The Experts below are selected from a list of 225 Experts worldwide ranked by ideXlab platform
Kyoko Kojima - One of the best experts on this subject based on the ideXlab platform.
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characterization of human p33 41 annexin iv a ca2 dependent Carbohydrate Binding Protein with monoclonal anti annexin iv antibodies as11 and as17
Biological & Pharmaceutical Bulletin, 1997Co-Authors: Ayano Satoh, Kyoko Kojima, Haruko Ogawa, Eiji Takayama, Yoshimoto Katsura, Tatsuo Kina, Isamu MatsumotoAbstract:p33/41 (annexin IV) is a member of the family of Ca2+-dependent phospholipid Binding Proteins known as annexins. We previously described that bovine kidney p33/41 (annexin IV) has Ca2+-dependent Carbohydrate Binding acrivity. In this study, we purified human p33/41 (annexin IV) from the HT29, human colon adenocarcianoma cell line, as well as the bovine kidney annexin by affinity chromatography. Then, we prepared recombinant human p33/41 (annexin IV) expressed in Escherichia coli. The apparent size and the Ca2+-dependent Carbohydrate Binding properties of purified recombinant p33/41 (annexin IV) were indistinguishable from those of the bovine kidney Protein. We also performed inhibition assays of Carbohydrate Binding and of phosphatidylserine/phosphatidylcholine liposome Binding of recombinant p33/41 (annexin IV) with anti-p33/41 monoclonal antibodies (AS11 and AS17). We determined the epitopes recognized by the monoclonal antibodies by Western blot analysis using deleted-recombinant p33/41 (annexin IV). The monoclonal antibodies recognized domain 1 and/or 2 of p33/41 (annexin IV). The results of the inhibition assays and the determination of the epitope showed that a Carbohydrate Binding site is located at domains 3 and 4 of p33/41 (annexin IV) and on the cell surface.
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Expression of Carbohydrate-Binding Protein p33/41 in Human Tumor Cell Lines
Journal of biochemistry, 1996Co-Authors: Ayano Satoh, Kyoko Kojima, Haruko Ogawa, Eiji Takayama, Takao Yamori, Shigeo Sato, Tokuichi Kawaguchi, Takashi Tsuruo, Yoshimoto Katsura, Tatsuo KinaAbstract:We previously reported a new type of lectin, p33/41 (annexin IV), which was isolated from a bovine tissue extract [Kojima, K. et al. (1992) J. Biol. Chem. 267, 20536-20539]. When the expression of p33/41 (annexin IV) was surveyed in the lysates of 39 human tumor cell lines by SDS-PAGE, followed by Western blot analysis with polyclonal anti-bovine p33/41 and monoclonal anti-annexin IV (Z016, Zymed) antibodies, 21 cell lines were found to be reactive with the polyclonal antibody, whereas all 39 cell lines were stained with Z016. These results together with those obtained with standard Proteins, annexins IV and V, suggested that the monoclonal antibody, Z016, recognizes annexin V, but not p33/41 (annexin IV). Therefore, we performed cDNA cloning of human p33/41 (annexin IV) to prepare a recombinant Protein and raised monoclonal antibodies against the Protein. Northern blot analysis with the cDNA as a probe showed that a human colon cancer cell line, HT29, contains p33/41 (annexin IV) mRNA of two sizes, 2.0 and 3.0 kb. The two monoclonal antibodies, AS11 and AS17, against the recombinant Protein generated were useful for flow cytometric analysis, ELISA, Western blot analysis and immunoprecipitation. Flow cytometric analysis with AS17 showed that p33/41 (annexin IV) is located in the cytoplasm of HT29 cells, but not on the cell surface. However, one of the cell surface Proteins first labeled with biotin and then solubilized with a detergent was immunoprecipitated with AS17. The results suggest the existence of a membrane spanning form of p33/41 (annexin IV).
