Abstract
Fractionation of octyl glucoside-solubilized proteins from young rat brain was monitored using rat brain neurons, which were cultured in microwells coated with various protein fractions to be studied. An adhesive protein that promotes neurite outgrowth in rat brain neurons was isolated by chromatography on heparin-Sepharose followed by Affi-Gel blue. The apparent molecular mass of the protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions was about 30 kilodaltons (p30). Under nonreducing conditions a closely spaced doublet band was observed corresponding to 27-28-kilodalton size. Gel filtration in the presence of 4 M urea indicated the molecular size of 58 kilodaltons suggesting a dimeric structure. Western blotting experiments using affinity-purified rabbit antibodies detected p30 as an immunochemically distinct protein in brain and in N18 neuroblastoma cells. The p30 protein was also detected in the N18 cells by lactoperoxidase-catalyzed cell surface iodination. Western blotting of heparin-binding proteins solubilized from brains of rats of various age groups indicated that p30 is clearly more abundant in perinatal brain as compared to adult tissue. The neuron-binding and neurite outgrowth-promoting properties of p30 as well as the developmental regulation of its content in brain tissue suggest a role in neuronal growth.
Highlights
From young rat brain was monitored using rat brain This study shows that a factor, which shares with laminin neurons, whichwere cultured in microwells coated the ability to bind to heparin, thecell adhesive activity, and with various protein fractions to bestudied
The p30 protein was clearly detected by Western blotting of the heparin-binding proteins solubilized by octyl glucoside from N18 cells that had been surface-labeled with lactoperoxidase-catalyzed iodination (Fig. 14A, lane I)
Previous studies have indicated that anadhesive and neurite-promoting activity can be solubilized by octyl glucoside from cell membranes of young rat brain [9, 12]
Summary
It was consistently found that the fractions eluted at low salt or the main heparin-binding protein eluting at about 0.5 M salt (fractionA in Fig. 1)displayed no neuritepromoting activity (panel A in Fig. 1).In contrast, fractions eluted at 0.75 to about 1 M NaCl just after the main protein peak (B,C, and D in Fig. 1) had a dramatic effect on the Neurite outgrowth may be enhanced by diffusible factors and by surface-bound substances [1, 2]. The neurofilament-positive processes extended up to 10-20 times the diameter of the round cell after 24 h on theactive surfaces (Fig. 1).A similar neurite-promotingactivity could be observed on surfaces coated with laminin, but the resultant cell morphology was different from thatcaused by the heparin-binding protein of brain.
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