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A Model System for Study of Subcellular Distribution of Cell Surface Antigens¹

Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27706

Abstract

A model system is described for the study of cell surface antigens and their subcellular distribution. The nuclear sediment of a rat liver homogenate was subjected to a rate zonal centrifugation for the partial separation of subcellular organelles. The problem of obtaining pure preparations of all subcellular organelles was circumvented by careful quantitation of enzyme markers for all the major membranous organelles in each fraction. Large plasma membrane fragments were isolated free of contaminating organelles as determined by enzyme markers. After quantitation of a cell surface antigen by a cytotoxicity inhibition assay with selected fractions, enzyme to antigen ratios were calculated. The antigen detected in this study was found to be limited to the plasma membrane. The ratios of the plasma membrane-enzyme-marker to antigen content were essentially constant in all fractions regardless of varying degrees of contamination by other subcellular organelles. This system offers several advantages, one of which is the estimation of a surface antigen on other subcellular organelles without the necessity of obtaining "pure" fractions of each organelle. In addition, the model eliminates repetitive sedimentation of the organelles with its consequent aggregation of membranes which would interfere with quantitative antibody absorptions. The system is also applicable to livers of other species and provides liver plasma membranes of good purity with only one gradient rate sedimentation.

Footnotes

¹ This investigation was supported by United States Public Health Service Grant AI 08897.

² Recipient of Postdoctoral Fellowship FO 2-AM 44427 National Institute of Arthritis and Metabolic Disease, National Institutes of Health.

³ Recipient of a Career Development Award PH 43-66-939.

⁴ Present address: School of Microbiology, University of Melbourne, Parkville, Victoria, Australia, 3052.