Micro-Scale Extraction and Analysis of Intact Carboxylesterase after Trapping on an Immunoaffinity Membrane Surface

Kimura, Akihiko; Shimazaki, Youji

doi:10.1007/s12010-014-0807-4

Cited by 5 publications

(1 citation statement)

References 30 publications

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“…To identify the protein components of the protein complexes, immunoblotting was performed, as previously reported 11) . Proteins separated by non-denaturing 2DE or SDS-PAGE were transferred from the gel to a polyvinylidene fluoride (PVDF) membrane using a semidry transblotting apparatus for immobilization 12) .…”

Section: Immunoblotting Analysismentioning

confidence: 99%

Electrophoretic extraction of protein complexes after separation and detection by a combined method of non-denaturing two-dimensional electrophoresis and reversible staining

Nakao

Shimazaki

2022

J. Electrophor

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A complex of carboxylesterase and transferrin was obtained after cytosolic proteins in the mouse liver were separated by a combination of non-denaturing two-dimensional electrophoresis (2DE) and reversible staining. At the site where the complex was separated on the 2DE gel, the complex retained esterase activity because the relative intensity of the fluorescent esterase substrate was 1.6-fold higher than that within the 2DE gel in the absence of protein. Furthermore, the complex was extracted from the 2DE gel and bound to antibody-conjugated protein G plus A (protein A/G) agarose by non-denaturing electrophoresis after separation and detection. The esterase activity was retained after the complex was extracted and bound to antibody-conjugated protein A/G agarose. These results indicate that intact protein complexes were separated, detected, and extracted by non-denaturing electrophoresis.

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Section: Immunoblotting Analysismentioning

confidence: 99%

Electrophoretic extraction of protein complexes after separation and detection by a combined method of non-denaturing two-dimensional electrophoresis and reversible staining

Nakao

Shimazaki

2022

J. Electrophor

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Pig Liver Cytosolic Carboxylesterase 1 and Its Inhibition by Remdesivir and Sofosbuvir

Yevgeniia,

Yuliia,

Irina

et al. 2024

ChemistrySelect

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Pig carboxylesterases (CESs) are recognized as enzymes that metabolize xenobiotics, and many drugs used in pig breeding that contain ester groups can be hydrolyzed by CESs. However, there are a limited number of publications focused on the molecular weight, inhibitor effects, and enzyme kinetics of porcine cytosolic carboxylesterase. This study presents the first investigation of the presence, kinetics, inhibition, and amino acid sequence of CES1, the major cytosolic CES isozyme in pig liver. The molecular weight of pig liver cytosolic CES1 was estimated to be approximately 179 kDa, and the amino acid sequence of the enzyme subunit was determined. The inhibition of CES1 by the antiviral drugs remdesivir and sofosbuvir was studied, and the IC50 values were determined. The results indicate that remdesivir and sofosbuvir can act as potent modulators of CES1. The inhibitors exhibited mixed‐type inhibition on pig liver cytosol CES1. It was found that the affinity of the remdesivir and sofosbuvir for free enzyme is greater than that for the enzyme‐substrate complex. A spectrofluorimetric method confirmed the inhibition findings, as the fluorescence intensity of remdesivir decreased upon interaction with the enzyme.

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Hydrolytic Activity of Esterase-Antibody Complexes Retained Within Gel Capsules After Complex Isolation

Shimazaki

Miyatsuka

2017

Appl Biochem Biotechnol

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Delipidation in biological samples is important for some diagnostic tests and protein analyses. Lipids in the samples can be hydrolyzed by native esterases (ESs) within gel capsules after ES, and ES-antibody complexes are specifically trapped, extracted, and separated. Acrylamide and agarose gel capsules containing complexes of ES antibody were produced after the complexes were extracted using protein A-immobilized membranes, separated by non-denaturing electrophoresis, and stained by colloidal silver using glucose as a reductant. ES activity of ES-antibody complexes within the gel capsule was significantly higher than that in the complexes with the control antibodies upon isolation, separation, and detection of the complex. In addition, lipids bound to human serum albumin decreased after human plasma was treated with gel capsules containing ES-antibody complexes. We demonstrate that the gel capsule containing ES-antibody complexes can be successfully isolated using techniques described in this study. Furthermore, delipidation of human plasma is obtained by incubation with the gel capsule. These results indicate that surplus materials such as lipids in biological samples can be removed or reduced by gel capsule containing enzymes.

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Micro-Scale Extraction and Analysis of Intact Carboxylesterase after Trapping on an Immunoaffinity Membrane Surface

Cited by 5 publications

References 30 publications

Electrophoretic extraction of protein complexes after separation and detection by a combined method of non-denaturing two-dimensional electrophoresis and reversible staining

Electrophoretic extraction of protein complexes after separation and detection by a combined method of non-denaturing two-dimensional electrophoresis and reversible staining

Pig Liver Cytosolic Carboxylesterase 1 and Its Inhibition by Remdesivir and Sofosbuvir

Hydrolytic Activity of Esterase-Antibody Complexes Retained Within Gel Capsules After Complex Isolation

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