Free-flow electrophoresis of the low-density structures that contain asialoglycoproteins in the rat liver

Abstract

Rats were given an intravenous dose (1–2 μg/100 g) of iodine-labelled asialotransferrin, asialofetuin, or asialoorosomucoid either alone or in combinations, and the distribution of the radioactivity in the liver, removed 10–20 min after the injection, was analyzed by free-flow electrophoresis in an Elphor VaP 11 apparatus. Liver homogenates were prepared for electrophoresis according to an elaborate ultracentrifugation scheme that is outlined in detail with respect to conditions and yields. The scheme involved differential centrifugation, followed by density gradient centrifugation in a linear sucrose gradient and gel filtration using Sepharose 2B. Two ligand-containing fractions were obtained during differential centrifugation, each associated with a different complement of subcellular marker enzymes. On free-flow electrophoresis, the ligand present in either fraction exhibited a major and a minor peak. They were incompletely separated, the minor peak shouldering on the major one. The major peak had a higher electrophoretic mobility than the peaks of the acid phosphatase and phosphodiesterase I activities, but it had the same mobility as the sialyltransferase activity. The minor, less electronegative peak comigrated with the peaks of acid phosphatase and phosphodiesterase I activities and also with the major protein component of the subcellular fraction. It is concluded that the asialoglycoprotein-transporting subcellular vesicles are heterogeneous in regard to charge and that their complete separation from subcellular marker enzymes cannot be accomplished by free-flow electrophoresis.