Are There Lipid Membrane-Domain Subtypes in Neurons with Different Roles in Calcium Signaling?

Lipid membrane nanodomains or lipid rafts are 10-200 nm diameter size cholesterol- and sphingolipid-enriched domains of the plasma membrane, gathering many proteins with different roles. Isolation and characterization of plasma membrane proteins by differential centrifugation and proteomic studies have revealed a remarkable diversity of proteins in these domains. The limited size of the lipid membrane nanodomain challenges the simple possibility that all of them can coexist within the same lipid membrane domain. As caveolin-1, flotillin isoforms and gangliosides are currently used as neuronal lipid membrane nanodomain markers, we first analyzed the structural features of these components forming nanodomains at the plasma membrane since they are relevant for building supramolecular complexes constituted by these molecular signatures. Among the proteins associated with neuronal lipid membrane nanodomains, there are a large number of proteins that play major roles in calcium signaling, such as ionotropic and metabotropic receptors for neurotransmitters, calcium channels, and calcium pumps. This review highlights a large variation between the calcium signaling proteins that have been reported to be associated with isolated caveolin-1 and flotillin-lipid membrane nanodomains. Since these calcium signaling proteins are scattered in different locations of the neuronal plasma membrane, i.e., in presynapses, postsynapses, axonal or dendritic trees, or in the neuronal soma, our analysis suggests that different lipid membrane-domain subtypes should exist in neurons. Furthermore, we conclude that classification of lipid membrane domains by their content in calcium signaling proteins sheds light on the roles of these domains for neuronal activities that are dependent upon the intracellular calcium concentration. Some examples described in this review include the synaptic and metabolic activity, secretion of neurotransmitters and neuromodulators, neuronal excitability (long-term potentiation and long-term depression), axonal and dendritic growth but also neuronal cell survival and death.

Formal Meta-Analysis of Hypoxic Gene Expression Profiles Reveals a Universal Gene Signature.

Integrating transcriptional profiles results in identifying gene expression signatures that are more robust than those obtained for individual datasets. However, a direct comparison of datasets derived from heterogeneous experimental conditions is problematic, hence their integration requires applying of specific meta-analysis techniques. The transcriptional response to hypoxia has been the focus of intense research due to its central role in tissue homeostasis and prevalent diseases. Accordingly, many studies have determined the gene expression profile of hypoxic cells. Yet, despite this wealth of information, little effort has been made to integrate these datasets to produce a robust hypoxic signature. We applied a formal meta-analysis procedure to datasets comprising 430 RNA-seq samples from 43 individual studies including 34 different cell types, to derive a pooled estimate of the effect of hypoxia on gene expression in human cell lines grown ingin vitro. This approach revealed that a large proportion of the transcriptome is significantly regulated by hypoxia (8556 out of 20,888 genes identified across studies). However, only a small fraction of the differentially expressed genes (1265 genes, 15%) show an effect size that, according to comparisons to gene pathways known to be regulated by hypoxia, is likely to be biologically relevant. By focusing on genes ubiquitously expressed, we identified a signature of 291 genes robustly and consistently regulated by hypoxia. Overall, we have developed a robust gene signature that characterizes the transcriptomic response of human cell lines exposed to hypoxia in vitro by applying a formal meta-analysis to gene expression profiles.

The Use of Flavylium Salts as Dynamic Inhibitor Moieties for Human Cb5R.

Cytochrome b5 reductase (Cb5R) is a flavoprotein that participates in the reduction of multiple biological redox partners. Co-localization of this protein with nitric oxide sources has been observed in neurons. In addition, the generation of superoxide anion radical by Cb5R has been observed. A search for specific inhibitors of Cb5R to understand the role of this protein in these new functions has been initiated. Previous studies have shown the ability of different flavonoids to inhibit Cb5R. Anthocyanins are a subgroup of flavonoids responsible for most red and blue colors found in flowers and fruits. Although usually represented by the flavylium cation form, these species are only stable at rather acidic pH values (pH ≤ 1). At higher pH values, the flavylium cation is involved in a dynamic reaction network comprising different neutral species with the potential ability to inhibit the activities of Cb5R. This study aims to provide insights into the molecular mechanism of interaction between flavonoids and Cb5R using flavylium salts as dynamic inhibitors. The outcome of this study might lead to the design of improved specific enzyme inhibitors in the future.

