Search

CcmK proteins are major constituents of icosahedral shells of β-carboxysomes, a bacterial microcompartment that plays a key role for CO2 fixation in nature. Supported by the characterization of bidimensional (2D) layers of packed CcmK hexamers in crystal and electron microscopy structures, CcmK are assumed to be the major components of icosahedral flat facets. Here, we reassessed the validity of this model by studying CcmK isoforms from Synechocystis sp. PCC6803. Native mass spectrometry studies confirmed that CcmK are hexamers in solution. Interestingly, potential pre-assembled intermediates were also detected with CcmK2. Atomic-force microscopy (AFM) imaging under quasi-physiological conditions confirmed the formation of canonical flat sheets with CcmK4. Conversely, CcmK2 formed both canonical and striped-patterned patches, while CcmK1 assembled into remarkable supra-hexameric curved honeycomb-like mosaics. Mutational studies ascribed the propensity of CcmK1 to form round assemblies to a combination of two features shared by at least one CcmK isoform in most β-cyanobacteria: a displacement of an α helical portion towards the hexamer edge, where a potential phosphate binding funnel forms between packed hexamers, and the presence of a short C-terminal extension in CcmK1. All-atom molecular dynamics supported a contribution of phosphate molecules sandwiched between hexamers to bend CcmK1 assemblies. Formation of supra-hexameric curved structures could be reproduced in coarse-grained simulations, provided that adhesion forces to the support were weak. Apart from uncovering unprecedented CcmK self-assembly features, our data suggest the possibility that transitions between curved and flat assemblies, following cargo maturation, could be important for the biogenesis of β-carboxysomes, possibly also of other BMC., PLoS ONE, 12 (9), ISSN:1932-6203

International audience; Archaeal initiation factor 2 (aIF2) is a protein involved in the initiation of protein biosynthesis. In its GTP-bound, "ON" conformation, aIF2 binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, "OFF" conformation, it dissociates from tRNA. To understand the specific binding of GTP and GDP and its dependence on the ON or OFF conformational state of aIF2, molecular dynamics free energy simulations (MDFE) are a tool of choice. However, the validity of the computed free energies depends on the simulation model, including the force field and the boundary conditions, and on the extent of conformational sampling in the simulations. aIF2 and other GTPases present specific difficulties; in particular, the nucleotide ligand coordinates a divalent Mg(2+) ion, which can polarize the electronic distribution of its environment. Thus, a force field with an explicit treatment of electronic polarizability could be necessary, rather than a simpler, fixed charge force field. Here, we begin by comparing a fixed charge force field to quantum chemical calculations and experiment for Mg(2+):phosphate binding in solution, with the force field giving large errors. Next, we consider GTP and GDP bound to aIF2 and we compare two fixed charge force fields to the recent, polarizable, AMOEBA force field, extended here in a simple, approximate manner to include GTP. We focus on a quantity that approximates the free energy to change GTP into GDP. Despite the errors seen for Mg(2+):phosphate binding in solution, we observe a substantial cancellation of errors when we compare the free energy change in the protein to that in solution, or when we compare the protein ON and OFF states. Finally, we have used the fixed charge force field to perform MDFE simulations and alchemically transform GTP into GDP in the protein and in solution. With a total of about 200 ns of molecular dynamics, we obtain good convergence and a reasonable statistical uncertainty, comparable to the force field uncertainty, and somewhat lower than the predicted GTP/GDP binding free energy differences. The sign and magnitudes of the differences can thus be interpreted at a semiquantitative level, and are found to be consistent with the experimental binding preferences of ON- and OFF-aIF2.

