LSA: a new liver-specific antigen in the rat. I. Purification and characterization.
A liver-specific antigen (LSA) was purified to homogeneity from rat liver by conventional methods of protein chemistry. By consecutive 100,000 g centrifugation, ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-200, ion-exchange chromatography on CM-cellulose and affinity chromatography on concanavalin-Sepharose, it has been possible to isolate a preparation that migrated as a single band on SDS-PAGE. This preparation gave a complete identity pattern with the original crude rat liver extract when tested by double immunodiffusion. This antigen has a molecular weight of 72.5 kD with an electrophoretic mobility in the region of alpha 2-globulins. The LSA proved to be thermolabile since exposure to 55 degrees C completely destroyed the antigen. Exposure of the LSA to different pH ranging from 4 to 10 had no detrimental effect on its antigenic activity. The amino acid composition of the LSA revealed that the acidic amino acids out-number the basic amino acids, with glutamic acid being the most abundant of them. Failure of beta-mercaptoethanol to split the LSA molecule suggests the absence of sulfhydryl groups related to its antigenic activity. Subcellular fractionation of rat liver revealed most of the antigenic activity in the 100,000 g supernate, i.e. the soluble cytoplasmic fraction of the liver (cytosol). By contrast, the LSA was absent from isolated Kupffer cells from rat liver. The absence of any carbohydrate or lipid from the purified preparation of this antigen, in conjunction with the destructive effects of trypsin suggest that the LSA is a protein or a moiety closely associated with proteins.
Mihas
A A
AA
Division of Digestive Diseases, University of Mississippi School of Medicine, Jackson.
Spenney
J G
JG
Saccomani
G
G
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000941:Antigens / Q000737:chemistry / Q000302:isolation & purification*; D002241:Carbohydrates / Q000032:analysis; D010450:Endopeptidases / Q000494:pharmacology; D005260:Female; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007123:Immunoelectrophoresis, Two-Dimensional; D008055:Lipids / Q000032:analysis; D008099:Liver / Q000276:immunology*; D008670:Metals / Q000737:chemistry; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000276:immunology; D013482:Superoxide Dismutase / Q000032:analysis
D000596:Amino Acids; D000941:Antigens; D002241:Carbohydrates; D008055:Lipids; D008670:Metals; D013482:Superoxide Dismutase; D010450:Endopeptidases