Detection and identification of NAD-catabolizing activities in rat tissue homogenates.
NAD may be degraded in several ways. A large number of investigations have shown that at least those catabolic routes which involve the formation of ADP-ribose are related to regulatory processes. In this study a rapid assay was utilized that permits identification of NAD-degrading enzymes directly in sodium dodecylsulfate polyacrylamide gels. Enzymatic activities were recovered by washing the gels in the presence of mild detergents such as lauryl dimethylamine N-oxide or Triton X-100. Subsequent incubation of the gels in the presence of the fluorescent analog 1,N6 etheno-NAD visualized NAD-degrading enzymes. Following excision of the fluorescent bands from the gels, the actual activity of the proteins was established by incubating the gel slices with 14C-labeled NAD and subsequent product analysis by thin layer chromatography (TLC). Homogenates from rat renal cortex and spleen were analyzed by this procedure. While in the spleen homogenate only a single band could be 'activity-stained', in the kidney three bands were detected. Kidney proteins with apparent molecular masses of about 210,000 and 105,000 Da were identified as phosphodiesterase and NAD pyrophosphatase (alkaline phosphodiesterase I), respectively. The third protein exhibited an apparent molecular mass of 41,000. The spleen protein (apparent molecular mass 45,000 Da) cleaved NAD to nicotinamide and ADP-ribose identifying it as NAD glycohydrolase. The procedure is suitable to screen for NAD-converting activities in crude extracts. It is specific for proteins which function as monomers or homo-oligomers.
Hagen
T
T
Institut für Biochemie, Freie Universität Berlin, Germany.
Ziegler
M
M
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D003902:Detergents; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007672:Kidney Cortex / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000032:analysis / Q000378:metabolism; D009536:Niacinamide / Q000378:metabolism; D009537:Nicotinamide Mononucleotide / Q000378:metabolism; D011755:Pyrophosphatases / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013154:Spleen / Q000201:enzymology*; D015032:Zinc / Q000494:pharmacology
D003902:Detergents; D009243:NAD; D009537:Nicotinamide Mononucleotide; D000246:Adenosine Diphosphate Ribose; D009536:Niacinamide; D000249:Adenosine Monophosphate; D004492:Edetic Acid; D009244:NAD+ Nucleosidase; D011755:Pyrophosphatases; C024289:NAD pyrophosphatase; D015032:Zinc