The use of 8-azido-ATP and 8-azido-ADP as photoaffinity labels of the ATP synthase in submitochondrial particles: evidence for a mechanism of ATP hydrolysis involving two independent catalytic sites?

8-Azido-ATP is a substrate for the ATP synthase in submitochondrial particles with a Vmax equal to 6% of the Vmax with ATP. The Km values for 8-azido-ATP are similar to those for ATP. ATP synthase in submitochondrial particles can bind maximally 2 mol 8-N-ATP or 8-N-ADP per mole and the inhibition of ATP hydrolysis by covalently bound N-ATP or N-ADP is proportional to the saturation of the enzyme with inhibitor, similar to the results obtained with isolated F1. Both 8-N-ATP and 8-N-ADP are bound mainly to the beta subunits and at all levels of saturation the distribution of the label is 77% to the beta and 23% to the alpha subunits. It is proposed that the binding of 8-azido-AXP itself is mainly to the beta subunit, but that part of the nitreno radicals formed during excitation with light reacts with an amino acid of the alpha subunit, due to the location of the binding site at an interface between a beta and an alpha subunit. Partial saturation with 8-N-ATP, under conditions that the concentration of 8-azido-ATP during the incubation is intermediate between the low and high Km values, does not abolish the apparent negative cooperativity of ATP hydrolysis. It is concluded that this apparent cooperativity is not due to the presence of two different catalytic sites, nor to a cooperativity between the two catalytic sites, but to interaction between the catalytic sites and regulatory sites.     

Regulation of ATP hydrolysis by the ε subunit, ζ subunit and Mg-ADP in the ATP synthase of Paracoccus denitrificans

F₁F_O-ATP synthase is a crucial metabolic enzyme that uses the proton motive force from respiration to regenerate ATP. For maximum thermodynamic efficiency ATP synthesis should be fully reversible, but the enzyme from Paracoccus denitrificans catalyzes ATP hydrolysis at far lower rates than it catalyzes ATP synthesis, an effect often attributed to its unique ζ subunit. Recently, we showed that deleting ζ increases hydrolysis only marginally, indicating that other common inhibitory mechanisms such as inhibition by the C-terminal domain of the ε subunit (ε-CTD) or Mg-ADP may be more important. Here, we created mutants lacking the ε-CTD, and double mutants lacking both the ε-CTD and ζ subunit. No substantial activation of ATP hydrolysis was observed in any of these strains. Instead, hydrolysis in even the double mutant strains could only be activated by oxyanions, the detergent lauryldimethylamine oxide, or a proton motive force, which are all considered to release Mg-ADP inhibition. Our results establish that P. denitrificans ATP synthase is regulated by a combination of the ε and ζ subunits and Mg-ADP inhibition.     

Mitochondrial ATP synthase catalytic mechanism: a novel visual comparative structural approach emphasizes pivotal roles for Mg²⁺ and P-loop residues in making ATP

The mitochondrial ATP synthase (F(o)F(1)) is one of the most abundant, important, and complex enzymes found in animals and humans. In earlier studies, we used the photosensitive phosphate analogue vanadate (V(i)) to study the enzyme's mechanism in the transition state. Significantly, these studies showed that Mg(2+) plays an important role in transition state formation during ATP synthesis. Additionally, in both MgADP·V(i)-F(1) and MgV(i)-F(1) complexes, photoactivation of orthovanadate (V(i)) induced cleavage at the third residue within the P-loop (GGAGVGKT), i.e., βA158, suggesting its proximity to the γ-phosphate during transition state formation. However, despite our recent release of the F(1)-ATPase structure containing V(i), the structural details regarding the role of Mg(2+) have remained elusive. Therefore, in this study, we sought to improve our understanding of the essential role of Mg(2+) during transition state formation. We utilized Protein Data Bank structural data representing different conformational intermediates of key steps in ATP synthesis to assemble a database of positional relationships between landmark residues of the catalytic site and the bound ligand. Applying novel bioinformatics methods, we combined the resulting interatomic spatial data with an animated model of the catalytic site to visualize the exact nature of the changes in these positional relationships during ATP synthesis. The results of these studies reported here show that the absence of Mg(2+) results in migration of inorganic phosphate (P(i)) from βA158 to a more medial position in the P-loop binding pocket, thereby disrupting essential placement and orientation of the P(i) needed to form the transition state structure and therefore MgATP.     