Structural Features of Cytochrome b5-Cytochrome b5 Reductase Complex Formation and Implications for the Intramolecular Dynamics of Cytochrome b5 Reductase.

Membrane cytochrome b5 reductase is a pleiotropic oxidoreductase that uses primarily soluble reduced nicotinamide adenine dinucleotide (NADH) as an electron donor to reduce multiple biological acceptors localized in cellular membranes. Some of the biological acceptors of the reductase and coupled redox proteins might eventually transfer electrons to oxygen to form reactive oxygen species. Additionally, an inefficient electron transfer to redox acceptors can lead to electron uncoupling and superoxide anion formation by the reductase. Many efforts have been made to characterize the involved catalytic domains in the electron transfer from the reduced flavoprotein to its electron acceptors, such as cytochrome b5, through a detailed description of the flavin and NADH-binding sites. This information might help to understand better the processes and modifications involved in reactive oxygen formation by the cytochrome b5 reductase. Nevertheless, more than half a century since this enzyme was first purified, the one-electron transfer process toward potential electron acceptors of the reductase is still only partially understood. New advances in computational analysis of protein structures allow predicting the intramolecular protein dynamics, identifying potential functional sites, or evaluating the effects of microenvironment changes in protein structure and dynamics. We applied this approach to characterize further the roles of amino acid domains within cytochrome b5 reductase structure, part of the catalytic domain, and several sensors and structural domains involved in the interactions with cytochrome b5 and other electron acceptors. The computational analysis results allowed us to rationalize some of the available spectroscopic data regarding ligand-induced conformational changes leading to an increase in the flavin adenine dinucleotide (FAD) solvent-exposed surface, which has been previously correlated with the formation of complexes with electron acceptors.

Targeting Lipid Peroxidation for Cancer Treatment.

Cancer is one of the highest prevalent diseases in humans. The chances of surviving cancer and its prognosis are very dependent on the affected tissue, body location, and stage at which the disease is diagnosed. Researchers and pharmaceutical companies worldwide are pursuing many attempts to look for compounds to treat this malignancy. Most of the current strategies to fight cancer implicate the use of compounds acting on DNA damage checkpoints, non-receptor tyrosine kinases activities, regulators of the hedgehog signaling pathways, and metabolic adaptations placed in cancer. In the last decade, the finding of a lipid peroxidation increase linked to 15-lipoxygenases isoform 1 (15-LOX-1) activity stimulation has been found in specific successful treatments against cancer. This discovery contrasts with the production of other lipid oxidation signatures generated by stimulation of other lipoxygenases such as 5-LOX and 12-LOX, and cyclooxygenase (COX-2) activities, which have been suggested as cancer biomarkers and which inhibitors present anti-tumoral and antiproliferative activities. These findings support the previously proposed role of lipid hydroperoxides and their metabolites as cancer cell mediators. Depletion or promotion of lipid peroxidation is generally related to a specific production source associated with a cancer stage or tissue in which cancer originates. This review highlights the potential therapeutical use of chemical derivatives to stimulate or block specific cellular routes to generate lipid hydroperoxides to treat this disease.

c-Src functionality controls self-renewal and glucose metabolism in MCF7 breast cancer stem cells.

Deregulation of Src kinases is associated with cancer. We previously showed that SrcDN conditional expression in MCF7 cells reduces tumorigenesis and causes tumor regression in mice. However, it remained unclear whether SrcDN affected breast cancer stem cell functionality or it reduced tumor mass. Here, we address this question by isolating an enriched population of Breast Cancer Stem Cells (BCSCs) from MCF7 cells with inducible expression of SrcDN. Induction of SrcDN inhibited self-renewal, and stem-cell marker expression (Nanog, Oct3-4, ALDH1, CD44). Quantitative proteomic analyses of mammospheres from MCF7-Tet-On-SrcDN cells (data are available via ProteomeXchange with identifier PXD017789, project DOI: 10.6019/PXD017789) and subsequent GSEA showed that SrcDN expression inhibited glycolysis. Indeed, induction of SrcDN inhibited expression and activity of hexokinase, pyruvate kinase and lactate dehydrogenase, resulting in diminished glucose consumption and lactate production, which restricted Warburg effect. Thus, c-Src functionality is important for breast cancer stem cell maintenance and renewal, and stem cell transcription factor expression, effects linked to glucose metabolism reduction.

Dissecting the function of Atg1 complex in Dictyostelium autophagy reveals a connection with the pentose phosphate pathway enzyme transketolase.

