The F P concentration reported

The F6-P concentration reported for a resting rabbit muscle [20] reduced the KAapp for aldolase–FBPase complex more than 50 times compared to KAapp for aldolase–FBPase in the absence of effectors (Fig. 1b and Table 1). A standard assay for determination of FBPase activity in a complex with aldolase was described by Rakus and Dzugaj [13] as a development of the method of Morikofer-Zwez [21]. In that method a great excess of aldolase over FBPase (more than 100 times) and high concentration of DHAP (0.5 mM) were used. However, the data on the effect of DHAP on FBPase–aldolase complex stability suggest that this Deoxycorticosterone acetate strongly destabilizes the complex. This finding explains why Rakus and Dzugaj [13] could observe only partial desensitization of FBPase by aldolase to AMP inhibition. It is presumed that under the assay conditions used in our previous report only a small part of FBPase molecules (ca. 1%) could interact with aldolase. In the present paper we used a great excess of FBPase over aldolase (1000 times) and a physiological concentration of DHAP (Fig. 2). Under such conditions more than 95% of aldolase should be saturated with FBPase during the reaction. The response of FBPase–aldolase complex activity to the increasing concentration of DHAP was biphasic (solid line, Fig. 3a). The rate of the reaction was linear up to 200 μM concentration of added DHAP (dashed line) and the second phase of the curve describing the dependence of FBPase velocity on DHAP concentration was hyperbolic (dotted line). FBPase in a complex with aldolase was inhibited by AMP, with I0.5 ca. 1.35 mM and without any positive cooperativity (Fig. 3b). Contrary to the inhibition of rabbit muscle FBPase in the absence of aldolase, we could not observe any significant inhibition of the complex under AMP concentrations below 100 μM (Fig. 3b, inset). The inhibition of FBPase–aldolase complex by F2,6-P2 was negatively cooperative (n was 0.65), and the concentration of F2,6-P2 which caused a two-fold inhibition of the complex was ca. 346 μM (Fig. 3c).
Discussion The glycolytic and glyconeogenic enzymes have been considered as soluble constituents of the cell. However, many studies indicate that glycolytic enzymes are reversibly associated with cellular structures and this association alters their properties [22]. On the other hand, only little information is available on the regulation and spatial organization of the enzymes of glyconeogenesis. [13], [23]. The basic problem of muscle glyconeogenesis is the apparent inactivity of muscle FBPase isozyme under physiological concentrations of AMP. Recently, we have shown that rabbit muscle FBPase is partially desensitized to AMP inhibition by muscle aldolase [13]. By now we have presented evidence that muscle FBPase in a complex with muscle aldolase is practically completely insensitive to AMP inhibition. In the presence of 20 mM AMP (about 1000 times higher than physiological concentration) only a five-fold inhibition of FBPase–aldolase complex was observed. The destabilization of aldolase–FBPase complex by DHAP, F6-P2 and F2,6-P2, which interact with active sites of aldolase and FBPase, was shown in the binding experiments (Fig. 1b and Table 1). F2,6-P2 is a competitive inhibitor of mammalian FBPases with inhibitory constant (Ki) below 1 μM [7], [8], [10], [14], [17]. In the presence of aldolase, the apparent Ki was about 350 μM. On the other hand, F2,6-P2 significantly decreased the affinity of FBPase to aldolase: in the presence of 5 μM F2,6-P2, the binding constant for a complex of aldolase–FBPase was lowered about 60 times. The similarity of the effects of AMP and F2,6-P2 on the kinetics and binding properties of the complex strongly suggests a similar mechanism of the inhibition. Both AMP and F2,6-P2 may interact with the active site of FBPase, destabilizing the aldolase–FBPase complex. Thus, AMP and F2,6-P2 decrease the concentration of the complex and, as a result, they can inhibit FBPase activity in a classic manner.