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. 2010 Nov 16;101(12):2612–2620. doi: 10.1111/j.1349-7006.2010.01701.x

Figure 2.

Figure 2

 (A) Analysis of recombinant human Lon by SDS‐PAGE. Crude lysate from Escherichia coli Rosetta cells containing human Lon expression plasmid with (lane 1) or without (lane 2) isopropyl‐β‐D‐thiogalactoside induction was incubated with a Ni‐NTA affinity agarose gel. After binding, bound fractions were eluted by imidazole (lane 4, 200 mM; lane 5, 250 mM) from the column. Affinity purified human Lon was concentrated (lane 6). The arrow shows the recombinant human Lon. M, molecular mass markers. (B) Substrate saturation curve for the recombinant human Lon, incubated with indicated concentrations of fluorogenic substrate (Glt‐AAF‐MNA,[S]) in a real‐time manner (0–30 min). The velocity (Y‐axis) of the reaction was determined at various Glt‐AAF‐MNA concentrations (X‐axis). The values of K m and k cat were 9.15 ± 1.95 μM and 68.3/min, respectively, by fitting the data with the Michaelis–Menten equation using the KaleidaGraph program.