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expression of Carbohydrate Binding Protein p33 41 in human tumor cell lines
Journal of Biochemistry, 1996Co-Authors: Ayano Satoh, Kyoko Kojima, Haruko Ogawa, Eiji Takayama, Takao Yamori, Shigeo Sato, Tokuichi Kawaguchi, Takashi Tsuruo, Yoshimoto Katsura, Tatsuo KinaAbstract:We previously reported a new type of lectin, p33/41 (annexin IV), which was isolated from a bovine tissue extract [Kojima, K. et al. (1992) J. Biol. Chem. 267, 20536-20539]. When the expression of p33/41 (annexin IV) was surveyed in the lysates of 39 human tumor cell lines by SDS-PAGE, followed by Western blot analysis with polyclonal anti-bovine p33/41 and monoclonal anti-annexin IV (Z016, Zymed) antibodies, 21 cell lines were found to be reactive with the polyclonal antibody, whereas all 39 cell lines were stained with Z016. These results together with those obtained with standard Proteins, annexins IV and V, suggested that the monoclonal antibody, Z016, recognizes annexin V, but not p33/41 (annexin IV). Therefore, we performed cDNA cloning of human p33/41 (annexin IV) to prepare a recombinant Protein and raised monoclonal antibodies against the Protein. Northern blot analysis with the cDNA as a probe showed that a human colon cancer cell line, HT29, contains p33/41 (annexin IV) mRNA of two sizes, 2.0 and 3.0 kb. The two monoclonal antibodies, AS11 and AS17, against the recombinant Protein generated were useful for flow cytometric analysis, ELISA, Western blot analysis and immunoprecipitation. Flow cytometric analysis with AS17 showed that p33/41 (annexin IV) is located in the cytoplasm of HT29 cells, but not on the cell surface. However, one of the cell surface Proteins first labeled with biotin and then solubilized with a detergent was immunoprecipitated with AS17. The results suggest the existence of a membrane spanning form of p33/41 (annexin IV).
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Highly polarized expression of Carbohydrate-Binding Protein p33/41 (annexin IV) on the apical plasma membrane of epithelial cells in renal proximal tubules.
FEBS letters, 1994Co-Authors: Kyoko Kojima, Haruko Ogawa, Hideko Utsumi, Isamu MatsumotoAbstract:p33/41 is a Ca2+-dependent Carbohydrate-Binding Protein and is identical to annexin IV, a member of the annexin Protein family. The localization of p33/41 in bovine kidney specimens was investigated immununohistochemically by use of specific polyclonal antibodies. The most interesting finding on immunostaining was that p33/41 was highly concentrated in the apical plasma membrane of the epithelial cells in the proximal tubules contrary to the distribution throughout the cytoplasm in the papillary ducts and papilla epithelium. The enrichment of p33/41 m the apical membrane was confirmed by immunoblotting of the brush border membrane fraction prepared from a kidney homogenate. Sequential extraction with EDTA and Triton X-100, and a partition experiment with Triton X-114 revealed that most p33/41 associates with the renal brush border membrane in a Ca2+-independent manner and is integrated into the membrane like intrinsic membrane Proteins.
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highly polarized expression of Carbohydrate Binding Protein p33 41 annexin iv on the apical plasma membrane of epithelial cells in renal proximal tubules
FEBS Letters, 1994Co-Authors: Kyoko Kojima, Haruko Ogawa, Hideko Utsumi, Isamu MatsumotoAbstract:p33/41 is a Ca2+-dependent Carbohydrate-Binding Protein and is identical to annexin IV, a member of the annexin Protein family. The localization of p33/41 in bovine kidney specimens was investigated immununohistochemically by use of specific polyclonal antibodies. The most interesting finding on immunostaining was that p33/41 was highly concentrated in the apical plasma membrane of the epithelial cells in the proximal tubules contrary to the distribution throughout the cytoplasm in the papillary ducts and papilla epithelium. The enrichment of p33/41 m the apical membrane was confirmed by immunoblotting of the brush border membrane fraction prepared from a kidney homogenate. Sequential extraction with EDTA and Triton X-100, and a partition experiment with Triton X-114 revealed that most p33/41 associates with the renal brush border membrane in a Ca2+-independent manner and is integrated into the membrane like intrinsic membrane Proteins.
