Prevalence of COL4A1 and COL4A2 mutations in severe fetal multifocal hemorrhagic and/or ischemic cerebral lesions.

OBJECTIVE: To establish the prevalence of COL4A1 and COL4A2 gene mutations in fetuses presenting with a phenotype suggestive of cerebral injury. METHODS: This was a single-center retrospective analysis of all cases of fetal cerebral anomalies suggestive of COL4A1 or COL4A2 gene mutation over the period 2009-2018. Inclusion criteria were: (1) severe and/or multifocal hemorrhagic cerebral lesions; (2) multifocal ischemic-hemorrhagic cerebral lesions. These anomalies could be of different ages and associated with schizencephaly or porencephaly. Between fetuses with and those without a mutation, we compared gestational age at the time of diagnosis, parity and fetal gender. RESULTS: Among the 956 cases of cerebral anomaly diagnosed in our center during the 10-year study period, 18 fetuses were identified for inclusion. A pathogenic COL4A1 gene mutation was found in five of these cases, among which four were de-novo mutations. A variant of unknown significance was found in four fetuses: in the COL4A1 gene in one case and in the COL4A2 gene in three cases. No COL4A1 or COL4A2 mutation was found in the remaining nine fetuses. The median (interquartile range) gestational age at diagnosis was significantly lower in cases with a mutation (24 (22-26) weeks) than in cases without a mutation (32 (29.5-34.5) weeks) (P = 0.03). CONCLUSIONS: A phenotype suggestive of cerebral injury was found in 18 of the 956 (1.9%) cases in our population, in 28% of which there was an associated COL4A1 or COL4A2 mutation. COL4A1 and COL4A2 gene mutations should be sought systematically in cases of severe and/or multifocal hemorrhagic or ischemic-hemorrhagic cerebral lesions, with or without schizencephaly or porencephaly. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

New insights in cerebral findings associated with fetal myelomeningocele: a retrospective cohort study in a single tertiary centre.

OBJECTIVE: To investigate cerebral anomalies other than Chiari type 2 malformation in fetuses with myelomeningocele (MMC). DESIGN: A retrospective cohort study in a single tertiary centre. SETTING: A review of associated cerebral anomalies in cases with prenatal diagnosis of myelomeningocele. POPULATION: Seventy cases of fetal myelomeningocele. METHODS: Ultrasound and MRI images were blindly reviewed. Postnatal imaging and results of the postmortem results were also reviewed. The association between cerebral anomalies and the following ultrasound findings was measured: level of the defect, ventriculomegaly, microcephaly and fetal talipes. MAIN OUTCOME MEASURES: A microcephaly was observed in 32/70 cases (46%) and a ventriculomegaly was observed in 39/70 cases (56%). Other cerebral anomalies were diagnosed in 47/70 (67%). RESULTS: Other cerebral anomalies were represented by 42/70 cases with abnormal CC (60%), 8/70 cases with perinodular heterotopia (PNH; 11%), 2/70 cases with abnormal gyration (3%). MRI performed only in fetal surgery cases confirmed the ulltrasound findings in all cases and provided additional findings in two cases (PNH). Risk ratios of fetal cerebral anomalies associated with MMC did not reach significance for microcephaly, ventriculomegaly, talipes or the level of the defect There was an overall good correlation between pre- and postnatal findings with a Kappa value of 0.79 [95% CI 0.57-1] and 82% agreement. CONCLUSION: Fetal brain anomalies other than Chiari type 2 malformation are frequently observed in fetuses with myelomeningocele, predominantly represented by CC anomalies. Whether these associated cerebral anomalies have an impact on selecting cases eligible for fetal surgery needs further evaluation. TWEETABLE ABSTRACT: Fetal cerebral anomalies other than Chiari type 2 malformation, microcephaly, and ventriculomegaly may be associated with MMC in up to 67% of the cases.

[Silent myocardial infarction diagnosed by myocardial perfusion SPECT in a young woman with systemic lupus erythematosus]. / Infarto de miocardio silente diagnosticado por SPECT de perfusión miocárdica en mujer joven con lupus eritematoso sistémico.

