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DOI： 10.1007/s44211-022-00229-w

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.2198/jelectroph.66.5

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Willey-VCH  

DOI： 10.1002/sscp.202100030

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Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.mimet.2020.106028

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Electrophoresis Society  

DOI： 10.2198/jelectroph.64.1

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DOI： 10.1002/sscp.201900049

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DOI： 10.1007/s12010-019-03050-w

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.mimet.2018.10.001

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley-VCH Verlag  

To separate and extract the native states of lysozyme from chicken egg white, a hybrid method for the mobilization of proteins after non-denaturing gel isoelectric focusing (IEF) combined with detection of lysozyme activity was developed. When the proteins in the chicken egg white were first separated using non-denaturing gel IEF, a lysozyme was obtained at the top of the gel column at the cathode end of the IEF. And, when the IEF-separated proteins of the egg white were mobilized by replacing the cathodic sodium hydroxide solution with phosphoric acid solution, an additional active state of the lysozyme that could be bound to proteins, such as ovotransferrin, was extracted from the solution. Furthermore, it was shown that the addition of lysozyme, obtained via IEF, to pure ovotransferrin generated a complex manifesting lysozyme activity, clearly indicating a successful reconstruction of the lysozyme-ovotransferrin complex in vitro. Therefore, the obtained results demonstrated that the native states of lysozymes, such as lysozyme and the lysozyme-ovotransferrin complex, can be effectively separated and extracted using non-denaturing gel IEF. Thus, this method can be applied to separate and extract different charge states of native proteins that retain their biological activities.

DOI： 10.1002/elps.201700445

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.15583/jpchrom.2017.020

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：HUMANA PRESS INC  

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.

DOI： 10.1007/s12010-016-2393-0

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

In this study, we firstly report inhibition of amyloid beta(42) (A beta(42)) fibrillation, which is related to the progression of Alzheimer's disease, by digestion using proteases, combined with A beta(42) isolation using an immunoaffinity membrane. Especially, digestion of A beta(42) with carboxypeptidase Y (CPY) is effective for the inhibition of A beta(42) fibrillation, indicating that carboxy-terminal digestion of A beta(42) is efficient for the prevention of its fibrillation. The combinational method of the immunoaffinity membrane with CPY digestion is applicable to suppress A beta(42) fibrillation.

DOI： 10.1246/cl.160613

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The purpose of this study was the measurement of enzyme activity within a single-drop of biological fluid after micropurification. Esterase and lactate dehydrogenase (LDH) retained their enzymatic activities after being captured by membrane-immobilized antibodies, which were prepared by non-denaturing two-dimensional electrophoresis, transferred to polyvinylidene difluoride and then stained by Ponceau S. The activities of both enzymes were also measured after being captured by antibodies and biotinylated antibodies bound to membrane-immobilized protein A or avidin, respectively. After esterase and LDH were captured from biological samples by membrane-immobilized protein A or avidin, their activities were semi-quantitatively measured on the surface of the membrane using fluorescence determination. More than 51% of enzyme activities were retained even after the enzymes were captured by biotinylated antibody bound to membrane-immobilized avidin and eluted by rinsing with 5 mu L of 1% Triton X-100, compared with the activities of the enzyme on the immunoaffinity membrane. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jchromb.2015.12.054

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：HUMANA PRESS INC  

Amyloid beta 1-40 peptide was specifically isolated and analyzed from human plasma spiked with amyloid beta using a combined method of biotinylated anti-amyloid beta antibody binding to membrane-immobilized avidin (immunoaffinity membrane) and matrix-assisted laser desorption /ionization time-of-flight mass spectrometry (MALDI-TOF MS). A solution of 10 mu L containing 13.6 ng to 2.9 mu g of amyloid beta peptide was examined in this method. After the isolated amyloid beta peptide from the spiked human plasma containing 2.9 mu g of amyloid beta peptide was incubated in the presence of trifluoroacetic acid, fibrillization of the peptides was observed using a thioflavin T assay. Furthermore, an immunoaffinity membrane present on the inner wall of a tube (diameter 2 mm) captured the amyloid beta peptide from the spiked human plasma. Our results indicate that the combination of the immunoaffinity membrane procedure and MALDI-TOF MS can be used to capture and analyze the target antigens such as amyloid beta in micro-spaces.

DOI： 10.1007/s12010-015-1837-2

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Monomeric molecules such as amyloid-beta can aggregate and transform into oligomeric and fibrous forms, which are implicated in the development and progression of Alzheimer's disease. Novel analytical techniques for the formation of oligomers are required to examine the neurotoxic amyloid-beta oligomers involving fibrils. After isolating amyloid-beta monomer 1-42 using a biotinylated antibody bound to membrane-immobilized avidin (immunoaffinity membrane), their masses were determined by MALDI-TOF MS. Fluorometric determination of more than 0.5 mu M of aggregated amyloid-beta in pipette droplets was performed after aggregation and dilution of 1 mM amyloid-beta. Thus, large (>105 nm) amyloid-beta oligomers in microliter volumes of fluids were isolated using the immunoaffinity membrane and quantitatively analyzed after removal of amyloid-beta monomers and small oligomers by non-denaturing electrophoresis. In addition, amyloid-beta oligomers were specifically isolated from a mixture of human plasma and aggregated amyloid-beta and then fluorometrically analyzed. Our results show that the combination of immunoaffinity membrane-binding and fluorescence determinations together with one drop analysis could be used to isolate and detect huge neurotoxic amyloid-beta oligomers such as fibrils in plasma samples. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jchromb.2014.09.021

