Inhibition of human poly(A)-specific ribonuclease (PARN) by purine nucleotides: kinetic analysis

Figures & data

Figure 1.  Effect of purine nucleotides on PARN activity. A. Relative activity (%) is presented as a function of increasing concentrations of adenosine nucleotides, 0–8 mM AMP (▴), 0–8 mM ADP (▪), or 0–4 mM ATP (•). Mg^{2 +} concentration is 1.5 mM. B. Same as in A, except that 0.5 mM Mg^{2 +} was used and the nucleotide concentrations are 0–8 mM AMP, 0–8 mM ADP and 0–2 mM ATP. C. Same as in A, except that 0–4 mM GMP (Δ), 0–8 Mm GDP (▪), or 4–16 mM GTP (○) was used. Reactions in the presence of PARN were performed as outlined in the text and Materials and Methods.

Figure 2.  Purine triphosphate nucleotides inhibit PARN. A. Michaelis-Menten plot showing the effect of ATP in PARN activity. Reactions were performed as described in Materials and Methods in the presence of 0 mM (▴), 0.25 mM (•) and 1 mM (▪) ATP. Representative of at least two independent experiments. B. Same as in A, except that GTP was used at 0 mM (Δ), 0.25 mM (○) and 1 mM (▪). C. Comparison of ATP and GTP inhibition effect on PARN activity. Reactions were performed in the presence of the indicated ATP (shaded bars) or GTP (white bars) concentrations. The data points are averages of at least three independent experiments.

Table I.  Inhibition of PARN by purine nucleotides.

Nucleotide	Ki mM	appKi, Mm
		1.5 mM Mg^2+	0.5 mM Mg^2+
GTP	3.3 ± 0.5^*	1.0 ± 0.1	0.5 ± 0.1
GDP	2.8 ± 0.8	3.3 ± 0.3	1.4 ± 0.5
GMP	4.5 ± 2.2	11.3 ± 3.2	4.8 ± 1.2
ATP	3.2 ± 0.8	1.0 ± 0.2	0.3 ± 0.1
ADP	0.5 ± 0.3	2.2 ± 0.7	1.0 ± 0.3
AMP	1.6 ± 0.3	4.7 ± 1.2	6.7 ± 2.2
dGTP	2.0 ± 0.1	n.d	n.d
dATP	2.8 ± 0.5	n.d	n.d

^* The values listed are averages of at least three independent experiments ± experimental error.

Figure 3.  Kinetic analysis of purine phosphate nucleotides inhibition of PARN: ATP (A) and GTP (B) are noncompetitive inhibitors; AMP (C) and ADP (D) are competitive inhibitors. Reactions were performed as described in Materials and Methods. Double reciprocal plots 1/v versus 1/[substrate] for PARN activity in the presence of GTP or ATP are shown. The ATP concentrations (filled markers in A) and GTP (empty markers in B) were 0 mM (triangle), 0.25 mM (circle), 1 mM (square) and 2 mM (inverted triangle). AMP concentrations were 0 mM (▴), 2 mM (•), 4 mM (▪) and 6 mM (▾). ADP concentrations were 0 mM (▴), 1 mM (•), 2 mM (▪) and 3 mM (▾). Representative of at least three independent experiments. Insets in A and B: The slopes (K_M/V_max) of the double reciprocal lines versus the nucleotide concentrations.

Figure 4.  Purine triphosphate deoxynucleotides inhibit PARN: dATP (A) and dGTP (B) are noncompetitive inhibitors. Double reciprocal plots 1/v versus 1/[substrate] for PARN activity in the presence of dATP or dGTP are shown. The dATP concentrations (filled markers in A) and dGTP (empty markers in A) were 0 mM (triangle), 0.25 mM (circle), 1 mM (square) and 2 mM (inverted triangle). Representative of at least three independent experiments. Insets: The slopes (K_M/V_max) of the double reciprocal lines versus the nucleotide concentrations.

Figure 5.  Effect of Mg^{2 +} in the inhibition of PARN by purine nucleotides. A. Relative activity inhibition by 1.5 mM ATP (grey bars) or 1.5 mM GTP (white bars) in the presence of 0, 1.5 and 15 mM Mg^{2 +} . B. Release of PARN activity inhibition by 1.5 mM AMP (grey bars) or 1.5 mM GMP (white bars) in the presence of 0, 1.5 and 15 mM Mg^{2 +} . The results are mean values from at least three independent experiments.