The β-glucuronidase catalyzed hydrolysis of a glucopyranosiduronamide and a glucopyranoside: Evidence for the oxocarbonium ion mechanism for bovine liver β-glucuronidase

Bovine β-glucuronidase (EC 3.2.1.31) catalyzes hydrolysis of ammonium 1-deoxy-1-(6-thiopurinyl)-β-D-glucopyranosiduronate (I), 1-deoxy-1-(6-thiopurinyl)-β-D-glucopyranosiduronamide (II), and 1-deoxy-1-(6-thiopurinyl)-β-D-glucopyranoside (III) to 6-mercaptopurine and the corresponding glucopyranose. Plots of log V_max and log V_max/Km $\text{vs.}\text{σ}{}_{\mathrm{I}}$, the comparative electronic substituent constant for -CO₂^?, -CONH₂, and -CH₂OH, gave slopes ?_I = ?5.1 (r=0.971) and ?_I = ?8.1 (r=0.998) respectively. These data, taken together with literature data, are interpreted to mean that the critical transition state has appreciable oxocarbonium ion character and that this transition state is primarily stabilized by the 6-carboxylate ion of the enzyme-bound substrate.     

Structure of theEscherichia coli ATP synthase and role of the γ and ε subunits in coupling catalytic site and proton channeling functions

The structure of theEscherichia coli ATP synthase has been studied by electron microscopy and a model developed in which the and subunits of the F₁ part are arranged hexagonally (in top view) alternating with one another and surrounding a central cavity of around 35 Å at its widest point. The and subunits are interdigitated in side view for around 60 Å of the 90 Å length of the molecule. The F₁ narrows and has three-fold symmetry at the end furthest from the F₀ part. The F₁ is linked to F₀ by a stalk approximately 45 Å long and 25–30 Å in diameter. The F₀ part is mostly buried in the lipid bilayer. The subunit provides a domain that extends into the central cavity of the F₁ part. The and subunits are in a different conformation when ATP+Mg²⁺ are present in catalytic sites than when ATP+EDTA are present. This is consistent with these two small subunits switching conformations as a function of whether or not phosphate is bound to the enzyme at the position of the phosphate of ATP. We suggest that this switching is the key to the coupling of catalytic site events with proton translocation in the F₀ part of the complex.     

New insights into the role of mast cells in autoimmunity: Evidence for a common mechanism of action?

Mast cells are classically considered innate immune cells that act as first responders in many microbial infections and have long been appreciated as potent contributors to allergic reactions. However, recent advances in the realm of autoimmunity have made it clear that these cells are also involved in the pathogenic responses that exacerbate disease. In the murine models of multiple sclerosis, rheumatoid arthritis and bullous pemphigoid, both the pathogenic role of mast cells and some of their mechanisms of action are shared. Similar to their role in infection and a subset of allergic responses, mast cells are required for the efficient recruitment of neutrophils to sites of inflammation. Although this mast cell-dependent neutrophil response is protective in infection settings, it is postulated that neutrophils promote local vascular permeability and facilitate the entry of inflammatory cells that enhance tissue destruction at target sites. However, there is still much to learn. There is little information regarding mechanisms of mast cell activation in disease. Nor is it known how many mast cell-derived mediators are relevant and whether interactions with other cells are implicated in these diseases including T cells, B cells and astrocytes. Here we review the current state of knowledge about mast cells in autoimmune disease. We also discuss findings regarding newly discovered mast cell actions and factors that modulate mast cell function. We speculate that much of this new information will ultimately contribute to a greater understanding of the full range of mast cell actions in autoimmunity. This article is part of a Special Issue entitled: Mast cells in inflammation.     

