Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain
Aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthas...
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| Autor*in: |
Madhavarao, C. N. [verfasserIn] Chinopoulos, C. [verfasserIn] Chandrasekaran, K. [verfasserIn] |
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| Format: |
E-Artikel |
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| Erschienen: |
Oxford, UK: Blackwell Science Ltd ; 2003 |
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| Schlagwörter: |
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| Umfang: |
Online-Ressource |
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| Reproduktion: |
2003 ; Blackwell Publishing Journal Backfiles 1879-2005 |
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| Übergeordnetes Werk: |
In: Journal of neurochemistry - Oxford : Wiley-Blackwell, 1956, 86(2003), 4, Seite 0 |
| Übergeordnetes Werk: |
volume:86 ; year:2003 ; number:4 ; pages:0 |
| Links: |
|---|
| DOI / URN: |
10.1046/j.1471-4159.2003.01905.x |
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| 520 | |a Aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp-NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp-NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated a single large protein (∼670 kDa), which on sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi-subunit complex Asp-NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) treatment followed by size-exclusion chromatography. Asp-NAT showed an order of magnitude difference in Km between l-aspartate (l-Asp, ∼0.5 mm) and acetyl CoA (∼0.05 mm). Asp-NAT showed high specificity towards l-Asp with 3% or less activity towards l-Glu, l-Asn, l-Gln and Asp-Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed. | ||
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10.1046/j.1471-4159.2003.01905.x doi (DE-627)NLEJ243128029 DE-627 ger DE-627 rakwb Madhavarao, C. N. verfasserin aut Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain Oxford, UK Blackwell Science Ltd 2003 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp-NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp-NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated a single large protein (∼670 kDa), which on sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi-subunit complex Asp-NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) treatment followed by size-exclusion chromatography. Asp-NAT showed an order of magnitude difference in Km between l-aspartate (l-Asp, ∼0.5 mm) and acetyl CoA (∼0.05 mm). Asp-NAT showed high specificity towards l-Asp with 3% or less activity towards l-Glu, l-Asn, l-Gln and Asp-Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed. 2003 Blackwell Publishing Journal Backfiles 1879-2005 |2003|||||||||| enzyme kinetics Chinopoulos, C. verfasserin aut Chandrasekaran, K. verfasserin aut Namboodiri, M. A. A. oth In Journal of neurochemistry Oxford : Wiley-Blackwell, 1956 86(2003), 4, Seite 0 Online-Ressource (DE-627)NLEJ243927584 (DE-600)2020528-4 1471-4159 nnns volume:86 year:2003 number:4 pages:0 http://dx.doi.org/10.1046/j.1471-4159.2003.01905.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 86 2003 4 0 |
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10.1046/j.1471-4159.2003.01905.x doi (DE-627)NLEJ243128029 DE-627 ger DE-627 rakwb Madhavarao, C. N. verfasserin aut Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain Oxford, UK Blackwell Science Ltd 2003 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp-NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp-NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated a single large protein (∼670 kDa), which on sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi-subunit complex Asp-NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) treatment followed by size-exclusion chromatography. Asp-NAT showed an order of magnitude difference in Km between l-aspartate (l-Asp, ∼0.5 mm) and acetyl CoA (∼0.05 mm). Asp-NAT showed high specificity towards l-Asp with 3% or less activity towards l-Glu, l-Asn, l-Gln and Asp-Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed. 2003 Blackwell Publishing Journal Backfiles 1879-2005 |2003|||||||||| enzyme kinetics Chinopoulos, C. verfasserin aut Chandrasekaran, K. verfasserin aut Namboodiri, M. A. A. oth In Journal of neurochemistry Oxford : Wiley-Blackwell, 1956 86(2003), 4, Seite 0 Online-Ressource (DE-627)NLEJ243927584 (DE-600)2020528-4 1471-4159 nnns volume:86 year:2003 number:4 pages:0 http://dx.doi.org/10.1046/j.1471-4159.2003.01905.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 86 2003 4 0 |
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10.1046/j.1471-4159.2003.01905.x doi (DE-627)NLEJ243128029 DE-627 ger DE-627 rakwb Madhavarao, C. N. verfasserin aut Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain Oxford, UK Blackwell Science Ltd 2003 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp-NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp-NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated a single large protein (∼670 kDa), which on sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi-subunit complex Asp-NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) treatment followed by size-exclusion chromatography. Asp-NAT showed an order of magnitude difference in Km between l-aspartate (l-Asp, ∼0.5 mm) and acetyl CoA (∼0.05 mm). Asp-NAT showed high specificity towards l-Asp with 3% or less activity towards l-Glu, l-Asn, l-Gln and Asp-Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed. 2003 Blackwell Publishing Journal Backfiles 1879-2005 |2003|||||||||| enzyme kinetics Chinopoulos, C. verfasserin aut Chandrasekaran, K. verfasserin aut Namboodiri, M. A. A. oth In Journal of neurochemistry Oxford : Wiley-Blackwell, 1956 86(2003), 4, Seite 0 Online-Ressource (DE-627)NLEJ243927584 (DE-600)2020528-4 1471-4159 nnns volume:86 year:2003 number:4 pages:0 http://dx.doi.org/10.1046/j.1471-4159.2003.01905.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 86 2003 4 0 |