Premature atherosclerosis and its consequent heart disease play a crucial role in patients with systemic lupus erythematosus, even in premenopausal women. It is one of the leading causes of death in long evolution lupus. We present the case of a 42-year-old premenopausal woman, smoker, with a history of hypertension, cholecystectomy and lupus for 23 years, treated with NSAID, steroids and antimalarial drugs. The patient consulted due to chest pain on moderate efforts. Due to the suspicion of ischemic heart disease, a cardiology study was initiated, performing a myocardial perfusion SPECT. This revealed an intense and extensive anterolateral perfusion defect, with very light reperfusion in rest images, consistent with the diagnosis of acute infarction in the apical region and ischemia in the territory of the left anterior descending artery, which was confirmed later by cardiac catheterization.

Effect of cattle grazing a species-rich mountain pasture under different stocking rates on the dynamics of diet selection and sward structure.

Although stocking rate is a key management variable influencing the structure and composition of pastures, only few studies have simultaneously analysed the seasonal patterns of pasture use by cattle, and the adjustments the animals make to maintain intake of a high-quality diet over the grazing season. Therefore, over a 3-year study, we recorded diet selection, plot use and impact of heifers on sward structure and quality under three different stocking rates (0.6, 1.0 and 1.4 livestock units (LU) per ha) in a species-rich mountain pasture of central France. Measurements were made on three occasions between early June and the end of September each year. Overall, heifers selected for bites dominated by legumes or forbs, and against reproductive grass, whatever the stocking rate or season. Selection for tall mixed (P < 0.05), short mixed (P < 0.05) and short pure grass bites (P < 0.01) was more pronounced in plots grazed at the lowest stocking rate. Although heifers' selection for short patches decreased at the end of the season (P < 0.001), they continued to graze previously grazed areas, thus exhibiting a typical 'patch grazing' pattern, with the animals that grazed at the lowest stocking rate tending to better maintain their selection for short patches in September (treatment × period: P = 0.078). Neither diet quality nor individual animal performance were affected by the different stocking rate treatments despite high variability in the quantity and quality of herbage offered and differences in diet selection. However, at the 1.4 LU per ha stocking rate, the quantity of forage available per animal at the end of the season, 0.79 t dry matter (DM) per ha of green leaves with the median of sward height at 4.6 cm, approached levels limiting cattle's ability to compensate for the effects of increasing stocking rate. In plots grazed at 0.6 LU per ha, the total herbage biomass remained higher than 3 t DM per ha with more than 30% of plot area still covered by reproductive grass patches at the end of the grazing season, which in the medium term should affect the botanical composition of these pastures. Sward heterogeneity was high in plots grazed at 1.0 LU per ha, with sufficient herbage availability (1.1 t DM per ha of green leaves) to maintain animal performance, and more than 15% of plot area was kept at a reproductive stage at the end of the grazing season. Hence, it could represent the optimal balance to satisfy both livestock production and conservation management objectives.

Dexamethasone increases RAMP1 and CRLR mRNA expressions in human vascular smooth muscle cells.

A recent report has shown that in vitro the RAMP2/CRLR complex is a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells. However, in vivo, it is well known that CGRP receptors are expressed in human coronary arteries and that a beneficial effect is observed in patients after CGRP infusion of patients with congestive cardiac failure. This contrast may be explained by the in vivo impregnation of major hormones, so we have tested if glucocorticoids were able in vitro to enhance the expression of the RAMP1/CRLR expression leading to functional CGRP receptors. The expression of RAMP1, RAMP2, CRLR, and adrenomedullin was evaluated by semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) using (33)P in human coronary arteries vascular smooth muscle cells (VSMC) cultured in the presence of dexamethasone. Under basal conditions, the CRLR mRNA was expressed, but RAMP2 mRNA was clearly more abundant than RAMP1 mRNA. Increases in CRLR and RAMP1 mRNA expressions occurred 4 h after treatment of VSMC with 10(-7) M dexamethasone and no change was found for RAMP2 mRNA. Adrenomedullin mRNA increased later, i.e., 8 and 16 h after dexamethasone treatment. The RAMP1 mRNA expression was elevated with doses of dexamethasone ranging from 10(-10) to 10(-7) M, thus a 5-fold increase in the ratio between RAMP1 and RAMP2 was observed with the lowest dose of dexamethasone and a 2-fold rise at 10(-7) M. CRLR mRNA levels were half-reduced with the two lowest doses of dexamethasone (10(-10) and 10(-9) M), but increased from 10(-8) to 10(-7) M. Thus, we suggest that, in vivo, glucocorticoids are involved in the expression of CGRP receptors by human coronary VSMC.

Increased production of hydrogen peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon aeration: involvement of an NADH oxidase in oxidative stress.

The growth of Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H(2)O(2) oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by L. delbrueckii subsp. bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen.

