A rat liver lysosomal membrane flavin-adenine dinucleotide phosphohydrolase: purification and characterization.
An enzyme hydrolyzing flavin-adenine dinucleotide (FAD) to flavin mononucleotide and AMP was identified and purified from rat liver lysosomal (Tritosomal) membranes. The purified enzyme showed a single band on silver-stained denaturing gels with an apparent Mr 70,000. Periodate-Schiff staining after denaturing gel electrophoresis of whole membrane preparations revealed that this enzyme is one of the major glycoproteins in lysosomal membranes. FAD appeared to be the preferred substrate for the purified enzyme; equivalent concentrations of NAD or CoA were hydrolyzed at about one-half of the FAD rate. Negligible activity (less than or equal to 16%) was noted with ATP, TTP, ADP, AMP, FMN, pyrophosphate, or p-nitrophenylphosphate. The enzyme was inhibited by EDTA or dithiothreitol. It was stimulated by Zn, and was not affected by Ca or Mg ions, nor by p-chloromercuribenzoate. The pH optimum for FAD hydrolysis was 8.5-9 with an apparent Km of 0.125 mM. Antibodies prepared against the purified enzyme partially (50%) inhibited FAD phosphohydrolase activity in lysosomal membrane preparations but had no effect on the soluble lysosomal acid pyrophosphatase known to hydrolyze FAD. This enzyme could not be detected immunochemically in preparations of microsomes, Golgi, plasma membranes, mitochondrial membranes, or the soluble lysosomal fraction, suggesting that the enzyme is different from either soluble lysosomal acid pyrophosphatase or other FAD hydrolyzing activities in the liver cell.
Shin
H J
HJ
Biology Department, University of Alabama, Tuscaloosa 35487.
Mego
J L
JL
D000249:Adenosine Monophosphate / Q000096:biosynthesis; D000818:Animals; D003065:Coenzyme A / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005486:Flavin Mononucleotide / Q000096:biosynthesis; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D009243:NAD / Q000378:metabolism; D011755:Pyrophosphatases / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D015032:Zinc / Q000494:pharmacology
D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D000249:Adenosine Monophosphate; D005486:Flavin Mononucleotide; D004492:Edetic Acid; D011755:Pyrophosphatases; C023417:FAD pyrophosphatase; D015032:Zinc; D003065:Coenzyme A; D004229:Dithiothreitol