Purification and characterization of the human neutrophil NADH-cytochrome b5 reductase.
NADH-cytochrome b5 reductase is the predominant NADH-diaphorase found in the human neutrophil (Blood 62:152, 1983). Although this reductase segregates with the light membranes of nitrogen-cavitated neutrophils separated on Percoll gradients (which include the plasma membrane markers alkaline phosphatase and NADPH-oxidase), it is approximately 95% excluded from plasma membrane-enriched phagocytic vacuoles. The reductase constitutes approximately 5% of the light membrane fraction FAD-flavoprotein (14.8 +/- 5.5 pmol/mg protein) and was found in equimolar concentration with a high potential b cytochrome also present in this light membrane fraction and tentatively identified as cytochrome b5. Isolation of the reductase from human neutrophils was accomplished by Triton X-114 solubilization of the light Percoll gradient membranes, followed by temperature-dependent phase separation and then affinity chromatography on AMP-Sepharose. The active preparation contained 1.3 mol FAD/mol protein, migrated on sodium dodecyl sulfate-polyacrylamide gels as a single band corresponding to an apparent mol wt of 45,000 daltons, exhibited a pl of 5.7 on chromatofocusing and was obtained in greater than 70% yield, with an overall purification of almost 900-fold. The purified enzyme was characterized by a high specificity for NADH as electron donor (Km = 6.4 mumol/L v Km greater than 1.6 mmol/L for NADPH) and exhibited a maximal turnover of ca. 30,000 min-1 at 22 degrees C with either ferricyanide or cytochrome b5 (Km = 10 nmol/L) as electron acceptor. Although the physical characterization and biochemical properties described here demonstrate that this neutrophil NADH b5 reductase is similar to the corresponding liver and erythrocyte enzymes, its unique function in the neutrophil has yet to be determined.
Tauber
A I
AI
Wright
J
J
Higson
F K
FK
Edelman
S A
SA
Waxman
D J
DJ
D002469:Cell Separation; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003579:Cytochrome Reductases / Q000097:blood / Q000302:isolation & purification*; D003573:Cytochrome b Group / Q000097:blood / Q000302:isolation & purification; D042966:Cytochrome-B(5) Reductase; D006801:Humans; D008099:Liver / Q000201:enzymology; D009504:Neutrophils / Q000201:enzymology*; D011205:Povidone; D012822:Silicon Dioxide; D013347:Subcellular Fractions / Q000201:enzymology
D003573:Cytochrome b Group; C016039:Percoll; D012822:Silicon Dioxide; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D011205:Povidone