Does cobalt really bind tightly to hemocyanin active sites?

The analysis of Co(II)-apoHc complexes of two arthropodan species (freshwater crayfish): Orconectes limosus and Astacus astacus enabled to reach some conclusions about possible cobalt binding sites in the hemocyanin molecules. The occurrence of binding sites for Co(II) at sites other than the active center has been demonstrated. We excluded the possibility of strong binding of EDTA-non-removable cobalt ions in the binding sites occupied by copper. There were no differences between apoHc and the Co(II)apoHc complex in terms of the amount of bound Cu(I) ions and the kinetics of Cu(I) ion reconstitution.Abbreviations He hemocyanin - apoHc apohemocyanin - oxyHc oxyhemocyanin - Co-Hc hemocyanin complex with cobalt ions Offprint requests to: E. Serafln     

How does BAFF activate B cells in patients with autoimmune diseases?

In a study in a recent issue of Arthritis Research &; Therapy, Yoshimoto and colleagues demonstrate that peripheral monocytes from patients with Sjögren's syndrome (SS) produce significantly higher amounts of B cell-activating factor (BAFF) and interleukin-6 (IL-6) in comparison with normal monocytes. This difference exists at baseline and is amplified after stimulation with interferon-gamma. Increased IL-6 secretion is partially suppressed by an anti-BAFF antibody, suggesting that signal transduction pathways mediated by BAFF are implicated in the regulation of IL-6 production by monocytes. The origin and pathways involved in this higher susceptibility to BAFF-driven IL-6 induction by monocytes of patients with SS are still unknown.     

Does pyrophosphate bind to the catalytic sites of mitochondrial F1-ATPase?

The interactions between the pyrophosphate (PPi) binding sites and the nucleotide binding sites on mitochondrial F1-ATPase have been investigated, using F1 preparations containing different numbers of catalytic and noncatalytic nucleotide-binding sites occupied by ligands. In all cases, the total number of moles of bound nucleotides and PPi per mole of F1 was less than or equal to six. F1 preparations containing either three or two filled noncatalytic sites and no filled catalytic sites (referred as F1[3,0] and F1[2,0]) were found to bind 3 mol of PPi/mol of F1. Tight binding of ADP-fluoroberyllate complexes to two of the catalytic sites of F1 converted the three heterogeneous PPi-binding sites into three homogeneous binding sites, each exhibiting the same affinity for PPi. The addition of PPi at saturating concentrations to F1 containing GDP bound to two catalytic sites (F1[2,2]) resulted in the release of 1 mol of GDP. Furthermore, the addition of PPi to F1 filled with ADP-fluoroberyllate at the catalytic sites resulted in the release of 1 mol of tightly bound ADP/mol of F1. Taken together, these results indicate that PPi binds to specific sites that interact with both the catalytic and the noncatalytic nucleotide-binding sites of F1.     

Does actin bind to the ends of thin filaments in skeletal muscle?

We examined whether or not purified actin binds to the ends of thin filaments in rabbit skeletal myofibrils. Phase-contrast, fluorescence, and electron microscopic observations revealed that actin does not bind to the ends of thin filaments of intact myofibrils. However, in I-Z-I brushes prepared by dissolving thick filaments at high ionic strength, marked binding of actin to the free ends, i.e., the pointed ends, of thin filaments was observed when actin was added at an early phase of polymerization. As the polymerization of actin proceeded, the binding efficiency decreased. The critical actin concentration for this binding was higher than that for polymerization in solution. The binding of G-actin was not observed at low ionic strength. On the basis of these results, we suggest that a particular structure suppressing the binding of actin is present at the free ends of thin filaments in intact myofibrils and that a part of the end structure population is eliminated or modified at high ionic strength so that further binding of actin becomes possible. The myofibril and I-Z-I brush appear to be useful systems for studies aimed at elucidating the organizational mechanisms of actin filaments in vivo.     

Does unpaired adenosine-66 from helix II of Escherichia coli 5S RNA bind to protein L18?

Adenosine-66 is unpaired within helix II of Escherichia coli 5S RNA and lies in the binding site of ribosomal protein L18. It has been proposed as a recognition site for protein L18. We have investigated further the structural importance of this nucleotide by deleting it. The 5S RNA gene of the rrnB operon of E. coli was subjected to primer-directed mutagenesis. To produce the deletion it was necessary to use simultaneously the mutagenic dodecamer dCGGCGCACGGCG and the universal M13 primer dCCCAGTCACGACGTT, and to employ forced annealing conditions. The mutated gene was expressed in an overproducing plasmid derived from pKK3535. Binding studies with protein L18 revealed that the protein bound much more weakly to the mutated 5S RNA. We consider the most likely explanation of this result is that L18 interacts with adenosine-66, and we present a tentative model for an interaction between the unpaired adenosine and the adjacent guanosine-67 of the RNA and glutamine-19 of the protein L18.     

IgE binding to proteins from sesame and assessment of allergenicity: implications for biotechnology?