The rate of ATP synthesis as a function of ΔpH in normal and dithiothreitol-modified chloroplasts

The rate of ATP synthesis catalyzed by normal and by dithiothreitol-modified ATPases is investigated as a function of ΔpH in spinach chloroplasts at constant pH_out. The transmembrane ΔpH was generated by an acid-base transition and the reaction time was limited to 150 ms by using a rapidly mixing quenched-flow apparatus. The result was that the functional dependence of the rate on ΔpH is shifted to lower ΔpH values and that the shape of this curve is altered after dithiothreitol modification. The maximal rate (400 ATP / CF₁ per s) is the same under both conditions.     

Regional distribution of adenosine uptake in guinea-pig brain slices and the effect of some inhibitors: Evidence for nitrobenzylthioinosine-sensitive and insensitive sites?

The accumulation of [2-³H]adenosine was measured in slices prepared from 7 regions of the guinea-pig central nervous system. There was a similar level of uptake in forebrain regions (cerebral cortex, striatum, hippocampus and midbrain), a lower level in the cerebellum, with lowest uptake in the pons-medulla and spinal cord. Uptake in all regions was strongly inhibited by the nucleoside transport inhibitor dipyridamole and by 5-iodotubercidin, an adenosine kinase inhibitor. The activity of adenosine kinase was similar in crude supernatants prepared from 8 regions of the guinea-pig and rat brain, with the exception of the spinal cord (lower activity than other regions in the guinea-pig CNS) and olfactory bulb (higher activity than other regions in the rat CNS). 5-Nitrobenzylthioinosine (NBMPR) and related thiopurines produced about 50% inhibition of adenosine uptake into guinea-pig cerebral cortex slices at 200 nM but increasing the concentration did not produce significant further inhibition. [³H]NBMPR has been proposed as a useful tight-binding ligand for nucleoside transport sites in various tissues but it is suggested that the distribution of such binding sites in different regions of the CNS may not directly reflect the adenosine uptake capacity of these regions1. Data suggest that there may be NBMPR-sensitive and -insensitive sites. Results confirm those of previous studies which suggest that intracellular adenosine kinase plays an important part in the uptake of adenosine in guinea-pig brain. The relatively homogeneous distribution of adenosine uptake activity in the brain contrasts with the heterogeneous distribution of A1-adenosine receptors in the CNS.     

Anticoagulation and sepsis: the opportunity for a new use of heparin?

Sepsis is a leading cause of morbidity and mortality worldwide, and the magnitude of the problem seems higher in developing countries. In the last two decades the accepted standard treatment has resulted in only a slight decrease in mortality, and that decrease has been overshadowed by an almost 300% increase in incidence. Recently has been documented the close relationship between infection, inflammation and coagulation in sepsis has been documented; and although clinically overt disseminated intravascular coagulation may occur in only 30% to 50% of septic patients, the activation of the coagulation cascade is an early and common response to the infectious challenge. Moreover most of the molecules involved in the pro-coagulant state that characterizes sepsis are also powerful generators or amplifiers of the inflammatory response. These findings have fostered a comprehensive body of research regarding biological products with anticoagulant activity, as additional therapies for patients with the most severe states of the sepsis syndrome. This review explains the biological and molecular aspects that support the potential use of anticoagulant treatments in sepsis. Furthermore, we analyze the evidence provided by experimental and pre-clinical studies, which suggest the usefulness of heparin as an effective complementary treatment throughout the clinical stages of the disease.     

