The environment of the high-affinity cation binding site on actin and the separation between cation and ATP sites as revealed by proton NMR and fluorescence spectroscopy

The lanthanide ions Lu3+ (diamagnetic) and Gd3+ (paramagnetic broadening probe) were used to displace Ca2+ from the high-affinity cation binding site on G-actin. The effects of these higher-affinity ions on the proton nuclear magnetic resonance spectrum of actin were recorded. The aliphatic proton envelope in the Gd-actin sample exhibited a complex array of changes due to the proximity of Gd to several aliphatic residues. No such changes were observed in the diamagnetic Lu-actin control spectrum. By contrast, the aromatic proton envelope remained largely unaffected in both Gd-actin and Lu-actin samples. However, the adenosine moiety on the actin-bound ATP became increasingly mobilized without the triphosphate chain being released from the ATP binding site. Maximum adenosine mobilization occurred with approximately 1 mol of lanthanide ion bound per mol of actin. The absence of changes in the aromatic proton envelope suggests that the high-affinity cation binding site is in a region well removed from the adenosine moiety of bound ATP as well as any aromatic side-chains. The separation of the ATP and cation sites was further explored using the fluorescent ATP analogues FTP and epsilon-ATP. Tb3+ bound to the high-affinity cation site was found to be separated by 16 A from the FTP chromophore bound to the nucleotide binding site on actin. Since this distance is greater than can be accommodated on a model of the Tb-ATP complex, we conclude that the sites are physically separate. This conclusion was further reinforced by experiments involving the quenching of epsilon-ATP fluorescence by Mn2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actin dynamics studied by solid-state NMR spectroscopy

Solid-state nuclear magnetic resonance spectroscopy was used to study the motion of ²H and ¹⁹F probes attached to the skeletal muscle actin residues Cys-10, Lys-61 and Cys-374. The probe resonances were observed in dried and hydrated G-actin, F-actin and F-actin-myosin subfragment-1 complexes. Restricted motion was exhibited by ¹⁹F probes attached to Cys-10 and Cys-374 on actin. The dynamics of probes attached to dry cysteine powder or F-actin were very similar and the binding of myosin had little effect indicating that the local probe environment imposes the major influence on motion in the solid state. Correlation times determined for the solid state probes indicated that they were undergoing some rapid internal motion in both G-actin and F-actin such as domain twisting. The probe size influenced the motion in G-actin and appeared to sense monomer rotation but not in F-actin where segmental mobility and intramonomer co-ordination appeared to dominate.     

Irreversible inhibition of fatty acid synthase from rat mammary gland with S-(4-bromo-2,3-dioxobutyl)-CoA. Effect on the partial reactions, protection by substrates and stoichiometry studies.

Fatty acid synthase from lactating rat mammary gland is rapidly and irreversibly inhibited by S-(4-bromo-2,3-dioxobutyl)-CoA. Of the seven partial reactions catalysed by the enzyme, the inhibition of the overall catalytic activity is closely paralleled only by inhibition of the beta-oxoacyl synthase (condensing) partial reaction. Three partial reactions. Beta-oxoacyl reductase, beta-hydroxyacyl dehydratase and enoyl reductase, are inhibited to a modest degree. The three partial reactions known to involve an acyl-CoA/CoA-binding site, acetyl acyltransferase, malonyl acyltransferase and palmitoyl thioesterase, are not inhibited by S-(4-bromo-2,3-dioxobutyl)-CoA. The modification process does not cause the enzyme to dissociate into catalytically incompetent monomers. Stoichiometric studies suggest that approx. 6 mol of reagent are incorporated per mol of totally inhibited enzyme (dimer). The formation of acylated enzyme from either acetyl-CoA or malonyl-CoA protects the enzyme equally well against S-(4-bromo-2,3-dioxobutyl)-CoA. Also, pretreatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid), a thiol-specific reagent reported to block essential thiol groups in the condensing partial reaction, protects against inhibition by the reagent. On the other hand, the presence of up to 770 microM-S-acetonyl-CoA or dethio-CoA does not protect the enzyme from irreversible inhibition. Together, the results suggest that the primary inhibitory process is a bimolecular reaction resulting in alkylation of essential thiol groups in the condensing partial reaction: this process does not require the obligatory formation of a Michaelis-Menten complex of enzyme and reagent before the alkylation reaction.     

Lateral conductivity of lamellar mesophases in lipid--water systems.