Haruko Ogawa - One of the best experts on this subject based on the ideXlab platform.
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characterization of human p33 41 annexin iv a ca2 dependent Carbohydrate Binding Protein with monoclonal anti annexin iv antibodies as11 and as17
Biological & Pharmaceutical Bulletin, 1997Co-Authors: Ayano Satoh, Kyoko Kojima, Haruko Ogawa, Eiji Takayama, Yoshimoto Katsura, Tatsuo Kina, Isamu MatsumotoAbstract:p33/41 (annexin IV) is a member of the family of Ca2+-dependent phospholipid Binding Proteins known as annexins. We previously described that bovine kidney p33/41 (annexin IV) has Ca2+-dependent Carbohydrate Binding acrivity. In this study, we purified human p33/41 (annexin IV) from the HT29, human colon adenocarcianoma cell line, as well as the bovine kidney annexin by affinity chromatography. Then, we prepared recombinant human p33/41 (annexin IV) expressed in Escherichia coli. The apparent size and the Ca2+-dependent Carbohydrate Binding properties of purified recombinant p33/41 (annexin IV) were indistinguishable from those of the bovine kidney Protein. We also performed inhibition assays of Carbohydrate Binding and of phosphatidylserine/phosphatidylcholine liposome Binding of recombinant p33/41 (annexin IV) with anti-p33/41 monoclonal antibodies (AS11 and AS17). We determined the epitopes recognized by the monoclonal antibodies by Western blot analysis using deleted-recombinant p33/41 (annexin IV). The monoclonal antibodies recognized domain 1 and/or 2 of p33/41 (annexin IV). The results of the inhibition assays and the determination of the epitope showed that a Carbohydrate Binding site is located at domains 3 and 4 of p33/41 (annexin IV) and on the cell surface.
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Expression of Carbohydrate-Binding Protein p33/41 in Human Tumor Cell Lines
Journal of biochemistry, 1996Co-Authors: Ayano Satoh, Kyoko Kojima, Haruko Ogawa, Eiji Takayama, Takao Yamori, Shigeo Sato, Tokuichi Kawaguchi, Takashi Tsuruo, Yoshimoto Katsura, Tatsuo KinaAbstract:We previously reported a new type of lectin, p33/41 (annexin IV), which was isolated from a bovine tissue extract [Kojima, K. et al. (1992) J. Biol. Chem. 267, 20536-20539]. When the expression of p33/41 (annexin IV) was surveyed in the lysates of 39 human tumor cell lines by SDS-PAGE, followed by Western blot analysis with polyclonal anti-bovine p33/41 and monoclonal anti-annexin IV (Z016, Zymed) antibodies, 21 cell lines were found to be reactive with the polyclonal antibody, whereas all 39 cell lines were stained with Z016. These results together with those obtained with standard Proteins, annexins IV and V, suggested that the monoclonal antibody, Z016, recognizes annexin V, but not p33/41 (annexin IV). Therefore, we performed cDNA cloning of human p33/41 (annexin IV) to prepare a recombinant Protein and raised monoclonal antibodies against the Protein. Northern blot analysis with the cDNA as a probe showed that a human colon cancer cell line, HT29, contains p33/41 (annexin IV) mRNA of two sizes, 2.0 and 3.0 kb. The two monoclonal antibodies, AS11 and AS17, against the recombinant Protein generated were useful for flow cytometric analysis, ELISA, Western blot analysis and immunoprecipitation. Flow cytometric analysis with AS17 showed that p33/41 (annexin IV) is located in the cytoplasm of HT29 cells, but not on the cell surface. However, one of the cell surface Proteins first labeled with biotin and then solubilized with a detergent was immunoprecipitated with AS17. The results suggest the existence of a membrane spanning form of p33/41 (annexin IV).
