Reacción #1365703

Ecuación de reacción

potassium phosphate

trifluoroacetic acid

CH3CN

CH3CN

→

phosphoserine

Condiciones de reacción

Tratamiento posterior

1. 1
   OtroSynapsin I was purified from rat and bovine brain
2. 2
   OtroMAP kinase, p44mpk, and the cyclin-dependent protein kinase (cdkl)-cyclin A complex were purified from sea star oocytes
3. 3
   workup.WAITdigested for 36 hours at 37° C.
4. 4
   workup.ADDITIONin a buffer containing 100 mM Tris (pH 8), 10% (vol/vol) CH3CN, 1%
5. 5
   workup.ADDITION1 M urea was added after 18 hours
6. 6
   Otrowere purified in a two-step procedure by reversed-phase
7. 7
   Otrowere isolated by linear gradient elution in the first chromatographic step [buffer 1
8. 8
   LavadoPeak 2 was eluted as a single 32P-

Procedimiento

In vitro Phosphorylation of Synapsin I by MAP Kinase and Identification of MAP Kinase-Dependent Phosphorylation Sites. Synapsin I was purified from rat and bovine brain as earlier described. MAP kinase, p44mpk, and the cyclin-dependent protein kinase (cdkl)-cyclin A complex were purified from sea star oocytes and assayed as earlier described by using 50 μM [γ-32P] ATP (DuPont/NEN) and 5 μM synapsin I. For stoichiometric phosphorylation, reactions were carried out for 2 hours with 7 μM synapsin I in the absence (MOCK-P) or presence of the indicted protein kinase. Samples were subjected to SDS/PAGE, followed by staining with Coomassie blue. Incorporation of 32P was quantitated by using a PhosphorImager (Molecular Dynamics). Two-dimensional phosphopeptide map analysis and phosphoamino acid analysis and in-gel MAP kinase assays were performed as earlier described. For sequence determination, rat synapsin I (340 μg) was stoichiometrically phosphorylated with p44mpk in the presence of trace amounts of [γ-32P]ATP and digested for 36 hours at 37° C. in a buffer containing 100 mM Tris (pH 8), 10% (vol/vol) CH3CN, 1% hydrogenated Triton X-100, and 17 μg each of trypsin and chymotrypsin; 1 M urea was added after 18 hours, 32P-labeled phosphopeptides were purified in a two-step procedure by reversed-phase HPLC using a C18 column (0.46×15 cm, Vydac, Hesperia, Calif.). Two major 32P-labeled peaks were isolated by linear gradient elution in the first chromatographic step [buffer 1:10 mM potassium phosphate [pH 2.2] with an increasing concentration of 40% CH3CN/20% isopropanol] Peaks 1 and 2 were further processed with a different buffer system (buffer 2:0.1% trifluoroacetic acid with increasing concentrations of 70% CH3CN). Peak 1 was resolved into two 32P-labeled phosphopeptides (peaks 1A and 1B), which appeared to be pure on the basis of absorbance profiles at 214 nm. Peak 2 was eluted as a single 32P-labeled peak in the second step. Each phosphopeptide was derivatized with ethanethiol prior to automated Edman degradation. The sequence obtained for peak 1A corresponded to residues 533-554 of rat synapsin I, with phosphoserine at residue 549: peak 1B corresponded to residues 54-76, with phosphoserine at residues 62 and 67; peak 2 corresponded to residues 54-73, with phosphoserine at residues 62 and 67.

Fuente