Systematic deletions and point mutations in the C-terminal extension of mammalian PFK (phosphofructokinase) led us to identify Leu-767 and Glu-768 of the M-type isoform (PFK-M) as the motifs responsible for the role of this region in inhibition by MgATP. These amino acids are the only residues of the C-terminus that are conserved in all mammalian isoforms, and were found to have a similar function in the C-type isoenzyme. Both residues in PFK-C and Leu-767 in PFK-M were also observed to be critical for inhibition by citrate, which is synergistic with that by MgATP. Binding studies utilizing titration of intrinsic protein fluorescence indicated that the C-terminal part of the enzyme participates in the signal transduction route from the MgATP inhibitory site to the catalytic site, but does not contribute to the binding of this inhibitor, whereas it is essential for the binding of citrate. Mutations of the identified structural motifs did not alter either the action of other allosteric effectors that also interact with MgATP, such as the inhibitor phosphoenolpyruvate and the strong activator fructose 2,6-bisphosphate, or the co-operative effect of fructose 6-phosphate. The latter data provide evidence that activation by fructose 2,6-bisphosphate and fructose 6-phosphate co-operativity are not linked to the same allosteric transition as that mediating inhibition by MgATP.
An allosteric phosphofructokinase (PFK) was created by sequence manipulation of the nonallosteric enzyme from the slime mold Dictyostelium discoideum (DdPFK). Most amino acid residues proposed as important for catalytic and allosteric sites are conserved in DdPFK except for a few of them, and their reversion did not modify its kinetic behavior. However, deletions at the unique C-terminal extension of this PFK produced a markedly allosteric enzyme. Thus, a mutant lacking the last 26 C-terminal residues exhibited hysteresis in the time course, intense cooperativity (n H ؍ 3.8), and a 200-fold decrease in the apparent affinity for fructose 6-phosphate (S 0.5 ؍ 4500 M), strong activation by fructose 2,6-bisphosphate (K act ؍ 0.1 M) and fructose 1,6-bisphosphate (K act ؍ 40 M), dependence on enzyme concentration, proton inhibition, and subunit association-dissociation in response to fructose 6-phosphate versus the nonhysteretic and hyperbolic wild-type enzyme (n H ؍ 1.0; K m ؍ 22 M) that remained as a stable tetramer. Systematic deletions and point mutations at the C-tail region of DdPFK identified the last C-terminal residue, Leu 834 , as critical to produce a nonallosteric enzyme. All allosteric mutants were practically insensitive to MgATP inhibition, suggesting that this effect does not involve the same allosteric transition as that responsible for fructose 6-phosphate cooperativity and fructose bisphosphate activation.
We present serological evidence that Dirofilaria immitis was the causative agent of subcutaneous nodules in two patients. ELISA and enzyme-linked immunoblot analysis showed that the two human sera had a reactivity pattern similar to that of D. immitis rabbit immune serum. The reaction pattern of the human sera was very different to that of Dirofilaria repens rabbit immune serum. In the light of our findings, the traditional view that subcutaneous filarial nodules are due to D. repens has to be revised.
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