Simultaneous Single-Sample Determination of NMNAT Isozyme Activities in Mouse Tissues

Giuseppe Orsomando,Giulio Magni,Silverio Ruggieri,Lucie Janeckova,Lucia Cialabrini,Adolfo Amici,Francesca Mazzola,Laura Conforti,Michael P Coleman,Vasu D Appanna

doi:10.1371/journal.pone.0053271

Abstract

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 (WldS) mouse. WldS 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 WldS.

Highlights

The reaction catalysed by nicotinamide mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1) is universal in living cells and the sole known source of pyridine dinucleotides
Human mitochondrial NMNAT3 is a homo-tetramer of 28 kDa subunits [19], whose expression pattern show little overlap with NMNAT2, and which is less abundant than NMNAT1 [13,14,19]
In the presence of various concentrations of the chloride salts of both Mg2+ and Zn2+ (Fig. 3B and C), the resulting metal-dependence of mNMNATs was largely similar to the corresponding human isozymes [13], with the exception of mNMNAT1 and Wallerian degeneration slow (WldS), whose Mg2+-dependence was superimposable with mNMNAT2 (Fig. 3B). These results show that the structurally-related mNMNAT1 and WldS enzymes are catalytically indistinguishable in all conditions tested

Summary

Introduction

The reaction catalysed by nicotinamide mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1) is universal in living cells and the sole known source of pyridine dinucleotides. The reverse reaction is most likely limited by the physiological absence of PPi. In mammals, where the nicotinamide salvage pathway generates most NAD, the metabolic flux is mainly controlled by the preceding enzyme in the salvage pathway, nicotinamide phosphoribosyltransferase [4], which can be inhibited by FK866 [5,6]. In mammals, where the nicotinamide salvage pathway generates most NAD, the metabolic flux is mainly controlled by the preceding enzyme in the salvage pathway, nicotinamide phosphoribosyltransferase [4], which can be inhibited by FK866 [5,6] This highly conserved enzyme [7,8,9,10] has three isoforms in mammals arising from multiple genes and showing distinctive oligomerization properties, subcellular localization, and tissue distribution [11,12,13,14]. One exception is human erythrocytes, which have a remarkably high level of NMNAT3 relative to NMNAT1 [20]

Methods

Results

Conclusion