Equipe AFEF ‘Architecture et Fonctionnement des Espèces fruitières’ ‘Team AFFS Architecture and Functioning of Fruit Species’ Contact: tjchiou@gate.sinica.edu.tw, manuel.echeverria@univ-perp.fr; International audience; Here we report on the characterization of rice osa-miR827 and its two target genes, OsSPX-MFS1 and OsSPX-MFS2, which encode SPX-MFS proteins predicted to be implicated in phosphate (Pi) sensing or transport. We first show by Northern blot analysis that osa-miR827 is strongly induced by Pi starvation in both shoots and roots. Hybridization of osa-miR827 in situ confirms its strong induction by Pi starvation, with signals concentrated in mesophyll, epidermis and ground tissues of roots. In parallel, we analyzed the responses of the two OsSPX-MFS1 and OsSPX-MFS2 gene targets to Pi starvation. OsSPX-MFS1 mRNA is mainly expressed in shoots under sufficient Pi supply while its expression is reduced on Pi starvation, revealing a direct relationship between induction of osa-miR827 and down-regulation of OsSPX-MFS1. In contrast, OsSPX-MFS2 responds in a diametrically opposed manner to Pi starvation. The accumulation of OsSPX-MFS2 mRNA is dramatically enhanced under Pi starvation, suggesting the involvement of complex regulation of osa-miR827 and its two target genes. We further produced transgenic rice lines overexpressing osa-miR827 and T-DNA knockout mutant lines in which the expression of osa-miR827 is abolished. Compared with wild-type controls, both target mRNAs exhibit similar changes, their expression being reduced and increased in overexpressing and knockout lines, respectively. This suggests that OsSPX-MFS1 and OsSPX-MFS2 are both negatively regulated by osa-miR827 abundance although they respond differently to external Pi conditions. We propose that this is a complex mechanism comprising fine tuning of spatial or temporal regulation of both targets by osa-miR827.

International audience; Bordetella pertussis endotoxin is a key modulator of the host immune response, mainly due to the role of its lipid A moiety in Toll-like receptor 4 (TLR4)-mediated signaling. We have previously demonstrated that the lipid A phosphate groups of B. pertussis BP338 can be substituted with glucosamine in a BvgAS-regulated manner. Here we examined the effect of this lipid A modification on the biological activity of B. pertussis endotoxin. We compared purified endotoxin and heat-killed B. pertussis BP338 whole cells that have modified lipid A phosphate groups to an isogenic mutant lacking this modification with respect to their capacities to induce the release of inflammatory cytokines by human and murine macrophages and to participate in the TLR4-mediated activation of NF-kappaB in transfected HEK-293 cells. We found inactivated B. pertussis cells to be stronger inducers of proinflammatory cytokines in THP-1-derived macrophages when lipid A was modified. Most notably, lack of lipid A modification abolished the ability of purified B. pertussis endotoxin to induce the release of inflammatory cytokines by human THP-1-derived macrophages but led to only slightly reduced inflammatory cytokine levels when stimulating murine (RAW 264.7) macrophages. Accordingly, upon stimulation of HEK-293 cells with inactivated bacteria and purified endotoxin, lack of lipid A modification led to impaired NF-kappaB activation only when human, and not when murine, TLR4-MD-2-CD14 was expressed. We speculate that in B. pertussis, lipid A modification has evolved to benefit the bacteria during human infection by modulating immune defenses rather than to evade innate immune recognition.

Adresse actuelle: I.M. van Aarle Unité de Microbiologie Université catholique de Louvain Place croix du Sud, 3, 1348 Louvain-la-Neuve Belgique; Expression of a mycorrhizal fungal-specific phosphate (P) transporter gene (HcPT1) was studied in mycelium of the ectomycorrhizal fungus Hebeloma cylindrosporum, by in situ reverse transcriptase polymerase chain reaction using amplification of complementary DNA sequences. The expression of HcPT1 was visualised under two different P treatments. Mycelium was transferred to liquid medium with or without P and incubated for 5 days. Under P starvation, mycelium growth and vitality was reduced and the expression of HcPT1 up regulated. Enzyme-labelled fluorescent substrate was used to detect gene expression in situ with epi-fluorescence microscopy and to visualise it at the level of the individual hyphae both in starved and non-starved hyphae. Up-regulation of HcPT1 was observed as a more intense fluorescent signal and from the larger proportion of hyphae that showed expression.