The network of protein-protein interactions of the Dictyostelium discoideum autophagy pathway was investigated by yeast two-hybrid screening of the conserved autophagic proteins Atg1 and Atg8. These analyses confirmed expected interactions described in other organisms and also identified novel interactors that highlight the complexity of autophagy regulation. The Atg1 kinase complex, an essential regulator of autophagy, was investigated in detail here. The composition of the Atg1 complex in D. discoideum is more similar to mammalian cells than to Saccharomyces cerevisiae as, besides Atg13, it contains Atg101, a protein not conserved in this yeast. We found that Atg101 interacts with Atg13 and genetic disruption of these proteins in Dictyostelium leads to an early block in autophagy, although the severity of the developmental phenotype and the degree of autophagic block is higher in Atg13-deficient cells. We have also identified a protein containing zinc-finger B-box and FNIP motifs that interacts with Atg101. Disruption of this protein increases autophagic flux, suggesting that it functions as a negative regulator of Atg101. We also describe the interaction of Atg1 kinase with the pentose phosphate pathway enzyme transketolase (TKT). We found changes in the activity of endogenous TKT activity in strains lacking or overexpressing Atg1, suggesting the presence of an unsuspected regulatory pathway between autophagy and the pentose phosphate pathway in Dictyostelium that seems to be conserved in mammalian cells.

Improvement and validation of d-xylose determination in urine and serum as a new tool for the noninvasive evaluation of lactase activity in humans.

BACKGROUND: The phloroglucinol assay is the current method for d-xylose determination in urine/plasma/serum. However, its sensitivity is limited when low amounts of d-xylose are to be measured, such as in the noninvasive evaluation of intestinal lactase with 4-galactosylxylose (gaxilose). An improved assay was therefore needed. METHODS: We developed and validated a modified version of the phloroglucinol-based assay for quantification of d-xylose in urine/serum samples. A method for gaxilose determination by gas chromatography (GC) was also optimized. RESULTS: Linearity ranged from 0.125 to 5.0 mg/l (5-200 mg/l in original sample). Accuracy at LOQ (0.125 mg/l) was 0.97/2.49% in spiked urine/serum; for other quality controls (QC), it was <1.27%. Intra- and interassay precision at LOQ were 6.02% and 6.45% for urine, and 8.86% and 10.00%, respectively, for serum; for other QC, precision was <2.15%. Linearity of gaxilose determination by GC was 3.90-195.17 for urine and 9.75-195.17 mg/l for serum with acceptable sensitivity and reproducibility. The method proved adequate for the d-xylose determination in healthy and hypolactasic subjects after oral administration of gaxilose. CONCLUSIONS: The modified method provides high sensitivity and robustness for d-xylose quantification in urine/serum for routine clinical use especially in the noninvasive diagnosis of intestinal lactase deficiency with the gaxilose test.

Noninvasive diagnosis of hypolactasia with 4-Galactosylxylose (Gaxilose): a multicentre, open-label, phase IIB-III nonrandomized trial.

GOALS AND BACKGROUND: Hypolactasia affects over half of the world population. Diagnosis remains problematic as currently available tests, such as the hydrogen breath test, have low reliability and lactose intolerance symptoms are unspecific. We evaluated the diagnostic performance and safety of a new noninvasive diagnostic test based on urine or serum measurement of D-xylose after lactase cleavage of orally administered 4-galactosylxylose (gaxilose). STUDY: In a multicentre, open-label, nonrandomized, phase IIb-III study, consecutive patients with symptoms suggestive of lactose intolerance sequentially underwent intestinal biopsy for direct measurement of lactase activity (reference standard), hydrogen breath test, and blood glucose test after lactose challenge, 4- and 5-hour urine-based gaxilose test, and blood-based gaxilose test. For the gaxilose tests, 0 to 4 and 4 to 5 hours urine samples were taken after a 0.45 g gaxilose dose, whereas serum samples were taken 90 minutes after a 2.7 g dose for D-xylose determination. Genetic testing of hypolactasia was also assessed. RESULTS: Of the 222 patients enrolled, 203 completed all diagnostic tests; 108 were hypolactasic according to biopsy. The sensitivities and specificities and positive and negative predictive values of the gaxilose tests were all >90% versus 69% to 85% for the hydrogen breath test and the blood glucose test. The area under the ROC curve was significantly higher for the gaxilose tests (>0.9, P≤0.007). These tests also had higher sensitivity than genetic testing for hypolactasia and were well tolerated. CONCLUSIONS: The diagnostic performance of the gaxilose tests is excellent and can substantially improve the diagnosis of hypolactasia.

