apoa iv  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis

doi: 10.1016/j.jbc.2025.108392

Figure Lengend Snippet: Protein folding and molecular tumbling analyses of apoA-IV polymorphisms. A , CD spectra for the average molar ellipticity versus wavelength of apoA-IV WT ( red ), apoA-IV Q360H ( blue ), and apoA-IV T347S ( green ) in PBS at 37 °C. B , displays a comparison of percentage α-helix content (fractional helicity) of apoA-IV polymorphisms. C , shows the thermal stability analysis of apoA-IV proteins. Dotted lines signify T m points of 53.2, 50.8, and 47.6 °C for apoA-IV WT ( red ), apoA-IV Q360H ( blue ), and apoA-IV T347S ( green ), respectively. D , shows protein folding stability analysis of apoA-IV proteins in PBS at 25 °C as a function of guanidine chloride concentration. E , exhibits molecular tumbling examination using fluorescence anisotropy of fluorophore-labeled apoA-IV variants in PBS at 37 °C. Fluorescence anisotropy measures the rotational mobility of labeled C terminus with polarized light. Bar graph demonstrates that apoA-IV Q360H tumbles faster than apoA-IV WT , and apoA-IV T347S tumbles faster than apoA-IV Q360H and apoA-IV WT . Mean normalized fluorescence anisotropy values are subtracted from an unconjugated fluorophore blank sample. Quantified values are listed in Table 1 . ∗ p < 0.05, ∗∗ p < 0.01, N = 20. apoA-IV, apolipoprotein A-IV.

Article Snippet: Recombinant His-tagged human apoA-IV WT , apoA-IV Q360H , and apoA-IV T347S protein isoforms (0.2 μM) were separately immobilized onto hydrated Ni–NTA biosensor probes using an Octet RH16 interferometer (Sartorius) and tilted-bottom microplates.

Techniques: Circular Dichroism, Comparison, Concentration Assay, Fluorescence, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis

doi: 10.1016/j.jbc.2025.108392

Figure Lengend Snippet: Quantified chemical and thermal denaturation data from CD spectroscopy

Article Snippet: Recombinant His-tagged human apoA-IV WT , apoA-IV Q360H , and apoA-IV T347S protein isoforms (0.2 μM) were separately immobilized onto hydrated Ni–NTA biosensor probes using an Octet RH16 interferometer (Sartorius) and tilted-bottom microplates.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis

doi: 10.1016/j.jbc.2025.108392

Figure Lengend Snippet: ApoA-IV polymorphisms inhibit human platelet aggregation . ADP-induced human platelet aggregation in platelet-rich plasma with and without the addition of 350 μg/ml (4 μM) apoA-IV WT ( red ), apoA-IV Q360H ( blue ), apoA-IV T347S ( green ), and PBS control ( gray ) using light transmission aggregometry. A , presents superimposed tracings of platelet aggregation in the presence of dimerized apoA-IV ( dark colors ). B , shows overlaid tracings of platelet aggregation in the presence of undimerized homogenous apoA-IV ( light colors ). C , displays bar graphs for comparative statistical analyses of acquired platelet aggregation results. Each bar represents mean ± SD of maximum platelet aggregation. ∗∗ p < 0.01, N = 6. apoA-IV, apolipoprotein A-IV.

Article Snippet: Recombinant His-tagged human apoA-IV WT , apoA-IV Q360H , and apoA-IV T347S protein isoforms (0.2 μM) were separately immobilized onto hydrated Ni–NTA biosensor probes using an Octet RH16 interferometer (Sartorius) and tilted-bottom microplates.

Techniques: Clinical Proteomics, Control, Transmission Assay

Journal: The Journal of Biological Chemistry

Article Title: Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis

doi: 10.1016/j.jbc.2025.108392

Figure Lengend Snippet: Direct binding affinity quantification of apoA-IV polymorphisms with purified human αIIbβ3. A , displays BLI sensograms for 0.2 μM apoA-IV polymorphisms as a function of activated human purified αIIbβ3 concentration in activation buffer at 1000 rpm, 37 °C ( B ) shows BLI kinetics sensograms for 0.2 μM apoA-IV WT , ( C ) apoA-IV Q360H , and ( D ) apoA-IV T347S polymorphisms with solutions of gel-filtered platelets in an activation buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM MnCl 2 , 30% [v/v] glycerol) at 1000 rpm, 37 °C (N = 3). Quantified binding affinity values are listed in Table 2 . apoA-IV, apolipoprotein A-IV; BLI, biolayer interferometry.