Context: Hyperostosis-hyperphosphatemia syndrome (HHS) is a rare metabolic disorder characterized by hyperphosphatemia and localized hyperostosis. HHS is caused by mutations in GALNT3, which encodes UDP-N-acetyl-α-d-galactosamine:polypeptide N- acetylgalactosaminyltransferase 3. Familial tumoral calcinosis (TC), characterized by ectopic calcifications and hyperphosphatemia, is caused by mutations in the GALNT3 or fibroblast growth factor 23 (FGF23) genes. Objective: Our objective was to identify mutations in FGF23 or GALNT3 and determine serum FGF23 levels in an HHS patient. Design: Mutation detection in FGF23 and GALNT3 was performed by DNA sequencing, and serum FGF23 concentrations were measured by ELISA. Patients or Other Participants: A 5-year-old French boy with HHS and his family members participated. Results: The patient presented with painful cortical lesions in his leg. Radiographs of the affected bone showed diaphyseal hyperostosis. The lesional tissue comprised trabeculae of immature, woven bone surrounded by fibrous tissue. Biochemistry revealed elevated phosphate, tubular maximum rate for phosphate reabsorption per deciliter of glomerular filtrate, and 1,25-dihydroxyvitamin D levels. The patient was a compound heterozygote for two novel GALNT3 mutations. His parents and brother were heterozygous for one of the mutations and had no biochemical abnormalities. Intact FGF23 level in the patient was low normal, whereas C-terminal FGF23 was elevated, a pattern similar to TC. Conclusion: The presence of GALNT3 mutations and elevated C-terminal, but low intact serum FGF23, levels in HHS resemble those seen in TC, suggesting that HHS and TC are different manifestations of the same disorder. The absence of biochemical abnormalities in the heterozygous individuals suggests that one normal allele is sufficient for secretion of intact FGF23.

Whereas increasing evidences suggest that inorganic phosphate (Pi) may act as a signaling molecule in mineralization-competent cells, its mechanisms of action remain largely unknown. The aims of the present work were to determine whether Pi regulates expression of matrix Gla protein (MGP), a mineralization inhibitor, in growth plate chondrocytes and to identify the involved signaling pathways. Chondrogenic ATDC5 cells and primary growth plate chondrocytes were used. Messenger RNA analysis was performed by RT-PCR and real-time quantitative PCR. Activation and role of mitogen-activated protein kinases (MAPK) were respectively determined by Western blotting and the use of specific inhibitors. Immunohistological detection of extracellular signal-regulated kinase 1 and 2 (ERK1/2) was performed in rib organ cultures from newborn mice. Results indicated that Pi markedly stimulated expression of MGP in ATDC5 cells and primary growth plate chondrocytes. Investigation of the involved intracellular signaling pathways revealed that Pi activated ERK1/2. The activation of ERK1/2 appeared cell-specific. Indeed, although Pi stimulated ERK1/2 in MC3T3-E1 osteoblasts and ST2 stromal cells, ERK1/2 phosphorylation could not be detected in L929 fibroblasts or C2C12 myogenic cells. Accordingly, immunohistological detection of ERK1/2 phosphorylation in rib growth plates revealed a marked signal in chondrocytes. Finally, a specific ERK1/2 inhibitor, UO126, blocked Pi-stimulated MGP expression in ATDC5 cells, indicating that ERK1/2 mediates, at least in part, the effects of Pi. These data demonstrate for the first time that Pi regulates MGP expression in growth plate chondrocytes, thereby suggesting a key role for Pi and ERK1/2 in the regulation of bone formation.

