Effect of ammonium, sodium, and potassium ions on rabbit muscle phosphofructokinase-1 and adenylate kinase activities

Figures & data

Table 1.  Activities of 30 nM, 70 nM and 140 nM PFK-1 and AK.

Concentration	PFK-1	AK
30 nM	eu/mL	eu/mL
Determined eu/mL	0.02	0.02
^a Estimated eu/mL	0.04	0.16
% Recovered	50%	13%
70 nM	eu/mL	eu/mL
Determined eu/mL	0.03	0.18
Estimated eu/mL	0.04	0.36
% Recovered	63%	50%
140 nM	eu/mL	eu/mL
Determined eu/ml	0.08	0.73
Estimated eu/mL	0.08	0.73
% Recovered	100%	100%

The final concentrations of PFK-1 and AK were prepared as described in Methods above and activities given were determined after 1 h incubation.

^aEstimates are based the dilutions of 3 µM enzyme activities.

Figure 1.  Effects of LDH and ammonium sulfate on 30 nM PFK-1 activity. Solutions were prepared as given in Methods. After 1h dilution to 30 nM PFK-1, salt-free LDH (▪) was added at the final concentrations indicated and activities determined after 1h. Commercial LDH as a suspension in 3.2M ammonium sulfate (□) was added to 30 nM PFK-1 at the same final concentrations as salt-free LDH. Ammonium sulfate alone was added (▴) at the same final concentrations occurring in commercial LDH (shown in the secondary X axis).

Figure 2.  Effect of 0.2M ammonium sulfate on activity at 30 nM, 70 nM, and 140 nM AK. As given in Methods, AK was diluted with 0.02 M potassium phosphate, pH 8 to concentrations shown and allowed to stand 1 h as given in Methods. No additions (□) and 0.2 M ammonium sulfate (▪) were then made, incubated for 1 h and AK activities were then determined.

Figures 3 A-F.  Relative activities of 30 nM (△), 70 nM (▴), and 140 nM (▪) PFK-1 and AK versus NH4+, Na+, and K+ion concentrations. The PFK-1 concentrations were derived from dilution of a 3 μM PFK-1stock solution and the preparation of samples is given in the Methods section. Figures 3 A-C shows that NH4+, Na+, and K+ salts in decreasing order of effectiveness, resulted in recovery of some AK activity losses due to its low concentration. PFK-1 was treated in the same manner as AK and Figures 3 D-E show that the presence of these salts results in losses of PFK-1 activities; K+, NH4+, and Na+ salts inhibited PFK-1 in decreasing order of effectiveness.

Figure 4.  Comparison of effects on 30 nM AK activity by sulfates and chlorides of NH4+, K+, and Na+ and the effect of substrates. A 30 nM AK solution was prepared as described in Methods (see Table 1) and solutions were made 0.2 molar with respect to each monovalent salts and 1 mmolar with respect to AMP or ATP.Mg when present. A 1 h incubation period followed and activities were then determined. The 30 nM AK control had an activity of 0.021 ± 0.002 eu/mL from 4 determinations.

Table 2.  The effect of 0.2 M sulfates of ammonium, potassium, and sodium on K_m and V_m values of 30 nM and 140 mM AK.

	Average kinetics values
	Km atp	Km amp	Vm atp	Vm amp
30 nM AK average Control Values	0.22 ± 0.05	0.21 ± 0.06	0.005 ± 0.002	0.007 ± 0.004
30 nM AK average Salt Values	0.33 ± 0.05	0.39 ± 0.02	0.014 ± 0.004	0.040 ± 0.011
140 nM AK average Control Values	0.22 ± 0.03	0.24 ± 0.02	0.20 ± 0.02	0.39 ± 0.06
140 nM AK average Salt Values	0.22 ± 0.04	0.25 ± 0.05	0.14 ± 0.03	0.22 ± 0.05

The K_m and V_m values were determined using Lineweaver-Burke plots [9] from a minimum of 4 independent trials for each salt and condition with standard deviations from the average of ± 10 percent or less. The average values of all the individual salts were then averaged and appear in Table 2 above; standard deviations of these latter values also determined and shown to provide some sense of the degree variation among values for the three salts.

Figure 5.  Effect of 0.2 M ammonium sulfate on 30 nM PFK-1 in the presence and absence of 5 μM aldolase. Dilutions to 30 nM PFK-1 (▪) were made with 0.1 M Tris-phosphate buffer, pH 8.0. The following were in 30 nM PFK-1 as final concentrations: 5 μM aldolase (□); 5 μM aldolase and 0.2 M ammonium sulfate (▴); and ammonium sulfate (△). The initial reading was at 13 minutes The 0-time value was based upon the estimated eu/mL values based on dilution shown in Table 1, Methods. Differences between aldolase alone (□) and aldolase and 0.2 M ammonium sulfate (▴) could not be shown to be significantly difference (p<0.01).

Lineweaver H, Burk D. Determination of enzyme dissociation constants. J Amer Chem Soc 1934; 56:658–666.

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