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Effect of ATP concentration on the rate of PFK catalyzed reaction at fixed concentration of F-6-P and Mg2+ (3.3 mM each). Inset shows the plot obtained on assaying the enzyme at variable inhibitory (>0.1 mM) concentration of ATP at two fixed concentrations of F-6-P such as 1.6 (•) and 3.3 (○) mM. Mg2+ concentration was constant (3.3 mM). Enzyme concentration was 10 μg/mL. Other conditions were same as in standard enzyme assay.

Effect of ATP concentration on the rate of PFK catalyzed reaction at fixed concentration of F-6-P and Mg2+ (3.3 mM each). Inset shows the plot obtained on assaying the enzyme at variable inhibitory (>0.1 mM) concentration of ATP at two fixed concentrations of F-6-P such as 1.6 (•) and 3.3 (○) mM. Mg2+ concentration was constant (3.3 mM). Enzyme concentration was 10 μg/mL. Other conditions were same as in standard enzyme assay.

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... To simulate reaction velocities in the absence of inhibition, f 1 , f 2 , and f 3 in Eq. (4) can be set to 1 to eliminate the pH effect and v S·E·S max replaced with v E·S max to eliminate the substrate inhibitor effect on production generation from S·E·S to give, Equation (11) represents the substrate effect on reaction velocity without inhibition and is plotted against ATP concentration in Fig. 5b. The plot is analogous to the Michaelis-Menten saturation curve, which exhibits a hyperbolic increase with substrate increase. ...
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A key player in energy metabolism is phosphofructokinase-1 (PFK1) whose activity and behavior strongly influence glycolysis and thus have implications in many areas. In this research, PFK1 assays were performed to convert F6P and ATP into F-1,6-P and ADP for varied pH and ATP concentrations. PFK1 activity was assessed by evaluating F-1,6-P generation velocity in two ways: (1) directly calculating the time slope from the first two or more datapoints of measured product concentration (the initial-velocity method), and (2) by fitting all the datapoints with a differential equation explicitly representing the effects of ATP and pH (the modeling method). Similar general trends of inhibition were shown by both methods, but the former gives only a qualitative picture while the modeling method yields the degree of inhibition because the model can separate the two simultaneous roles of ATP as both a substrate of reaction and an inhibitor of PFK1. Analysis based on the model suggests that the ATP affinity is much greater to the PFK1 catalytic site than to the inhibitory site, but the inhibited ATP-PFK1-ATP complex is much slower than the uninhibited PFK1-ATP complex in product generation, leading to reduced overall reaction velocity when ATP concentration increases. The initial-velocity method is simple and useful for general observation of enzyme activity while the modeling method has advantages in quantifying the inhibition effects and providing insights into the process.
... The protein content was measured using a BCA1 kit (Sigma, St. Louis, MO). The activity of PFK was measured spectrophotometrically following the procedure reported in [100]. ALDO activity was measured by using an Aldolase Activity Colorimetric Assay Kit (BioVision, Milpitas, CA). ...
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... The HK activity was measured with the Hexokinase Colorimetric Assay Kit (Sigma-Aldrich). The activities of the phosphofructokinease-1 (PFK1) assay and enolase were measured spectrophotometrically as reported in [77]. The activity of PK was detected with the Enzymatic Assay of Pyruvate Kinase kit (Sigma-Aldrich). ...
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... Enzymatic activities were measured on 10 µl cell lysates, incubated for 5 min at 37 • C. The protein content was measured using the BCA1 Kit (Sigma, St. Louis, MO, United States). The activity of phosphofructokinase-1 (PFK1) assay was measured spectrophotometrically as reported in the study by Sharma (2011). The activities of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), enolase (ENO), pyruvate kinase (PK), and lactate dehydrogenase (LDH) were measured spectrophotometrically according to the study by Riganti (Riganti et al., 2002;Capello et al., 2016). ...
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... Enzymatic activities were measured on 10 µL cell lysates, incubated for 5 min at 37 • C. The protein content was measured using the BCA1 Kit (Sigma Aldrich). The activity of phosphofructokinase-1 (PFK1) was measured spectrophotometrically as reported in Sharma (2011) [24]. The activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase were measured spectrophotometrically according to Beutler (1975) [25]. ...
... Enzymatic activities were measured on 10 µL cell lysates, incubated for 5 min at 37 • C. The protein content was measured using the BCA1 Kit (Sigma Aldrich). The activity of phosphofructokinase-1 (PFK1) was measured spectrophotometrically as reported in Sharma (2011) [24]. The activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase were measured spectrophotometrically according to Beutler (1975) [25]. ...
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... Thus, V polyP , which represents the contribution to dD[ATP]/dt from polyP buffering, is approximated by proportional feedback. Figure 6 uses experimental data for: rat muscle (Beis and Newholme, 1975;Bosca et al., 1985;Kushmerick et al., 1992), rabbit muscle (Kemp, 1970;Storey and Hochachka, 1974;Pettigrew and Friedens, 1978), rabbit liver (Ennor and Rosenburg, 1951;Kemp, 1970), desert locust (Walker and Bailey, 1969;Beis and Newholme, 1975), lobster muscle (Beis and Newholme, 1975;Sugden and Newholme, 1975), yeast (Kopperschlager et al., 1968), Escherichia coli (Zheng and Kemp, 1992), and Setaria cervi (Sharma, 2011). The normalized ÀDPFK activity/D[ATP] ratio is determined graphically from the slope of ATP concentration versus PFK activity plots when PFK activity is at half maximum (see STAR Methods B.2); the ratio of ATP to pCr concentrations is determined at steady state. ...
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... The results were expressed as nanomoles of NADH/min/mg cell proteins. PFK1 assay was performed according to ref. 59. The results were expressed as nanomoles of NAD + /min/mg cell proteins. ...
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... The uptake of glucose was measured as described earlier [48] and expressed as pmol 2-deoxy-D-[ 3 H]glucose/mg cell proteins. PFK1 assay was performed according to [49]. The activities of GAPDH, enolase, LDH were measured as reported in [50]. ...
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... The higher PFK activity after 3 h of hypoxia, is very important since PFK is a main element in regulation of the glycolytic flux (Banaszak et al., 2011;Sharma, 2011), indicating that ATP production is accelerating to meet the energy needs of the cell. It is evident that PFK is prime to sense the "energy level" in shrimp. ...