Feb 25, 1976

Transport mechanisms in isolated plasma membranes. Nucleoside processing by membrane vesicles from mouse fibroblast cells grown in defined medium

The Journal of Biological Chemistry
C C Li, J Hochstadt

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 r...Continue Reading

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Mentioned in this Paper

Ethylmaleimide
DRUG Screen Quant Caffeine
Nucleotidases
Specimen Type - Fibroblasts
Adenosine
Adenosine Triphosphatases
6-methyladenine
Ribonucleosides
Inosine
Potassium

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