Purification and characterization of flavine-adenine dinucleotide phosphohydrolase from rat liver lysosomal membranes.
An enzyme hydrolyzing flavine-adenine dinucleotide (FAD) to flavine mononucleotide (FMN) and adenosine monophosphate (AMP) was purified about 460-fold over the isolated lysosomal membranes with 9% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and the absence of SDS. Purification procedures included: preparation of crude lysosomal membranes, solubilization with Triton X-100, WGA-Sepharose, Con A-Sepharose, hydroxylapatite chromatography, gel filtration with Superdex 200, DEAE ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme, estimated by gel filtration with Superdex 200, was approximately 560 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular weight of 140,000. The pH optimum for FAD hydrolysis was 8.5 with an apparent Km of 0.1 mM and the isoelectric point was pH 7.3. The activity was inhibited by o-phenanthroline, EDTA, DTT, and NEM and was slightly stimulated by Zn ion, but was not affected by Ca or Mg ions. The purified FADase contained N-linked complex type oligosaccharide chains lacking neuraminic acids. The NH2 terminal 21 amino acid residues of the purified FADase were Ser-Pro-Cys-Val-Cys-Asp-Pro-Val-Val-Val-Cys-Lys-Val-Val-Pro-Cys-Thr-Leu- Ala-Leu .
Kim
J K
JK
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka.
Ezaki
J
J
Himeno
M
M
Kato
K
K
Kim
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011755:Pyrophosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013194:Staining and Labeling; D013379:Substrate Specificity
D008670:Metals; D011755:Pyrophosphatases; C023417:FAD pyrophosphatase