Article Figures & Data
Figures
- Fig. 1.
Gene organization of the pur cluster ofS. alboniger. This figure is modified from Tercero et al. (32). Size of the genes is indicated by thick arrows. Transcripts are indicated below by thin arrows. The first 8 nucleotides of pur7 overlap with napH, which encodes an N -acetylpuromycin- N -acetylhydrolase (17). pur10, pac, dmpM, andpur8 encode NAD-dependent ATP dehydrogenase (23), puromycin- N -acetyltransferase (14), acetyl- O -demethylpuromycin- O -methyltransferase (15), and a transmembrane protein which confers resistance to puromycin (33), respectively. pur6,pur4, pur5, and pur3 encode putative tyrosinyltransferase, aminotransferase, methyltransferase, and monophosphatase activities, respectively (32).
- Fig. 2.
Purification of recombinant Pur7. A Coomassie-stained SDS–11% polyacrylamide gel is shown. Lanes: 1, crude extract ofE. coli BL21(DE3)pLysS(pRSETb); 2, crude extract of E. coli BL21(DE3)pLysS(pUR7.EX) 2 h after addition of IPTG; 3, first eluate from the Ni2+-NTA agarose column; 4, eluate with sodium phosphate buffer; 5, eluate with 20 mM imidazole-containing buffer; 6, eluate with 500 mM imidazole-containing buffer; M, molecular mass markers (sizes are in kilodaltons). The arrow indicates recombinant Pur7.
- Fig. 3.
3′-Amino-3′-dATP pyrophosphohydrolase activity. (A) Reactions were carried out with supernatants from cell extracts ofE. coli BL21(DE3)pLysS transformed with either pRSETb (○) or pUR7.EX (□). (B) Reactions were carried out with purified Pur7 (1 mU) in the presence (□) or absence (◊) of inorganic pyrophosphatase. The latter, as a negative control, shows that Pur7 does not release orthophosphate. Other details are described in Materials and Methods.
- Fig. 4.
Reaction catalyzed by Pur7.
- Fig. 5.
Effect of pH and cations on the reaction catalyzed by Pur7. Assays were performed as described in Material and Methods, except that the pH (A) or cations (B) of the reactions, which contained 1 mU of Pur7, were changed as indicated. In panel A, buffers used were 50 mM Tris-HCl (○) or 80 mM glycine (□). In panel B, salts used were MgCl2 (○), CaCl2 (◊), NH4Cl (×), or MnCl2 (▵).
Tables
- Table 1.
Effects of purification of recombinant Pur7
Treatment of Pur7 Protein (mg) Activity (U) Specific activity (U/mg) Fold purification Yield (%) None (crude extract) 124 25.3 0.2 1 100 Ni2+purification 1.8 32.9 18.3 91.5 130 - Table 2.
Substrate specificity of recombinant Pur7
Substratea Specific activity (U/mg of protein) 3′-Amino-3′-dATP 54.6 3′-Amino-3′-dTTP 20.2 2′-Amino-dATP <0.1 8-Oxo-dGTP <0.1 dPTP <0.1 3′-Azido-3′-dTTP <0.1 3′-Fluoro-3′-dTTP <0.1 3′-Deoxy-ATP (cordycepin triphosphate) <0.1 Nucleoside triphosphates <0.1 2′-Deoxy-nucleoside triphosphates <0.1 ↵a Substrate concentrations were 2 mM except in the cases of 3′-amino-3′-dATP and 3′-amino-3′-dTTP, for which concentrations were 1 mM.