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A microfluidic device containing membrane-immobilized anti-esterase (ES) antibodies and anti-lactate dehydrogenase (LDH) antibodies was prepared. The membrane was prepared by transferring antibodies that had been separated by non-denaturing two-dimensional electrophoresis to a polyvinylidene difluoride membrane, which was then stained and cut into small pieces (16 mm(2)). In this microfluidic device, > 0.014 Unit mL(-1) of the purified porcine carboxylesterase was specifically captured by membrane-immobilized anti- ES antibodies and > 147 Unit mL(-1) of purified porcine LDH was specifically captured by membrane-immobilized anti-LDH antibodies. Furthermore, ES and LDH in micro-scale aliquots of porcine liver cytosol were successively captured by membrane-immobilized antibodies in the device, and the enzyme activities were quantitatively analyzed by spectrofluorometry. The results indicate that the microfluidic device containing membrane-immobilized antibodies can be used to investigate the activities of several types of intact enzymes. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.talanta.2014.03.001

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：HUMANA PRESS INC  

Porcine liver carboxylesterase was captured using an immunoaffinity membrane, which was prepared by separating an anti-porcine esterase antibody using non-denaturing two-dimensional electrophoresis, followed by transfer to a polyvinylidene difluoride membrane and staining. The activity of this esterase was 0.008 units after it was captured in the tiny spaces (4 mm(2)) of this membrane and eluted by rinsing with 5 mu L of aspartic acid solution. The molecular mass of the eluted esterase was m/z 61,885 according to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after the purification of this enzyme from the porcine liver cytosol. The purified enzyme's activity was inhibited by 6,9-diamino-2-ethoxyacridine, and this inhibition was retained even after extracting the enzyme from the immunoaffinity membrane. These results indicate that micro-scale extraction and analysis of a carboxylesterase are possible when the enzyme is trapped using an immunoaffinity membrane and eluted with aspartic acid.

DOI： 10.1007/s12010-014-0807-4

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：INFORMA HEALTHCARE  

Non-denaturing electrophoresis can be used to screen enzymes that self-regulate their activities by using a combination of enzymes and their inhibitors. Furthermore, this technique can be applied to develop enzyme reactors that self-regulate their activities. After separation of proteins from mouse liver cytosol by non-denaturing isoelectric focusing, lactate dehydrogense (LDH) and esterase activities were qualitatively and quantitatively examined using a combination of two-dimensional electrophoresis (2-DE) and non-denaturing stacking gel electrophoresis. Activities of mouse liver-derived LDH and carboxylesterase were reversibly inhibited by oxamate and 6,9-diamino-2-ethoxyacridine (acrinol), respectively, in the stacking gels and recovered when the enzymes migrated towards the separation gels. After separation and immobilization of the enzymes, their activities were inhibited by inhibitors and recovered after inhibitor removal. These results indicate that non-denaturing electrophoresis can be applied to select enzymes that self-regulate their activities and subsequently aid in the development of enzyme reactors that can control the enzyme activities.

DOI： 10.3109/14756366.2012.693918

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Background: The functions of proteins can be retained following separation by non-denaturing two-dimensional electrophoresis (2-DE). The trypsin inhibition activities can then be examined following the separation and immobilization of the proteins under non-denaturing conditions.
Methods: Human plasma proteins were separated using 2-DE and transferred onto a polyvinylidene difluoride membrane and stained using Ponceau S. The trypsin inhibition activity of the membrane-bound proteins was qualitatively examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The activities were also quantitatively examined by analyzing the release of the azo-chromophore when azocasein was the substrate.
Results: Ttypsin activity was inhibited by the haptoglobin and alpha(2)-macroglobulin spots located on the membrane, whereas the protease activity was retained for the spots containing albumin and transferrin. The inhibition activities of the alpha(2)-macroglobulin and haptoglobin spots were 4.81- and 4.83-fold higher, respectively, when compared with the inhibition activity of the albumin spot. An axis of the relative activities of trypsin inhibition was added to the 2-DE pattern of human plasma proteins to construct a non-denaturing 3-D map of human plasma proteins.
Conclusion: This 3-D map should represent a suitable diagnostic tool for the qualitative and quantitative analyses of the ttypsin inhibition activities of proteins. (C) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.cca.2013.07.004

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A combination of methods is required to achieve separation of intact proteins and subsequently perform structure analysis to examine their unstable or external structures. The aim of this study was to develop a method of structure analysis in intact proteins after purification. Transferrin from human plasma was trapped by membrane-immobilized anti-transferrin antibody, which was produced by non-denaturing two-dimensional electrophoresis (2-DE), and transferred to a polyvinylidene fluoride (PVDF) membrane and stained with Ponceau S. The antigen transferrin was eluted by rinsing the membrane with trifluoroacetic acid (TFA) or aspartic acid. In addition, a method was established by which the purified human transferrin was enzymatically digested on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) target plate. Thus, after purification of the human transferrin antigen from tens of microlitres of human plasma using an immunoaffinity membrane, transferrin polypeptide fragments were obtained on the plate following digestion with pepsin in the presence of 0.1% TFA or endoproteinase Lys-C or Lys-C/trypsin with 0.001% sodium dodecyl sulphate (SDS). The results indicated that the combined methods of isolation using an immunoaffinity membrane and enzymatic digestion on a MALDI-TOF MS plate could be applied to the purification and microanalysis of antigens. This approach would be particularly applicable to the analysis of the primary structure and the less stable and highly accessible regions of antigens from limited sample volumes. (c) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jpba.2013.05.031

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Apolipoprotein A-1 (apo A-1), a major component of high density lipoprotein (HDL), was efficiently digested by membrane-immobilized trypsin after HDL was treated with membrane-immobilized esterase. Compared to treatment with membrane-immobilized trypsin alone, the relative amounts of apo A-1 polypeptides, m/z 1723.78 and m/z 1568.82, increased by 2.7- and 3.9-fold, respectively, when HDL was treated with membrane-immobilized esterase and trypsin. Furthermore, the efficient digestion of apo A-1 by trypsin was inhibited when HDL was treated with membrane-immobilized esterase in the presence of an esterase inhibitor, 6,9-diamino-2-ethoxyacridine (acrinol). The data indicate that the lipid components of lipoproteins are released by membrane-immobilized esterase. This method can be used to investigate the structure and function of other apolipoproteins. (C) 2012 Elsevier By. All rights reserved.

