Simultaneous single-sample determination of NMNAT isozyme activities in mouse tissues.
A novel assay procedure has been developed to allow simultaneous activity discrimination in crude tissue extracts of the three known mammalian nicotinamide mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1) isozymes. These enzymes catalyse the same key reaction for NAD biosynthesis in different...
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0053271&type=printable |
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| author | Giuseppe Orsomando Lucia Cialabrini Adolfo Amici Francesca Mazzola Silverio Ruggieri Laura Conforti Lucie Janeckova Michael P Coleman Giulio Magni |
| author_facet | Giuseppe Orsomando Lucia Cialabrini Adolfo Amici Francesca Mazzola Silverio Ruggieri Laura Conforti Lucie Janeckova Michael P Coleman Giulio Magni |
| author_sort | Giuseppe Orsomando |
| collection | DOAJ |
| description | A novel assay procedure has been developed to allow simultaneous activity discrimination in crude tissue extracts of the three known mammalian nicotinamide mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1) isozymes. These enzymes catalyse the same key reaction for NAD biosynthesis in different cellular compartments. The present method has been optimized for NMNAT isozymes derived from Mus musculus, a species often used as a model for NAD-biosynthesis-related physiology and disorders, such as peripheral neuropathies. Suitable assay conditions were initially assessed by exploiting the metal-ion dependence of each isozyme recombinantly expressed in bacteria, and further tested after mixing them in vitro. The variable contributions of the three individual isozymes to total NAD synthesis in the complex mixture was calculated by measuring reaction rates under three selected assay conditions, generating three linear simultaneous equations that can be solved by a substitution matrix calculation. Final assay validation was achieved in a tissue extract by comparing the activity and expression levels of individual isozymes, considering their distinctive catalytic efficiencies. Furthermore, considering the key role played by NMNAT activity in preserving axon integrity and physiological function, this assay procedure was applied to both liver and brain extracts from wild-type and Wallerian degeneration slow (Wld(S)) mouse. Wld(S) is a spontaneous mutation causing overexpression of NMNAT1 as a fusion protein, which protects injured axons through a gain-of-function. The results validate our method as a reliable determination of the contributions of the three isozymes to cellular NAD synthesis in different organelles and tissues, and in mutant animals such as Wld(S). |
| format | Article |
| id | doaj-art-e23865cca4fc4efbad968e00b4db03b7 |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-e23865cca4fc4efbad968e00b4db03b72025-08-20T02:32:14ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-01712e5327110.1371/journal.pone.0053271Simultaneous single-sample determination of NMNAT isozyme activities in mouse tissues.Giuseppe OrsomandoLucia CialabriniAdolfo AmiciFrancesca MazzolaSilverio RuggieriLaura ConfortiLucie JaneckovaMichael P ColemanGiulio MagniA novel assay procedure has been developed to allow simultaneous activity discrimination in crude tissue extracts of the three known mammalian nicotinamide mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1) isozymes. These enzymes catalyse the same key reaction for NAD biosynthesis in different cellular compartments. The present method has been optimized for NMNAT isozymes derived from Mus musculus, a species often used as a model for NAD-biosynthesis-related physiology and disorders, such as peripheral neuropathies. Suitable assay conditions were initially assessed by exploiting the metal-ion dependence of each isozyme recombinantly expressed in bacteria, and further tested after mixing them in vitro. The variable contributions of the three individual isozymes to total NAD synthesis in the complex mixture was calculated by measuring reaction rates under three selected assay conditions, generating three linear simultaneous equations that can be solved by a substitution matrix calculation. Final assay validation was achieved in a tissue extract by comparing the activity and expression levels of individual isozymes, considering their distinctive catalytic efficiencies. Furthermore, considering the key role played by NMNAT activity in preserving axon integrity and physiological function, this assay procedure was applied to both liver and brain extracts from wild-type and Wallerian degeneration slow (Wld(S)) mouse. Wld(S) is a spontaneous mutation causing overexpression of NMNAT1 as a fusion protein, which protects injured axons through a gain-of-function. The results validate our method as a reliable determination of the contributions of the three isozymes to cellular NAD synthesis in different organelles and tissues, and in mutant animals such as Wld(S).https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0053271&type=printable |
| spellingShingle | Giuseppe Orsomando Lucia Cialabrini Adolfo Amici Francesca Mazzola Silverio Ruggieri Laura Conforti Lucie Janeckova Michael P Coleman Giulio Magni Simultaneous single-sample determination of NMNAT isozyme activities in mouse tissues. PLoS ONE |
| title | Simultaneous single-sample determination of NMNAT isozyme activities in mouse tissues. |
| title_full | Simultaneous single-sample determination of NMNAT isozyme activities in mouse tissues. |
| title_fullStr | Simultaneous single-sample determination of NMNAT isozyme activities in mouse tissues. |
| title_full_unstemmed | Simultaneous single-sample determination of NMNAT isozyme activities in mouse tissues. |
| title_short | Simultaneous single-sample determination of NMNAT isozyme activities in mouse tissues. |
| title_sort | simultaneous single sample determination of nmnat isozyme activities in mouse tissues |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0053271&type=printable |
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