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

Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University Portland, Oregon 97201
Department of Cell Biology and Anatomy, Oregon Health Sciences University Portland, Oregon 97201
Department of Zoology, University of Arizona Tucson, Arizonia 85721
Oregon Regional Primate Center Beaverton, Oregon 97006
Department of Pharmacology, University of Rochester Medical Center Rochester, New York 14642

Address requests for reprints to: Dr. Olivier Civelli, Vollum Institute for Advanced Biomedical Research, L-474, Oregon Health Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201.

Abstract

A 23-kDa (p23k) rat brain protein was stereospecifically eluted from a 14β-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% -helices and 18% β-pleated sheets. Northern blot analysis revealed p23k transcripts in rat brain, liver, and the mouse x rat neuroblastoma-glioma NG 108–14 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 β-endorphin.

FOOTNOTES

This work was supported by a research grant from the NIDDK(DK-3731;to O.C).

^* Holder of a fellowship from the NIH.

Received for publication April 6, 1990. Revision received June 14, 1990. Accepted for publication June 18, 1990.

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