Immunological studies of a nerve specific protein

Axoplasmic transport of a brain-specific soluble protein☆

“…However, recent studies in our laboratory [33] on physical and chemical characteristics of the 14-3-2 protein have shown that it is composed of two identical subunits and that it can undergo conformational alterations which may play a role in its function in the nervous system. Thus, one can speculate that the 14-3-2 protein may be involved in slowly developing biological processes which are considered to participate in long-term memory storage [ 49] .…”

“…Tritiated proline was used as a precursor since it has been found to produce more efficient labeling of the transported protein in both the goldfish and the chick optic systems [ 31,32), with relatively little incorporation into the brain as a whole. Furthermore, the 14-3-2 protein contains 17 residues of proline [ 33] so that reasonable amounts of isotope incorporation into newly synthesized 14-3-2 protein would be expected. The chicks were lightly anesthetized with halothane and the injection was made into the left eye by means of a 0.…”

“…After aliquots were taken, the samples were lyophilized, dissolved in 0.5 ml of Tris/phosphate (pH 7.2) and subjected to Sephadex G-150 (superfine) chromatography in order to eliminate a large proportion of non-(14-3-2)-labeled material. Radioactive fractions corresponding to the position of elution of pure 14-3-2 protein prepared from rat brain [ 33] were obtained from columns (0.5 X 20 cm) equilibrated with 10 mM Tris/phosphate (pH 7 .2). Elution was carried out with the same buffer.…”

“…10 µg of pure 14-3-2 protein and 50 µl of specific antiserum (diluted 1 : 5 with the phosphate/NaCl buffer) were added to each sample. The carrier 14-3-2 protein had been isolated and purified from rat brain and antiserum was produced in New Zealand white rabbits [33]. The antiserum to the rat 14-3-2 protein produced a single sharp precipitin band by Ouchterlony double diffusion when tested against the high-speed supernatant fractions from chick retinae and optic lobes as well as rat high-speed supernatant and purified rat 14-3-2 protein.…”

Biochemical Maturation of the Non-Innervated Chick Optic Lobe

“…NSP was determined by a radioimmunoassay method previously described [25). Chick optic Jobes were homogenized in 2.5 ml of 10 mM Tris-HCI, pH 7.6 and the suspension centrifuged (100,000 g, 60 min).…”