Glycine → 5'-Atp

Beteiligte Stoffe

Reaktionsschritte

This type of gel allows for separating proteins upon their molecular weight and electrical charge while preserving their activity in such a way that this activity can be measured after migration. Two polyacrylamide preparations were poured between two glass plates to form a gradient and polymerized. The 4% acrylamide solution was composed of: 4.5 mL of separating buffer (Tris 1.5 M pH 8.8+0.4% Triton X-100™), 2.5 mL acrylamide 30%, 180 μL Na deoxycholate 10%, water up to 18 mL, 60 μL APS 10% and 7 μL TEMED. The 7.5% acrylamide solution was composed of the same ingredients except for the volume of acrylamide: 4.5 mL. A stacking gel was extemporaneously prepared and poured at the top of the separating gel, the stacking gel was composed of: 2.5 mL of stacking buffer (Tris-base 0.5 M pH 6.8), 6.1 mL of water, 1.34 acrylamide 30%, 0.1 mL Na deoxycholate 10%, 0.1 mL Triton X-100™, 50 μL APS 10% and 10 μL TEMED. Wells are formed in this layer during polymerization. Two volumes of the sample obtained after DEAE-agarose or Affigel Blue columns were added to one volume of sample buffer of the following composition to obtain about 100 μg proteins: 0.07% (v/v) Triton X-100™, 1.5% (w/v) Na deoxycholate, 10% glycerol, 65 mM Tris-base and 0.005% bromophenol blue. The suspended sample was allowed to stand 10 minutes on ice and centrifuged. The supernatant was loaded on gel. The proteins were migrated at 4° C. at a 20 mAmp power in reservoir buffer (0.1% Triton X-100, 0.1% sodium deoxycholate, 192 mM glycine and 25 mM Tris pH 8.3). For revealing activity in the separated bands, the latter were placed in a dosage buffer (Tris-base 66.7 mM, imidazole 66.7 mM, CaCl2 10 mM, pH 7.5). After preliminary incubation for 30 minutes at 37° C., the substrate (ADP or ATP) 5 mM was added. After 2 to 10 minute incubation, a white calcium phosphate precipitate significative of ATP diphosphohydrolase activity is formed. Three bands are seen for the aorta enzyme and one for the pancreas (these bands were all revealed on gel by silver overstaining). For further characterization, the most active band was loaded on an SDS-PAGE according to Laemmli (1970) and a single band appeared on the gel after silver nitrate staining, which is indicative of an enzyme purification to homogeneity after the non-denaturing gel. FIG. 1 shows the high sensitivity of detection conferred by the use of silver staining compared to a conventional Coomassie blue staining (see lanes 4 and 5). The active band purified from the gel has a molecular weight of 78 KDa when migrated on SDS-PAGE.

Herkunft & Nachweise