The NADH oxidase of Streptococcus pneumoniae: its involvement in competence and virulence.

A soluble flavoprotein that reoxidizes NADH and reduces molecular oxygen to water was purified from the facultative anaerobic human pathogen Streptococcus pneumoniae. The nucleotide sequence of nox, the gene which encodes it, has been determined and was characterized at the functional and physiological level. Several nox mutants were obtained by insertion, nonsense or missense mutation. In extracts from these strains, no NADH oxidase activity could be measured, suggesting that a single enzyme encoded by nox, having a C44 in its active site, was utilizing O2 to oxidize NADH in S. pneumoniae. The growth rate and yield of the NADH oxidase-deficient strains were not changed under aerobic or anaerobic conditions, but the efficiency of development of competence for genetic transformation during growth was markedly altered. Conditions that triggered competence induction did not affect the amount of Nox, as measured using Western blotting, indicating that nox does not belong to the competence-regulated genetic network. The decrease in competence efficiency due to the nox mutations was similar to that due to the absence of oxygen in the nox+ strain, suggesting that input of oxygen into the metabolism via NADH oxidase was important for controlling competence development throughout growth. This was not related to regulation of nox expression by O2. Interestingly, the virulence and persistence in mice of a blood isolate was attenuated by a nox insertion mutation. Global cellular responses of S. pneumoniae, such as competence for genetic exchange or virulence in a mammalian host, could thus be modulated by oxygen via the NADH oxidase activity of the bacteria, although the bacterial energetic metabolism is essentially anaerobic. The enzymatic activity of the NADH oxidase coded by nox was probably involved in transducing the external signal, corresponding to O2 availability, to the cell metabolism and physiology; thus, this enzyme may function as an oxygen sensor. This work establishes, for the first time, the role of O2 in the regulation of pneumococcal transformability and virulence.

Olfactory receptors, Golf alpha and adenylyl cyclase mRNA expressions in the rat heart during ontogenic development.

Golf alpha (a G heterotrimeric protein which shares a high homology with Gs alpha) expression was studied in the rat heart before birth and until weaning. Since Golf alpha in the neuro-olfactory epithelium is coupled to olfactory receptors and type III adenylyl cyclase, we looked for the presence of such molecules in the heart. Golf alpha mRNA was detected in the rat heart, highest levels being found in 21-day old fetuses until 3 days post partum. The protein amounts measured by Western blots paralleled the Golf alpha mRNA levels. Immunohistochemical studies revealed the presence of Golf alpha in atrial and ventricular cardiomyocytes. OL1 and latrophilin mRNAs, G protein-coupled olfactory receptors, were expressed at early postnatal stages. Adenylyl cyclase mRNAs for type II, type III, type V and type VI were expressed before birth and until weaning. Elements for an unexpected signaling pathway involving odorant receptors like OL1 and latrophilin, Golf alpha and type III adenylyl cyclase were expressed in rat heart, and appeared developmentally regulated.

Galphaolf identification by RT-PCR in purified normal pancreatic B cells and in islets from rat models of non-insulin-dependent diabetes.

Recent reports using immunohistochemistry have shown that Galphaolf which shares 88% homology with Galphas was expressed in pancreatic islets. To test the specificity of the expression of this G protein isotype in rat islet cells, B and non-B cells were separated by flow cytometry. The expression of Galphaolf and adenylyl cyclases (AC) of types II, III, V, and VI was evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR). Since alterations in the expression of AC III were recently reported in the GK rat (a model of non-insulin-dependent diabetes mellitus, NIDDM), we also have analyzed the mRNA expression of Galphaolf and AC isoforms in pancreatic islets from GK rats and from adult rats neonatally treated by streptozotocin (nSTZ rats), another model of NIDDM. Southern blots of amplicons generated with specific primers of Galphaolf revealed the presence of a 540-bp band only in B cells. AC of types II, III, V, and VI were expressed both in B and non-B cells. However, AC III mRNA was clearly more abundant in non-B than in B cells. Moreover, in B cells the expression of AC VI was higher than that of AC V, whereas equal expressions of AC V and AC VI were found in non-B cells. In GK rat islets, the mRNA expressions of Galphaolf, AC II, and AC III were clearly increased and no change in AC V and AC VI was found. In nSTZ rat islets, Galphaolf expression was barely detectable, but AC II and AC III mRNA levels were higher than those observed in controls. In conclusion, Galphaolf mRNA appeared specifically expressed in islet B cells and was increased in GK islets. The steady-state mRNA levels of AC II and AC III were clearly increased in the islets of the two rat models of NIDDM. Thus, alterations in the expression of G protein isotypes and AC isoforms could contribute to the diabetic phenotype.