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expression of Carbohydrate Binding Protein p33 41 in human tumor cell lines
Journal of Biochemistry, 1996Co-Authors: Ayano Satoh, Kyoko Kojima, Haruko Ogawa, Eiji Takayama, Takao Yamori, Shigeo Sato, Tokuichi Kawaguchi, Takashi Tsuruo, Yoshimoto Katsura, Tatsuo KinaAbstract:We previously reported a new type of lectin, p33/41 (annexin IV), which was isolated from a bovine tissue extract [Kojima, K. et al. (1992) J. Biol. Chem. 267, 20536-20539]. When the expression of p33/41 (annexin IV) was surveyed in the lysates of 39 human tumor cell lines by SDS-PAGE, followed by Western blot analysis with polyclonal anti-bovine p33/41 and monoclonal anti-annexin IV (Z016, Zymed) antibodies, 21 cell lines were found to be reactive with the polyclonal antibody, whereas all 39 cell lines were stained with Z016. These results together with those obtained with standard Proteins, annexins IV and V, suggested that the monoclonal antibody, Z016, recognizes annexin V, but not p33/41 (annexin IV). Therefore, we performed cDNA cloning of human p33/41 (annexin IV) to prepare a recombinant Protein and raised monoclonal antibodies against the Protein. Northern blot analysis with the cDNA as a probe showed that a human colon cancer cell line, HT29, contains p33/41 (annexin IV) mRNA of two sizes, 2.0 and 3.0 kb. The two monoclonal antibodies, AS11 and AS17, against the recombinant Protein generated were useful for flow cytometric analysis, ELISA, Western blot analysis and immunoprecipitation. Flow cytometric analysis with AS17 showed that p33/41 (annexin IV) is located in the cytoplasm of HT29 cells, but not on the cell surface. However, one of the cell surface Proteins first labeled with biotin and then solubilized with a detergent was immunoprecipitated with AS17. The results suggest the existence of a membrane spanning form of p33/41 (annexin IV).
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Highly polarized expression of Carbohydrate-Binding Protein p33/41 (annexin IV) on the apical plasma membrane of epithelial cells in renal proximal tubules.
FEBS letters, 1994Co-Authors: Kyoko Kojima, Haruko Ogawa, Hideko Utsumi, Isamu MatsumotoAbstract:p33/41 is a Ca2+-dependent Carbohydrate-Binding Protein and is identical to annexin IV, a member of the annexin Protein family. The localization of p33/41 in bovine kidney specimens was investigated immununohistochemically by use of specific polyclonal antibodies. The most interesting finding on immunostaining was that p33/41 was highly concentrated in the apical plasma membrane of the epithelial cells in the proximal tubules contrary to the distribution throughout the cytoplasm in the papillary ducts and papilla epithelium. The enrichment of p33/41 m the apical membrane was confirmed by immunoblotting of the brush border membrane fraction prepared from a kidney homogenate. Sequential extraction with EDTA and Triton X-100, and a partition experiment with Triton X-114 revealed that most p33/41 associates with the renal brush border membrane in a Ca2+-independent manner and is integrated into the membrane like intrinsic membrane Proteins.
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highly polarized expression of Carbohydrate Binding Protein p33 41 annexin iv on the apical plasma membrane of epithelial cells in renal proximal tubules
FEBS Letters, 1994Co-Authors: Kyoko Kojima, Haruko Ogawa, Hideko Utsumi, Isamu MatsumotoAbstract:p33/41 is a Ca2+-dependent Carbohydrate-Binding Protein and is identical to annexin IV, a member of the annexin Protein family. The localization of p33/41 in bovine kidney specimens was investigated immununohistochemically by use of specific polyclonal antibodies. The most interesting finding on immunostaining was that p33/41 was highly concentrated in the apical plasma membrane of the epithelial cells in the proximal tubules contrary to the distribution throughout the cytoplasm in the papillary ducts and papilla epithelium. The enrichment of p33/41 m the apical membrane was confirmed by immunoblotting of the brush border membrane fraction prepared from a kidney homogenate. Sequential extraction with EDTA and Triton X-100, and a partition experiment with Triton X-114 revealed that most p33/41 associates with the renal brush border membrane in a Ca2+-independent manner and is integrated into the membrane like intrinsic membrane Proteins.
