the accompanying anion and to the temperature of incubation. The results demonstrate that partially purified membrane proteins of mouse fibroblast cells can be incorporated into the liposomes that have the characteristics of Na+-stimulated and electrochemically sensitive active transport of alpha-aminoisobutyric acid.

Aminoisobutyric Acids↗