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10.1046/j.1471-4159.2003.01905.x doi (DE-627)NLEJ243128029 DE-627 ger DE-627 rakwb Madhavarao, C. N. verfasserin aut Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain Oxford, UK Blackwell Science Ltd 2003 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp-NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp-NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated a single large protein (∼670 kDa), which on sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi-subunit complex Asp-NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) treatment followed by size-exclusion chromatography. Asp-NAT showed an order of magnitude difference in Km between l-aspartate (l-Asp, ∼0.5 mm) and acetyl CoA (∼0.05 mm). Asp-NAT showed high specificity towards l-Asp with 3% or less activity towards l-Glu, l-Asn, l-Gln and Asp-Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed. 2003 Blackwell Publishing Journal Backfiles 1879-2005 |2003|||||||||| enzyme kinetics Chinopoulos, C. verfasserin aut Chandrasekaran, K. verfasserin aut Namboodiri, M. A. A. oth In Journal of neurochemistry Oxford : Wiley-Blackwell, 1956 86(2003), 4, Seite 0 Online-Ressource (DE-627)NLEJ243927584 (DE-600)2020528-4 1471-4159 nnns volume:86 year:2003 number:4 pages:0 http://dx.doi.org/10.1046/j.1471-4159.2003.01905.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 86 2003 4 0 |
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10.1046/j.1471-4159.2003.01905.x doi (DE-627)NLEJ243128029 DE-627 ger DE-627 rakwb Madhavarao, C. N. verfasserin aut Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain Oxford, UK Blackwell Science Ltd 2003 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp-NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp-NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated a single large protein (∼670 kDa), which on sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi-subunit complex Asp-NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) treatment followed by size-exclusion chromatography. Asp-NAT showed an order of magnitude difference in Km between l-aspartate (l-Asp, ∼0.5 mm) and acetyl CoA (∼0.05 mm). Asp-NAT showed high specificity towards l-Asp with 3% or less activity towards l-Glu, l-Asn, l-Gln and Asp-Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed. 2003 Blackwell Publishing Journal Backfiles 1879-2005 |2003|||||||||| enzyme kinetics Chinopoulos, C. verfasserin aut Chandrasekaran, K. verfasserin aut Namboodiri, M. A. A. oth In Journal of neurochemistry Oxford : Wiley-Blackwell, 1956 86(2003), 4, Seite 0 Online-Ressource (DE-627)NLEJ243927584 (DE-600)2020528-4 1471-4159 nnns volume:86 year:2003 number:4 pages:0 http://dx.doi.org/10.1046/j.1471-4159.2003.01905.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 86 2003 4 0 |
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Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain |
| abstract |
Aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp-NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp-NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated a single large protein (∼670 kDa), which on sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi-subunit complex Asp-NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) treatment followed by size-exclusion chromatography. Asp-NAT showed an order of magnitude difference in Km between l-aspartate (l-Asp, ∼0.5 mm) and acetyl CoA (∼0.05 mm). Asp-NAT showed high specificity towards l-Asp with 3% or less activity towards l-Glu, l-Asn, l-Gln and Asp-Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed. |
| abstractGer |
Aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp-NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp-NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated a single large protein (∼670 kDa), which on sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi-subunit complex Asp-NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) treatment followed by size-exclusion chromatography. Asp-NAT showed an order of magnitude difference in Km between l-aspartate (l-Asp, ∼0.5 mm) and acetyl CoA (∼0.05 mm). Asp-NAT showed high specificity towards l-Asp with 3% or less activity towards l-Glu, l-Asn, l-Gln and Asp-Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed. |
| abstract_unstemmed |
Aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp-NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp-NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated a single large protein (∼670 kDa), which on sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi-subunit complex Asp-NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) treatment followed by size-exclusion chromatography. Asp-NAT showed an order of magnitude difference in Km between l-aspartate (l-Asp, ∼0.5 mm) and acetyl CoA (∼0.05 mm). Asp-NAT showed high specificity towards l-Asp with 3% or less activity towards l-Glu, l-Asn, l-Gln and Asp-Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed. |
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| title_short |
Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain |
| url |
http://dx.doi.org/10.1046/j.1471-4159.2003.01905.x |
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| author2 |
Chinopoulos, C. Chandrasekaran, K. Namboodiri, M. A. A. |
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Chinopoulos, C. Chandrasekaran, K. Namboodiri, M. A. A. |
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10.1046/j.1471-4159.2003.01905.x |
| up_date |
2024-07-06T04:23:15.788Z |
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