Characterization and partial purification of antigenic components solubilized by a reversible chemical modification from rat-thymocyte membrane.

Abstract

Antigenic components were solubilized from washed rat thymocyte membranes by reaction with citraconic anhydride. The product remained soluble after partial decitraconylation. The weight composition of the soluble material was 60% protein, 10% RNA, 8% DNA, 2% carbohydrates and 9% phospholipids. The antigenic activity of the soluble material denned as the capacity to neutralise the cytotoxic activity of an antithymocyte serum, is not affected by exposure to temperatures up to 45°C, by exposure to pH values ranging from 3 to 10, and by removal of the nucleic acid components. Proteolytic digestion with trypsin brought about a 30% loss of antigenie activity, while digestion with papain abolished all the antigenic activity. Periodate oxidation destroyed 65% of the antigenic activity. Reduction of the oxidized mixture with sodium borohydride restored part of the lost activity. The oxidized and reduced mixture contained 50% of the original antigenic activity. Treatment with chaotropic agents only slightly affected the antigenic activity of the solubilized material. However, when mercaptoethanol was added to the chaotropic agents, most of the antigenic activity was lost. Exposure of the solubilized material to organic solvents resulted in partial loss of antigenic activity. Thus, it seems that most of the antigenic determinants are conformation‐dependent.Gel filtration of nuclease‐treated solubilized membrane preparation on columns of Sepharose 6 B eluted with Tris‐saline buffer, fractionated the antigenic material into 3 main components. Amino acid analysis showed that each of the components contained about 17% basic, 23% acidic and 31% hydrophobia amino acid residues. Significant differences were observed in the carbohydrate and lipid contents of these peaks. The material contained in the high molecular weight component, eluting near the void volume, had a specific activity up to 10‐times higher than that of the unfractionated mixture.Electrophoresis on polyacrylamide gels containing sodium dodecylsulfate and mercaptoethanol revealed that a fully citraconylated preparation (which is antigenically inactive) contained the same electrophoretic components as those present in a decitraconylated, antigenically active, preparation. The fractions obtained by gel filtration on Sepharose 6B contained components of various molecular weight. The high molecular weight peak (which contained most of the antigenic activity) displayed one major band with a molecular weight of about 70000. In the absence of reducing agents bands of higher molecular weight were observed. The data suggest that peak I contained aggregated material.Indeed, upon gel filtration on Sepharose 4B eluted with 1% sodium dodecylsulphate, two fractions of a lower molecular weight were obtained. The larger molecular weight component on dodecylsulfate polyacrylamide gel electrophoresis displayed one major band of molecular weight of 70000, while the smaller molecular weight component displayed one major electrophoretic band of molecular weight of about 60000, and an unresolved mixture of smaller molecular weight components, some of which may be phospholipid.Antisera elicited by the purified antigenic components were active against thymocytes in the cytotoxic assay, and were able to prolong skin graft survival in an allogeneic system.