Whereas increasing evidence suggests that inorganic phosphate (Pi) may act as a signaling molecule in mineralization-competent cells, its mechanisms of action remain largely unknown. The aims of the present work were to determine whether Pi regulates expression of matrix Gla protein (MGP), a mineralization inhibitor, in growth plate chondrocytes and to identify the involved signaling pathways. Chondrogenic ATDC5 cells and primary growth plate chondrocytes were used. Messenger RNA and protein analyses were performed by quantitative PCR and Western blotting, respectively. The activation and role of MAPKs were, respectively, determined by Western blotting and the use of specific inhibitors. Immunohistological detection of ERK1/2 was performed in rib organ cultures from newborn mice. The results indicate that Pi markedly stimulates expression of MGP in ATDC5 cells and primary growth plate chondrocytes. Investigation of the involved intracellular signaling pathways reveals that Pi activates ERK1/2 in a cell-specific manner, because the stimulation was observed in ATDC5 and primary chondrocytes, MC3T3-E1 osteoblasts, and ST2 stromal cells, but not in L929 fibroblasts or C2C12 myogenic cells. Accordingly, immunohistological detection of ERK1/2 phosphorylation in rib growth plates revealed a marked signal in chondrocytes. Finally, a specific ERK1/2 inhibitor, UO126, blocks Pi-stimulated MGP expression in ATDC5 cells, indicating that ERK1/2 mediates, mainly, the effects of Pi. These data demonstrate, for the first time, that Pi regulates MGP expression in growth plate chondrocytes, thereby suggesting a key role for Pi and ERK1/2 in the regulation of bone formation.

The physiological role of proteins phosphorylated on serine/threonine/tyrosine (Ser/Thr/Tyr) residues or the identity of the corresponding kinases and phosphatases is generally poorly understood in bacteria. As a first step in analysing the importance of such phosphorylation, we sought to establish the nature of the Ser/Thr/Tyr phosphoproteome in Bacillus subtilis, using in vivo labelling with [(32)P]-orthophosphate, one-unit pH 2-DE, combined with MS. Highly reproducible 2-D profiles of phosphoproteins were obtained with early stationary-phase cells. The 2-D profiles contained at least 80 clearly labelled spots in the pH range 4-7. Forty-six spots were analysed by MS (confirmed in most cases by LC-MS/MS), identifying a total of 29 different proteins, with 19 identified for the first time as bacterial phosphoproteins. These phosphoproteins are implicated in a wide variety of cellular processes, including carbon and energy metabolism, transport, stress and development. Significant changes to the profiles were obtained as a result of cold, heat or osmotic shock, demonstrating that, in stationary-phase cells, the phosphoproteome is dynamic. An initial comparative study indicated that at least 25 [(32)P]-labelled spots were also stained by Pro-Q Diamond, with apparently six additional phosphoproteins uniquely detected by Pro-Q.

International audience; In Pseudomonas aeruginosa, rhamnolipid production is controlled by the quorum-sensing system RhlRI, which itself depends on LasRI. These systems use cell-to-cell signal molecules: N-butyryl-l-homoserine lactone (C(4)-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3OC(12)-HSL), respectively. Whereas both HSLs were produced in M63 medium, rhamnolipid synthesis was not achieved. Phosphate limitation reduced the HSL concentrations, while allowing rhamnolipid production. Hyperosmotic shock applied during the exponential growth phase stopped the accumulation of 3OC(12)-HSL, and prevented C(4)-HSL and rhamnolipid production. These defects result from lower expression of genes involved in C(4)-HSL and rhamnolipid syntheses. The osmoprotectant glycine betaine partially restored C(4)-HSL and rhamnolipid production

International audience; The proton-pumping and the ATP hydrolysis activities of the ATP synthase of Rhodobacter capsulatus have been compared as a function of the ADP and P(i) concentrations. The proton pumping was measured either with the transmembrane pH difference probe, 9-amino-6-chloro-2-methoxyacridine, or with the transmembrane electric potential difference probe, bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol, obtaining consistent results. The comparison indicates that an intrinsic uncoupling of ATP synthase is induced when the concentration of either ligand is decreased. The half-maximal effect was found in the submicromolar range for ADP and at about 70 microM for P(i). It is proposed that a switch from a partially uncoupled state of ATP synthase to the coupled state is induced by the simultaneous binding of ADP and P(i).