Separation of tubulin subunits under nondenaturing conditions.

The dissociation and separation of the tubulin alpha- and beta-subunits have been achieved by binding alpha-subunits to an immunoadsorbent gel and selectively inducing release of free beta-subunits. The immunoadsorbent gel was prepared by coupling the monoclonal antibody YL1/2 to Sepharose 4B which specifically recognizes the C-terminal end of tyrosinated alpha-subunits. Extensive tubulin subunit dissociation and separation occurred in Tris buffer at neutral pH but was greatly enhanced at basic pHs (8. 0-8.5). The binding of colchicine to heterodimeric tubulin resulted in a marked protection against dissociation. The dissociation of tubulin subunits was accompanied by loss of colchicine binding capacity, and ability to polymerize into microtubules. As shown by circular dichroism, loss of functional properties was not due to extensive denaturation of tubulin, as tubulin retained most of its secondary structure. Neither of the separated alpha- or beta-subunits was able to bind colchicine, but functional tubulin that was able to bind colchicine could be reconstituted from the dissociated subunits by changing the buffer to a neutral mixture of Tris and Pipes. The yield of reconstitution, as estimated from kinetic measurements of colchicine binding capacity, amounted to about 25%. Such a yield can probably be improved with minor changes in experimental conditions. The quantitative dissociation of tubulin into separated "native" alpha- and beta-subunits should provide a powerful tool for further studies on the properties of the individual tubulin subunits and the structure-function relationships of the tubulins.

Stimulatory transducing systems in pancreatic islet cells.

We have determined the cellular distribution of different alpha subtypes of G proteins and adenylyl cyclase (AC) isoforms in endocrine, exocrine, and established pancreatic cell lines. VIP, PACAP, and tGLP-1 receptor proteins are expressed to varying extents in A and B cells, whereas the expression of G alpha subunits is cell specific. Thus, G(olf) alpha is detected in normal rodent B cells and immortalized pancreatic B cell lines, whereas Gs alpha is more ubiquitously expressed. The cellular density of AC isoforms labeling (I, II, III, IV, V/VI) is also islet cell-specific and their distribution is age- and species-dependent. The identification of numerous signaling molecule subtypes, together with the discovery of their specific subcellular distribution, will help the functional characterization of their intraregulatory pathways, leading to the extrusion of insulin or glucagon secretory granules, and those leading to differentiation and apoptosis of islet cells.

Crystal structures of the small G protein Rap2A in complex with its substrate GTP, with GDP and with GTPgammaS.

The small G protein Rap2A has been crystallized in complex with GDP, GTP and GTPgammaS. The Rap2A-GTP complex is the first structure of a small G protein with its natural ligand GTP. It shows that the hydroxyl group of Tyr32 forms a hydrogen bond with the gamma-phosphate of GTP and with Gly13. This interaction does not exist in the Rap2A-GTPgammaS complex. Tyr32 is conserved in many small G proteins, which probably also form this hydrogen bond with GTP. In addition, Tyr32 is structurally equivalent to a conserved arginine that binds GTP in trimeric G proteins. The actual participation of Tyr32 in GTP hydrolysis is not yet clear, but several possible roles are discussed. The conformational changes between the GDP and GTP complexes are located essentially in the switch I and II regions as described for the related oncoprotein H-Ras. However, the mobile segments vary in length and in the amplitude of movement. This suggests that even though similar regions might be involved in the GDP-GTP cycle of small G proteins, the details of the changes will be different for each G protein and will ensure the specificity of its interaction with a given set of cellular proteins.

Allosteric activation increases the maximum velocity of E. coli phosphofructokinase.