Phase I and phase IB clinical trials for the noninvasive evaluation of intestinal lactase with 4-galactosylxylose (gaxilose).

GOALS AND BACKGROUND: Hypolactasia is widespread, yet reliable diagnostic tests are lacking. A new test based on oral administration of 4-galactosylxylose (gaxilose) and urine or serum measurement of D-xylose after cleavage by intestinal lactase is under clinical development. We investigated the optimal dose of gaxilose and calculate cutoff values of D-xylose for that dose. STUDY: In the randomized, dose-finding, phase I study, urine and serum pharmacokinetics of D-xylose were determined after oral administration of 6 ascending doses of gaxilose (and placebo) to 12 healthy adult volunteers. In the open, parallel, phase Ib study, 30 volunteers received the doses established for the urine and blood tests and D-xylose was measured. Cutoff values were calculated as 1.96 × SD below the mean value. Safety was assessed through reporting of adverse events. RESULTS: Gaxilose administration showed a progressive, dose-dependent increase in D-xylose in urine and serum. An optimal gaxilose dose of 0.45 g and urine collection periods of 4 and 5 hours were selected for further studies. For the blood test, a 2.7 g dose was selected and C max measured at 90 minutes. The calculated cutoff values of D-xylose for normal lactase activity were 27.58 and 37.87 mg for the 4- and 5-hour urine tests, respectively, and 0.97 mg/dL for the blood test. There were no treatment-related adverse events. CONCLUSIONS: The methodology described provides a simple, safe test for the evaluation of lactase activity in vivo. Further evaluation of the test as a noninvasive diagnosis of hypolactasia is ongoing in patients with lactose intolerance.

Distinct functional roles of the two terminal halves of eukaryotic phosphofructokinase.

Eukaryotic PFK (phosphofructokinase), a key regulatory enzyme in glycolysis, has homologous N- and C-terminal domains thought to result from duplication, fusion and divergence of an ancestral prokaryotic gene. It has been suggested that both the active site and the Fru-2,6-P2 (fructose 2,6-bisphosphate) allosteric site are formed by opposing N- and C-termini of subunits orientated antiparallel in a dimer. In contrast, we show in the present study that in fact the N-terminal halves form the active site, since expression of the N-terminal half of the enzymes from Dictyostelium discoideum and human muscle in PFK-deficient yeast restored growth on glucose. However, the N-terminus alone was not stable in vitro. The C-terminus is not catalytic, but is needed for stability of the enzyme, as is the connecting peptide that normally joins the two domains (here included in the N-terminus). Co-expression of homologous, but not heterologous, N- and C-termini yielded stable fully active enzymes in vitro with sizes and kinetic properties similar to those of the wild-type tetrameric enzymes. This indicates that the separately translated domains can fold sufficiently well to bind to each other, that such binding of complementary domains is stable and that the alignment is sufficiently accurate and tight as to preserve metabolite binding sites and allosteric interactions.

Electron microscopy analysis of mammalian phosphofructokinase reveals an unusual 3-dimensional structure with significant implications for enzyme function.

Phosphofructokinase is a sophisticated allosteric enzyme that is fundamental for the control of glycolysis. The structure of the bacterial enzyme is well characterized. However, little is known about the structural organization of the more complex enzyme from mammals. We have obtained the structure of human muscle phosphofructokinase in the presence of fructose 6-phosphate at a resolution of 1.8 nm by electron microscopy (EM). Particles of the tetrameric enzyme corresponded to an elongated molecule (14.5 × 9 nm) arranged into 2 dimeric subdomains. Image analysis and 3-dimensional reconstruction showed the presence of a prominent channel in one of the dimers but not in the opposite one, revealing that they are in greatly different conformations. Fitting of bacterial structures into the EM model suggested disruption of the fructose 6-phosphate catalytic and the fructose 2,6-bisphophate allosteric sites in the cavity-containing dimer. Therefore, the reported structure might have major implications for the function of mammalian phosphofructokinase.

Subunit interactions and composition of the fructose 6-phosphate catalytic site and the fructose 2,6-bisphosphate allosteric site of mammalian phosphofructokinase.

