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Characterization of Single‐Stranded cAMP Response Element Binding Protein (ssCRE‐BP) from Mouse Cerebellum
Author(s) -
OSUGI TAKESHI,
DING YUN,
MIKI NAOMASA
Publication year - 1996
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.1996.tb17430.x
Subject(s) - fusion protein , microbiology and biotechnology , complementary dna , recombinant dna , affinity chromatography , biology , biochemistry , cerebellum , binding protein , gene , molecular cloning , western blot , chemistry , enzyme , neuroscience
SUMMARY It has been speculated that opiate tolerance and dependence may occur at the level of gene expression. Our previous studies have shown that the binding activity of a nuclear factor (ssCRE‐BP) to single‐stranded CRE of somatostatin gene is altered by long‐term treatment with morphine in the mouse cerebellum. ssCREBP was purified from the mouse cerebellum by a combination of chromatography on DNA affinity agarose and Mono Q HR. The native protein exhibited a molecular size of 110–150 kDa by gel filtration, and two polypeptides of about 35–40 kDa were observed on SDS‐PAGE. The cloning and sequencing of a cDNA encoding ssCRE‐BP showed that the protein possesses a glycine‐rich domain and a glutamine‐rich domain in the amino terminus and the carboxyl terminus, respectively. To investigate the function of ssCRE‐BP in the brain, recombinant glutathione‐ S ‐transferase (GST) fusion proteins containing ssCRE‐BP were expressed in bacterial systems. Rabbit anti‐ssCRE‐BP antibodies were raised against a GST‐ssCRE‐BP fusion protein. Using the antibodies in western blot analysis, a polypeptide of ∼66 kDa was detected in the brain. These findings indicate that ssCRE‐BP is involved in opiate tolerance and dependence.