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 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)

Catabolite inactivation of bakers'-yeast uridine nucleosidase. Isolation and partial purification of a specific proteolytic inactivase.

When bakers’ yeast cells are grown in culture media containing ethanol instead of glucose, uridine nucleosidase specific activity increased up to 10 times its original value. Addition of glucose to ethanol- grown cells caused a rapid drop of uridine nucleosidase activity. This inactivation could be prevented by the presence of phenylmethylsulfonyl fluoride. Experiments with cycloheximide indicated that both the inactivation and the reappearance of the enzyme activity require protein synthesis. In addition a fraction has been isolated and partially purified from bakers’ yeast that is able to inactivate uridine nucleosidase in vitro. The inactivating fraction is not dialysable, and can be purified using conventional protein purification procedure, suggesting its protein character. The pH optimum of the inactivating activity is around 5 and the factor shows a molecular weight of 75000. Glucose-6- phosphate dehydrogenase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, cytidine de- aminase, cytosine deaminase, 3-phosphoglycerate kinase and glutathione reductase were not inac- tivated by the preparation. Bakers’ yeast boiled crude extract supernatant contained an inhibitor of the inactivating factor. The inactivating protein is endowed also with proteolytic activity and the inhibition exerted by phenylmethylsulfonyl fluoride and by the endogeneous inhibitor indicate that the factor is a protease