Purification and characterization of PCPP-260: a Purkinje cell-enriched cyclic AMP-regulated membrane phosphoprotein of Mr 260,000.
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 alpha-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.
Weeks
G
G
Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021.
Picciotto
M
M
Nairn
A C
AC
Walaas
S I
SI
Greengard
P
P
D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D011689:Purkinje Cells / Q000032:analysis*; D013347:Subcellular Fractions / Q000032:analysis*
D008565:Membrane Proteins; D010750:Phosphoproteins; D000242:Cyclic AMP