Several mutations that cause a decrease of 25 to 65% of the catalytic activity, were introduced at different positions in the phosphofructokinase from Escherichia coli, and the influence of the allosteric activator GDP on these mutants was measured. In the case of the wild-type enzyme, GDP converts the highly cooperative saturation towards fructose-6-phosphate into a hyperbolic saturation with almost no change in the maximum velocity. The mutants Glu148 --> Leu, Leu178 --> Val and Leu178 --> Trp are still cooperative for fructose-6-phosphate, and their cooperativity is also abolished or markedly decreased by GDP. In addition, GDP acts on these mutants as an activator of maximum velocity, and increases their catalytic rate constants by 35 to 65% depending on the mutation. The Leu178 --> Val mutant is even as active as the wild-type enzyme in the presence of GDP. The Thr125 --> Ser mutation decreases the maximum velocity by 60% and also suppresses the cooperativity towards fructose-6-phosphate. Accordingly, the only effect of GDP on the Thr125 --> Ser mutant is on its maximum velocity and not on its affinity for fructose-6-phosphate. However, the maximum velocity of this mutant is not increased by GDP but reduced by 33%. These results show that GDP affects the maximum velocity of these mutants and suggest that the activation by GDP of the wild-type enzyme measured by steady-state kinetics could be partially due to an increase of the maximum velocity, and not exclusively to an increase of the affinity for fructose-6-phosphate.

A zinc-dependent proteinase from the cell wall of Lactobacillus delbrueckii subsp. bulgaricus.

A zinc-dependent proteinase was extracted from the cell wall of Lactobacillus delbrueckii subsp. bulgaricus and partially purified despite a marked unstability. The caseinolytic activity was associated with a polypeptide chain of 65 kDa that belonged to the M1 family of zinc-dependent proteases. This zinc-dependent proteinase could degrade intact caseins, with a significant preference for beta-casein. The pH-profile of its activity indicated that its relative contribution to the caseinolytic activity increased at acidic pH, suggesting that this zinc proteinase could be involved in the late stages of milk fermentation.

Decrease in CGRP and CT levels either contained in or released by CA-77 C cells after combined treatments with 1,25-dihydroxyvitamin D3 analogues and 9-cis retinoic acid.

This study examined the action of 9-cis retinoic acid and 1,25-dihydroxyvitamin D3 analogues (KH 1060, EB 1089 and MC 903) on the release of calcitonin (CT) and calcitonin gene-related peptide (CGRP) in the rat C cell line CA-77. This cell line mainly secretes CGRP. Using radioimmunoassays (RIAs) for CT and CGRP, we measured the release of both peptides in the culture medium as well as the amount of these proteins contained in the CA-77 C cells. 9-cis retinoic acid decreased the release of both CGRP and CT dose-dependently in the range between 1 nM and 1 microM. The half-effective dose was 10 nM. The treatment of CA-77 C cells with 0.1 microM calcitriol alone only slightly decreased the release of both CT and CGRP. The increase in the amount of CT and CGRP released by the action of 1 microM dexamethasone was reduced by 1 microM 9-cis retinoic acid, and this effect was enhanced by the addition of 0.1 microM calcitriol or KH 1060, EB 1089 and MC 903. When the C cells were continuously stimulated by dexamethasone, after 6 days of exposure to the combined treatment with calcitriol analogues + 9-cis retinoic acid, there was a greater decrease in the amount of CGRP contained in the C cells than after treatment with 9-cis retinoic alone. Our data suggested that combined treatment with retinoic acid and calcitriol analogues exerted a stronger inhibition on the amounts of the two peptides either contained in the cells or released in the medium than each hormone alone.

Expression of glucagon-like peptide 1 receptor in a murine C cell line: regulation of calcitonin gene by glucagon-like peptide 1.

We have characterized, by RT-PCR amplification using specific primers, the presence of glucagon-like peptide-1 (GLP-I) receptor mRNA in CA-77 cells, a C cell line derived from a rat medullary thyroid carcinoma. Down-regulation of the GLP-1 receptor mRNA was observed after exposure of CA-77 C cells with GLP-1 (7-37). Increased secretion of both calcitonin gene-related peptide (CGRP) and calcitonin (CT) occurred after treatment with GLP-1 (7-37) associated with elevated steady-state levels of CGRP and CT mRNA. GLP-1 (7-37) increased cAMP formation in CA-77 cells in a dose-dependent manner; exendin (9-39), a GLP-1 receptor antagonist, inhibited cAMP production. The GLP-1 peptide which is produced by intestinal cells could be involved in the control of CT secretion through an entero-thyroidal axis implying GLP-1 receptor and increased CT gene expression.

The genes for phosphofructokinase and pyruvate kinase of Lactobacillus delbrueckii subsp. bulgaricus constitute an operon.

In Lactobacillus delbrueckii subsp. bulgaricus, the pyk gene coding for pyruvate kinase and the pfk gene coding for phosphofructokinase formed a bicistronic operon transcribed into a 2.9-kb RNA. The nucleotide sequence of the pyk gene indicated that the encoded protein possessed an extra C-terminal domain with a potential phosphoenolpyruvate-dependent autophosphorylation site.

