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Transport mechanisms in isolated plasma membranes. Nucleoside processing by membrane vesicles from mouse fibroblast
Abstract:
Plasma membrane vesicles were isolated from a subline of L929 mouse fibroblasts grown on defined medium in the absence of serum. These vesicles were not significantly contaminated by mitochondria or endoplasmic reticulum. The isolation procedure, a modification of that originally developed by McKeel and Jarett (McKeel, D.W., and Jarett, L. (1970) J. Cell Biol. 44, 417-432) employs mechanical homogenization in isotonic medium followed by differential centrifugation. The resultant plasma membrane vesicles take up radioactivity when exposed to uniformly labeled nucleosides. Two subfractions of the plasma membrane were isolated, distinguished by their differing activity of 5'-nucleotidase and (Na+,K+)-stimulated ATPase, two well known plasma membrane enzyme markers. Uptake of nucleoside radioactivity was extensively studied in one subfraction; it was linear with time and membrane concentration over ranges used for the studies. Apparent Km values for uptake of radioactivity from adenosine, inosine, and uridine were 7.1 +/- 26 muM, respectively. Uptake of radioactivity from all three nucleosides exhibits a broad pH optimum from pH 7 to pH 9, but falls off rapidly at lower pH. N-Ethylmaleimide was an effective inhibitor of uptake of radioactivity from all three nucleosides; uptake of radioactivity from uridine is more sensitive than uptake of radioactivity from the purine nucleosides. Adenosine inhibited uptake of radioactivity from inosine more than from uridine. Inosine inhibited the uptake of radioactivity from adenosine, but uridine did not. Caffeine and 6-methylaminopurine riboside (6-N-methyladenosine differentially inhibit uptake of radioactivity from adenosine and inosine, and thus the vesicles apparently possess seperate transport systems for uptake of radioactivity from purine nucleosides and from uridine.
Insights
Isolated plasma membrane vesicles from L929 mouse fibroblasts efficiently transport nucleosides. Studies reveal distinct transport systems for purine nucleosides and uridine, with varying sensitivities to inhibitors and pH.
Area of Science:
- Cell Biology
- Membrane Transport
- Biochemistry
Background:
- Plasma membrane vesicles are crucial for studying cellular transport mechanisms.
- Previous methods for vesicle isolation required optimization for specific cell types and research goals.
Purpose of the Study:
- To isolate and characterize plasma membrane vesicles from L929 mouse fibroblasts for nucleoside uptake studies.
- To investigate the transport kinetics and specificity of nucleoside uptake in these vesicles.
Main Methods:
- Isolation of plasma membrane vesicles using mechanical homogenization and differential centrifugation.
- Enzymatic characterization of vesicle subfractions using 5'-nucleotidase and (Na+,K+)-stimulated ATPase assays.
- Quantification of radiolabeled nucleoside uptake and inhibition studies.
Main Results:
- Isolated vesicles showed minimal contamination from other organelles.
- Nucleoside uptake was linear with time and membrane concentration.
- Apparent Km values for adenosine, inosine, and uridine uptake were determined.
- Uptake exhibited a broad pH optimum (pH 7-9) and was inhibited by N-Ethylmaleimide.
- Differential inhibition patterns indicated separate transport systems for purine nucleosides and uridine.
Conclusions:
- L929 mouse fibroblast plasma membrane vesicles provide a viable model for studying nucleoside transport.
- Evidence suggests the presence of distinct transporter proteins for purine nucleosides and uridine.
- Inhibitor studies highlight differences in the transport mechanisms for these nucleosides.