Characterization of the regulatory subunit of Yarrowia lipolytica cAMP - dependent protein kinase evidences of a monomeric protein

cAMP-dependent protein kinase [PKA] catalytic [C] and regulatory [R] subunits from Yarrowia lipolytica are encoded by single genes, TPK1 and RKA1, respectively. Here we performed the heterologous expression, purification and characterization of the R subunit from Y. lipolytica yeast cells, and explo...

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Detalles Bibliográficos
Otros Autores: Kronberg, María Florencia, Giacometti, Romina, Ruiz Herrera, José, Passeron, Susana
Formato: Artículo
Lenguaje:Inglés
Materias:
AUTOPHOSPHORYLATION
PKA
REGULATORY SUBUNIT
YARROWIA LIPOLYTICA
CYCLIC AMP DEPENDENT PROTEIN KINASE
HOLOENZYME
MONOMER
AMINO TERMINAL SEQUENCE
CENTRIFUGATION
CONTROLLED STUDY
DIMERIZATION
GEL FILTRATION
HYDRODYNAMICS
IN VIVO STUDY
MASS SPECTROMETRY
NONHUMAN
PROTEIN ANALYSIS
PROTEIN EXPRESSION
PROTEIN INTERACTION
PROTEIN PHOSPHORYLATION
PROTEIN PURIFICATION
AMINO ACID SEQUENCE
CLONING, MOLECULAR
CYCLIC AMP-DEPENDENT PROTEIN KINASES
GENE EXPRESSION
HOLOENZYMES
MOLECULAR SEQUENCE DATA
PHOSPHORYLATION
PROTEIN MULTIMERIZATION
PROTEIN SUBUNITS
RECOMBINANT PROTEINS
YARROWIA
Acceso en línea:http://ri.agro.uba.ar/files/intranet/articulo/2011Kronberg.pdf
LINK AL EDITOR
Aporte de:Registro referencial: Solicitar el recurso aquí
Biblioteca Central (FAUBA) de Universidad de Buenos Aires
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520 |a cAMP-dependent protein kinase [PKA] catalytic [C] and regulatory [R] subunits from Yarrowia lipolytica are encoded by single genes, TPK1 and RKA1, respectively. Here we performed the heterologous expression, purification and characterization of the R subunit from Y. lipolytica yeast cells, and explored the main biochemical features of the PKA. The purified recombinant R, active and capable to interact with C subunit was used to prepare highly specific polyclonal antiserum. Sucrose-gradient centrifugation and gel filtration analysis of both recombinant and native R revealed the monomeric nature of this subunit. Hydrodynamic parameters of the holoenzyme indicated that Y. lipolytica PKA is a dimer of 90 kDa composed of an R subunit of 42 kDa and a C subunit of 39 kDa. The identification of the N-terminal sequence was carried out by mass spectrometry analysis of the purified native R subunit. The differences between N-terminal sequences of R subunits from Y. lipolytica and other organisms, particularly a short linker that spans the inhibitory site, were discussed as the possible cause of the lack of dimerization. R was identified as a type II subunit since our results indicated that it was phosphorylated in vivo by C at S124 identified by anti-phospho-PKA substrate [RRXS/T] antibody. 
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773 |t Archives of Biochemistry and Biophysics  |g Vol.509, no.1 (2011), p.66-75 
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