Mammalian phosphofructokinase originated by duplication, fusion, and divergence of a primitive prokaryotic gene, with the duplicated fructose 6-phosphate catalytic site in the C-terminal half becoming an allosteric site for the activator fructose 2,6-bisphosphate. It has been suggested that both sites are shared across the interface between subunits aligned in an antiparallel orientation, the N-terminal half of one subunit facing the C-terminal half of the other. The composition of these binding sites and the way in which subunits interact to form the dimer within the tetrameric enzyme have been reexamined by systematic point mutations to alanine of key amino acid residues of human muscle phosphofructokinase. We found that residues His-199, His-298, Arg-201, and Arg-292 contribute to the catalytic site and not to the allosteric site, because their mutation decreased the affinity for fructose 6-phosphate without affecting the activation by fructose 2,6-bisphosphate or its binding affinity. In contrast, residues Arg-566, Arg-655, and His-661 were critical components of the fructose bisphosphate allosteric site, because their mutation strongly reduced the action and affinity of the activator, with no alteration of substrate binding to the active site. Our results suggest that mammalian phosphofructokinase subunits associate with the N-terminal halves facing each other to form the two catalytic sites/dimer and the C-terminal halves forming the allosteric sites. Additionally, mutation of certain residues eliminated activation by fructose 1,6-bisphosphate, but not its binding, with little effect on activation by fructose 2,6-bisphosphate, indicating a divergence in the signal transduction route despite their binding to the same site.

The serum levels of the EGF-like homeotic protein dlk1 correlate with different metabolic parameters in two hormonally different children populations in Spain.

BACKGROUND: The Dlk1 gene encodes for dlk1, a transmembrane protein belonging to the EGF-like repeat-containing family. Dlk1 has been shown to act as a regulator of adipogenesis. Fc-dlk1 transgenic mice show a decrease in adipose tissue and glucose tolerance, hypertriglyceridaemia and lower insulin sensitivity. Dlk1-deficient mice show growth retardation, increased serum lipid metabolites and develop obesity. These data advocate for a role of dlk1 in the maintenance of lipid homeostasis, and suggest that dlk1 levels may influence the development of cardiovascular disease. AIM AND METHODS: In this study, we analysed whether dlk1 serum levels could be indicative of the different hormonal or metabolic status shown by two Spanish children populations (6-8 years-old), Orense and Murcia. We determined dlk1 serum levels by ELISA assay, using an antibody raised against the recombinant protein, and performed a correlation analysis against measurements of several hormonal and biochemical parameters in samples from 494 subjects. RESULTS: We found a statistically significant positive correlation between serum levels of dlk1 and those of glucose (P < 0.05), total cholesterol (P < 0.01) and high-density lipoprotein-cholesterol (HDL-C) (P < 0.01) in children from Murcia, but not from Orense's population, where dehydroepiandrosterone-sulphate (DHEA-S) levels were significantly higher (P < 0.01) and dlk1 correlated positively with insulin (P < 0.01), homeostasis model assessment (HOMA) (P < 0.01) and free fatty acids (FFA) (P < 0.05). CONCLUSIONS: dlk1 serum levels appear related to the anabolic status of the children in association with changes in the levels of DHEA-S, which have been associated with hyperinsulinaemia and diabetes. Monitoring dlk1 levels may be important to evaluate the metabolic and hormonal stage of child development.

Chimeric phosphofructokinases involving exchange of the N- and C-terminal halves of mammalian isozymes: implications for ligand binding sites.

Two phosphofructokinase (PFK) chimeras were constructed by exchanging the N- and C-terminal halves of the mammalian M- and C-type isozymes, to investigate the contribution of each terminus to the catalytic site and the fructose-2,6-P(2)/fructose-1,6-P(2) allosteric site. The homogeneously-purified chimeric enzymes organized into tetramers, and exhibited kinetic properties for fructose-6-P and MgATP similar to those of the native enzyme that furnished the N-terminal domain in each case, whereas their fructose-2,6-P(2) activatory characteristics coincided with those of the isozyme that provided the C-terminal half. This reflected the role of each domain in the formation of the corresponding binding site. Grafting the N-terminus of PFK-M onto the C-terminus of the fructose-1,6-P(2) insensitive PFK-C restored transduction of this signal to the catalytic site, which significance is also discussed.

Noninvasive evaluation of intestinal lactase with 4-galactosylxylose: comparison with 3- and 2-galactosylxylose and optimization of the method in rats.

