Charles T. Ladoulis

Lymphocyte plasma membranes I. Thymic and splenic membranes from inbred rats

Abstract Thymic and splenic lymphocyte plasma membranes were isolated from inbred ACI and F344 rats. Chemical and enzymic analyses indicated a highly purified plasma membrane preparation with a high cholesterol to phospholipid ratio and over 12-fold enrichment of the membrane enzyme 5′-nucleotidase. Polyacrylamide-gel electrophoresis of externally radioiodinated membranes showed a major 117 000 dalton glycoprotein in both thymic and splenic lymphocytes. Thymocytes differ from splenic lymphocytes by having an unique external glycoprotein of 27 000 daltons and by the relative lack of membrane components above 200 000 daltons.

Lymphocyte plasma membranes. III. Composition of lymphocyte plasma membranes from normal and immunized rats.

Isolated plasma membranes of thymic and splenic lymphocytes from unimmunized and immunized rats of the inbred ACI and F344 strains were analyzed for chemical and enzymatic composition, for membrane protein patterns by polyacrylamide gel electrophoresis and for membrane-associated immunoglobulins. After immunization, the thymic and splenic lymphocyte membranes from F344 rat contained less carbohydrate and higher phospholipid contents than control animals. In both ACI and F344 inbred rat strains the membrane phospholipid to cholesterol weight ratio increased significantly after immunization. The electrophoretic patterns of solubilized membrane proteins and of iodinated external membrane proteins were similar in unimmunized and immunized animals. When thymic and splenic lymphocytes of normal or immunized animals were surface radioidinated, solubilized in Triton X-100, NP-40 or 10 M urea in 1.5 M acetic acid and analyzed by immunoprecipitation, labeled IgM immunoglobulin was recovered from thymic lymphocytes but both labeled IgG and IgM were recovered from splenic lymphocytes. However, when unlabeled isolated plasma membranes were solubilized in 1 percent Triton X-100 and analyzed by immunodiffusion in agarose gels both IgG and IgM were identified in thymic and splenic cells.

Lymphocyte plasma membranes—V. Immunoglobulins on isolated plasma membranes of the thymic and splenic lymphocytes of the rat

Abstract The plasma membranes from thymic and splenic lymphocytes of inbred ACI rats were isolated and compared for immunoglobulin content before and after immunization of the animals with poly(Glu 52 Lys 33 Tyr 15 ). The highly purified plasma membranes were solubilized in the nonionic detergent Triton X-100 and analyzed by double immunodiffusion with antisera specific for heavy chains of rat IgM and IgG immunoglobulins. Both the thymic and splenic lymphocyte plasma membranes from unimmunized and immunized animals had IgM and IgG immunoglobulins. The IgM content was lowest in thymocyte plasma membranes from unimmunized animals. Immunization resulted in an increase in the IgM content of the thymocyte plasma membranes only. Both thymic and splenic cell membranes appeared to have similar amounts of IgG before and after immunization. Semiquantitative estimates indicated that the content of IgG in thymocyte membranes was about 1% of the total membrane protein. Since our previous studies of rat thymocytes by surface radioiodination demonstrated IgM but no IgG immunoglobulin, the present findings suggest that IgG in rat thymocyte membranes may lie within the plasma membrane matrix.

Lymphocyte plasma membranes. VI. Plasma membrane glycoproteins of thymic and splenic lymphocytes from inbred rats.

Plasma membranes of splenic and thymic lymphocytes from ACI rats were analyzed for their protein and glycoprotein components by surface radioiodination with 125I and SDS-polyacrylamide gel electrophoresis. The glycoproteins were extracted with lithium diiodosalicylate, characterized and assayed with antisera to thymic antigen. Plasma membranes of both cell types showed more than 25 proteins of which 10--15 were glycoproteins. Both cells showed five major glycoproteins but their apparent molecular weights or intensities differed. Surface radioiodination showed a 120 000 daltons component, common to both cell types, and a 27 000 daltons thymus-specific component as the most exposed surface glycoproteins. Lithium diiodosalicylate extracts of the plasma membranes contained almost all of the glycoprotein components and comprised 5-6 percent of the total membrane protein and 40-50 percent of the total membrane carbohydrate, with sialic acid content in thymus twice that of the spleen cells. About 1 percent of the total plasma membrane protein and 7 percent of the total isolated glycoproteins from thymocytes were reactive with rabbit anti-rat thymocyte antiserum and the immune precipitates showed two components with apparent molecular weights of 72 000 and 27 000.

Effects of Detergents on Isolated Rat Lymphocyte Plasma Membranes

For several years we have been studying the genetic control of the immune response in inbred rats by immunochemical procedures for assaying antibody and by kinetic analysis of the production of antibody forming cells. These studies have been carried out using a chemically defined synthetic polypeptide antigen poly(Glu52Lys33Tyr15) in high and low responder strains of rats (1). We are now purifying lymphoid cells in order to explore the chemical basis of the genetic control mechanisms by isolating and characterizing the membrane components which may be involved in the interaction with antigen. Since the membrane proteins and glycoproteins are integral components of the liquid crystalline structure of the plasma membrane, their isolation depends upon dissociation from their native semicrystalline environment into a liquid phase suitable for studies of their binding activity, molecular structure and biological function. The methods of dissociation employ certain assumptions about the effects of solubilization on the structure and function of membrane components. Although the various detergents used for isolation of membrane components are classified as anionic, cationic or nonionic, there is no reliable means of predicting the results of solubilization either in terms of the degree of solubilization or with respect to their selectivity for different macromolecules. For this reason, the selection of these agents is based upon empirical results.