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Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone: Flavinylation of the enzyme in hepatoc...
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| Zeitschriftentitel: | European Journal of Biochemistry |
|---|---|
| Personen und Körperschaften: | , , |
| In: | European Journal of Biochemistry, 198, 1991, 3, S. 793-799 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
Wiley
|
| Schlagwörter: |
| author_facet |
LANG, Helmut POLSTER, Martin BRANDSCH, Roderich LANG, Helmut POLSTER, Martin BRANDSCH, Roderich |
|---|---|
| author |
LANG, Helmut POLSTER, Martin BRANDSCH, Roderich |
| spellingShingle |
LANG, Helmut POLSTER, Martin BRANDSCH, Roderich European Journal of Biochemistry Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone Biochemistry |
| author_sort |
lang, helmut |
| spelling |
LANG, Helmut POLSTER, Martin BRANDSCH, Roderich 0014-2956 1432-1033 Wiley Biochemistry http://dx.doi.org/10.1111/j.1432-1033.1991.tb16083.x <jats:p>Dimethylglycine dehydrogenase (Me<jats:sub>2</jats:sub>GlyDH), an enzyme of choline catabolism specifically expressed in the mammalian liver, was analyzed in rat hepatocytes in culture. This mitochondrial enzyme carries the FAD cofactor covalently attached to the polypeptide chain by its riboflavin 8α position to N<jats:sup>π</jats:sup> of histidine [Cook, R., Misono, K.S. & Wagner, C. (1980) <jats:italic>J. Biol. Chem. 259</jats:italic>, 12475–12480]. Subcellular fractionation of [<jats:sup>14</jats:sup>C]riboflavin‐labelled hepatocytes and immunoprecipitation with Me<jats:sub>2</jats:sub>GlyDH‐specific antiserum identified a [<jats:sup>14</jats:sup>C]riboflavin‐labelled polypeptide of the size of mature Me<jats:sub>2</jats:sub>GlyDH only in the mitochondrial fraction. Immunoprecipitation of extracts from [<jats:sup>35</jats:sup>S]Met‐labelled hepatocytes revealed a putative precursor protein to the mature Me<jats:sub>2</jats:sub>GlyDH in the cytoplasmic fraction. These Me<jats:sub>2</jats:sub>GlyDH polypeptides were not expressed in cells of the rat hepatoma cell line FAO. A Me<jats:sub>2</jats:sub>GlyDH cDNA clone of apparent full length was isolated from a rat liver cDNA bank constructed in the plasmid vector pcD‐X [Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T. & Arai, K. (1987) <jats:italic>Methods Enzymol. 154</jats:italic>, 3–28]. The nucleotide sequence of the cDNA contains an open reading frame encoding a protein of 96059 Da. This molecular mass agrees well with the migration on SDS/PAGE of the assumed Me<jats:sub>2</jats:sub>GlyDH precursor immunoprecipitated from the cytoplasm of [<jats:sup>35</jats:sup>S]Met‐labelled cells. Proteolytic cleavage at the putative mitochondrial processing protease‐recognition site Arg(‐2)‐Ala(‐1)‐Glu(+1) would lead to the formation of a protein of 91391 Da, which is in good agreement with the estimated 90 kDa of mature Me<jats:sub>2</jats:sub>GlyDH [Wittwer, A. J. & Wagner, C. (1981) <jats:italic>J. Biol. Chem. 256</jats:italic>, 4102–4108], and a 43‐amino‐acid leader peptide. The N‐terminus of Me<jats:sub>2</jats:sub>GlyDH contains a conserved amino acid sequence which forms the dinucleotide‐binding site in many enzymes with noncovalently bound FAD. Close to the modified histidine there is an amino acid sequence resembling a sequence conserved in thymidylate synthases and shown in these enzymes to be involved in the binding of the pteroyl polyglutamate cofactor.</jats:p> Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone European Journal of Biochemistry |
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Wiley, 1991 |
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Wiley, 1991 |
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European Journal of Biochemistry |
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49 |
| title_sub |
Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone |
| title |
Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone |
| title_unstemmed |
Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone |
| title_full |
Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone |
| title_fullStr |
Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone |
| title_full_unstemmed |
Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone |
| title_short |
Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone |
| title_sort |
rat liver dimethylglycine dehydrogenase : flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cdna clone |
| topic |
Biochemistry |