When Bacillus subtilis is subjected to phosphate starvation, the Pho regulon is activated by the PhoP-PhoR two-component signal transduction system to elicit specific responses to this nutrient limitation. The response regulator, PhoP, and its cognate histidine sensor kinase, PhoR, are encoded by the phoPR operon that is transcribed as a 2.7-kb bicistronic mRNA. The phoPR operon is transcribed from two σ A -dependent promoters, P 1 and P 2 . Under conditions where the Pho regulon was not induced (i.e., phosphate-replete conditions or phoR- null mutant), a low level of phoPR transcription was detected only from promoter P 1 . During phosphate starvation-induced transition from exponential to stationary phase, the expression of the phoPR operon was up-regulated in a phosphorylated PhoP (PhoP∼P)-dependent manner; in addition to P 1 , the P 2 promoter becomes active. In vitro gel shift assays and DNase I footprinting experiments showed that both PhoP and PhoP∼P could bind to the control region of the phoPR operon. The data indicate that while low-level constitutive expression of phoPR is required under phosphate-replete conditions for signal perception and transduction, autoinduction is required to provide sufficient PhoP∼P to induce other members of the Pho regulon. The extent to which promoters P 1 and P 2 are activated appears to be influenced by the presence of other sigma factors, possibly the result of sigma factor competition. For example, phoPR is hyperinduced in a sigB mutant and, later in stationary phase, in sigH , sigF , and sigE mutants. The data point to a complex regulatory network in which other stress responses and post-exponential-phase processes influence the expression of phoPR and, thereby, the magnitude of the Pho regulon response.

International audience; The X-ray structure of Mycobacterium tuberculosis TMP kinase at 1.95 A resolution is described as a binary complex with its natural substrate TMP. Its main features involve: (i) a clear magnesium-binding site; (ii) an alpha-helical conformation for the so-called LID region; and (iii) a high density of positive charges in the active site. There is a network of interactions involving highly conserved side-chains of the protein, the magnesium ion, a sulphate ion mimicking the beta phosphate group of ATP and the TMP molecule itself. All these interactions conspire in stabilizing what appears to be the closed form of the enzyme. A complete multialignment of all (32) known sequences of TMP kinases is presented. Subtle differences in the TMP binding site were noted, as compared to the Escherichia coli, yeast and human enzyme structures, which have been reported recently. These differences could be used to design specific inhibitors of this essential enzyme of nucleotide metabolism. Two cases of compensatory mutations were detected in the TMP binding site of eukaryotic and prokaryotic enzymes. In addition, an intriguing high value of the electric field is reported in the vicinity of the phosphate group of TMP and the putative binding site of the gamma phosphate group of ATP.

International audience; Polyunsaturated fatty acid (PUFA) deficiency affects respiratory rate both in isolated mitochondria and in hepatocytes, an effect that is normally ascribed to major changes in membrane composition causing, in turn, protonophoriclike effects. In this study, we have compared the properties of hepatocytes isolated from PUFA-deficient rats with those from control animals treated with concentrations of the protonophoric uncoupler 2,4-dinitrophenol (DNP). Despite identical respiratory rate and in situ mitochondrial membrane potential (delta psi), mitochondrial and cytosolic ATP/ADP-Pi ratios were significantly higher in PUFA-deficient cells than in control cells treated with DNP. We show that PUFA-deficient cells display an increase of phosphorylation efficiency, a higher mitochondrial ATP/ADP-Pi ratio being maintained despite the lower delta psi. This is achieved by (1) decreasing mitochondrial Pi accumulation, (2) increasing ATP synthase activity, and (3) by increasing the flux control coefficient of adenine nucleotide translocation. As a consequence, oxidative phosphorylation efficiency was only slightly affected in PUFA-deficient animals as compared to protonophoric uncoupling (DNP). Thus, the energy waste induced by PUFA deficiency on the processes that generate the proton motive force (pmf) is compensated in vivo by powerful adaptive mechanisms that act on the processes that use the pmf to synthesize ATP.

