The subunit structure of bovine brain 14-3-2

Two aspects of the subunit structure of the bovine brain specific protein 14-3-2 have been examined. On the one hand, native 14-3-2 has been separated into two fractions by hydroxylapatite chromatography. One eluted at the same position when chromatographed on the same column, while the other redistributed into the same two fractions again. Amino acid analysis of these two forms of 14-3-2 gave results that were not significantly different under the condition of analysis. Furthermore, when each peak was subjected to cyanogen bromide cleavage, very similar elution profiles of the resultant fragments were obtained. The two pools also cross-reacted with antiserum to 14-3-2. Reaction of purified 14-3-2 with dimethylsuberimidate caused the formation of covalently bound protein units of 100,000 molecular weight when measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis, as opposed to the 50,000 minimal molecular weight normally detected. On the other hand, analysis of the soluble tryptic peptides of S-[¹⁴C]carboxymethyl 14-3-2 yielded only three distinct radioactive peptides, each with one residue of S-carboxymethylcysteine whereas 8 are expected on the basis of the amino acid composition of the 50,000 molecular weight polypeptide chains. Thermolysin digestion of a similarly modified 14-3-2 preparation yielded all of the radioactivity in 5 S-carboxymethylcysteine-containing peptides. The partial amino acid sequence of these peptides indicates that they represent 4 unique areas of the polypeptide chain. Since 8 such peptides were expected, that is, double the number found, the minimum structural unit of the protein must be of 25,000 molecular weight. The results of these experiments do not permit distinction between a duplication of the structure within a single polypeptide chain or the alternate possibility of two polypeptide chains bound by unusually strong non-covalent bonds. These results suggest that 14-3-2 is a covalently linked dimer of 25,000 mol.wt. units that can aggregate to form larger species of 100,000 mol. wt. and higher.