DOI： 10.1016/j.jpba.2012.07.032

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Adrenocorticotropic hormone (ACTH) and transferrin were trapped by biotinylated anti-ACTH antibody and anti-transferrin antibody, respectively, bound to membrane-immobilized avidin. Polypeptides with the sequences SYSMEHFR. SYSMEHFRWGKPVGK and SYSMEHFRWGKPVGKK were bound to the biotinylated anti-ACTH antibody on the membrane-immobilized avidin after the trapped ACTH was digested with trypsin on the membrane and non-binding polypeptides were washed from the membrane. Further, the polypeptides with the sequence SYSMEHFRWGKPVGK and SYSMEHFRWGKPVGKK were trapped by anti-ACTH antibody bound to membrane-immobilized protein A. The antibody recognized the WGKPVGK region of the antigen, ACTH. Polypeptide with the sequence SMGGKEDLIWELLNQAQEHFGKDK was bound to the biotinylated anti-transferrin antibody on the membrane-immobilized avidin after the trapped transferrin was digested with trypsin on the membrane and non-binding polypeptides were washed from the membrane. Further, the polypeptide with the sequence SMGGKEDLIWELLNQAQEHFGKDK was trapped by anti-transferrin antibody bound to membrane-immobilized protein A. The antibody recognized the SMGGKEDLIWELLNQAQEHFGKDK region of the antigen, transferrin. These results thus indicate that the combined methods of membrane-immobilized avidin and protein A can be applied to examine the epitopes of antigens. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.pep.2012.03.017

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Avidin from egg white was migrated toward a cathode of nondenaturing electrophoresis and then immobilized on a polyvinylidene difluoride membrane. Adrenocorticotropic hormone (ACTH) was specifically captured after the biotinylated anti-ACTH antibody was bound to the membrane-immobilized avidin, and the captured ACTH was digested by the biotinylated trypsin on the membrane after extraction. The digested polypeptides from the ACTH were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). These results indicate that target substances can be specifically trapped and digested on membrane-immobilized avidin. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2011.12.023

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Background: Specific proteins in biological fluids can be captured on an immunoaffinity membrane after polyclonal anti-porcine liver esterase antibodies are separated by non-denaturing 2-dimensional electrophoresis (2-DE) and transferred onto the membrane. The enzymatic activities of these captured proteins can then be monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Methods: Polyclonal anti-porcine liver esterase antibody was separated by non-denaturing 2-DE, transferred onto a polyvinylidene difluoride membrane and stained with Ponceau S. Esterase activity was examined by enzyme activity staining and MALDI-TOF MS after antigens, including purified carboxylesterase from porcine liver and cytosolic esterase from porcine retina, were captured on the immunoaffinity membrane.
Results: Esterase activity was detected on the immunoaffinity membrane after the enzyme was captured. Phosphatidylcholine hydrolysis by the esterase was monitored after the esterase was captured onto the membrane and attached to the target plate for MALDI-TOF MS.
Conclusions: This method could be used to analyze changes in enzymatic activity under biological conditions such as health and disease conditions using immunoaffinity membranes and MALDI-TOF MS. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.cca.2011.10.015

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Haptoglobin is known to bind to hemoglobin during intravascular hemolysis. Membrane-immobilized anti-haptoglobin antibody, which was produced after antibody was isolated by non-denaturing two-dimensional electrophoresis, was transferred to a polyvinylidene difluoride membrane and was stained using Ponceau S. The proteins bound to the membrane-immobilized anti-haptoglobin antibody were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. Hemoglobin was specifically obtained when the membrane-immobilized anti-haptoglobin antibody was incubated with human serum obtained from hemolysis blood. Furthermore, hemoglobin in the flowing fluid was captured by the membrane-immobilized anti-haptoglobin antibody and analyzed directly. The results indicate that hemolysis can be examined by direct trapping and analysis of hemoglobin under physiological conditions. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jpba.2011.07.027

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：HUMANA PRESS INC  

An inhibitor, 9-amino-1,2,3,4-tetra hydroacridine (tacrine), is a reversible inhibitor of esterases. The reversible inhibition of the enzyme activity is thought to be examined after separation and immobilization of the enzyme under non-denaturing conditions. Hydrolytic changes of phosphatidylcholine by carboxylesterase were obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after the esterase was separated by non-denaturing two-dimensional electrophoresis, was immobilized to membranes and was stained by Ponceau S. The changes were inhibited after the enzyme on the membrane was treated by tacrine. Furthermore, the hydrolytic activity of the esterase was recovered after the inhibitor was washed with aspartic acid solution. These results indicate that the phosphatidylcholine hydrolysis activity of the isolated and immobilized enzyme is reversibly inhibited under non-denaturing conditions. Furthermore, this method can be developed to the production of an enzyme reactor able to regulate amounts of lipids.

