Purification, redox and spectroscopic properties of the tetraheme cytochrome c isolated from Rubrivivax gelatinosus.
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The tetraheme cytochrome c subunit of the Rubrivivax gelatinosus reaction center was isolated in the presence of octyl beta-D-thioglucoside by ammonium sulfate precipitation and solubilization at pH 9 in a solution of Deriphat 160. Several biochemical properties of this purified cytochrome were characterized. In particular, it forms small oligomers and its N-terminal amino acid is blocked. In the presence or absence of diaminodurene, ascorbate and dithionite, different oxidation/reduction states of the isolated cytochrome were studied by absorption, EPR and resonance Raman spectroscopies. All the data show two hemes quickly reduced by ascorbate, one heme slowly reduced by ascorbate and one heme only reduced by dithionite. The quickly ascorbate-reduced hemes have paramagnetic properties very similar to those of the two low-potential hemes of the reaction center-bound cytochrome (gz = 3.34), but their alpha band is split with two components peaking at 552 nm and 554 nm in the reduced state. Their axial ligands did not change, being His/Met and His/His, as indicated by the resonance Raman spectra. The slowly ascorbate-reduced heme and the dithionite-reduced heme are assigned to the two high-potential hemes of the bound cytochrome. Their alpha band was blue-shifted at 551 nm and the gz values decreased to 2.96, although the axial ligations (His/Met) were conserved. It was concluded that the estimated 300 mV potential drop of these hemes reflected changes in their solvent accessibility, while the reduction in gz indicates an increased symmetry of their cooordination spheres. These structural modifications impaired the cytochrome's essential function as the electron donor to the photooxidized bacteriochlorophyll dimer of the reaction center. In contrast to its native state, the isolated cytochrome was unable to reduce efficiently the reaction center purified from a Rubrivivax gelatinosus mutant in which the tetraheme was absent. Despite the conformational changes of the cytochrome, its four hemes are still divided into two groups with a pair of low-potential hemes and a pair of high-potential hemes. (+info)
Solubilization of diglyceride acyltransferase from the membrane of Mycobacterium smegmatis.
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Diglyceride acyltransferase [acyl-CoA : 1,2-diacylglycerol O-acyltransferase, EC 2.3.1.20] was found to be localized in the membrane of Mycobacterium smegmatis, and this enzyme could be solubilized from the membrane by treatment with aqueous acetone. The solubilized enzyme required either 1,2-diolein or 1, 3-diolein as an acceptor for palmitoyl-CoA. The apparent Km value for 1,2- or 1,3-diolein and that for palmitoyl-CoA were about 1.4 X 10(-5) M and 6 X 10(-6) M, respectively. Several sulfhydryl reagents were inhibitory to the enzyme activity, suggesting the existence of a thiol group(s) in its active site. The solubilized enzyme, which was more labile than that membrane-bound one, could be stabilized to some extent with antichaotropic salts such as phosphate, pyrophosphate, and sulfate. (+info)
Ligand binding to the amino-terminal domain of the mGluR4 subtype of metabotropic glutamate receptor.
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The metabotropic glutamate receptor (mGluR) 4 subtype of metabotropic glutamate receptor is a presynaptic receptor that modulates neurotransmitter release. We have characterized the properties of a truncated, epitope-tagged construct containing part of the extracellular amino-terminal domain of mGluR4. The truncated receptor was secreted into the cell culture medium of transfected human embryonic kidney cells. The oligomeric structure of the soluble truncated receptor was assessed by gel electrophoresis. In the presence of high concentrations of a reducing agent, the truncated receptor migrated as a monomer; at lower concentrations of the reducing agent, only higher molecular weight oligomers were observed. Competition binding experiments using the radiolabeled agonist [3H]L-2-amino-4-phosphonobutyric acid revealed that the rank order of potency of metabotropic ligands at the truncated receptor was similar to that of the full-length membrane-bound receptor. However, the truncated receptor displayed higher affinities for agonists and lower affinities for antagonists compared with the full-length receptor. Deglycosylation produced a shift in the relative molecular weight of the soluble protein from Mr = 71,000 to Mr = 63,000; deglycosylation had no effect on the binding of [3H]L-2-amino-4-phosphonobutyric acid, indicating that the asparagine-linked carbohydrates are not necessary for agonist binding. These results demonstrate that although the primary determinants of ligand binding to mGluR4 are contained within the first 548 amino acids of the receptor, additional amino acids located downstream of this region may influence the affinity of ligands for the binding site. (+info)
Molecular uncoupling of fractalkine-mediated cell adhesion and signal transduction. Rapid flow arrest of CX3CR1-expressing cells is independent of G-protein activation.
