Acidic Proteins from Human Brain: Purification and Properties of Glu-20 and Glu-35 Proteins, and Immunochemical Comparison with Glu-50 Protein

抄録

In a continuation of previous work [Nomata et al. (1983) J. Biochem. 93, 825-831], this paper reports the purification and properties of the two other acidic proteins from human brain, and the immunochemical comparison of the three proteins. The protein described previously was named Glu-50 protein, because 50%. of its amino acid residues were glutamic acid. By analogy, the proteins reported here were named Glu-20 and Glu-35 proteins on the basis of their glutamic acid contents. Both proteins were purified to a homogenous state from human brain by ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography and gel filtration on Sephadex G-75 and G-100, like Glu-50 protein. The molecular weights of the Glu-20 and Glu-35 proteins in the native state were 61, 000 and 48, 000 daltons and the values in SDS medium were 60, 000 and 16, 000, respectively, suggesting that Glu-20 protein is a monomer, and Glu-35 a trimer. The isoelectric points of Glu-20 and Glu-35 proteins were pH 4.2 and 3.6, respectively. Glu-20 protein contained 20.5%. Glu, 18.4%. Asp and 10.5% Lys, and Glu-35 protein contained 33.9% Glu and 17.2% Asp. The N-terminal amino acids of both proteins were Gly, whereas the C-termini of Glu-20 and Glu-35 proteins were Leu and Lys, respectively. Glu-20 protein gave a slightly positive periodic acid-Schiff reaction, suggesting that it is a glycoprotein, but Glu-35 protein gave a negative reaction.
The three acidic proteins, Glu-50, Glu-35, and Glu-20 proteins, were compared immunochemically. Antibodies could be produced in rabbits against Glu-50 and Glu-20 proteins only after immunization with the performate-oxidized proteins, but no antibody could be produced against Glu-35 by similar immunization. Anti Glu-50 antibody reacted only with Glu-50 protein, not with Glu-35 or Glu-20 protein, calmodulin, or S-100 protein. Similarly, anti Glu-20 antibody was specific for Glu-20 protein and did not cross-react with Glu-35 or Glu-50 protein, calmodulin, or S-100. Anti S-100 antibody did not react with Glu-50, Glu-35, or Glu-20 protein. The cross-reactivities of these antibodies were tested with extracts of various tissues of a goat. Anti Glu-50 antibody reacted only with an extract of the brain, not with extracts of other tissues, whereas anti Glu-20 antibody reacted with all the tissue extracts examined.