DOI： 10.1007/s12010-011-9233-z

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We simultaneously separated antibodies for transferrin, the third component of complement (C3), haptoglobin and transthyretin by multi-sample non-denaturing two-dimensional electrophoresis (2-DE), transferred them to a polyvinylidene difluoride (PVDF) membrane and then stained them using direct blue 71 to obtain membrane-immobilized antibodies. The antigens, transferrin, C3. haptoglobin and transthyretin were specifically bound to the membrane-immobilized antibodies and were eluted only after rinsing the membrane with acid solution. The antigens specifically bound to the membrane-immobilized antibodies were separated by SDS-PAGE and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, transferrin and transthyretin were trapped and eluted by each membrane-immobilized antibody and detected by MALDI-TOF MS directly without separations. Using membrane-immobilized anti-transferrin antibody, transferrin in flowing blood was directly trapped and analyzed. The results indicated that membrane-immobilized antibodies are simultaneously produced, and that the immunoaffinity membranes can capture specific substances in flowing fluids. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jchromb.2010.08.042

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Carboxylesterase and sorbitol dehydrogenase are separated by non-denaturing two-dimensional electrophoresis (2-DE) of isoelectric focusing separation using 5% carrier ampholyte (pH 6-8) and 1.25% carrier ampholyte (pH 3-10) and size separation. Furthermore, activities of sorbitol, malate and lactate dehydrogenases are sequentially examined when the enzymes are separated by 2-DE and are sequentially reacted to sorbitol, malic and lactic acid, respectively, in the presence of nicotinamide adenine dinucleotide, nitro blue tetrazolium and phenazine methosulphate. Several kinds of enzymes including lactate dehydrogenize isozymes can be simultaneously separated using 2-DE. Furthermore, the binding differences between lactate dehydrogenase isozymes and concanavalin A (con A) can be examined using a combination of 2-DE and non-denaturing stacking gel electrophoresis. The results of this study indicate that non-denaturing 2-DE can be applied to both enzyme separation and isozyme heterogeneity analysis. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.talanta.2010.06.028

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DOI： 10.1016/j.cca.2010.03.017

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BENTHAM SCIENCE PUBL LTD  

Mouse liver cytosolic malate dehydrogenase was separated by non-denaturing two-dimensional electrophoresis and identified. Furthermore, the activity of the enzyme was preserved even after separation, electroblotting onto a membrane and staining with Ponceau S in acidic buffer solution (pH 5.1). Using the membrane-immobilized enzyme, the malic acid content was estimated by measuring absorbance changes due to the conversion of nicotinamide adenine dinucleotide (NAD) to NADH. These results indicate that enzyme reactors can be systematically produced after purification, immobilization and staining with Ponceau S.

DOI： 10.2174/092986610791498867

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Activities of carboxylesterase and malate dehydrogenase on membranes were retained after enzymes of mouse liver cytosol were separated by non-denaturing, two-dimensional electrophoresis (2-DE), stained using imidazole and zinc salts and electroblotted on to membranes. Furthermore, hydrolytic changes of phosphatidylcholine by the esterase were examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) after separation, and reversible staining and immobilization to membranes. Hydrolytic activity of the esterase on the membranes was 20% of the original activity of the tissue homogenate. The present method can be applied to the production of several types of enzyme reactors on membranes.

DOI： 10.1007/s10529-009-0041-2

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Membrane-immobilized anti-transferrin antibody, which was produced after antibody was separated using non-denaturing two-dimensional electrophoresis (2DE), was transferred to a polyvinylidene difluoride (PVDF) membrane and was stained by direct blue 71. The antigen, transferrin, specifically bound to the membrane-unmobilized antibody and was eluted only after rinsing the membrane with glutamic acid or aspartic acid Solution. The antigen was analyzed directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) when the membrane was incubated with human plasma after the removal Of human serum albumin using polyethylene glycol. The transferrin extracted by glutamic acid or aspartic acid solution retained the binding of Fe(3+) in the presence of the carbonate anion. We found that immunoaffinity membranes can be useful for simple and rapid removal and extraction of intact proteins after antibody separation and blotting under non-denaturing conditions. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aca.2009.04.007

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We detected mouse liver malate, sorbitol and aldehyde dehydrogenases by negative staining, analysis of malate and sorbitol dehydrogenase activities using each substrate, and electron transfers including nicotinamide adenine dinucleotide (NAD) and nitroblue tetrazolium in non-denaturing two-dimensional electrophoresis (2-DE) gel. Dehydrogenases were also identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) after 2-DE separation and protein detection by negative staining. Spots of dehydrogenases separated by 2-DE were excised, and simultaneously transferred and immobilized on polyvinylidene difuoride (PVDF) resin by electrophoresis. The dehydrogenase activities remained intact after immobilization. In conclusion, resin-immobilized dehydrogenases can be simultaneously obtained after separation by non-denaturing 2-DE, detection by negative staining and transferring to resins. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aca.2008.04.046

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Background: Enzyme activity is normally lost when formaldehyde is used as a reductant for silver staining after separation by electrophoresis. Hydrolytic activity of esterases can be examined on membranes without impairing enzyme activity when another reductant such as glucose is used for silver staining of the enzyme after separation by non-denaturing two-dimensional electrophoresis (2-DE) and subsequent transfer.
Methods: The hydrolysis of lipids in human high density lipoprotein (HDL) by esterases first separated on a polyvinylidene fluoride membrane using non-denaturing 2-DE and silver stained using glucose as a reductant was examined.
Results: Esterase activity was retained after glucose was used as a silver reductant for silver staining after separation using non-denaturing 2-DE. Lipids of HDL were removed by the esterases retained on the membrane after esterases were separated by 2-DE.
Conclusion: The results indicated that hydrolytic enzyme activity is retained after separation, staining and immobilization. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.cca.2007.12.017

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The reaction from retinal to retinoic acid catalyzed by retinal dehydrogenase on a polyvinylidene difluoride (PVDF) membrane was examined using laser desorption ionization time of flight mass spectrometry (LDI-TOF MS) when the enzyme was separated by non-denaturing two-dimensional electrophoresis (2-DE), transferred onto the membrane, and stained without impairing the enzyme activity. Furthermore, the enzyme was analyzed by de novo sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after proteins from mouse liver were separated by non-denaturing 2-DE, blotted onto the membrane, and stained. The results indicated that the reported methods could be applied for the direct examination of changes in retinoid catalyzed by enzymes on such membranes. (C) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jchromb.2007.10.028

