Thermal Stability of the Human Immunodeficiency Virus Type 1 (HIV-1) Receptors, CD4 and CXCR4, Reconstituted in Proteoliposomes

BACKGROUND: The entry of human immunodeficiency virus (HIV-1) into host cells involves the interaction of the viral exterior envelope glycoprotein, gp120, and receptors on the target cell. The HIV-1 receptors are CD4 and one of two chemokine receptors, CCR5 or CXCR4. METHODOLOGY/PRINCIPAL FINDINGS: We created proteoliposomes that contain CD4, the primary HIV-1 receptor, and one of the coreceptors, CXCR4. Antibodies against CD4 and CXCR4 specifically bound the proteoliposomes. CXCL12, the natural ligand for CXCR4, and the small-molecule CXCR4 antagonist, AMD3100, bound the proteoliposomes with affinities close to those associated with the binding of these molecules to cells expressing CXCR4 and CD4. The HIV-1 gp120 exterior envelope glycoprotein bound tightly to proteoliposomes expressing only CD4 and, in the presence of soluble CD4, bound weakly to proteoliposomes expressing only CXCR4. The thermal stability of CD4 and CXCR4 inserted into liposomes was examined. Thermal denaturation of CXCR4 followed second-order kinetics, with an activation energy (E(a)) of 269 kJ/mol (64.3 kcal/mol) and an inactivation temperature (T(i)) of 56°C. Thermal inactivation of CD4 exhibited a reaction order of 1.3, an E(a) of 278 kJ/mol (66.5 kcal/mol), and a T(i) of 52.2°C. The second-order denaturation kinetics of CXCR4 is unusual among G protein-coupled receptors, and may result from dimeric interactions between CXCR4 molecules. CONCLUSIONS/SIGNIFICANCE: Our studies with proteoliposomes containing the native HIV-1 receptors allowed an examination of the binding of biologically important ligands and revealed the higher-order denaturation kinetics of these receptors. CD4/CXCR4-proteoliposomes may be useful for the study of virus-target cell interactions and for the identification of inhibitors

Selective Determination of Lysine in Dry-Cured Meats Using a Sensor Based on Lysine-α-Oxidase Immobilised on a Nylon Membrane

An enzymatic sensor employing lysine oxidase (LOx) with the immobilised enzyme system by crosslinking with glutaraldehyde using an immunodyne ABC nylon membrane, in combination with an oxygen electrode, has been optimised to determine the lysine content in dry-cured ham and dry-fermented sausage at different cured times. The amperometric signal obtained due to the oxygen depletion (consumed oxygen) during the lysine oxidation was recorded at 5 s in the immobilised enzyme sensor, and the reaction rates (slope) were related to the lysine content. A linear relationship between the consumed oxygen as a function of time (mg O2/l/s) and the lysine concentration in the range 10–250 μM (R2 = 0.9946) for the immobilised enzyme system was found. The immobilised enzyme sensor showed a high specificity and sensibility. Nevertheless, the stability of the immobilised enzyme at the assay temperature was very poor, and thus, a new membrane was required for each analysis. The analysis of lysine with the immobilised enzyme system in cured meat samples revealed very good agreement with the determination performed through standard HPLC methodology, which validated the use of this sensor as an alternative technique to evaluate cured meat quality.This study was funded by Grant Prometeo 2012/001 from Conselleria d’Educació of Generalitat Valenciana (Valencia, Spain) and the scholarship to F. A. Jadán Piedra from Secretaria de Educación Superior, Ciencia, Tecnología e Innovación (SENESCYT) of Ecuador.Peer reviewe