Purification, cloning, and tissue distribution of a 23-kDa rat protein isolated by morphine affinity chromatography.
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 x 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.
Grandy
D K
DK
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland.
Hanneman
E
E
Bunzow
J
J
Shih
M
M
Machida
C A
CA
Bidlack
J M
JM
Civelli
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000032:analysis; D001483:Base Sequence; D001923:Brain Chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D005971:Glutamates / Q000032:analysis; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000737:chemistry; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009022:Morphine Derivatives; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification*; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D051381:Rats; D011919:Rats, Inbred Strains; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
D005971:Glutamates; D009022:Morphine Derivatives; D009419:Nerve Tissue Proteins; D015345:Oligonucleotide Probes; D001224:Aspartic Acid; D018698:Glutamic Acid; C030452:14 beta-bromoacetamidomorphine; D004247:DNA