Fig. 2.

Enzymatic properties of Pyk2 from C. glutamicum. a SDS-PAGE of purified Pyk2. Lane 1, supernatant of crude extract from E. coli BL21(DE3)/pET-28a; Lane 2, supernatant of crude extract from E. coli BL21(DE3)/pET-28a-pyk2; Lane 3, Pyk2 purified by His-tagged affinity chromatography; and Lane 4, Pyk2 purified by ion exchange chromatography. b Molecular weight determination by gel filtration chromatography. The molecular weight of a protein could be determined from the calibration curve (plot of K_av versus the logarithm of molecular weight). The K_av value was calculated from the measured elution volume using the following equation: K_av = (V_e − V_o)/(V_c − V_o), where V_e is the elution volume, V_o is the column void volume, and V_c is the geometric column volume. The logarithm of the molecular weight was represented as lgMW. The standard proteins were shown as follows (●): 1, ovalbumin (44 kDa); 2, conalbumin (75 kDa); 3, aldolase (158 kDa); 4, ferritin (440 kDa); and 5, thyroglobulin (669 kDa). The native molecular weight of Pyk2 was determined to be a homohexamer of 430,881 (arrow). c The enzyme activity of Pyk2 at different temperatures. The maximum value of 2.19 ± 0.09 U/mg was set as relative 100%. d Enzyme activities of Pyk2 at various pHs (The maximum value of 2.43 ± 0.02 U/mg was set as relative 100%). e Enzyme activities of Pyk2 in the presence of various monovalent ions (The maximum value of 1.77 ± 0.05 U/mg was set as relative 100%). The enzyme was incubated in 100 mM Tris-HCl buffer (pH 7.4) and 5 mM Mn²⁺ with 50 mM or 100 mM monovalent ions. The data were derived from at least three replicate experiments, and the error bars represent the standard deviations