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

After separation by microscale non-denaturing two-dimensional gel electrophoresis (2DE) and transferring to a blotting membrane, major proteins are detected by a staining of direct blue 71 in a neutral solution. The carboxylesterase on the membrane hydrolyzes phosphatidylcholine after the spot of carboxylesterase is excised from the membrane, and incubated with phosphatidylcholine. Lipids of human serum proteins and the purified human high density lipoprotein (HDL) are removed by enzymatic hydrolysis when human serum proteins and the purified HDL are respectively incubated with the spot of carboxylesterase on the membrane. These results indicate that carboxylesterase on the membrane hydrolyzes not only lipids such as phosphatidylcholine but also lipids of lipoproteins such as HDL after separation by the 2DE, transferring to the membrane and staining without impairing the activity. These results also indicate that a micro-immobilized enzyme reactor on the membrane can be produced when biological enzymes are separated by microscale 2DE, transferred to the membrane and stained without impairing their activities.

DOI： 10.1002/bit.21437

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

This study reports the initial separation of phospholipase C-alpha from porcine retina using non-denaturing two-dimensional gel electrophoresis (2-DE). Detection was by negative staining and then its hydrolytic activity was estimated using alpha-naphthyl acetate in a 2-DE gel. A spot of phospholipase Calpha separated by 2-DE was excised. It was then electrophoretically transferred to an anion-exchange solid phase column after 40 mg, equal to dry weight of the solid resin from the cartridge (Accell (TM) Plus QMA, Waters Corporation), was packed into a disposable 1 ml syringe to make an anion-exchange solid phase column. Phosphatidylcholine was hydrolyzed in the anion-exchange solid phase column containing phospholipase C-alpha. The results indicated that a column with hydrolytic activity could be produced once lipases separated by non-denaturing 2-DE were transferred to the solid phase column. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbbm.2006.11.010

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Hydrolysis of retinyl esters and phospholipids is important for visual functions of the animal retina. This study aimed to examine hydrolytic activity of an enzyme with native substrates such as retinyl esters and phospholipids responsible for this function in porcine retina. After cytosolic proteins were extracted from porcine retina, the proteins were separated using non-denaturing two-dimensional electrophoresis (2DE). Some major proteins and phospholipase Cot were identified by matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) or electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). The phospholipase C alpha showed hydrolytic activities with not only alpha-naphtyl acetate but also with retinyl palmitate and phosphatidylcholine when effects of different substrates were investigated using enzyme activity staining on 2DE or MALDI-TOF-MS. Results indicated that hydrolytic activity of the enzyme with non-native and native substrates could be examined using a combination of non-denaturing 2DE and MALDI-TOF-MS. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jchromb.2006.05.038

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To examine the activities and identity of enzymes associated with organelles such as microsomes and mitochondria, proteins from mouse liver were extracted using the non-ionic detergents Nonidet P-40 (NP-40), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene isooctylphenyl ester (Triton X), n -octyl β-D-glucoside (octyl glycoside) or anionic detergent sodium dodecylsulfate (SDS) after the removal of cytosolic proteins. The proteins extracted by detergents were separated by non-denaturing two-dimensional electrophoresis (2-DE). The activities of esterase and aldehyde dehydrogenase were retained by non-denaturing 2-DE after treatment with each non-ionic detergent, but the activities were reduced or lost when the proteins were extracted with more than 0.5% SDS. For proteomic analysis of the organelle-associated proteins in mouse liver, proteins were separated by non-denaturing 2-DE and were identified using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after the proteins were solubilized by octyl glycoside, NP-40 and 0. 1 % SDS. Several organelle-associated proteins such as carboxylesterase, aldehyde dehydrogenase, glucose regulated protein and HSP60 were identified. These results indicate that the activities and identity of detergent-soluble enzymes can be examined by this non-denaturing 2-DE and mass spectrometry. © 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbapap.2005.02.011

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

DOI： 10.1016/j.ab.2004.01.028

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

After cytosol proteins in the mouse liver were separated by nondenaturing two-dimensional electrophoresis (2-DE), activities of several enzymes, such as fructose bisphosphatase, sorbitol dehydrogenase and malate dehydrogenase, transferase and sorbitol dehydrogenase, or several dehydrogenases, were analyzed on the same 2-D gel. Further, peptidase (or protease) activity can be examined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) when peptides such as angiotensin and adenocorticotropic hormone are incubated in the presence of the cytosol protein separated by nondenaturing 2-DE. Sequence structures of proteins on the 2-D gel were analyzed by peptide mass fingerprinting using MALDI-TOF-MS or by peptide sequencing using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The combination of activity and sequence structure accurately verified the position and activity range of the separated enzymes on the nondenaturing 2-D gel. From these results, we created a nondenaturing 2-D enzyme profile involving activities and sequence structure of cytosol proteins from mouse liver. This profile can be used for checking whether activities of enzymes were specifically or nonspecifically inhibited by inhibitors.

