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Abstract
Using a sterol-specific auxotroph of LM cells (mouse fibroblasts), we obtained cells with progressively lower sterol contents. 3-O-Methylglucose and thymidine were shown to enter the cells by saturable as well as by nonsaturable processes and it was possible to measure these processes separately at various stages of sterol depletion. Sterol depletion inhibited the saturable as well as the nonsaturable components for both 3-O-methylglucose and thymidine uptake. The extent of inhibition of the saturable process was much more than that of the nonsaturable component at any stage of the sterol depletion. Furthermore, in the course of progressive sterol depletion the saturable component of 3-O-methylglucose or thymidine uptake was inhibited well before the nonsaturable process. Sterol depletion produced reductions in the Vmax values but no change in the Km values for the saturable uptake of these two substrates. The depletion of cellular sterol to 75% resulted in 70% and 90% reduction of the saturable uptake of 3-O-methylglucose and thymidine, respectively, and 30% and 50% reduction of the nonsaturable uptake of 3-O-methylglucose and thymidine, respectively. DNA and protein synthesis, however, were not affected at all when the saturable component of thymidine uptake was inhibited by 50% demonstrating that the observed decrease of transport activity was selective and not a reflection of a gross alteration in cellular physiology.
Highlights
Using a sterol-specific auxotroph of LM cells, we obtained cells with progressively lower sterol contents. 3-0-Methylglucose and thymidine were shown to enter the cells by saturable as well as by nonsaturable processes and it was possible to measure these processes separately at various stages of sterol depletion
The data indicate that 3-0-methylglucose uptake was linear for at least 60 s using 2 X lo6 cells (Fig. 1) and that thymidine uptake was linear over the same time interval using 1 x lo6 cells (Fig. 2)
As we show below (Fig. 6), the fraction of 3-0-methylglucose uptake which is inhibited competitively by glucose represents a saturable component of 3-0-methylglucose uptake while the remainder is a nonsaturable process
Summary
Using a sterol-specific auxotroph of LM cells (mouse fibroblasts), we obtained cells with progressively lower sterol contents. 3-0-Methylglucose and thymidine were shown to enter the cells by saturable as well as by nonsaturable processes and it was possible to measure these processes separately at various stages of sterol depletion. The extent of inhibition of the saturable process was much more than that of the nonsaturable component at any stage of the sterol depletion. The depletion of cellular sterol to 75% resulted in 70% and 90% reduction of the saturable uptake of 3-0-methylglucose and thymidine, respectively, and. DNA and protein synthesis, were not affected at all when the saturable component of thymidine uptake was inhibited by 50% demonstrating that the observed decrease of transport activity was selective and not a reflection of a gross alteration in cellular physiology. Physical studies have shown that sterols modulate interactions between fatty acyl chains in membrane polar lipids These lipid-lipid interactions may have a great influence on various biological functions, these effects have not been fully explored or well characterized. While it is known that the amount of sterols found in various membranes of different cells and tissues is not constant, it is not clear how the content of sterols in different membranes is regulated or how sterols are transported from the site of synthesis in the endoplasmic reticulum to the other membranes within a cell
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