The use of AlbuMAX II(®) as a blood or serum alternative for the culture of Helicobacter pylori

Background: Growth of Helicobacter pyloriin vitro depends on supplementation of the medium with blood or serum. However, these supplements often require frozen storage and can show batch‐to‐batch variation, resulting in differences in bacterial growth. In this study, we introduce the use of a commercially available, lipid‐rich supplement called AlbuMAX II^® (Gibco BRL, Grand Island, NY, USA) for use as a serum/blood replacement for H. pylori culture. Materials and Methods: The growth of H. pylori on solid and liquid media was examined by comparing growth after supplementation with horse blood, fetal calf serum, β‐cyclodextrin or AlbuMAX II^® (Gibco BRL). Human gastric adenocarcinoma (AGS) cellular responses to H. pylori were measured by NF‐κB luciferase assays and IL‐8 ELISA. Results: We show that the growth of H. pylori on both solid and liquid media containing AlbuMAX II^® (Gibco BRL) were comparable to levels obtained on blood agar or liquid media supplemented with serum. Growth was consistently higher in media supplemented with AlbuMAX II^® (Gibco BRL) than media containing β‐cyclodextrin. Furthermore, bacteria grown in AlbuMAX II^® (Gibco BRL) induced proinflammatory responses in AGS cells. Conclusions: AlbuMAX II^® (Gibco BRL) can be used as a serum/blood replacement for the cultivation of H. pylori in solid and liquid media. This medium could be useful for an improved understanding of H. pylori metabolism or for antigen production. Furthermore, AlbuMAX II^® (Gibco BRL) may be suitable for use in remote locations, particularly in areas where frozen storage of serum may be a problem.     

The uptake of Aspergillus ochraceus spores on diatomaceous particles and their use in the 11α-hydroxylation of progesterone

Summary The entrapment of Aspergillus ochraceus spores on to diatomaceous earth particles occurs rapidly, the number of spores entrapped at equilibrium being dependent upon the initial spore:particle ratio. The rate of agitation during spore uptake markedly affected entrapment. Immobilized spores carried out the 11-hydroxylation of progesterone as effectively as free spores.     

Galactosyltransferase activities in subcellular fractions of two YC8 lymphoma cell variants: does a relationship exist between antigenic determinants and acceptor sites for galactose?

Experiments have been undertaken to correlate physiological changes, observed in two YC8 cells variants (P and L) and some of their immunological and enzymatic properties. These cell lines show different responses towards antilymphocyte and anti-Moloney sera. Subcellular fractionations have been made. The A fractions (d: 1.14/1.16) have the highest ouabain-inhibited Mg²⁺-stimulated (Na⁺-K⁺)-dependent ATPase and galactosyltransferase activities. Some properties of the latter enzyme have been studied: whereas optima pH and requirements for Mn²⁺ ions have been found to be the same for both cell line enzymes, on the contrary, different kinetic parameters have been shown with respect to sugar donor (UDP-galactose) on endogeneous or exogeneous (ovomucoid) acceptors. Apparent Km for UDP-galactose is 1.7 × 10⁻⁶ M (P-cells) and 3.3 × 10⁻⁶ M (L-cells), on endogeneous acceptors, and P-cell V max < L-cell V max; on ovomucoid it is 0.61 × 10⁻⁶ M, for both cell lines. These results suggest the presence on L-cells of more endogeneous acceptor sites, the higher affinity of P-cells for UDP-galactose being balanced by less endogeneous acceptor sites for galactose. When ovomucoid is added, galactose transfer on endogeneous acceptor sites of both cells is negligible. Apparent Km for ovomucoid is 8.6 × 10⁻⁵ M (P-cells) and 4.3 × 10⁻⁵ M (L-cells). These data support the above-mentioned hypothesis: L-cell enzymes would be more rapidly saturated than P-cell enzymes because of the higher number of endogeneous sites on L-cells.This supposed acquired character of L-cells as well as their immunological behaviour could explain the modified properties of L-cells as compared to P-cells.     