Successful prediction of the potential allergenicity of a protein may be a key factor in the development of novel, genetically modified foods. The use of the decision tree approach for the prediction of allergenicity is discussed. The methods currently used for identifying allergenic proteins (including use of IgE from patient sera for recognition of proteins) are reviewed. Finally, a specific review of the literature concerning identification of allergens from sesame leads to the conclusion that in the absence of validated animal models, identification of allergenicity (and, consequently, prediction of allergenicity) may be problematic.     

Barley γ3-hordein: Glycosylation at an atypical site,disulfide bridge analysis,and reactivity with IgE from patients allergic to wheat

Post translational modifications of a seed storage protein, barley γ3-hordein, were determined using immunochemical and mass spectrometry methods. IgE reactivity towards this protein was measured using sera from patients diagnosed with allergies to wheat. N-glycosylation was found at an atypical Asn-Leu-Cys site. The observed glycan contains xylose. This indicates that at least some γ3-hordein molecules trafficked through the Golgi apparatus. Disulfide bridges in native γ3-hordein were almost the same as those found in wheat γ46-gliadin, except the bridge involving the cysteine included in the glycosylation site. IgE reacted more strongly towards the recombinant than the natural γ3-hordein protein. IgE binding to γ3-hordein increased when the protein sample was reduced. Glycosylation and disulfide bridges therefore decrease epitope accessibility. Thus the IgE from patients sensitized to wheat cross-react with γ3-hordein due to sequence homology with wheat allergens rather than through shared carbohydrate determinants.     

How to regulate a gene: to repress or to activate?

Gene-expression responses to an input can depend on growth conditions; in this issue, Sasson et al. (2012) show that this dependence is lower when the input results in a high degree of promoter occupancy.     

Mammalian ABCG-transporters,sterols and lipids: To bind perchance to transport?

Members of the ATP binding cassette (ABC) transporter family perform a critical function in maintaining lipid homeostasis in cells as well as the transport of drugs. In this review, we provide an update on the ABCG-transporter subfamily member proteins, which include the homodimers ABCG1, ABCG2 and ABCG4 as well as the heterodimeric complex formed between ABCG5 and ABCG8. This review focusses on progress made in this field of research with respect to their function in health and disease and the recognised transporter substrates. We also provide an update on post-translational regulation, including by transporter substrates, and well as the involvement of microRNA as regulators of transporter expression and activity. In addition, we describe progress made in identifying structural elements that have been recognised as important for transport activity. We furthermore discuss the role of lipids such as cholesterol on the transport function of ABCG2, traditionally thought of as a drug transporter, and provide a model of potential cholesterol binding sites for ABCG2.     

Is it ethical for patients with renal disease to purchase kidneys from the world's poor?

BACKGROUND TO THE DEBATE: In many countries, the number of patients waiting for a kidney transplant is increasing. But there is a widespread and serious shortage of kidneys for transplantation, a shortage that can lead to suffering and death. One approach to tackling the shortage is for a patient with renal disease to buy a kidney from a living donor, who is often in a developing country, a sale that could--in theory at least--help to lift the donor out of poverty. Such kidney sales are almost universally illegal. Proponents of kidney sales argue that since the practice is widespread, it would be safer to formally regulate it, and that society should respect people's autonomous control over their bodies. Critics express concern about the potential for exploitation and coercion of the poor, and about the psychological and physical after-effects on the donors of this illegal kidney trade.     

Mechanism of adenylate kinase. Does adenosine 5'-triphosphate bind to the adenosine 5'-monophosphate site?

Although the substrate binding properties of adenylate kinase (AK) have been studied extensively by various biochemical and biophysical techniques, it remains controversial whether uncomplexed adenosine 5'-triphosphate (ATP) binds to the adenosine 5'-monophosphate (AMP) site of AK. We present two sets of experiments which argue against binding of ATP to the AMP site. (a) 31P nuclear magnetic resonance titration of ATP with AK indicated a 1:1 stoichiometry on the basis of changes in coupling constants and line widths. This ruled out binding of ATP to both sites. (b) ATP and MgATP were found to behave similarly by protecting AK from spontaneous inactivation while AMP showed only a small degree of protection. Such inactivation could also be protected or reversed by dithioerythritol and is most likely due to oxidation of sulfhydryl groups, one of which (cysteine-25) is located near the MgATP site. The results support binding of ATP to the MgATP site predominantly, instead of the AMP site, in the absence of Mg2+.     

Cell wall growth during elongation and division: one ring to bind them?

The role of the cell division protein FtsZ in bacterial cell wall (CW) synthesis is believed to be restricted to localizing proteins involved in the synthesis of the septal wall. In this issue of Molecular Microbiology, the groups of Christine Jacobs-Wagner and Waldemar Vollmer provide compelling evidence that in Caulobacter crescentus, FtsZ plays an additional role in CW synthesis in non-dividing cells. During elongation (cell growth) FtsZ is responsible for the incorporation of CW material in a zone at the midcell by recruiting MurG, a protein involved in peptidoglycan (PG) precursor synthesis. This resembles earlier findings of FtsZ mediated PG synthesis activity in Escherichia coli. A role of FtsZ in PG synthesis during elongation forces a rethink of the current model of CW synthesis in rod-shaped bacteria.