Article Snippet: Recombinant His-tagged human apoA-IV WT , apoA-IV Q360H , and apoA-IV T347S protein isoforms (0.2 μM) were separately immobilized onto hydrated Ni–NTA biosensor probes using an Octet RH16 interferometer (Sartorius) and tilted-bottom microplates.

Techniques: Binding Assay, Purification, Concentration Assay, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis

doi: 10.1016/j.jbc.2025.108392

Figure Lengend Snippet: Quantified direct binding affinity data for apoA-IV polymorphisms and human platelets and α IIb β 3 titrations using BLI

Article Snippet: Recombinant His-tagged human apoA-IV WT , apoA-IV Q360H , and apoA-IV T347S protein isoforms (0.2 μM) were separately immobilized onto hydrated Ni–NTA biosensor probes using an Octet RH16 interferometer (Sartorius) and tilted-bottom microplates.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis

doi: 10.1016/j.jbc.2025.108392

Figure Lengend Snippet: Dissociation of apoA-IV variants in PBS using ITC methods

Article Snippet: Recombinant His-tagged human apoA-IV WT , apoA-IV Q360H , and apoA-IV T347S protein isoforms (0.2 μM) were separately immobilized onto hydrated Ni–NTA biosensor probes using an Octet RH16 interferometer (Sartorius) and tilted-bottom microplates.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis

doi: 10.1016/j.jbc.2025.108392

Figure Lengend Snippet: Isothermal titration calorimetry thermograms of apoA-IV polymorphisms. Comparative analysis of multimer dissociation parameters of ( A ) apoA-IV WT , ( B ) apoA-IV Q360H , and ( C ) apoA-IV T347S in PBS (N = 3). apoA-IV, apolipoprotein A-IV.

Article Snippet: Recombinant His-tagged human apoA-IV WT , apoA-IV Q360H , and apoA-IV T347S protein isoforms (0.2 μM) were separately immobilized onto hydrated Ni–NTA biosensor probes using an Octet RH16 interferometer (Sartorius) and tilted-bottom microplates.

Techniques: Isothermal Titration Calorimetry

Journal: The Journal of Biological Chemistry

Article Title: Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis

doi: 10.1016/j.jbc.2025.108392

Figure Lengend Snippet: Characterization of molecular size and multimer formation of studied apoA-IV polymorphism. A , shows molecular size analysis of apoA-IV WT ( red ), apoA-IV Q360H ( blue ), and apoA-IV T347S ( green ) using dynamic laser light scattering. B , demonstrates variations in multimer formation properties of apoA-IV on 8% PAGE under nondenaturing (pH 7.4) conditions at 25 °C. M denotes protein molecular weight marker. Electrophoresis was performed on the displayed lanes within a single gel, and the spacer lanes were removed to enhance clarity. C , shows multiangle light scattering demonstrated multimer diffusion properties of apoA-IV variants in PBS (pH 7.4) at 25 °C. Numeric annotations 1 to 4 represent eluates corresponding to monomer, dimer, trimer, and tetramer, respectively, N = 4. apoA-IV, apolipoprotein A-IV.

Article Snippet: Recombinant His-tagged human apoA-IV WT , apoA-IV Q360H , and apoA-IV T347S protein isoforms (0.2 μM) were separately immobilized onto hydrated Ni–NTA biosensor probes using an Octet RH16 interferometer (Sartorius) and tilted-bottom microplates.

Techniques: Molecular Weight, Marker, Electrophoresis, Multi-Angle Light Scattering, Diffusion-based Assay

Journal: The Journal of Biological Chemistry

Article Title: Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis

doi: 10.1016/j.jbc.2025.108392

Figure Lengend Snippet: Intrinsic fluorescence enhancement and quenching titrations demonstrating the conformational changes of examined apoA-IV polymorphisms. A , shows fluorescence emission increase and redshifted spectra of apoA-IV WT ( red ), apoA-IV Q360H ( blue ), and apoA-IV T347S ( green ) as a function of titrations with phospholipid (DMPC). B , exhibits fluorescence emission quenching and blueshifted spectra of apoA-IV polymorphisms unbound to DMPC as a function of sodium iodide concentration. C , shows overlaid Stern–Volmer plots for fluorescence quenching analysis. D , displays fluorescence emission quenching and blueshifted spectra of apoA-IV polymorphisms bound to DMPC as a function of sodium iodide concentration. Data acquired in 20 mM Hepes, pH 7.4, 140 mM NaCl at 37 °C. apoA-IV, apolipoprotein A-IV; DMPC, 1,2-dimyristoyl- sn -glycero-3-phosphocholine.