Isamu Matsumoto - One of the best experts on this subject based on the ideXlab platform.
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characterization of human p33 41 annexin iv a ca2 dependent Carbohydrate Binding Protein with monoclonal anti annexin iv antibodies as11 and as17
Biological & Pharmaceutical Bulletin, 1997Co-Authors: Ayano Satoh, Kyoko Kojima, Haruko Ogawa, Eiji Takayama, Yoshimoto Katsura, Tatsuo Kina, Isamu MatsumotoAbstract:p33/41 (annexin IV) is a member of the family of Ca2+-dependent phospholipid Binding Proteins known as annexins. We previously described that bovine kidney p33/41 (annexin IV) has Ca2+-dependent Carbohydrate Binding acrivity. In this study, we purified human p33/41 (annexin IV) from the HT29, human colon adenocarcianoma cell line, as well as the bovine kidney annexin by affinity chromatography. Then, we prepared recombinant human p33/41 (annexin IV) expressed in Escherichia coli. The apparent size and the Ca2+-dependent Carbohydrate Binding properties of purified recombinant p33/41 (annexin IV) were indistinguishable from those of the bovine kidney Protein. We also performed inhibition assays of Carbohydrate Binding and of phosphatidylserine/phosphatidylcholine liposome Binding of recombinant p33/41 (annexin IV) with anti-p33/41 monoclonal antibodies (AS11 and AS17). We determined the epitopes recognized by the monoclonal antibodies by Western blot analysis using deleted-recombinant p33/41 (annexin IV). The monoclonal antibodies recognized domain 1 and/or 2 of p33/41 (annexin IV). The results of the inhibition assays and the determination of the epitope showed that a Carbohydrate Binding site is located at domains 3 and 4 of p33/41 (annexin IV) and on the cell surface.
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Highly polarized expression of Carbohydrate-Binding Protein p33/41 (annexin IV) on the apical plasma membrane of epithelial cells in renal proximal tubules.
FEBS letters, 1994Co-Authors: Kyoko Kojima, Haruko Ogawa, Hideko Utsumi, Isamu MatsumotoAbstract:p33/41 is a Ca2+-dependent Carbohydrate-Binding Protein and is identical to annexin IV, a member of the annexin Protein family. The localization of p33/41 in bovine kidney specimens was investigated immununohistochemically by use of specific polyclonal antibodies. The most interesting finding on immunostaining was that p33/41 was highly concentrated in the apical plasma membrane of the epithelial cells in the proximal tubules contrary to the distribution throughout the cytoplasm in the papillary ducts and papilla epithelium. The enrichment of p33/41 m the apical membrane was confirmed by immunoblotting of the brush border membrane fraction prepared from a kidney homogenate. Sequential extraction with EDTA and Triton X-100, and a partition experiment with Triton X-114 revealed that most p33/41 associates with the renal brush border membrane in a Ca2+-independent manner and is integrated into the membrane like intrinsic membrane Proteins.
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highly polarized expression of Carbohydrate Binding Protein p33 41 annexin iv on the apical plasma membrane of epithelial cells in renal proximal tubules
FEBS Letters, 1994Co-Authors: Kyoko Kojima, Haruko Ogawa, Hideko Utsumi, Isamu MatsumotoAbstract:p33/41 is a Ca2+-dependent Carbohydrate-Binding Protein and is identical to annexin IV, a member of the annexin Protein family. The localization of p33/41 in bovine kidney specimens was investigated immununohistochemically by use of specific polyclonal antibodies. The most interesting finding on immunostaining was that p33/41 was highly concentrated in the apical plasma membrane of the epithelial cells in the proximal tubules contrary to the distribution throughout the cytoplasm in the papillary ducts and papilla epithelium. The enrichment of p33/41 m the apical membrane was confirmed by immunoblotting of the brush border membrane fraction prepared from a kidney homogenate. Sequential extraction with EDTA and Triton X-100, and a partition experiment with Triton X-114 revealed that most p33/41 associates with the renal brush border membrane in a Ca2+-independent manner and is integrated into the membrane like intrinsic membrane Proteins.
