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. 2003 Feb;185(4):1299–1315. doi: 10.1128/JB.185.4.1299-1315.2003

FIG. 7.

FIG. 7.

Gel filtration assay for PII binding to NRII mutants. PII (48 μM) and the indicated NRII protein (12 μM) were mixed on ice in reaction mixtures containing 1 mM MgCl2, 50 μM free 2-ketoglutarate, and 0.5 mM free ATP, as indicated in Materials and Methods. The bulk of the reaction mixture was loaded onto a Sephadex G-100 column equilibrated in buffer containing 1 mM MgCl2, 50 μM 2-ketoglutarate, and 0.5 mM ATP and eluted at 4°C. Fractions were collected and analyzed on SDS-15% polyacrylamide gels stained with Coomassie brilliant blue R-250. Fraction numbers are indicated above the lanes, and IN indicates the column input. (A) NRII (I141V); (B) NRII (S227R); (C) NRII (Y302N); (D) NRII (A129T/I221V); (E) NRII (L16R).