The critical micelle concentrations of aqueous solutions of N^α-acyl-L-histidine have been determined by the spectral shift method with Rhodamine 6G and by the light scattering method. With the spectral shift method critical micelle concentrations of 40, 9.0, 1.0, 0.11, and 0.012 mM were obtained for N^α-acyl-L-histidine containing saturated acids of 8, 10, 12, 14, and 16 carbons respectively, at 45°C and pH 8.6 in the absence of added salt. For the homologs containing 10, 12, and 14 carbon acids, critical micelle concentration of 9.0, 1.0, and 0.11 mM were determined by the light scattering method.The light scattering studies yield micelle weights of 60, 66, and 84 thousand for the C-10, C-12, and C-14 homologs, respectively.N^α-acyl-L-histidine is an unusual surfactant in that the hydrophilic portion of the molecule is relatively large and contains both an ionic group (carboxylate group) and a nonionic group (imidazole side-chain). The bulky hydrophilic group of N^α-acyl-L-histidine causes this molecule to exhibit physico-chemical behavior which is not typical of that exhibited by most ionic surfactants. In particular, the dependence of the critical micelle concentration on the acyl chain length and on the concentration of added salt is atypical.Chemical shift measurements (by NMR) on the C-2 and C-5 protons of imidazole in micellar N^α-dodecanoyl-L-histidine indicate that the imidazole group is, indeed, positioned at the water-micelle interface.     

NMR of a synthetic peptide spanning the triphosphate binding site of adenosine 5'-triphosphate in actin

The amino acid residues 114-118 in actin were found to be implicated strongly in the binding of nucleotide, and as would be expected for such an important binding site, they are located in a completely conserved region of the actin sequence. A 19-residue peptide with the actin sequence 106-124 was synthesized in order to span the putative triphosphate binding site. Proton NMR spectra of the actin peptide 114-118 in the presence and absence of ATP indicated that Arg-116 and Lys-118 are particularly involved in binding ATP. A strong binding of ATP to the peptide 106-124 also was measured. Tripolyphosphate bound to the peptide 106-124 somewhat more weakly than ATP. Binding involved residues 115-118 and 121-124, indicating the presence of a reverse turn between these segments. Proton resonances were assigned by using two-dimensional double quantum correlated spectroscopy, one-dimensional spin decoupling techniques, one-dimensional nuclear Overhauser enhancement difference spectroscopy, and pH titration. The alpha CH resonances of Ala-3 and Asn-6 are markedly shifted downfield with respect to values in small unstructured peptides due to their close proximity to the side chains of Pro-4 and Pro-7, respectively. Several other resonances display chemical shifts which are indicative of a structured environment. Assignment of the amide proton resonances in H2O and measurements of the coupling constant 3JHNCH and the chemical shifts of the amide protons reveal that much of the synthetic peptide, particularly the backbone, exhibits a highly structured environment and represents a good model for the triphosphate binding site in actin.     

A new genus of hell ants from the Cretaceous (Hymenoptera: Formicidae: Haidomyrmecini) with a novel head structure

An unusual Cretaceous trap jaw ant is described from Burmese amber dated to the Late Cretaceous. Linguamyrmex vladi gen.n. sp.n. is distinguished by an unusual suite of morphological characters indicating specialized predatory behaviour and an adaptive strategy no longer found among modern ant lineages. The clypeus, highly modified as in other closely related haidomyrmecine hell ants, is equipped with a paddle‐like projection similar to Ceratomyrmex. X‐ray imaging reveals that this clypeal paddle is reinforced, most probably with sequestered metals. Presumably this fortified clypeal structure was utilized in tandem with scythe‐like mandibles to pin and potentially puncture soft‐bodied prey. This unique taxon, which stresses the diversity of stem‐group ants, is discussed in the context of modern and other Cretaceous trap jaw ant species. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:40D636A3‐4D88‐470A‐BC5B‐85ABFD1A49E2 .     

Spatial relationship between the nucleotide-binding site, Lys-61 and Cys-374 in actin and a conformational change induced by myosin subfragment-1 binding