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Fractalkine is a novel multidomain protein expressed on the surface of activated endothelial cells. Cells expressing the chemokine receptor CX3CR1 adhere to fractalkine with high affinity, but it is not known if adherence requires G-protein activation and signal transduction. To investigate the cell adhesion properties of fractalkine, we created mutated forms of CX3CR1 that have little or no ability to transduce intracellular signals. Cells expressing signaling-incompetent forms of CX3CR1 bound rapidly and with high affinity to immobilized fractalkine in both static and flow assays. Video microscopy revealed that CX3CR1-expressing cells bound more rapidly to fractalkine than to VCAM-1 (60 versus 190 ms). Unlike VCAM-1, fractalkine did not mediate cell rolling, and after capture on fractalkine, cells did not dislodge. Finally, soluble fractalkine induced intracellular calcium fluxes and chemotaxis, but it did not activate integrins. Taken together these data provide strong evidence that CX3CR1, a seven-transmembrane domain receptor, mediates robust cell adhesion to fractalkine in the absence of G-protein activation and suggest a novel role for this receptor as an adhesion molecule. (+info)
Unaltered cleavage and secretion of angiotensin-converting enzyme in tumor necrosis factor-alpha-converting enzyme-deficient mice.
(61/12820)
Mammalian angiotensin-converting enzyme (ACE) is one of several biologically important ectoproteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic cleavage. It has been suggested that a common proteolytic system is responsible for the cleavage of a diverse group of membrane ectoproteins, and tumor necrosis factor-alpha-converting enzyme (TACE), a recently purified disintegrin-metalloprotease, has been implicated in the proteolytic cleavage of several cell surface proteins. Mice devoid of TACE have been developed by gene targeting. Such mice could provide a useful system to determine if TACE is responsible for the cleavage of other ectoproteins. Cultured fibroblasts without TACE activity, when transfected with cDNA encoding for the testicular isozyme of ACE (ACET), synthesized and secreted ACET normally after a proteolytic cleavage near the C terminus. In addition, similar quantities of the soluble, C-terminally truncated somatic isozyme of ACE (ACEP) were present in the serum of wild-type and TACE-deficient mice. These results demonstrate that TACE is not essential in the generation of soluble ACE under physiological conditions. Finally, we also report solubilization of ACE-secretase, the enzyme that cleaves ACE, from mouse ACE89 cells and from rabbit lung. We demonstrate that soluble ACE-secretase from both sources failed to cleave its substrate in solution, suggesting a requirement for anchoring to the membrane. (+info)
N-Substituted analogues of S-nitroso-N-acetyl-D,L-penicillamine: chemical stability and prolonged nitric oxide mediated vasodilatation in isolated rat femoral arteries.
