Interactions between calmodulin (CaM) and several hydrophobic fluorescent probes were characterized in order to determine if CaM expresses hydrophobic binding sites in the presence of Ca2+. Several classes of fluorescent probes capable of sensing exposure of hydrophobic binding sites on proteins were found to bind to CaM, and these interactions were greatly enhanced by Ca2+. In the presence of Ca2+, the fluorescence intensity of 9-anthroylcholine (9AC) was increased 24-fold by CaM, with a shift in the fluorescence emission maximum from 514 to 486 nm. The fluorescence intensity of 8-anilino-1-naphthalenesulfonate (Ans) was enhanced 27-fold with an emission maximum shift from 540 to 488 nm in the presence of CaM and Ca2+. Similar results were obtained with the uncharged fluorescent ligand, N-phyenyl-1-naphthylamine. With all three fluorescent dyes, the fluorescence changes caused by CaM in the absence of Ca2+ were minor compared to those observed with CaM and Ca2+. Direct binding studies using equilibrium dialysis demonstrated that CaM can bind four to six molecules of 9AC or two to three molecules of Ans in a calcium-dependent manner. The effects of various amphiphilic compounds on the Ca2+-dependent complex formation between CaM and the Ca2+-sensitive phosphodiesterase or troponin I were investigated. Trifluoperazine (TFP) and 9AC inhibited CaM stimulation of the Ca2+-sensitive phosphodiesterase. The Ca2+-dependent binding of the phosphodiesterase to CaM-Sepharose was also inhibited by TFP, 9AC, and Ans. Furthermore, binding of CaM to troponin I-Sepharose was inhibited by these ligands. Consistent with these data was the observation that troponin I antagonized binding of 9AC to CaM. These data indicate that binding of Ca2+ to CaM results in exposure of a domain with considerable hydrophobic character, and binding of hydrophobic ligands to this domain antagonizes CaM-protein interactions. It is proposed that this hydrophobic domain may serve as the interface for the Ca2+-dependent binding of CaM to the phosphodiesterase or troponin I.

No abstract

The sensitivity to regulation of proteins undergoing covalent modification can be greatly increased when the substrates saturate the converter enzymes. This phenomenon, termed zero-order ultrasensitivity, has been found to occur in the reversible phosphorylation of isocitrate dehydrogenase. The possibility that this enhanced sensitivity is a common feature of covalent regulatory systems is discussed.

The csrA gene encodes a small RNA-binding protein, which acts as a global regulator in Escherichia coli and other bacteria (T. Romeo, Mol. Microbiol. 29:1321-1330, 1998). Its key regulatory role in central carbon metabolism, both as an activator of glycolysis and as a potent repressor of glycogen biosynthesis and gluconeogenesis, prompted us to examine the involvement of csrA in acetate metabolism and the tricarboxylic acid (TCA) cycle. We found that growth of csrA rpoS mutant strains was very poor on acetate as a sole carbon source. Surprisingly, growth also was inhibited specifically by the addition of modest amounts of acetate to rich media (e.g., tryptone broth). Cultures grown in the presence of >25 mM acetate consisted substantially of glycogen biosynthesis (glg) mutants, which were no longer inhibited by acetate. Several classes of glg mutations were mapped to known and novel loci. Several hypotheses were examined to provide further insight into the effects of acetate on growth and metabolism in these strains. We determined that csrA positively regulates acs (acetylcoenzyme A synthetase; Acs) expression and isocitrate lyase activity without affecting key TCA cycle enzymes or phosphotransacetylase. TCA cycle intermediates or pyruvate, but not glucose, galactose, or glycerol, restored growth and prevented the glg mutations in the presence of acetate. Furthermore, amino acid uptake was inhibited by acetate specifically in the csrA rpoS strain. We conclude that central carbon flux imbalance, inhibition of amino acid uptake, and a deficiency in acetate metabolism apparently are combined to cause metabolic stress by depleting the TCA cycle.

In Escherichia coli, expression of the glyoxylate bypass operon appears to be controlled, in part, by the product of icIR+. Mutations (17,19). This bypass is essential for growth on acetate, since it yields C4 acids while avoiding the net loss of the acetate carbons as carbon dioxide in the Krebs cycle (Fig. 1). After induction, the flow of isocitrate through the glyoxylate bypass is regulated, in part, by the phosphorylation of isocitrate dehydrogenase (IDH), the Krebs cycle enzyme that competes with isocitrate lyase (8,12,24). During growth on acetate, ca. 70% of the IDH is maintained in the inactive phosphorylated form (22,23,32), reducing the activity of this enzyme and so forcing isocitrate through the bypass (24, 32). The phosphorylation and dephosphorylation of IDH are catalyzed by a single bifunctional enzyme, IDH kinase/phosphatase (20, 21).The metabolic and regulatory proteins of the glyoxylate bypass reside in the same operon, which maps at 91 min on the E. coli chromosome (4,5,23,26 When required, ampicillin (200 ,ug/ml) tetracycline (12.5 ,ug/ml), or kanamycin (50 ,ug/ml) was included in the growth media.Measurement of enzymatic activities. Cultures were grown at 37°C in a gyratory incubator to mid-log phase and were then harvested by centrifugation at 4,000 x g for 10 min. The cells were suspended in 10 ml of extraction buffer (25 mM N-morpholinepropanesulfonate [pH 7.5], 2 mM ,-mercaptoethanol, 1 mM EDTA) and then pelleted again by centrifugation. Cell pellets were stored at -80°C. For assay, the samples were thawed, suspended in 5 ml of extraction buffer, and disrupted by sonication. Cellular debris was removed by centrifuged at 22,000 x g for 20 min, and the samples were assayed for IDH phosphatase activity. Samples derived from cultures harboring plasmid were also assayed for ,-lactamase activity to ensure that the plasmid had not been lost during growth.The activity of IDH phosphatase was measured by monitoring the release of [32P]phosphate from [32P]phospho-IDH,