Affinity purification of ?-endorphin-like material from NG108CC15 hybrid cells by means of the monoclonal ?-endorphin antibody 3-E7.
Neuroblastoma x glioma hybrid cells (NG108CC15) were examined for the presence of ?-endorphin-like material. In order to differentiate this ?-endorphin-like material from crude cell extract, a procedure for immunoaffinity chromatography was developed. The monoclonal antibody 3-E7 employed possesses the unique property of recognizing the N-terminal sequence of virtually all endogenous opioid peptides, but not their precursors. By means of this immunoaffinity procedure about 90% of exogenous ?-endorphin was recovered from 10 ml phosphate buffered saline samples. Affinity chromatography served as first-step purification of crude NG108CC15 cell extract for the separation and concentration of ?-endorphin-like material. The eluate of the immunoaffinity gel was subjected either to Sephadex gel filtration or to high pressure liquid chromatography. Under either condition, immunoreactive ?-endorphin which eluted with synthetic ?-endorphin was detected. The concentration in six different batches varied from 4 to 17 fmol/10(8) cells. This would be 10-200-fold lower than that observed for the enkephalins or dynorphin A/?-neo-endorphin. It is concluded that the utilization of the monoclonal antibody 3-E7 for a first-step purification of cell extracts was an essential pre-requisite for the separation of ?-endorphin-like material from the hybrid cells. The presence of enkephalin-like material, of dynorphin A/?-neo-endorphin-like material and of ?-endorphin immunoreactive material suggests that NG108CC15 cells are able to generate opioid peptides related to the precursors pre-proenkephalin A, pre-proenkephalin B and pro-opiomelanocortin.
Dandekar
T
T
Department of Neuropharmacology, Max-Planck-Institut für Psychiatrie, Am Klopferspitz 18A, 8033 Martinsried, F.R.G.
Gramsch
C
C
Houghten
R A
RA
Schulz
R
R