International audience; NF-kappaB is involved in the transcriptional regulation of a large number of genes, in particular those of human immunodeficiency virus (HIV). Recently, we used NMR spectroscopy and molecular modelling to study the solution structure of a native duplex related to the HIV-1 kappaB site, together with a mutated duplex for which a three base-pair change abolishes NF-kappaB binding. The native duplex shows unusual dynamics of the four steps surrounding the kappaB site. Here, we explore the intrinsic properties of the NMR-refined structures of both duplexes in order to understand why the native sequence is recognised by NF-kappaB among other DNA sequences. We establish that only the native kappaB site can adopt a conformation where its structure (curvature and base displacement), the accessibility and the electrostatic potentials of key atoms become very favourable for binding the large loops of NF-kappaB, in contrast to the mutated duplex. Finally, we show that the neutralisation of phosphate groups contacted by NF-kappaB favours a more canonical DNA structure. These findings lead to a new hypothesis for specific recognition through the phosphodiester backbone dynamics of the sequences flanking a binding site. Such unusual behaviour confers upon the overall duplex properties that can be used by NF-kappaB to select its binding site. Thus, the selectivity determinants for NF-kappaB binding appear to depend on deformability of an "extended" consensus sequence.

International audience; Carotid blood flow was measured in rats by implanted transit-time ultrasonic flowprobes during hyperbaric experiments at up to 70 bar (7 MPa) using an helium-oxygen hyperoxic (partial pressure of O2 = 400 mbar) mixture. Before the hyperbaric experiment, an intracerebroventricular injection of phosphate saline buffered solution (PBS) or aminophylline, an adenosine receptor blocker, in PBS was given. Throughout the hyperbaric experiment carotid blood flow increased with ambient pressure in both PBS, i.e. control, and aminophylline treated rats. The increase in carotid blood flow was significantly attenuated in aminophylline treated rats. Additional experiments showed that the increased carotid blood flow was independent of hyperoxia as well as of temperature. The hypothesis that the hyperbaric dependent increase in carotid blood flow was mediated by brain adenosine receptors and its implication regarding a cerebral vasodilatation are discussed.

International audience; We recently characterized the association of the 44-kDa mitogen-activated protein kinase, also known as extracellular-regulated kinase 1 (ERK1), with the 90-kDa ribosomal S6 kinase (pp90rsk), one of its putative substrates in intact PC12 cells. Using antibodies to ERK1 that precipitate a functional ERK1.pp90rsk phosphoprotein complex, we demonstrate here the regulation of both kinases by various stimuli. In mouse fibroblasts expressing human insulin receptors, insulin and vanadate swiftly stimulated ERK1 activity within 5 min. While the hormonal effect was short-lived, vanadate led to a first peak followed by a progressively increasing second phase. In PC12 cells, epidermal growth factor, which is a growth promoting factor, provokes a rapid but evanescent activation of ERK1. In contrast, nerve growth factor (NGF), which acts as a neuronal differentiation factor for PC12 cells, induced a swift monophasic response followed by a sustained second phase. This strikingly different pattern of ERK1 stimulation by NGF and epidermal growth factor was associated to a contrasting effect on ERK1 cellular translocation. Thus, NGF induced a nuclear translocation of ERK1, while epidermal growth factor was without noticeable effect on ERK1 localization. In both cell systems all effectors tested stimulated ERK1 phosphorylation on both threonine and tyrosine residues in an 1:1 ratio. During ERK1 inactivation, phosphothreonine and phosphotyrosine were dephosphorylated in a similar fashion. Concurrent with ERK1 activation was the de novo appearance of phosphothreonine and an increase in phosphoserine on pp90rsk. The pp90rsk phosphothreonine content paralleled the ERK1 activity more closely than the phosphoserine level. These results provide compelling evidence that in fibroblasts and PC12 cells ERK1 plays a direct role in the phosphorylation of pp90rsk and that pp90rsk represents a physiologically relevant substrate of extracellular-regulated kinases. Finally, we would like to suggest that the differentiating action of NGF in PC12 cells might be due, at least in part, to the conjunction of its sustained and robust stimulation of ERK1 and pp90rsk, and of its induction of ERK1 nuclear translocation.