DOI： 10.1002/pmic.200300702

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Esterase and transferase activities were analyzed simultaneously after cytosol proteins in the bovine retina were separated by microscale non-denaturing two-dimensional electrophoresis (2-DE). Esterase activity was specifically inhibited by an esterase inhibitor, 9-amino-1,2,3,4-tetra hydroacridine (tacrine), and transferase activity was specifically inhibited by a glutathione S-transterase (GST) inhibitor, 2-phenyl-1,2-benziso selenazol-3(2H)-one (ebselen). Both esterase and transferase were precipitated when ammonium sulfate was added to the cytosol up to 50% saturation (50% AS fraction), and were detected in the 50% AS fraction by using the 2-DE. After the cytosol proteins in the 50% AS fraction were separated by using non-denaturing 2-DE, polypeptides of the separated proteins were identified by peptide mass fingerprinting and post-source decay analysis by using MALDI-MS, or by immunoreactivity by using a specific antibody. The spots of esterase and transferase activities in the 2-DE pattern were identified as phosphodiesterase and GST, respectively. This simultaneous analysis of enzyme activities can be applied to screen-specific or non-specific medicines which affect enzyme activities. (C) 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbapap.2003.09.007

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Enzyme activities such as of fructose bisphosphatase, malate dehydrogenase and carbonic anhydrase were analyzed after cytosol proteins in the mouse liver and were separated using nondenaturing two-dimensional electrophoresis (2-DE). The activities of both fructose bisphosphatase and malate dehydrogenase were inhibited by thyroxine, and fructose bisphosphatase activity was specifically inhibited by adenosine monophosphate in nondenaturing 2-DE. Furthermore, polypeptides of the separated proteins were analyzed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or by peptide sequencing using electrospray ionization-tandem mass spectrometry, or both. Proteins separated by 2-DE were identified. These results indicate that the function of proteins such as enzyme activity, and their sequence structure can be analyzed, for example by peptide mapping and peptide sequencing, after the proteins have been separated by nondenaturing 2-DE. Present results also indicate analysis of enzyme activity using nondenaturing 2-DE can be applied to screen substances which affect enzyme activity.

DOI： 10.1002/pmic.200300467

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The Cu,Zn-superoxide dismutase (SOD) activity in bovine retina cytosol was separated from retinal pigment using short-length non-denaturing isoelectric focusing (IEF) (15-mm long x 1.3-mm i.d. column) and detected using non-denaturing two-dimensional electrophoresis (2-DE), After the SOD and pigment in the retina cytosol are separated, SOD activity can be quantified by water-soluble tetrazolium salt. We also found that SOD separated by this IEF retained its native function. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0304-4165(02)00259-3

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC ANALYTICAL CHEMISTRY  

Non-denaturing two-dimensional electrophoresis (2-DE) can be applied to the separation of soluble proteins with high resolution. The separated proteins retain their physiological functions. The present study indicates that soluble proteins of the bovine retina were separated by non-denaturing 2-DE, and the enzyme activities of esterase, dehydrogenase, superoxide dismutase and transferase were detected in the presence of each enzyme-specific substrate and chromophore. While the activities of esterase and dehydrogenase were inhibited by a cholinesterase inhibitor, 9-amino-1,2,3,4-tetra hydroacridine (tacrine), the transferase activity was not affected. The enzymes separated by non-denaturing 2-DE were analyzed by matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS). These results indicate that non-denaturing 2-DE is applicable to analyzing of the functions and structures of soluble proteins.

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DOI： 10.2116/bunsekikagaku.51.367

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The third component of complement (C3) of a newt, Cynops pyrrhogaster, was purified using a fast protein liquid chromatography technique. The purified newt C3 consists of two polypeptide chains (the molecular masses of the alpha and beta -chains of C3 were 120,000 and 70,000. respectively) linked by disulfide: bonds. The alpha -chain retained an internal thiolester bond that was cleaved with methylamine, acid the N-terminal amino acid sequence of the alpha -chain was XVQLIDAKAGKAAKF. Digestion of newt C3 with trypsin yielded fragments that induced significant histamine release from newt peritoneal cells. These results: indicate that newt C3 retains structural and functional properties shared with mammalian C3. (C) 2001 Elsevier science Ltd. All rights reserved.

DOI： 10.1016/S0145-305X(01)00007-6

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publisher：Japanese Electrophoresis Society  

After the albumin content of human plasma proteins was decreased by the addition of polyethylene glycol, the plasma proteins were separated in non-denaturing isoelectric focusing (IEF) gel. The separated proteins in the IEF gel were digested with one of three enzymes; trypsin, <i>Achromobacter</i> protease I (API) or <i>Staphylococcus aureus</i> V8 protease (V8). Proteins in the gel were boiled in the presence of mercaptoethanol/SDS, and constituent polypeptides of proteins were separated by SDS-PAGE. The electrophoretic pattern of polypeptide spots were compared to that of non-denaturing IEF/SDS-PAGE. The α chain of the third component of complement (C3α) was effectively digested with trypsin, but albumin and IgG were not effectively fragmented with trypsin. On the other hand, albumin was digested with API and V8 protease, but C3α was not effectively fragmented with these enzymes. These results indicate that trypsin is the most effective enzyme for the fragmentation of C3 in the IEF gel.

DOI： 10.2198/sbk.45.89

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

DOI： 10.1016/S0009-8981(00)00329-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Phorbol ester treatment induces the phosphorylation of SNAP-25 at Ser(187) and the potentiation. of Ca2+-induced dopamine (DA) and acetylcholine (Ach) release from PC12 cells. In order to evaluate the functional consequences of phosphorylation, quantitative analysis was carried out using an. anti-phosphopeptide antibody that specifically recognizes SNAP-25 phosphorylated at Ser(187), DA and ACh release, assayed in low-K+ as well as high-K+ solution, increased by treating the cells with phorbol-12-myristate-13-acetate (PMA); however, the stimulation of high-K+-dependent release occurred at lower concentrations and with shorter exposures to PMA than that of the basal release in low-K+-solution, The PMA-induced phosphorylation of SNAP-25 did not correlate with the potentiation of high-K+-dependent neurotransmitter release. The potentiation of high-K+-dependent DA release by phorbol 12,13-diacetate (PDA), a water soluble phorbol ester, almost completely disappeared within 1 min after washing PDA in the presence of okadaic acid, conditions under which the phosphorylation of SNAP-25 persisted for at least 15 min, PMA-induced phosphorylation of SNAP-25 was inhibited by staurosporine, however, the potentiation of high-K+-dependent PA release was suppressed only partially. These results indicate that protein kinase activation does not account for a large fraction of the phorbol ester-induced potentiation of depolarization-dependent neurotransmitter release from PC12 cells.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Electrophoresis Society  