The spatial relationship between Lys-61, the nucleotide binding site and Cys-374 was studied. Lys-61 was labelled with fluorescein-5-isothiocyanate as a resonance energy acceptor, the nucleotide-binding site was labelled with the fluorescent ATP analogues epsilon ATP or formycin-A 5'-triphosphate (FTP) and Cys-374 was labelled with 5-(2-[(iodoacetyl)amino]ethyl)aminonaphthalene-1-sulfonic acid (1,5-IAEDANS) as a resonance energy donor. The distances between the nucleotide binding site and Lys-61 or between Lys-61 and Cys-374 were calculated to be 3.5 +/- 0.3 nm and 4.60 +/- 0.03 nm, respectively. (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) On the other hand, when doubly-labelled actin with 1,5-IAEDANS at Cys-374 and FITC at Lys-61 was polymerized in the presence of a twofold molar excess of phalloidin [Miki, M. (1987) Eur. J. Biochem. 164, 229-235], the fluorescence of 1,5-IAEDANS bound to actin was quenched significantly. This could be attributed to inter-monomer energy transfer. The inter-monomer distance between FITC attached to Lys-61 in a monomer and 1,5-IAEDANS attached to Cys-374 in its nearest-neighbour monomer in an F-actin filament was calculated to be 3.34 +/- 0.06 nm, assuming that the likely change in the intra-monomer distance does not change during polymerization by more than 0.4 nm. One possible spatial relationship between Lys-61, Cys-374 and the nucleotide binding site in an F-actin filament is proposed. The effect of myosin subfragment-1 (S1) binding on the energy transfer efficiency was studied. The fluorescence intensity of AEDANS-FITC-actin decreased by 30% upon interaction with S1. The fluorescence intensity of AEDANS-FITC-actin polymer in the presence of phalloidin increased by 21% upon interaction with S1. The addition of ATP led to the fluorescence intensity returning to the initial level. Assuming that the change of fluorescence intensity can be attributed to conformational change in the actin molecule induced by S1 binding, the intra-monomer distance was reduced by 0.4 nm and the inter-monomer distance was increased by 0.2 nm.     

Experimental Arthritis in Mice with Mycoplasma pulmonis

A Mycoplasma pulmonis strain, recovered from the arthritic joints of mice employed in the serial passage of a chemically induced tumor, was found to be arthritogenic for mice under experimental conditions. Some joint involvement occurred in all mice challenged intravenously with this strain, and M. pulmonis was recovered frequently from the enlarged joints. The arthritis was migratory, appearing first in the radiocarpal joints and later in the tibiotarsal joints. There was little evidence of a generalized mycoplasmal infection as a consequence of the experimental challenge. Histopathologically, the early stages of the infection in the joints was characterized by an inflammatory response in the synovium and periarticular tissues. Exudate in the joint space contained about equal numbers of polymorphonuclear and mononuclear cells. The polyarthritis resolved slowly, but some residual joint enlargement was noted for as long as 4 months. Two other M. pulmonis strains were also observed to be arthritogenic for mice. Rats were not susceptible to M. pulmonis challenge. Characteristics of the nonsuppurative M. pulmonis arthritis in mice were compared to M. arthritidis joint infections in rats.     

F2-Isoprostanes in HDL are bound to neutral lipids and phospholipids

Low HDL cholesterol (HDL-C) is a risk factor for coronary artery disease (CAD). However, interventions that raise HDL-C have failed to reduce cardiovascular events. We previously reported that HDL is the main carrier of plasma F₂-isoprostanes (F₂-IsoPs) that are markers of oxidative stress formed upon oxidation of arachidonic acid. F₂-IsoPs are predominantly associated with phospholipids. However, there is evidence that F₂-IsoPs in the liver of rats treated with carbon tetrachloride associate with the neutral lipids. To date it is not known whether F₂-IsoPs are found in the neutral lipids in HDL in humans. Possible candidate neutral lipids include cholesteryl esters, triglycerides, diglycerides, and monoglycerides. This study aimed to identify the lipid classes within native and oxidized HDL that contain F₂-IsoPs. We showed that F₂-IsoPs in HDL are bound to neutral lipids as well as phospholipids. HDL-3 contained the highest concentration of F₂-IsoPs in all lipid classes before and after in vitro oxidation. Using targeted LC/MS and high resolution MS, we were unable to provide conclusive evidence for the presence of the synthesized standards 15(R)-15-F_2t-isoP cholesterol and 1-ent-15(RS)-15-F_2t-isoprostanoyl-sn-glycerol in the neutral lipids of HDL. Our findings show that oxidized lipids such as F₂-IsoPs are found in the core and surface of HDL. However, the exact molecular species remain to be definitively characterized. Future studies are required to determine whether the presence of F₂-IsoPs in neutral lipids alters HDL function.     

Purification and characterization of human plasma lecithin:cholesterol acyltransferase.

A highly purified (approximately 12 000-fold) homogeneous preparation of human plasma lecithin:cholesterol acyltransferase (LCAT) with 16% yield was obtained by a combination of density ultracentrifugation, high density lipoprotein affinity column chromatography, hydroxylapatite chromatography, and finally chromatography on anti-apolipoprotein D immunoglobulin-Sepharose columns to remove apolipoprotein D. This enzyme preparation was homogeneous by the following criteria: a single band by polyacrylamide gel electrophoresis in 8 M urea; a single band on sodium dodecyl sulfate gel electrophoresis with an apparent molecular weight of 68 000 +/- 1600; a single protein peak with a molecular weight of 70 000 on a calibrated Sephadex G-100 column. Its amino acid composition was different from human serum albumin and all other apoproteins isolated from lipoprotein fractions.