International audience; After studying the effects of almitrine, a new kind of ATPase/ATP synthase inhibitor, on two kinds of isolated mammalian mitochondrion, we have observed that: (1) Almitrine inhibits oligomycin-sensitive ATPase; it decreases the ATP/O value of oxidative phosphorylations without any change in the magnitude of delta mu H+. (2) Almitrine increases the mechanistic H+/ATP stoichiometry of ATPase as shown by measuring either (i) the extent of potassium acetate and of potassium phosphate accumulation sustained by ATP utilisation, or (ii) the electrical charge/ATP (K+/ATP) ratio at steady-state of ATPase activity. (3) Rat liver mitochondria are at least 10-times more sensitive to almitrine than beef heart mitochondria. (4) The change in H+/ATP stoichiometry induced by almitrine depends on the magnitude of the flux through ATPase. The inhibitory effect of almitrine on ATPase/ATP synthase complex, as a consequence of such an H+/ATP stoichiometry change, is discussed.

International audience; We present here an improvement in the classical molybdate method for inorganic phosphate determination. This method has high sensitivity, with 1 nmol of Pi giving an absorbance change of 0.060 A850 unit. It is highly reproducible and the color remains stable for at least 3.5 h. In addition the use of sodium dodecyl sulfate makes it possible to stop enzymatic reactions without organic phosphate hydrolysis. It also shows an extremely low interference by highly concentrated solutions of different origins. Of special interest is its high tolerance to protein, permitting as much as 50 mg/ml of human serum protein in the sample without precipitation or color interference. For these reasons, it proves to be very useful in the determination of ATPases in tissues such as the ocular lens with low specific activity.

Source gallica.bnf.fr / Bibliothèque nationale de France; International audience; Some small peptides, ACTH sequences, are able to modify the 32P incorporation in brain proteins in vitro. The possibility that these peptides play a role in the regulation mechanism of some brain functions through differentiated protein-kinases could be considered.

Human lactotransferrin binds 2 Fe3+ tightly at two specific sites. In order to demonstrate differences between the stability of the two iron-binding sites, the removal of iron was studied in buffers in the pH range 8-3 varying the ionic strength and with or without metal chelators such as phosphate ions and EDTA. The results show that in the presence of formate and acetate buffers of ionic strength 0.1–0.4 and in a pH range of 5–3, the two Fe3+ from human lactotransferrin are removed stimultaneously. Addition of 4 mM EDTA to buffers of ionic strength 0.1 and in the pH range 8–3 shows that between pH 5–4.3 the iron from only one of the binding sites, called the ‘acid labile’ site, is removed. Addition of 0.2 M phosphate ions to buffers of ionic strength 0.2 and in pH range 8–3 containing 4 mM EDTA shows that Fe3+ from the ‘acid labile’ site may be completely removed at pH 6. Removal of Fe3+ from the ‘acid stable’ site is obtained at pH 4. The differential behavior of the two iron binding sites was also shown by saturation experiments in the presence of citrate/bicarbonate buffers at different pH values. In a pH range 6.2–4.8, 50% saturation was obtained, but at pH 6.35 complete saturation was achieved. When saturation of partially saturated samples of human lactotransferrin was performed with 59Fe it was demonstrated that in the pH range 6.2–4.8 iron is bound only to the ‘acid labile’ site.