Isolation and characterization of the nervous system-specific protein 14-3-2 from rat brain. Purification, subunit composition, and comparison to the beef brain protein.

Abstract

A procedure is described for the isolation of the nervous system-specific protein designated 14-3-2 from rat brain. The methods utilized are salt precipitation, DEAE-cellulose ion exchange chromatography, Sephadex G-150 gel filtration, and column isoelectric focusing. The native 14-3-2 protein has an isoelectric point of 4.7 in the absence of denaturing agents and 5.0 in the presence of 2.0 M urea. The protein, as isolated, appears homogeneous since it migrates as a single band on Tris-glycine (pH 8.9), sodium dodecyl sulfate (pH 7.2), and 8 M urea (pH 4.0) polyacrylamide gels. Sedimentation velocity and equilibrium data indicate a homogeneous component of molecular weight 78,000. Sedimentation of 14-3-2 in 6 M guanidine HCl containing 0.02% glutathione yielded a molecular weight of 39,000, indicating the dimeric nature of the protein as isolated. The rat brain protein seems to be composed of one subunit type, since polyacrylamide gel electrophoresis in 8 M urea yields a single protein component. Sodium dodecyl sulfate gel electrophoresis of rat brain 14-3-2 produced one sharp band with a relative mobility corresponding to a molecular weight of 48,000. Specific anti-14-3-2 serum has been prepared from both New Zealand white rabbits and goats. Rat 14-3-2 is very similar in amino acid composition to the beef brain protein and to antigen alpha. The antigenic properties of rat and beef 14-3-2 are also similar, since beef 14-3-2 antiserum reacts well with rat 14-3-2 and vice versa. Electrophoretic mobilities of denatured rat and beef 14-3-2 (0.1% sodium dodecyl sulfate and 8 M urea) are identical. Despite these similarities the two proteins are completely resolved on Tris-glycine gels. The sedimentation behavior of the beef and rat proteins are also different, indicating a difference in the association state and conformation of the two preparations.