Purification and characterization of PCPP‐260: A Purkinje cell‐enriched cyclic amp‐regulated membrane phosphoprotein of Mr 260,000

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Abstract
PCPP‐260 (Purkinje cell phosphoprotein of Mr 260,000), a substrate for cAMP‐dependent protein kinase, appears to be an integral membrane protein highly enriched in Purkinje cells of the mammalian cerebellum (Walaas et al.: J. Neurosci., 3:291–301, 1983; Walaas et al.: J. Neurosci., 6:954–961, 1986). PCPP‐260 has now been purified from a crude particulate fraction of bovine cerebellum, using the ionic detergent N‐lauryl sarcosine (NLS) as solubilizing agent, and monitoring the purification by silver stain and autoradiography of 32P‐phosphorylated samples, after separation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Concanavalin A was found to bind to PCPP‐260, suggesting that it is a glycoprotein. PCPP‐260 was therefore extracted, retained on a column of concanavalin A‐agarose, and eluted by α‐methyl mannoside. Further chromatography on Sephacryl S‐400 yielded a preparation that was purified approximately 250‐fold relative to the initial particulate fraction and that was at least 95% pure. The protein was estimated to represent approximately 0.4% of total membrane protein in the cerebellum. Peptide mapping and phosphoamino acid analysis following phosphorylation of the protein by cAMP‐dependent protein kinase showed one major tryptic phosphopeptide containing phosphoserine. A similar, less prominent protein was also found in membranes from other brain regions but could not be detected in liver membranes. The availability of highly purified PCPP‐260 should facilitate the investigation of its possible functional roles in the nervous system.