Purification, Cloning, and Tissue Distribution of a 23-kDa Rat Protein Isolated by Morphine Affinity Chromatography

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Abstract
A 23-kDa (p23k) rat brain protein was stereospecifically eluted from a 14.beta.-bromoacetamidomorphine affinity column, purified to apparent homogeneity by reverse phase HPLC, and partially sequenced. Three degenerate oligodeoxynucleotide probes were synthesized based on this partial amino acid sequence. A rat brain cDNA library was screened using these probes, and a full-length cDNA was isolated. The deduced protein, 187 amino acids long, is rich in glutamic and aspartic acid residues, endowing p23k with a net negative charge at neutral pH. The protein lacks a signal sequence as well as any transmembrane domains. Based on predictions of secondary structure, p23k is a globular protein composed of 30% .alpha.-helices and 18% .beta.-pleated sheets. Northern blot analysis revealed p23k transcripts in rat brain, liver, and the mouse .times. rat neuroblastoma-glioma NG108-14 cell line. Although not an opioid receptor itself, this protein may be associated with such a receptor or be related to a protein that has been shown to be cross-linked to the opioid peptide .beta.-endorphin.

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