Samuel W. Luborsky

Detergent solubilized and molecular weight estimation of tumor specific surface antigen from SV40 virus transformed cells

Mild detergent treatment of SV40 transformed mouse embryo fibroblasts, followed by (NH4)2SO4 precipitation of solubilized proteins and rate zonal centrifugation in a sucrose gradient, provided direct and rapid determination of the sedimentation coefficient (4S) of tumor specific surface antigen(s) (TSSA), as detected by an antibody dependent, cell lysis assay. A molecular weight range of 50,000–60,000 was estimated for the TSSA. These results agree with those also obtained from Sephadex G-150 exclusion chromatography.

Ultracentrifugation Studies of Polyelectrolytes in Polyglucose Density Gradient

Abstract An ideal solute for density gradient ultracentrifugation of polymers in aqueous solution should be inert and readily soluble in water to form an extended range of solution densities of low viscosity. High molecular weight is an added attraction because osmotic effects are minimized. Highly branched spherical synthetic polysaccharides fulfill these requirements. High degree of branching is a consequence of the condensation of polyfunctional monomers. Density and relative viscosity of solutions of polyglucose, sucrose, and of a natural sucrose polymer, Ficoll, are compared. The behavior of various polyelectrolytes was studied in low viscosity polyglucose density gradients in equilibrium buoyant density measurement in the ultra-centrifuge. Macromolecules or macromolecular complexes attain low apparent equilibrium buoyant density, probably because of an excluded volume effect of the solute. This allows sedimentation to isopycnic position of complex biopolymers in inert polyglucose solutions, which ot...

Inactivation and dissociation of β-galactosidase by amines

Abstract In addition to the inactivation and partial dissociation of β-galactosidase by mercaptoethanol, mercaptoethylamine or dithiothreitol, we found that ethanolamine or ethylenediamine rapidly inactivates the enzyme and causes quantitative dissociation of the 16S tetramer to a single 6S component. These results suggest that the thiol function of mercaptoethanol or mercaptoethylamine may be acting primarily as an electron donor in chelation rather than as a disulfide reducing agent.

SUBCELLULAR DISTRIBUTION OF SV40 T ANTIGEN SPECIES IN VARIOUS CELL LINES

Publisher Summary This chapter discusses the subcellular distribution of SV40 T antigen species in various cell lines. It describes the presence of various SV40 virus transformed cell lines of the SV40 T antigen (TA) on the plasma membrane and the subcellular size distribution of TA species. The cells were labeled with 35 S methionine, suspended in hypotonic solution, dounced, and the nuclei, membranes, and cytoplasm fractionated in a two-phase system. TA was immunoprecipitated with anti TA serum in the presence of Sepharose linked to protein A of Staph, aureus, and analyzed by electrophoresis on SDS-polyacrylamide gels and fluorography. SV40 transformed cells were examined from normal rat kidney, Balb/c and AL/N mice, cloned and mass cultures. Both 94 K and 56 K molecular weight (MW) species were found in the nuclei of the four cell lines examined, while the membrane fractions contained the 94 K dalton species and only low levels of the 56 K species. The cytoplasm fractions contained little radioactive label specifically immunoprecipitated by the anti TA serum. Detection of the small TA species was variable,and rendered more difficult by generally containing least label. The chapter also describes control mixing experiments carried out to clarify the nature and purity of the cell fractions.

PERTINENCE OF SURFACE MEMBRANE CHANGES IN SPONTANEOUSLY AND VIRALLY TRANSFORMED CELLS TO THE BALANCE BETWEEN TUMORIGENICITY AND IMMUNE REJECTION

Spontaneously transformed highly tumorigenic AL/N strain mouse cells and their SV40 virus transformed clonal derivatives were compared. Glycoproteins were labeled by various metabolic precursors (amino acids, glucosamine, mannosamine, etc.). The flow of such labels into various subcellular components was followed. Cell surface membranes were obtained and the components of these membranes were separated by polyacrylamide gel electrophoresis. The biosynthesis of a prominent glycoprotein component with an apparent molecular weight of 180 K dalton was reduced in the SV40 transformed cells, when all the different kinds of cells were grown in their logarithmic phase. Three types of SV40 specific antigens were detected by immunologic assays: (1) T antigens, (2) tumor specific surface antigens (TSSA), and (3) tumor specific transplantation antigens (TSTA). A rapid and sensitive microassay for TSSA guided the development of a mild and efficient detergent (Triton X-100) solubilization technique, and the purification of the antigens that appear to co-purify in various procedures. The antigens expressed on the cell surface that are responsible for immunologic recognition and rejection of the SV40 transformed cells represented a very minor fraction of the cell surface membrane components. The microassay for TSSA allowed partial purification and characterization of the SV40 specific cell surface antigen (s). The(se) SV40 specific cell surface antigen (s) in the AL/N strain mouse functionally opposes cellular tumorigenicity.