BACKGROUND: Urinary excretion of D-xylose by suckling rats after ingestion of a mixture of 4-, 3-, and 2-galactosylxyloses reflects lactase activity in vivo. We aimed to select the most convenient of these disaccharides for detecting changes of the enzyme activity in vivo and to optimize the method. METHODS: 4-, 3-, and 2-galactosylxyloses were synthesized and purified, then orally administered to suckling rats of different ages. D-Xylose was measured colorimetrically by the phloroglucinol reaction in urine and plasma. Lactase activity was determined in extracts of small intestine mucosa with lactose, galactosylxyloses, and phlorizin as substrates. RESULTS: D-Xylose appeared in the urine in a dose-dependent manner after ingestion of any of the 3 galactosylxylose disaccharides. Correlation between D-xylose elimination and intestinal lactase activity was highest with 4-galactosylxylose (r = 0.97; n = 24), lower with 2-galactosylxylose (r = 0.89; n = 24), and lowest with 3-galactosylxylose (r = 0.34; n = 23). The kinetic properties of intestinal lactase accounted for these differences. D-Xylose concentration in plasma after administration of 4-galactosylxylose also correlated with lactase activity (r = 0.93; n = 33). CONCLUSIONS: 4-Galactosylxylose is the most suitable compound for the evaluation of lactase activity in vivo. Measurement of the derived D-xylose in either urine or blood gives an estimate of the total lactose digestive capacity of the small intestine. The optimized method holds promise for development of a simple, low-cost, and reliable new test for the noninvasive diagnosis of hypolactasia.

The dimorphic yeast Yarrowia lipolytica possesses an atypical phosphofructokinase: characterization of the enzyme and its encoding gene.

The phosphofructokinase from the non-conventional yeast Yarrowia lipolytica (YlPfk) was purified to homogeneity, and its encoding gene isolated. YlPfk is an octamer of 869 kDa composed of a single type of subunit, and shows atypical kinetic characteristics. It did not exhibit cooperative kinetics for fructose 6-phosphate (Hill coefficient, h 1.1; S0.5 52 microM), it was inhibited moderately by MgATP (Ki 3.5 mM), and it was strongly inhibited by phosphoenolpyruvate (Ki 61 microM). Fructose 2,6-bisphosphate did not activate the enzyme, and AMP and ADP were also without effect. The gene YlPFK1 has no introns, and encodes a putative protein of 953 aa, with a molecular mass consistent with the subunit size found after purification. Disruption of the gene abolished growth in glucose and Pfk activity, while reintroduction of the gene restored both properties. This indicates that Y. lipolytica has only one gene encoding Pfk, and supports the finding that the enzyme consists of identical subunits. Glucose did not interfere with growth of the Ylpfk1 disruptant in permissive carbon sources. The unusual kinetic characteristics of YlPfk, and the intracellular concentrations of glycolytic intermediates during growth in glucose, suggest that YlPfk may play an important role in the regulation of glucose metabolism in Y. lipolytica, different from the role played by the enzyme in Saccharomyces cerevisiae.

Characterization of the aspartate kinase from Saccharomyces cerevisiae and of its interaction with threonine.

Aspartate kinase (AK) from Saccharomyces cerevisiae has been characterized to elucidate its quaternary structure and the effect of the allosteric inhibitor threonine on the enzyme conformation. The homogeneously purified enzyme was inhibited by threonine (K(i) 1.4 mM) and was found to bind this compound (K(d) 0.97 mM) in a hyperbolic manner. Gel filtration and native gel electrophoresis indicated that yeast AK is a homohexamer of 346 kDa composed by 58 kDa subunits. Threonine caused a decrease in the apparent molecular mass of AK as evidenced by size-exclusion chromatography (from 345 to 280 kDa) and blue native gel electrophoresis (from 346 to 297 kDa); no other molecular species were detected. This shift in the hydrodynamic size was threonine-specific and was reversed by rechromatography in the absence of threonine. No change in the apparent molecular mass was induced by threonine in an AK mutant insensitive to inhibition by this amino acid, which was observed to be unable to bind threonine. These results indicate that the allosteric transition elicited by binding of threonine to yeast AK involves a large conformational change of the protein that isomerizes from a relaxed active conformation to a more compact inactive one of smaller molecular dimensions.

Identification of C-terminal motifs responsible for transmission of inhibition by ATP of mammalian phosphofructokinase, and their contribution to other allosteric effects.

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.