The use of “second rate” raw materials during Middle Palaeolithic. Technological and functional analysis of two sites in north-eastern Iberia

In recent years many issues arose regarding the technological and functional analysis of lithic assemblages dominated by “second rate raw materials” like vein quartz, metamorphic or volcanic rocks. Nevertheless, the technological study of this kind of assemblages and their consequent interpretation seems to be still based on the techno-typological criteria elaborated for flint artefacts, thus leading to the common inference that these lithic assemblages are archaic and mainly linked to opportunistic production systems. Pedra Dreta and Can Garriga, located in the north-east of the Iberian Peninsula, present two small lithic assemblages dominated by local raw materials (vein quartz, quartzite, porphyry, syenite, etc.) and by opportunistic reduction sequences but with the presence of Levallois and discoid châine operatoires. The technological analysis is aimed to answer two main issues that emerged during the study and that can contribute to the study of non-flint lithic assemblages: 1. Which criteria should we adopt to distinguish between discoid and recurrent centripetal Levallois products? 2. Is it possible that the Levallois products are the result rather of the opportunistic and expedient reduction sequences that are dominant in these assemblages than of a real use of the Levallois method? A use-wear analysis has also been conducted for the first time on Pedra Dreta and Can Garriga lithic assemblages. The peculiarities of the lithic raw materials employed made necessary the adoption of specific methodologies and the realization of a particular procedure for the analysis of porphyry's tools.     

Modulation of nucleotide binding to the catalytic sites of thermophilic F1-ATPase by the ε subunit: Implication for the role of the ε subunit in ATP synthesis

Effect of ε subunit on the nucleotide binding to the catalytic sites of F₁-ATPase from the thermophilic Bacillus PS3 (TF₁) has been tested by using α₃β₃γ and α₃β₃γε complexes of TF₁ containing βTyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the ε subunit weakened ADP binding to each catalytic site, especially to the highest affinity site. This effect was also observed when GDP or IDP was used. The ratio of the affinity of the lowest to the highest nucleotide binding sites had changed two orders of magnitude by the ε subunit. The differences may relate to the energy required for the binding change in the ATP synthesis reaction and contribute to the efficient ATP synthesis.     

Urocortin: a mechanism for the sustained activation of the HPA axis in the late-gestation ovine fetus?

We hypothesized that urocortin might be produced in the pituitary of the late-gestation ovine fetus in a manner that could contribute to the regulation of ACTH output. We used in situ hybridization and immunohistochemistry to identify urocortin mRNA and protein in late-gestation fetal pituitary tissue. Levels of urocortin mRNA rose during late gestation and were associated temporally with rising concentrations of pituitary proopiomelanocortin (POMC) mRNA. Urocortin was localized both to cells expressing ACTH and to non-ACTH cells by use of dual immunofluorescence histochemistry. Transfection of pituitary cultures with urocortin antisense probe reduced ACTH output, whereas added urocortin stimulated ACTH output from cultured pituitary cells. Cortisol infusion for 96 h in chronically catheterized late-gestation fetal sheep significantly stimulated levels of pituitary urocortin mRNA. We conclude that urocortin is expressed in the ovine fetal pituitary and localizes with, and can stimulate output of, ACTH. Regulation of urocortin by cortisol suggests a mechanism to override negative feedback and sustain feedforward of fetal hypothalamic-pituitary-adrenal function, leading to birth.     

The preparation of a photoreactive analogue of 2′,3′-dideoxyuridine 5′-triphosphate and its use for photoaffinity modification of human replication protein A

A new reagent for photoaffinity modification of biopolymers, 5-[E-N-(2-nitro-5-azidobenzoyl)-3-amino-1-propen-1-yl]-2′,3′-dideoxyuridine 5′-triphosphate (NAB-ddUTP), was synthesized. Like a similar derivative of 2′-deoxyuridine 5′-triphosphate (NAB-dUTP), it was shown to be able to effectively substitute for dTTP in the synthesis of DNA catalyzed by eukaryotic DNA polymerase β and to terminate DNA synthesis. A 5′-³²P-labeled primer with a photoreactive group at the 3′-terminus was derived from NAB-ddUTP and used for photoaffinity labeling of the human replication protein A (RPA). The covalent attachment of RPA p32 and p70 subunits to the labeled primers was demonstrated. NAB-ddUTP is a promising tool for studying the interaction of proteins of the replicative complex with NA in cellular extracts and living cells during the termination of DNA synthesis.