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http://dx.doi.org/10.1111/j.1432-1033.1991.tb16083.x |
| publishDate |
1991 |
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793-799 |
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<jats:p>Dimethylglycine dehydrogenase (Me<jats:sub>2</jats:sub>GlyDH), an enzyme of choline catabolism specifically expressed in the mammalian liver, was analyzed in rat hepatocytes in culture. This mitochondrial enzyme carries the FAD cofactor covalently attached to the polypeptide chain by its riboflavin 8α position to N<jats:sup>π</jats:sup> of histidine [Cook, R., Misono, K.S. & Wagner, C. (1980) <jats:italic>J. Biol. Chem. 259</jats:italic>, 12475–12480]. Subcellular fractionation of [<jats:sup>14</jats:sup>C]riboflavin‐labelled hepatocytes and immunoprecipitation with Me<jats:sub>2</jats:sub>GlyDH‐specific antiserum identified a [<jats:sup>14</jats:sup>C]riboflavin‐labelled polypeptide of the size of mature Me<jats:sub>2</jats:sub>GlyDH only in the mitochondrial fraction. Immunoprecipitation of extracts from [<jats:sup>35</jats:sup>S]Met‐labelled hepatocytes revealed a putative precursor protein to the mature Me<jats:sub>2</jats:sub>GlyDH in the cytoplasmic fraction. These Me<jats:sub>2</jats:sub>GlyDH polypeptides were not expressed in cells of the rat hepatoma cell line FAO. A Me<jats:sub>2</jats:sub>GlyDH cDNA clone of apparent full length was isolated from a rat liver cDNA bank constructed in the plasmid vector pcD‐X [Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T. & Arai, K. (1987) <jats:italic>Methods Enzymol. 154</jats:italic>, 3–28]. The nucleotide sequence of the cDNA contains an open reading frame encoding a protein of 96059 Da. This molecular mass agrees well with the migration on SDS/PAGE of the assumed Me<jats:sub>2</jats:sub>GlyDH precursor immunoprecipitated from the cytoplasm of [<jats:sup>35</jats:sup>S]Met‐labelled cells. Proteolytic cleavage at the putative mitochondrial processing protease‐recognition site Arg(‐2)‐Ala(‐1)‐Glu(+1) would lead to the formation of a protein of 91391 Da, which is in good agreement with the estimated 90 kDa of mature Me<jats:sub>2</jats:sub>GlyDH [Wittwer, A. J. & Wagner, C. (1981) <jats:italic>J. Biol. Chem. 256</jats:italic>, 4102–4108], and a 43‐amino‐acid leader peptide. The N‐terminus of Me<jats:sub>2</jats:sub>GlyDH contains a conserved amino acid sequence which forms the dinucleotide‐binding site in many enzymes with noncovalently bound FAD. Close to the modified histidine there is an amino acid sequence resembling a sequence conserved in thymidylate synthases and shown in these enzymes to be involved in the binding of the pteroyl polyglutamate cofactor.</jats:p> |
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| description | <jats:p>Dimethylglycine dehydrogenase (Me<jats:sub>2</jats:sub>GlyDH), an enzyme of choline catabolism specifically expressed in the mammalian liver, was analyzed in rat hepatocytes in culture. This mitochondrial enzyme carries the FAD cofactor covalently attached to the polypeptide chain by its riboflavin 8α position to N<jats:sup>π</jats:sup> of histidine [Cook, R., Misono, K.S. & Wagner, C. (1980) <jats:italic>J. Biol. Chem. 259</jats:italic>, 12475–12480]. Subcellular fractionation of [<jats:sup>14</jats:sup>C]riboflavin‐labelled hepatocytes and immunoprecipitation with Me<jats:sub>2</jats:sub>GlyDH‐specific antiserum identified a [<jats:sup>14</jats:sup>C]riboflavin‐labelled polypeptide of the size of mature Me<jats:sub>2</jats:sub>GlyDH only in the mitochondrial fraction. Immunoprecipitation of extracts from [<jats:sup>35</jats:sup>S]Met‐labelled hepatocytes revealed a putative precursor protein to the mature Me<jats:sub>2</jats:sub>GlyDH in the cytoplasmic fraction. These Me<jats:sub>2</jats:sub>GlyDH polypeptides were not expressed in cells of the rat hepatoma cell line FAO. A Me<jats:sub>2</jats:sub>GlyDH cDNA clone of apparent full length was isolated from a rat liver cDNA bank constructed in the plasmid vector pcD‐X [Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T. & Arai, K. (1987) <jats:italic>Methods Enzymol. 