We have reported that the third component of complement (C3) is observed in the pattern of microscale non-denaturing two-dimensional electrophoresis (2-DE) after removal of IgG and IgA using protein A agarose and antibodies.¹⁾ For the investigation of constitutive polypeptides of the C3, the constituent polypeptides of human plasma proteins were analyzed using non-denaturing isoelectric focusing (IEF) in the first dimension and treated with mercaptoethanol/SDS, and then subjected to the second dimensional SDS electrophoresis (non-denaturing IEF/reduced-SDS PAGE). The α chain of C3 spot was detected in the pattern of non-denaturing IEF/reduced-SDS PAGE, but, the β chain of C3 spot was hidden by albumin spot. After albumin was removed using polyethylene glycol, both C3α and C3β spots were observed in the pattern of the non-denaturing IEF/reduced-SDS PAGE. The combining analysis of C3 in the non-denaturing 2-DE and the constituent polypeptides in the non-denaturing IEF/reduced-SDS PAGE can be useful for the analysis of C3 and its fragments during the activation of complements.

DOI： 10.2198/sbk.44.21

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Specific proteins in small amounts of human plasma were subtracted from patterns of non-denaturing two-dimensional electrophoresis (non-denaturing 2-DE) by layering a 7 mu l aliquot of Protein A agarose-antibody complex on the top of an isoelectric focusing (IEF) gel before the loading of a plasma sample. The Protein A agarose suspension was recovered after non-denaturing IEF and was mixed with 8 M urea-5% 2-mercaptoethanol-1% NP-40 to extract the antibody and the specific plasma protein from Protein A agarose. The extract was then subjected to denaturing two-dimensional electrophoresis (denaturing 2-DE) and the location of the specific polypeptide was determined. The technique can be applied to the extraction and analysis of proteins in small amounts of samples. (C) 1999 Elsevier Science BN. All rights reserved.

DOI： 10.1016/S0165-022X(99)00014-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Heterotrimeric GTP-binding proteins (G proteins) play an important role in phototransduction. The presence of G-protein subclasses has been reported in photoreceptive membranes, e.g., the Gi subgroup (transducin) in vertebrate rods, and the Gq subgroup in the eyes of the Arthropoda and the Mollusca. We examined the immunoreactivity and distribution of a Gq homologue in the cerebral ocelli of Perinereis brevicirris (Polychaeta, Annelida) using an anti-GqC antibody raised against a conserved sequence at the C-terminal of the a-subunit of Gq (Gq-alpha). The anti-GqC antibody labeled a 48-kDa band on the Western blot of proteins from the Perinereis ocelli. The anti-GtC antibody, which is raised against the C-terminal sequence of bovine transducin cc-subunit (Gt-alpha), did not cross-react to the ocellar proteins of Perinereis. The rhabdomeric layers of the anterior and posterior ocelli were strongly labeled by anti-GqC on light-microscopic immunohistology. Immunoelectron microscopy showed that the Gq molecules were specifically localized in the photoreceptive membrane of the rhabdomeric microvilli. These results suggest that the Gq protein plays a role in the phototransduction of the Perinereis ocelli.

DOI： 10.1007/s004410051302

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Protein spots, such as immunoglobulin G (IgG), A (IgA) or alpha(2)-macroglobulin, on the patterns of non-denaturing two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) were selectively removed by the application of protein A or antibody;conjugated protein A agarose to the first-dimension gel top at the cathode side. A protein residing at pI 5.8 and with a molecular mass of about 200 kDa was clearly detected when the IgG and IgA spots were removed from the 2-D PAGE pattern. (C) 1998 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0165-022X(98)00004-9

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Electrophoresis Society  

A comparative study of vertebrate plasma proteins was performed by two-dimensional electrophoresis (2-D PAGE) without denaturing agents in order to obtain a perspective of the variation of plasma protein composition in different vertebrates. Plasma from raccoon, chicken, turtle, newt, goldfish and human was subjected to the analysis. The 2-D PAGE patterns offered information on vertebrate plasma proteins in their physiological conditions: their isoelectric point (pI), approximate molecular weight (<i>M. W.</i>) and quantity. Each vertebrate plasma protein showed a characteristic protein distribution, reflecting the evolutionary changes in DNA sequence and the expression of plasma protein. On the other hand, for some plasma proteins, the structural relationships between human and other vertebrates were suggested immunochemically. A series of spots detected at pI 6.2-8.3, <i>M. W</i>. 850-kDa, observed in the 2-D PAGE patterns of raccoon, chicken and turtle plasma was immunochemically stained by antihuman immunoglobulin M antibody. And, a series of spots detected at pI 4.8-5.5, <i>M. W</i>. 500-kDa in the patterns of raccoon, chicken and newt plasma was immunochemically stained by anti-human α₂-macroglobulin antibody. These results indicate that a perspective of the variation of plasma protein composition in different vertebrates may be obtained by 2-D PAGE without denaturing agents.

DOI： 10.2198/sbk.42.13

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Electrophoresis Society  

Using microscale multisample two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in the absence of denaturing agents, eight protein samples can be simultaneously analyzed and compared. In the present study, this method was applied for the analysis of chicken serum, chicken egg yolk and chicken egg albumen proteins, and the 2-D PAGE pattern of chicken yolk and albumen were compared with that of chicken serum. The spot locations (pI and <i>Mr</i>) of yolk proteins resembled with that of chicken serum, but those of albumen proteins were considerably different from those of chicken serum. Employing the techniques of electrophoretic blotting and immunochemical staining, immunoglobulin G (IgG), third component of complement (C3) and transferrin were identified in the patterns of chicken serum and yolk proteins.

