Biomaterial Database

Characterization of the phosphorylation site of PP59, a substrate for cyclic AMP-dependent protein kinase that is enriched in the synaptic membrane fraction of rat cerebellum. 1996

R C Rich, and D W Aswad

School of Biological Sciences, University of California, Irvine 92697-4550, USA.

We have identified previously a synaptic membrane-associated protein, PP59, that serves as a substrate for cyclic AMP-dependent protein kinase and is enriched in rat cerebellum. We show here that PP59 can be extracted from synaptic plasma membranes with a combination of 2% Triton X-100 plus 1 M KCl. A 290-fold purification of PP59 was achieved by selective solubilization, followed by continuous-elution preparative gel electrophoresis. To determine the amino acid sequence surrounding the cyclic AMP-dependent protein kinase phosphorylation site within PP59, the partially purified 32P-phosphorylated protein was digested with chymotrypsin, and radiolabeled peptides were purified by sequential reversed-phase HPLC in two different solvent systems. Automated Edman degradation revealed a single phosphorylation site contained within the sequence Ala-Arg-Glu-Arg-Ser-Asp-Ser(P)-Thr-Gly-Ser-Ser-Ser-Val-Tyr. No strong sequence homology to this peptide fragment with other known peptides or proteins in the SwissProt, PIR, or GenPept databases could be found. A synthetic peptide containing this unique 14-amino acid sequence was used to develop polyclonal anti-peptide antibodies that were affinity-purified and shown to recognize intact PP59 as determined by western blotting. These antibodies specifically inhibited the phosphorylation of PP59 by cyclic AMP-dependent protein kinase in an in vitro phosphorylation assay containing synaptic plasma membranes.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008297	Male		Males
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010455	Peptides	Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are considered to be larger versions of peptides that can form into complex structures such as ENZYMES and RECEPTORS.	Peptide,Polypeptide,Polypeptides
D010766	Phosphorylation	The introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety.	Phosphorylations
D002458	Cell Fractionation	Techniques to partition various components of the cell into SUBCELLULAR FRACTIONS.	Cell Fractionations,Fractionation, Cell,Fractionations, Cell
D002531	Cerebellum	The part of brain that lies behind the BRAIN STEM in the posterior base of skull (CRANIAL FOSSA, POSTERIOR). It is also known as the "little brain" with convolutions similar to those of CEREBRAL CORTEX, inner white matter, and deep cerebellar nuclei. Its function is to coordinate voluntary movements, maintain balance, and learn motor skills.	Cerebella,Corpus Cerebelli,Parencephalon,Cerebellums,Parencephalons
D002851	Chromatography, High Pressure Liquid	Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed.	Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D004591	Electrophoresis, Polyacrylamide Gel	Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.	Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein