Preparation of two plasma membrane fractions from ascites tumour cells by gel chromatography on Sephacryl S-1000

Abstract

Two plasma membrane fractions from ascites tumour cells with differences in vesicle size were isolated by gel-exclusion chromatography on Sephacryl S-1000. Fraction 1 appeared in the void volume and had a vesicle diameter in the range 300–400 nm. Fraction 3, with an equilibrium constant ( K d) of 0.58, consisted of vesicles between 100 and 200 nm in diameter as measured by routine size analysis with the electron microscope and by calibration of the column with latex beads. The appearance of two plasma membrane fractions could also be confirmed by iodination of the surface membrane prior to fractionation. This gel chromatographic procedure represents a rapid and convenient method for the isolation of membrane material, which was enriched between five- and fourteen-fold based on the specific activity of the membrane-bound marker enzymes. Fraction 1 contained small amounts of lysosomal and Golgi membranes, and fraction 3 some material of the Golgi apparatus and the endoplasmic reticulum. The major portion of the contaminating membraneous material remained on the column and could be eluted with high salt buffer. The two plasma membrane fractions revealed some differences in 5′-nucleotidase specific activity and in the protein pattern, especially in the higher molecular weight range, as shown by sodium dodecyl sulphate gel electrophoresis.