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Previous studies show that linking acetylated glucosamine to S-nitroso-N-acetyl-D,L-penicillamine (SNAP) stabilizes the molecule and causes it to elicit unusually prolonged vasodilator effects in endothelium-denuded, isolated rat femoral arteries. Here we studied the propanoyl (SNPP; 3 carbon side-chain), valeryl (SNVP; 5C) and heptanoyl (SNHP; 7C) N-substituted analogues of SNAP (2C), to further investigate other molecular characteristics that might influence chemical stability and duration of vascular action of S-nitrosothiols. Spectrophotometric analysis revealed that SNVP was the most stable analogue in solution. Decomposition of all four compounds was accelerated by Cu(II) and cysteine, and neocuproine, a specific Cu(I) chelator, slowed decomposition of SNHP. Generation of NO from the compounds was confirmed by electrochemical detection at 37 degrees C. Bolus injections of SNAP (10 microl; 10(-8)-10(-3) M) into the perfusate of precontracted, isolated rat femoral arteries taken from adult male Wistar rats (400-500 g), caused concentration-dependent, transient vasodilatations irrespective of endothelial integrity. Equivalent vasodilatations induced by SNVP and SNHP were transient in endothelium-intact vessels but failed to recover to pre-injection pressures at moderate and high concentrations (10(-6)-10(-3) M) in those denuded of endothelium. This sustained effect (> 1 h) was most prevalent with SNHP and was largely reversed by the NO scavenger, haemoglobin. We suggest that increased lipophilicity of SNAP analogues with longer sidechains facilitates their retention by endothelium-denuded vessels; subsequent slow decomposition within the tissue generates sufficient NO to cause prolonged vasodilatation. This is a potentially useful characteristic for targeting NO delivery to areas of endothelial damage. (+info)
A human histocompatibility leukocyte antigen (HLA)-G-specific receptor expressed on all natural killer cells.
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Human natural killer (NK) cells express several killer cell immunoglobulin (Ig)-like receptors (KIRs) that inhibit their cytotoxicity upon recognition of human histocompatibility leukocyte antigen (HLA) class I molecules on target cells. Additional members of the KIR family, including some that deliver activation signals, have unknown ligand specificity and function. One such KIR, denoted KIR2DL4, is structurally divergent from other KIRs in the configuration of its two extracellular Ig domains and of its transmembrane and cytoplasmic domains. Here we show that recombinant soluble KIR2DL4 binds to cells expressing HLA-G but not to cells expressing other HLA class I molecules. Unlike other HLA class I-specific KIRs, which are clonally distributed on NK cells, KIR2DL4 is expressed at the surface of all NK cells. Furthermore, functional transfer of KIR2DL4 into the cell line NK-92 resulted in inhibition of lysis of target cells that express HLA-G, but not target cells that express other class I molecules including HLA-E. Therefore, given that HLA-G expression is restricted to fetal trophoblast cells, KIR2DL4 may provide important signals to maternal NK decidual cells that interact with trophoblast cells at the maternal-fetal interface during pregnancy. (+info)
Role of the myxoma virus soluble CC-chemokine inhibitor glycoprotein, M-T1, during myxoma virus pathogenesis.
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Myxoma virus is a poxvirus that causes a virulent systemic disease called myxomatosis in European rabbits. Like many poxviruses, myxoma virus encodes a variety of secreted proteins that subvert the antiviral activities of host cytokines. It was recently demonstrated that the myxoma virus M-T1 glycoprotein is a member of a large poxvirus family of secreted proteins that bind CC-chemokines and inhibit their chemoattractant activities in vitro. To determine the biological role of M-T1 in contributing to myxoma virus virulence, we constructed a recombinant M-T1-deletion mutant virus that was defective in M-T1 expression. Here, we demonstrate that M-T1 is expressed continuously during the course of myxoma virus infection as a highly stable 43-kDa glycoprotein and is dispensable for virus replication in vitro. Deletion of M-T1 had no significant effects on disease progression or in the overall mortality rate of infected European rabbits but heightened the localized cellular inflammation in primary tissue sites during the initial 2 to 3 days of infection. In the absence of M-T1 expression, deep dermal tissues surrounding the primary site of virus inoculation showed a dramatic increase in infiltrating leukocytes, particularly monocytes/macrophages, but these phagocytes remained relatively ineffective at clearing virus infection, likely due to the concerted properties of other secreted myxoma virus proteins. We conclude that M-T1 inhibits the chemotactic signals required for the influx of monocytes/macrophages during the acute-phase response of myxoma virus infection in vivo, as predicted by its ability to bind and inhibit CC-chemokines in vitro. (+info)