DOI： 10.2198/sbk.42.155

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Formoguanamine hydrochloride (FG) is known as a potent chemical to induce blindness in chick eyes by disrupting the pigment epithelium and visual cells in the retina. In this study, we examined the effect of FG on the structure and function of :he compound eyes of the butterfly, Papilio xuthus (Lepidoptera, Insecta). We administrated 2 mg FG per 1 g body weight of the pupa at about the first one third of the whole pupal period, because accomplishment of morphogenesis of the compound eye occurs in the last half of the pupal period. As a result, unusual membranous structures such as trophospongium-like structures and myeloid bodies were observed in the cytoplasm of the retinular cells besides the normal rhabdom. This result suggests that FG treatment influences on some steps in the formation of rhabdom membranes. However, the amount of chromophore, 3-hydroxyretinal and the responses to white light recorded by the ERG method from FG-treated specimens were not different from the control animals.

DOI： 10.2108/zsj.14.29

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Protein kinase C-mediated phosphorylation of a 25-kDa synaptosome-associated protein (SNAP-25) was examined in living PC12 cells, Phorbol 12-myristate 13 acetate treatment enhanced high potassium-induced [H-3]-norepinephrine release, and a 28-kDa protein recognized by an anti-SNAP-25 antibody was phosphorylated on Ser residues. The molecular size of the phosphorylated band decreased slightly following treatment with Clostridium botulinum type A neurotoxin, whereas the band disappeared after treatment with botulinum type E neurotoxin, indicating that the 28-kDa protein was SNAP-25, A phosphorylation is likely to occur at Ser(187), as this is the only Ser residue located between the cleavage sites of botulinum type A and E neurotoxins, SNAP-25 of PC12 cells was phosphorylated by purified protein kinase C in vitro, and the amount of syntaxin co immunoprecipitated with SNAP-25 was decreased by phosphorylation, These results suggest that the phosphorylation of SNAP-25 may be involved in protein kinase C-mediated regulation of catecholamine release from PC12 cells.

DOI： 10.1074/jbc.271.24.14548

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

1. After the intact compound eyes of the butterfly Papilio xuthus were adapted to darkness, white, blue (lambda max 460 nm) or orange light (lambda max 580 nm), the eyes were separated into the distal (primary pigment cells, the dioptric apparatus and ca. 30% of retinal tissue) and the proximal layers (the rest of the retinal tissues). Each layer was separated into a supernatant and a precipitate. Both in white and blue light-adapted eyes, the amount of 11-cis 3-hydroxyretinal increased in the supernatant of the distal layer (Sup-DL) much more than it did in dark-adapted eyes. No increase was observed in the Sup-DL of orange light-adapted eyes.
2. When all-trans retinol (non-native chemical) was added to the Sup-DL, it was converted to all-trans retinal under the darkness, and to all-trans and 11-cis retinal by blue light irradiation. When all-trans retinal was added to the Sup-DL, the isomerization of all-trans retinal to 11-cis retinal was accelerated by the blue light.
3. The Sup-DL was separated into ammonium sulfate soluble (AS-sup) and insoluble (AS-ppt) fractions. The AS-ppt fraction contained 3-hydroxyretinal but no 3-hydroxyretinol. Blue light irradiation to the AS-ppt fraction induced an increase in 11-cis 3-hydroxyretinal, with a concomitant decrease in all-trans 3-hydroxyretinal.
These results indicate that both the oxidation of all-trans 3-hydroxyretinol to all-trans 3-hydroxyretinal and the light-dependent isomerization of all-trans 3-hydroxyretinal to 11-cis isomer take place in the tissues of the distal layer of the eyes.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The metabolism of 3-hydroxyretinoids in the cytosol of the compound eyes of a species of butterfly, Papilio xuthus, was investigated. The cytosol was found to contain 25-30% of the total 3-hydroxy-retinal and 70-82% of the total 3-hydroxyretinol in the eye. These percentages of 3-hydroxyretinoids in the cytosol were found to be constant regardless of whether the eyes are light-adapted or dark-adapted. 3-Hydroxyretinal can be newly synthesized in the cytosol of light-adapted eves. Blue light specifically increases the amount of 11-cis and all-trans 3-hydroxyretinal ca 2.5 and 1.8 times respectively, compared to pre-irradiation. When 3-hydroxyretinal was synthesized, 3-hydroxyretinol was decreased or disappeared in the cytosol. When retinol (non-native chemical) was added to the cytosol, it was converted into retinal. This result indicates that an oxidative system exists in the compound eye which can convert 3-hydroxyretinol to 3-hydroxyretinal.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

As is known from the literature, the intact compound eyes of the grapsid crab, Hemigrapsus sanguineus, show clear circadian changes in rhabdom sizes even in constant darkness.
In order to identify the location controlling the circadian changes in rhabdom sizes, we incubated isolated compound eyes (distal to the basement membranes) and eye-stalks (compound eye with optic ganglions) for 6 to 8 hr in physiological saline at 20-degrees-C under a LD 12:12 light regime and in continuous darkness (DD). The rhabdom sizes were compared at different times using the electron microscope. There were no significant differences in rhabdom sizes between isolated eye-stalks and compound eyes at each time examined during a day. This indicates that optic ganglions do not directly participate in regulations of rhabdom sizes. The isolated compound eyes showed significant decreases in rhabdom sizes at the subjective dawn under DD. This fact indicates that the breakdown of rhabdoms was endogenously controlled by the biological clock within the retina. However, no distinct increases of rhabdom sizes in the isolated compound eyes were observed at the subjective dusk under DD.

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Event date： 2022.11

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