Tetramer-dimer equilibrium of phosphofructokinase and formation of hybrid tetramers.

Moderate concentrations of KSCN inactivate the allosteric phosphofructokinase from Escherichia coli by dissociating the subunit interface that contains the binding site for the substrate fructose-6-phosphate. At a given KSCN concentration, the activity varies with the concentration of protein as expected from a simple equilibrium between active tetramers and inactive dimers. The equilibrium constants for the dissociation of a tetramer into dimers have been determined in 0.4 M KSCN for the wild-type enzyme and the noncooperative mutant T125S, the hypercooperative mutant E148A-R152A, and the inactive mutant D127S. The stability of the tetrameric structure is decreased by the mutations E148A-R152A that are in the interface and increased by the mutation T125S that does not belong to it. There could be an inverse correlation between the cooperativity of the saturation by fructose-6-phosphate (in absence of any effector) and the stability of the interface that contains its binding site. Hybrid tetramers can be formed upon reassociation of a dimer from an active phosphofructokinase (wild-type, T125S, or E148-R152A) with a dimer from the inactive D127S mutant, and their stability and cooperativity toward fructose-6-phosphate have been measured without purifying them. The results indicate that the formation of a hybrid interface involves some flexibility of the two dimers and that the allosteric coupling between distant sites could be related to the plasticity and instability of the interactions across this interface.

Slow ligand-induced transitions in the allosteric phosphofructokinase from Escherichia coli.

The fluorescence of the unique tryptophan residue of the allosteric phosphofructokinase from Escherichia coli varies upon binding of any ligand, whether substrate or effector, suggesting that the protein undergoes a conformational change. This fluorescent probe has been exploited to determine the rates of the structural transitions that occur upon ligand binding and that are responsible for the remarkable allosteric behavior of this enzyme. The kinetics of fluorescence changes measured after rapidly mixing phosphofructokinase with one of its ligands show the presence of several allosteric transitions with widely different rates, ranging from a few hundred s-1 to less than 0.1 s-1. The rate of each conformational change increases with the concentration of the ligand used to trigger it, suggesting that ligands induce a conformational change and do not displace a pre-existing equilibrium. The hypothesis that each ligand stabilizes a different conformational state of the protein is confirmed by the kinetics of displacement of one ligand by another: for instance, the binary complexes between phosphofructokinase and either its substrate, fructose-6-phosphate, or its allosteric activator, ADP, have the same low fluorescence and should be in the same active state, but they show different rates of conformational transition upon binding the inhibitor phosphoenolpyruvate. It appears that phosphofructokinase can exist in more than two states. Some conformational changes between these multiple states are slow enough to play an important role in the kinetics of the reaction catalyzed by phosphofructokinase, and could even explain part of its allosteric behavior. These results show that steady-state measurements are not sufficient to analyze the regulatory properties of E. coli phosphofructokinase.

Role of residue 161 in the allosteric transitions of two bacterial phosphofructokinases.

The loop between alpha-helix 6 and beta-strand F of the phosphofructokinase (PFK) from Bacillus stearothermophilus is proposed to be important in the allosteric transition of the enzyme [Schirmer, T., & Evans, P.R. (1990) Nature 343, 140-145]. Except for residue 161, the amino acids within the loop are similar between B. stearothermophilus PFK (BsPFK) and the PFK from Escherichia coli (EcPFK). In the former enzyme, residue 161 is a glutamate, while in the latter it is a glutamine. We have used site-directed mutagenesis to investigate the importance of residue 161 for the allosteric regulation of the two enzymes by phosphoenolpyruvate (PEP), an inhibitor, and GDP, an activator. In BsPFK, glutamate 161 has been changed to a glutamine and an alanine, while in EcPFK, glutamine 161 has been changed to a glutamate, an arginine, and an alanine. The kinetic parameters of the mutant enzymes were similar to those of the respective wild types, indicating that residue 161 is not directly involved in substrate binding and catalysis. One of the EcPFK mutants, Q161A, though activated normally by GDP, was completely insensitive to PEP. This indicates that the hydrogen-bonding ability of residue 161 is critical for PEP inhibition of EcPFK and suggests that GDP activation and PEP inhibition follow different structural pathways in EcPFK. The BsPFK mutant enzymes were less sensitive to PEP inhibition and more sensitive to GDP activation, suggesting that inhibition and activation are opposed and follow a common structural pathway in agreement with a concerted allosteric mechanism.