Article Snippet: Recombinant His-tagged human apoA-IV WT , apoA-IV Q360H , and apoA-IV T347S protein isoforms (0.2 μM) were separately immobilized onto hydrated Ni–NTA biosensor probes using an Octet RH16 interferometer (Sartorius) and tilted-bottom microplates.

Techniques: Fluorescence, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis

doi: 10.1016/j.jbc.2025.108392

Figure Lengend Snippet: Stern–Volmer constants ( K SV ) of apoA-IV polymorphisms quantified from intrinsic fluorescence quenching titrations

Article Snippet: Recombinant His-tagged human apoA-IV WT , apoA-IV Q360H , and apoA-IV T347S protein isoforms (0.2 μM) were separately immobilized onto hydrated Ni–NTA biosensor probes using an Octet RH16 interferometer (Sartorius) and tilted-bottom microplates.

Techniques: Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis

doi: 10.1016/j.jbc.2025.108392

Figure Lengend Snippet: Comparison of overall in silico computational structure elucidation of studied apoA-IV polymorphisms in their monomeric conformations. A , demonstrates amphipathic nature of apoA-IV WT , apoA-IV Q360H , and apoA-IV T347S white color denotes hydrophilic surfaces, and blue shades signify hydrophilic regions. B , shows differences in spatial orientations of N and C termini of apoA-IV WT ( red ), apoA-IV Q360H ( blue ), and apoA-IV T347S ( olive ). Proximity and side-chain interaction of aspartic acid (D5) and arginine (R326) residues are denoted on each structure. apoA-IV, apolipoprotein A-IV.

Article Snippet: Recombinant His-tagged human apoA-IV WT , apoA-IV Q360H , and apoA-IV T347S protein isoforms (0.2 μM) were separately immobilized onto hydrated Ni–NTA biosensor probes using an Octet RH16 interferometer (Sartorius) and tilted-bottom microplates.

Techniques: Comparison, In Silico

Journal: The Journal of Biological Chemistry

Article Title: Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis

doi: 10.1016/j.jbc.2025.108392

Figure Lengend Snippet: Comparison of C-terminal conformations of examined apoA-IV polymorphisms. A , demonstrates superimposed comparison of apoA-IV WT ( red ) with apoA-IV T347S ( olive ) on the left and superimposed comparison of apoA-IV WT ( red ) with apoA-IV Q360H ( blue ) on the right . Distance variations in the spatial orientations are denoted on superimposed structures. B , shows differences in spatial orientations and interactions of apoA-IV WT ( light red ), apoA-IV Q360H ( gold ), and apoA-IV T347S ( purple ) with the headpiece structure of GPIIbIIIa (αIIbβ3). Protein surface representation of GPIIbIIIa (Protein Data Bank code: 3ZE2 ) are shown in green and blue , respectively. apoA-IV, apolipoprotein A-IV.

Article Snippet: Recombinant His-tagged human apoA-IV WT , apoA-IV Q360H , and apoA-IV T347S protein isoforms (0.2 μM) were separately immobilized onto hydrated Ni–NTA biosensor probes using an Octet RH16 interferometer (Sartorius) and tilted-bottom microplates.

Techniques: Comparison

Journal: The Journal of Biological Chemistry

Article Title: Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis

doi: 10.1016/j.jbc.2025.108392

Figure Lengend Snippet: Structural elucidation of direct interactions of studied apoA-IV polymorphisms. A , demonstrates differences in the RGD motif region ( orange ) on GPIIbIIIa headpiece domain (Protein Data Bank code: 3ZE2 , gold ) covered by the binding sites of apoA-IV WT ( red ), apoA-IV Q360H ( blue ), and apoA-IV T347S ( olive ). B , shows zoomed in ribbon representations for intermolecular helix–helix interactions of folded apoA-IV WT ( red ), apoA-IV Q360H ( blue ), and apoA-IV T347S ( olive ) structures. apoA-IV, apolipoprotein A-IV.

Article Snippet: Recombinant His-tagged human apoA-IV WT , apoA-IV Q360H , and apoA-IV T347S protein isoforms (0.2 μM) were separately immobilized onto hydrated Ni–NTA biosensor probes using an Octet RH16 interferometer (Sartorius) and tilted-bottom microplates.

Techniques: Binding Assay