Stimulation of rat liver mitochondrial adenosine triphosphatase by anions.

The hydrolysis of MgATP by isolated rat liver mitochondrial ATPase (EC 3.6.1.3) at pH 8.0 was stimulated by various anions. The rate of hydrolysis was increased from 18 to 170 mumol per min per mg, a 9.4-fold stimulation, by HSeO3 at 1 mM MgATP. In the absence of a stimulatory anion, reciprocal plots of initial velocity studies with MgATP as the variable substrate were curved (Hill coefficient approximately 0.5). With the addition of anion, the reciprocal plots became linear. When the substrate was MgITP or MgGTP with the isolated enzyme or MgATP with submitochondrial particles, no curvature of the reciprocal plots was observed. With purified ATPase, anions stimulated the hydrolysis of MgITP, MgGTP, MgUTP or MgCTP only slightly. With submitochondrial particles the stimulation by anions of MgATP hydrolysis was limited to approximately 2-fold. These data are interpreted to indicate the existence of two substrate sites for MgATP and an anion-binding site on the isolated enzyme.

These data are interpreted to indicate the existence of two substrate sites for MgATP and an anion-binding site on the isolated enzyme.
During the past decade, it has become increasingly apparent that anions affect the properties of numerous enzymes.
Particularly prominent among these are ATPases. Both stimulatory and inhibitory effects of various anions on ATPase activity have been reported for rat liver mitochondria (l), rat liver (2, 3) and beef heart (4) submitochondrial particles, isolated ATPase from beef heart (5), rat liver (6,7) and yeast (8) mitochondria, coupling factor 1 from chloroplasts (9), and various microsomal ATPases from pancreas (lo), gastric mucosa (1 l-131, and salivary gland (14). Although the degree of stimulation or inhibition and the detailed anion specificity differ among these preparations, there appear to be a considerable number of similarities which suggest a common mechanism of action. In this study the influence of various anions on the ATPase activity of isolated mitochondrial ATPase and submitochondrial particles from rat liver was investigated.
The  reaction was followed by observing the disappearance of NADH at 340 nm (315 nm with chromate, 300 nm with 2,4-dinitrophenol) with a Beckman DU modified with a Gilford model 2220 adapter and a Hewlett-Packard 7101B strip chart recorder. Linear double reciprocal and Hill plots were constructed from the data using a weighted least squares fit. The weighting factor was the reciprocal of the variance (17). The fold activation and K, for an anion were determined as follows: usine 1 mM MaATP. plots of l/(v -v0) versus l/concentration of a&e anion species (where v = velocity in the presence and v,, = velocity in the absence of anion) were constructed ( Fig. 1); the y intercept represented l/(Vm., v,J and fold activation was defined as Vmax/vO; slope/intercept was defined as the K, for the anion. The stability constant for MgATP in 50 mM HEPS-KOH, pH 8.0, at 30" was found to be 167,000 using the method of Burton 08).

Anion E$ects on Hydrolysis
of MgATP-As shown in Table I, the hydrolysis of MgATP by isolated rat liver mitochondrial ATPase was stimulated by several anions. In the absence of an activating anion, reciprocal plots of initial velocit.ies with varying MgATP concentration were curved (Hill coefficient approximately 0.5) (Fig. 2). With increasing concentration of an activating anion, the initial velocity of the reaction increased and the reciprocal plots became linear (Hill coefficient 1.0) (Fig. 2). This phenomenon was observed with each of the anions listed in Table I Considering the activating anions, there appears to be an optimal anion concentration for stimulation. Concentrations above this optimum cause lesser degrees of stimulation and reciprocal plots (l/v versus l/MgATP) at these supraoptimal anion concentrations result in patterns analogous to those of noncompetitive substrate inhibition in that both slope and intercept show the inhibitory effect (Fig. 3). It should be noted that the reciprocal plots remain linear even at supraoptimal anion concentrations.
Rat liver mitochondrial ATl'ase was found to be very sensitive to inhibition by three other anions: N3-, OCN-, and SCN-. In contrast to nitrate and phosphate considered above, these anions produce a greater percentage of inhibition in the absence of activating anions than in the presence of these anions. In the absence of an activating anion, reciprocal plots varying MgATP at different fixed levels of one of these inhibitors resulted in a series of curves with increasing y intercept values (Fig. 4~). The HiI1 coefficients of these curves were constant with values between 0.5 and 0.6. This was in marked contrast to the situation for activating anions which cause the reciprocal plots to become more linear with increasing anion concentration.
In the concentration.
presence of 10 mM HC03, the pattern of inhibition of these anions was noncompetitive versus MgATP (Fig. 4b). Using 1 mM MgATP and varying HC03 concentration, at different fixed levels of inhibitor, resulted in a competitive pattern of inhibition of these anions versus HC03 (Fig. 5). The various Ki values for these types of experiments with N3, OCN-, and SCN-are listed in Table II. ciprocal plots (Fig. 6).   (Fig. 7a), while with 20 mM HC03, they appeared to be competitive versus MgITP (Fig. 76) (Fig. 8). The K, for MgATP was 50 PM without anion and 67 L(M with 20 mM HC03-. The inhibition by phosphate of MgATP hydrolysis by submitochondrlal particles was competitive versus MgATP in the absence of HC03 (Ki = 49 mM) (Fig. 8a) and noncompetitive in the presence of 20 mM HC03-(Kii = 96 mM, Kis = 21 mM) (Fig. 8b). there are two possibilities. One would be the existence of two independent active sites which have different kinetic parameters (23). Interestingly, such a proposal has been made in the case of myofibrillar ATPase (24). The other would invoke the existence of two equivalent sites which are interdependent (negative cooperativity) (23,25). Another alternative would be the existence of only one active site and a separate modifying site (26,27). Several studies have been reported concerning the number of nucleotide binding sites of the isolated ATl'ase. Hilborn and Hammes (28) suggest the existence of one "tight" and one "loose" binding site for ADP on beef heart Fr, but report less than one ATP bound per mol of Fr, probably due to the absence of Mg* in their experiments. Harris et al. (29), also using beef heart Fr, observed five tight binding sites, three specific for ATP and two for ADP.

DISCUSSION
Garrett and Penefsky (30)  acid. The only activating species which do not fit into these groups are Br-and 2,4-dinitrophenol, although, in the latter case, the structure around the 2-nitro and the ionized -OH is analogous to the structure of the active species of malonate.
Size also appears to be one of the criteria for activation as can be seen by comparing the extent of activation and the K, values of those anions that are included in the pyramidal group above. Interestingly, nitrate inhibits rather than activates mitochondrial ATPase. This singly charged anion is of comparable size but lacks the second, protonated acid group found in most of the anions in this group that activate ATPase. Activation by Br-and not by I-or Cl-supports the importance of size. On the basis of K,, there appears to be some enhancement of binding afforded by the additional carboxyl group on maleate and malonate as compared to HCOJ. This also suggests that there are two binding sites, one that accepts a negatively charged group and the second which accepts a protonated, in this case, carboxyl group since the data indicate the active species of malonate and maleate is the monoprotonated anion. At pH 8 and 9 succinate did not stimulate significantly. This is consistent with the active species of dicarboxylic acids being the monoprotonated anion for the pKz of succinic acid is 5.64 (21).
The situation with submitochondrial particles would appear to be somewhat different.
Although the structural requirements for activation are, for the most part, retained, other factors apparently control the extent of activation since HC03 and HSOs, which produce a 5.8-and 8.3-fold activation of the isolated enzyme, respectively, produce approximately a 2-fold stimulation