Tatsuo Kina - One of the best experts on this subject based on the ideXlab platform.
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characterization of human p33 41 annexin iv a ca2 dependent Carbohydrate Binding Protein with monoclonal anti annexin iv antibodies as11 and as17
Biological & Pharmaceutical Bulletin, 1997Co-Authors: Ayano Satoh, Kyoko Kojima, Haruko Ogawa, Eiji Takayama, Yoshimoto Katsura, Tatsuo Kina, Isamu MatsumotoAbstract:p33/41 (annexin IV) is a member of the family of Ca2+-dependent phospholipid Binding Proteins known as annexins. We previously described that bovine kidney p33/41 (annexin IV) has Ca2+-dependent Carbohydrate Binding acrivity. In this study, we purified human p33/41 (annexin IV) from the HT29, human colon adenocarcianoma cell line, as well as the bovine kidney annexin by affinity chromatography. Then, we prepared recombinant human p33/41 (annexin IV) expressed in Escherichia coli. The apparent size and the Ca2+-dependent Carbohydrate Binding properties of purified recombinant p33/41 (annexin IV) were indistinguishable from those of the bovine kidney Protein. We also performed inhibition assays of Carbohydrate Binding and of phosphatidylserine/phosphatidylcholine liposome Binding of recombinant p33/41 (annexin IV) with anti-p33/41 monoclonal antibodies (AS11 and AS17). We determined the epitopes recognized by the monoclonal antibodies by Western blot analysis using deleted-recombinant p33/41 (annexin IV). The monoclonal antibodies recognized domain 1 and/or 2 of p33/41 (annexin IV). The results of the inhibition assays and the determination of the epitope showed that a Carbohydrate Binding site is located at domains 3 and 4 of p33/41 (annexin IV) and on the cell surface.
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Expression of Carbohydrate-Binding Protein p33/41 in Human Tumor Cell Lines
Journal of biochemistry, 1996Co-Authors: Ayano Satoh, Kyoko Kojima, Haruko Ogawa, Eiji Takayama, Takao Yamori, Shigeo Sato, Tokuichi Kawaguchi, Takashi Tsuruo, Yoshimoto Katsura, Tatsuo KinaAbstract:We previously reported a new type of lectin, p33/41 (annexin IV), which was isolated from a bovine tissue extract [Kojima, K. et al. (1992) J. Biol. Chem. 267, 20536-20539]. When the expression of p33/41 (annexin IV) was surveyed in the lysates of 39 human tumor cell lines by SDS-PAGE, followed by Western blot analysis with polyclonal anti-bovine p33/41 and monoclonal anti-annexin IV (Z016, Zymed) antibodies, 21 cell lines were found to be reactive with the polyclonal antibody, whereas all 39 cell lines were stained with Z016. These results together with those obtained with standard Proteins, annexins IV and V, suggested that the monoclonal antibody, Z016, recognizes annexin V, but not p33/41 (annexin IV). Therefore, we performed cDNA cloning of human p33/41 (annexin IV) to prepare a recombinant Protein and raised monoclonal antibodies against the Protein. Northern blot analysis with the cDNA as a probe showed that a human colon cancer cell line, HT29, contains p33/41 (annexin IV) mRNA of two sizes, 2.0 and 3.0 kb. The two monoclonal antibodies, AS11 and AS17, against the recombinant Protein generated were useful for flow cytometric analysis, ELISA, Western blot analysis and immunoprecipitation. Flow cytometric analysis with AS17 showed that p33/41 (annexin IV) is located in the cytoplasm of HT29 cells, but not on the cell surface. However, one of the cell surface Proteins first labeled with biotin and then solubilized with a detergent was immunoprecipitated with AS17. The results suggest the existence of a membrane spanning form of p33/41 (annexin IV).
