RESUMEN
Sequencing of phosphoserine-containing peptides yields normally no identifiable PTH-derivatives at those positions where phosphoserine is located. Here a new method is described which allows identification of the position of phosphoserine by chemical modification just before sequence analysis. In a one-step microbatch reaction, phosphoserine present in the intact peptide can be transformed quantitatively into stable derivatives such as beta-methylaminoalanine (MAA), S-ethanolcysteine or S-ethylcysteine. These derivatives are detectable during microsequencing with less than 100 pmol peptide using an Applied Biosystems gas-phase sequencer equipped with an on-line PTH amino acid analyzer.
Asunto(s)
Fosfoproteínas/análisis , Fosfoserina/análisis , Serina/análogos & derivados , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Microquímica , Oligopéptidos/análisisAsunto(s)
Oligopéptidos/química , Fosfopéptidos/química , Fosfoproteínas/química , Tirosina/análogos & derivados , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión/métodos , Indicadores y Reactivos , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Fosfopéptidos/aislamiento & purificación , Radioisótopos de Fósforo , Fosfotirosina , Técnica de Dilución de Radioisótopos , Espectrofotometría Ultravioleta/métodos , Tirosina/análisisAsunto(s)
Cisteína/análogos & derivados , Oligopéptidos/química , Fosfopéptidos/química , Fosfoproteínas/química , Fosfoserina/análisis , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión/métodos , Hidrólisis , Indicadores y Reactivos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Miocardio/metabolismo , Oligopéptidos/síntesis química , Oligopéptidos/aislamiento & purificación , Fosfopéptidos/aislamiento & purificación , Fosfoproteínas/aislamiento & purificación , Fosforilasa Quinasa/química , Fosforilasa Quinasa/aislamiento & purificación , Fosfotreonina/análisis , Troponina/química , Troponina IRESUMEN
The intact ion yields of phosphotyrosine, phosphothreonine and mono- and diphosphoserine residue-containing peptides have been compared with the non-phosphorylated sequences using plasma desorption mass spectrometry. Equimolar mixtures of the phosphorylated (Mp) and non-phosphorylated peptides (M) were also analysed. The positive mass spectra of these mixtures show a higher intensity of the [M + H]+ compared with the [Mp + H]+. In the negative mass spectrum, the bias towards the [M - H]- compared with the [Mp - H]- was reduced, but the spectra generally did not accurately reflect the stoichiometry.
Asunto(s)
Fosfopéptidos/análisis , Secuencia de Aminoácidos , Espectrometría de Masas , Datos de Secuencia Molecular , Oligopéptidos/análisis , Fosforilación , Fosfotreonina/análisis , Fosfotirosina , Tirosina/análogos & derivados , Tirosina/análisisRESUMEN
A method is described that permits the phosphoserine content of proteins and peptides to be determined in picomolar amounts. A micro-batch reaction first converts phosphoserine into S-ethylcysteine. Hydrolysis with 6 M hydrochloric acid then yields the free amino acid, which is coupled with phenyl isothiocyanate to give the corresponding phenylthiocarbamylamino acid. This derivative is determined quantitatively in the range 10-20 pmol by reversed-phase high-performance liquid chromatography. The method works well with either small peptides or proteins in the low picomole range.
Asunto(s)
Fosfoserina/análisis , Serina/análogos & derivados , Aminoácidos/análisis , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Cisteína/análogos & derivados , Indicadores y Reactivos , Oligopéptidos/análisis , Péptidos/análisis , Fosforilación , Proteínas/análisis , Troponina/análisisRESUMEN
We have isolated and sequenced a cDNA encoding the alpha subunit of phosphorylase kinase from rabbit fast-twitch skeletal muscle. The cDNA molecule consists of 388 nucleotides of 5'-nontranslated sequence, the complete coding sequence of 3711 nucleotides, and 342 nucleotides of 3'-nontranslated sequence followed by a poly(dA) tract. It encodes a polypeptide of 1237 amino acids and a deduced molecular mass of 138,422 Da. Nearly half of the deduced amino acid sequence is confirmed by peptide sequencing. Seven positions of endogenously phosphorylated serine residues and autophosphorylation sites, identified by peptide sequencing, could be assigned. They cluster in a segment of only 60 amino acids. RNA blot hybridization analysis demonstrates a predominant RNA species of approximately equal to 4500 nucleotides and a less abundant RNA of 8700 nucleotides.