154</jats:italic>, 3–28]. The nucleotide sequence of the cDNA contains an open reading frame encoding a protein of 96059 Da. This molecular mass agrees well with the migration on SDS/PAGE of the assumed Me<jats:sub>2</jats:sub>GlyDH precursor immunoprecipitated from the cytoplasm of [<jats:sup>35</jats:sup>S]Met‐labelled cells. Proteolytic cleavage at the putative mitochondrial processing protease‐recognition site Arg(‐2)‐Ala(‐1)‐Glu(+1) would lead to the formation of a protein of 91391 Da, which is in good agreement with the estimated 90 kDa of mature Me<jats:sub>2</jats:sub>GlyDH [Wittwer, A. J. & Wagner, C. (1981) <jats:italic>J. Biol. Chem. 256</jats:italic>, 4102–4108], and a 43‐amino‐acid leader peptide. The N‐terminus of Me<jats:sub>2</jats:sub>GlyDH contains a conserved amino acid sequence which forms the dinucleotide‐binding site in many enzymes with noncovalently bound FAD. Close to the modified histidine there is an amino acid sequence resembling a sequence conserved in thymidylate synthases and shown in these enzymes to be involved in the binding of the pteroyl polyglutamate cofactor.</jats:p> |
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| imprint_str_mv | Wiley, 1991 |
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| spelling | LANG, Helmut POLSTER, Martin BRANDSCH, Roderich 0014-2956 1432-1033 Wiley Biochemistry http://dx.doi.org/10.1111/j.1432-1033.1991.tb16083.x <jats:p>Dimethylglycine dehydrogenase (Me<jats:sub>2</jats:sub>GlyDH), an enzyme of choline catabolism specifically expressed in the mammalian liver, was analyzed in rat hepatocytes in culture. This mitochondrial enzyme carries the FAD cofactor covalently attached to the polypeptide chain by its riboflavin 8α position to N<jats:sup>π</jats:sup> of histidine [Cook, R., Misono, K.S. & Wagner, C. (1980) <jats:italic>J. Biol. Chem. 259</jats:italic>, 12475–12480]. Subcellular fractionation of [<jats:sup>14</jats:sup>C]riboflavin‐labelled hepatocytes and immunoprecipitation with Me<jats:sub>2</jats:sub>GlyDH‐specific antiserum identified a [<jats:sup>14</jats:sup>C]riboflavin‐labelled polypeptide of the size of mature Me<jats:sub>2</jats:sub>GlyDH only in the mitochondrial fraction. Immunoprecipitation of extracts from [<jats:sup>35</jats:sup>S]Met‐labelled hepatocytes revealed a putative precursor protein to the mature Me<jats:sub>2</jats:sub>GlyDH in the cytoplasmic fraction. These Me<jats:sub>2</jats:sub>GlyDH polypeptides were not expressed in cells of the rat hepatoma cell line FAO. A Me<jats:sub>2</jats:sub>GlyDH cDNA clone of apparent full length was isolated from a rat liver cDNA bank constructed in the plasmid vector pcD‐X [Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T. & Arai, K. (1987) <jats:italic>Methods Enzymol. 154</jats:italic>, 3–28]. The nucleotide sequence of the cDNA contains an open reading frame encoding a protein of 96059 Da. This molecular mass agrees well with the migration on SDS/PAGE of the assumed Me<jats:sub>2</jats:sub>GlyDH precursor immunoprecipitated from the cytoplasm of [<jats:sup>35</jats:sup>S]Met‐labelled cells. Proteolytic cleavage at the putative mitochondrial processing protease‐recognition site Arg(‐2)‐Ala(‐1)‐Glu(+1) would lead to the formation of a protein of 91391 Da, which is in good agreement with the estimated 90 kDa of mature Me<jats:sub>2</jats:sub>GlyDH [Wittwer, A. J. & Wagner, C. (1981) <jats:italic>J. Biol. Chem. 256</jats:italic>, 4102–4108], and a 43‐amino‐acid leader peptide. The N‐terminus of Me<jats:sub>2</jats:sub>GlyDH contains a conserved amino acid sequence which forms the dinucleotide‐binding site in many enzymes with noncovalently bound FAD. Close to the modified histidine there is an amino acid sequence resembling a sequence conserved in thymidylate synthases and shown in these enzymes to be involved in the binding of the pteroyl polyglutamate cofactor.</jats:p> Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone European Journal of Biochemistry |
| spellingShingle | LANG, Helmut, POLSTER, Martin, BRANDSCH, Roderich, European Journal of Biochemistry, Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone, Biochemistry |
| title | Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone |
| title_full | Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone |
| title_fullStr | Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone |
| title_full_unstemmed | Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone |
| title_short | Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone |
| title_sort | rat liver dimethylglycine dehydrogenase : flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cdna clone |
| title_sub | Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone |
| title_unstemmed | Rat liver dimethylglycine dehydrogenase : Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone |
| topic | Biochemistry |
| url | http://dx.doi.org/10.1111/j.1432-1033.1991.tb16083.x |