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expression of Carbohydrate Binding Protein p33 41 in human tumor cell lines
Journal of Biochemistry, 1996Co-Authors: Ayano Satoh, Kyoko Kojima, Haruko Ogawa, Eiji Takayama, Takao Yamori, Shigeo Sato, Tokuichi Kawaguchi, Takashi Tsuruo, Yoshimoto Katsura, Tatsuo KinaAbstract:We previously reported a new type of lectin, p33/41 (annexin IV), which was isolated from a bovine tissue extract [Kojima, K. et al. (1992) J. Biol. Chem. 267, 20536-20539]. When the expression of p33/41 (annexin IV) was surveyed in the lysates of 39 human tumor cell lines by SDS-PAGE, followed by Western blot analysis with polyclonal anti-bovine p33/41 and monoclonal anti-annexin IV (Z016, Zymed) antibodies, 21 cell lines were found to be reactive with the polyclonal antibody, whereas all 39 cell lines were stained with Z016. These results together with those obtained with standard Proteins, annexins IV and V, suggested that the monoclonal antibody, Z016, recognizes annexin V, but not p33/41 (annexin IV). Therefore, we performed cDNA cloning of human p33/41 (annexin IV) to prepare a recombinant Protein and raised monoclonal antibodies against the Protein. Northern blot analysis with the cDNA as a probe showed that a human colon cancer cell line, HT29, contains p33/41 (annexin IV) mRNA of two sizes, 2.0 and 3.0 kb. The two monoclonal antibodies, AS11 and AS17, against the recombinant Protein generated were useful for flow cytometric analysis, ELISA, Western blot analysis and immunoprecipitation. Flow cytometric analysis with AS17 showed that p33/41 (annexin IV) is located in the cytoplasm of HT29 cells, but not on the cell surface. However, one of the cell surface Proteins first labeled with biotin and then solubilized with a detergent was immunoprecipitated with AS17. The results suggest the existence of a membrane spanning form of p33/41 (annexin IV).
Jörn Kalinowski - One of the best experts on this subject based on the ideXlab platform.
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Absence of the highly expressed small Carbohydrate-Binding Protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110
Applied Microbiology and Biotechnology, 2020Co-Authors: Lena Schaffert, Susanne Schneiker-bekel, Jessica Gierhake, Julian Droste, Marcus Persicke, Winfried Rosen, Alfred Pühler, Jörn KalinowskiAbstract:Actinoplanes sp. SE50/110 (ATCC 31044) is the wild type of industrial producer strains of acarbose. Acarbose has been used since the early 1990s as an inhibitor of intestinal human α-glucosidases in the medical treatment of type II diabetes mellitus. The small secreted Protein Cgt, which consists of a single Carbohydrate-Binding module (CBM) 20-domain, was found to be highly expressed in Actinoplanes sp. SE50/110 in previous studies, but neither its function nor a possible role in the acarbose formation was explored, yet. Here, we demonstrated the starch-Binding function of the Cgt Protein in a Binding assay. Transcription analysis showed that the cgt gene was strongly repressed in the presence of glucose or lactose. Due to this and its high abundance in the extracellular proteome of Actinoplanes , a functional role within the sugar metabolism or in the environmental stress protection was assumed. However, the gene deletion mutant ∆ cgt , constructed by CRISPR/Cas9 technology, displayed no apparent phenotype in screening experiments testing for pH and osmolality stress, limited carbon source starch, and the excess of seven different sugars in liquid culture and further 97 carbon sources in the Omnilog Phenotypic Microarray System of Biolog. Therefore, a protective function as a surface Protein or a function within the retainment and the utilization of carbon sources could not be experimentally validated. Remarkably, enhanced production of acarbose was determined yielding into 8–16% higher product titers when grown in maltose-containing medium.