Asunto(s)
Clonación Molecular , ADN/análisis , Músculos/enzimología , Fosforilasa Quinasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Peso Molecular , Fosforilasa Quinasa/análisis , Poli A/análisis , ARN Mensajero/análisis , ConejosRESUMEN
The primary structure of the alpha and beta subunits of phosphorylase kinase reveals that both proteins contain a carboxyl-terminal CA1A2X motif (where C is cysteine, A1 and A2 are aliphatic amino acids, and X is an uncharged amino acid), the recognition signal for a protein polyisoprenyltransferase. The product, a polyisoprenylated cysteine, can be detected by phenylthiocarbamoylamino acid analysis and by microsequencing following conversion to S-ethylcysteine. Mass spectrometry confirms a covalently linked farnesyl residue in both subunits. Tandem mass spectrometry localizes these modifications at the cysteine residues present in the carboxyl-terminal CAMQ and CLVS sequences of the alpha and beta subunits, respectively. Membrane association of phosphorylase kinase, probably mediated by these farnesyl residues, is discussed.
Asunto(s)
Cisteína/análogos & derivados , Fosforilasa Quinasa/química , Secuencia de Aminoácidos , Animales , Cisteína/química , Cisteína/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Músculos/enzimología , Fragmentos de Péptidos/química , Prenilación de Proteína , ConejosRESUMEN
Identification of O-phosphorylated amino acids within the primary structure of regulatory proteins is important in understanding the mechanisms by which their functions are regulated. In many cases radioactive labeling with [32P]phosphate is tedious or sometimes impossible. Therefore, we have established a series of new non-radioactive methods that permit the localization of phosphoserine, phosphothreonine, and phosphotyrosine. After partial hydrolysis of a phosphopeptide or phosphoprotein, phosphoserine, phosphothreonine, or phosphotyrosine are determined by capillary electrophoresis as their dabsyl-derivatives. Chemical modification transforms phosphoserine or phosphothreonine to S-ethyl-cysteine or beta-methyl-S-ethyl-cysteine, respectively, allowing their localization during sequence analysis. We apply solid-phase sequencing to overcome the limitations of the gas-phase sequenator in the case of phosphotyrosine-containing peptides. Liquid chromatography on-line connected to an electrospray mass spectrometer is a powerful new method of increasing importance in the protein chemistry field. It is especially well suited for identification of phosphoserine- or phosphothreonine-containing peptides in a proteolytic digest of a phosphoprotein. In this article we will describe how to work with these new methods practically.
Asunto(s)
Aminoácidos/análisis , Fosfoproteínas/análisis , Secuencia de Aminoácidos , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosfoserina/análisis , Fosfotreonina/análisis , Fosfotirosina , Tirosina/análogos & derivados , Tirosina/análisisRESUMEN
The octapeptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu, corresponding to the 14-21 sequence of bovine beta-casein A2 and 11 shorter and/or modified derivatives were synthesized and used as model substrates for three casein kinases: rat liver casein kinases 2 and 1 and a casein kinase isolated from the golgi-enriched fraction of lactating mammary gland (GEF-casein kinase). Casein kinase-2 readily phosphorylates the octapeptide at its Ser-4 residue with a Vmax value comparable to those obtained with protein substrates and Km values of 85 microM and 11 microM in the absence and presence of polylysine, respectively. These are the most favourable kinetic parameters reported so far with peptide substrates of casein kinase-2. Stepwise shortening of the octapeptide from its N terminus promotes both a gradual decrease of Vmax and an increase of Km, this being especially dramatic in passing from the hexapeptide Leu-Ser-Ser-Ser-Glu-Glu (Km 210 microM) to the pentapeptide Ser-Ser-Ser-Glu-Glu (Km 2630 microM). The tetrapeptide Ser-Ser-Glu-Glu is the shortest derivative still phosphorylated by casein kinase-2, albeit very slowly, and the tripeptides Ser-Glu-Glu and Glu-Leu-Ser were not substrates at all. Furthermore, the pentapeptide Ser-Ser-Ser-Glu-Glu was found to be a better substrate than Ser-Ser-Ala-Glu-Glu, Ser-Ala-Ser-Glu-Glu and Ser-Ala-Ala-Glu-Glu by virtue of its lower Km value. These data, while confirming that the motif Ser-Xaa-Xaa-Glu is specifically recognized by casein kinase-2, strongly suggest that additional local structural features can improve the phosphorylation efficiency of serine-containing peptides which are devoid of the large acidic clusters recurrent in many phosphorylation sites of casein kinase 2. In particular, predictive structural analysis as well as NMR and C18 reverse-phase HPLC elution profile data support the hypothesis that a beta-turn conformation is responsible for the remarkable suitability of the octapeptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu and some of its shorter derivatives to phosphorylation mediated by casein kinase-2. While neither the peptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu nor any of its derivatives were affected by casein kinase-1, a rapid phosphorylation of the octapeptide by GEF-casein kinase at Ser-5 (not Ser-4) was obtained.(ABSTRACT TRUNCATED AT 400 WORDS)