Purification and characterization of the human neutrophil NADH- cytochrome b5 reductase

Abstract 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.

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Purification and characterization of the human neutrophil NADH- cytochrome b5 reductase

Blood ◽

10.1182/blood.v66.3.673.bloodjournal663673 ◽

1985 ◽

Vol 66 (3) ◽

pp. 673-678

Author(s):

AI Tauber ◽

J Wright ◽

FK Higson ◽

SA Edelman ◽

DJ Waxman

Keyword(s):

Plasma Membrane ◽

Human Neutrophil ◽

Membrane Fraction ◽

Cytochrome B5 ◽

Sodium Dodecyl ◽

High Specificity ◽

Biochemical Properties ◽

Human Neutrophils ◽

Cytochrome B5 Reductase ◽

Nadh Cytochrome

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.

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Distribution of the integral membrane protein NADH-cytochrome b5 reductase in rat liver cells, studied with a quantitative radioimmunoblotting assay

Biochemical Journal ◽

10.1042/bj2390393 ◽

1986 ◽

Vol 239 (2) ◽

pp. 393-403 ◽

Cited By ~ 33

Author(s):

N Borgese ◽

G Pietrini

Keyword(s):

Enzyme Activity ◽

Plasma Membrane ◽

Membrane Protein ◽

Rat Liver ◽

Cytochrome B5 ◽

Integral Membrane Protein ◽

Subcellular Fractions ◽

Outer Membranes ◽

Cytochrome B5 Reductase ◽

Nadh Cytochrome

The intracellular localization of the post-translationally inserted integral membrane protein, NADH-cytochrome b5 reductase, was investigated, using a quantitative radioimmunoblotting method to determine its concentration in rat liver subcellular fractions. Subcellular fractions enriched in rough or smooth microsomes, Golgi, lysosomes, plasma membrane and mitochondrial inner or outer membranes were characterized by marker enzyme analysis and electron microscopy. Reductase levels were determined both with the NADH-cytochrome c reductase activity assay, and by radioimmunoblotting, and the results of the two methods were compared. When measured as antigen, the reductase was relatively less concentrated in microsomal subfractions, and more concentrated in fractions containing outer mitochondrial membranes, lysosomes and plasma membrane than when measured as enzyme activity. Rough and smooth microsomes had 4-5-fold lower concentrations, on a phospholipid basis than did mitochondrial outer membranes. Fractions containing Golgi, lysosomes and plasma membrane had approximately 14-, approximately 16, and approximately 9-fold lower concentrations of antigen than did mitochondrial outer membranes, respectively, and much of the antigen in these fractions could be accounted for by cross-contamination. No enzyme activity or antigen was detected in mitochondrial inner membranes. Our results indicate that the enzyme activity data do not precisely reflect the true enzyme localization, and show an extremely uneven distribution of reductase among different cellular membranes.

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Properties of NADH-cytochrome-b5 reductase from human neutrophils

Blood ◽

10.1182/blood.v62.1.152.bloodjournal621152 ◽

1983 ◽

Vol 62 (1) ◽

pp. 152-157

Author(s):

JA Badwey ◽

AI Tauber ◽

ML Karnovsky

Keyword(s):

Reductase Activity ◽

Cytochrome B5 ◽

Extraction Procedure ◽

Human Neutrophils ◽

Ph Optimum ◽

Beef Liver ◽

Cytochrome B5 Reductase ◽

Ferricyanide Reductase ◽

Membrane Bound ◽

Nadh Cytochrome

An NADH-ferricyanide reductase activity of ca. 170 nmole ferricyanide reduced/min/10(7) cells is present in the membrane fraction of human neutrophils. This membrane-bound activity constitutes ca. 85% of the total NADH-ferricyanide reductase activity that is present in these cells. The enzyme(s) readily utilize(s) purified cytochrome-b5 from beef liver as an electron acceptor. No other physiologic electron acceptors tested (e.g., ubiquinone-30, menadione) were active. The specificities of electron donors (e.g., NADH congruent to deamino-NADH much greater than NADPH) and acceptors (e.g., Fe(CN)6–3 greater than 2,6-dichlorophenol-indophenol much greater than O2) for the enzyme(s) in unfractionated membranes, along with action of inhibitors (e.g., ADP, p-chloromercuribenzoate) and the pH optimum, indicate that virtually all of the membrane-bound ferricyanide reductase activity in these cells is NADH-cytochrome-b5 reductase. This reductase, however, is only slightly solubilized (ca. 10%) by a phosphate buffer extraction procedure that is effective with the liver enzyme.

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Properties of NADH-cytochrome-b5 reductase from human neutrophils

Blood ◽

10.1182/blood.v62.1.152.152 ◽

1983 ◽

Vol 62 (1) ◽

pp. 152-157 ◽

Cited By ~ 20

Author(s):

JA Badwey ◽

AI Tauber ◽

ML Karnovsky

Keyword(s):

Reductase Activity ◽

Cytochrome B5 ◽

Extraction Procedure ◽

Human Neutrophils ◽

Ph Optimum ◽

Beef Liver ◽

Cytochrome B5 Reductase ◽

Ferricyanide Reductase ◽

Membrane Bound ◽

Nadh Cytochrome

Abstract An NADH-ferricyanide reductase activity of ca. 170 nmole ferricyanide reduced/min/10(7) cells is present in the membrane fraction of human neutrophils. This membrane-bound activity constitutes ca. 85% of the total NADH-ferricyanide reductase activity that is present in these cells. The enzyme(s) readily utilize(s) purified cytochrome-b5 from beef liver as an electron acceptor. No other physiologic electron acceptors tested (e.g., ubiquinone-30, menadione) were active. The specificities of electron donors (e.g., NADH congruent to deamino-NADH much greater than NADPH) and acceptors (e.g., Fe(CN)6–3 greater than 2,6-dichlorophenol-indophenol much greater than O2) for the enzyme(s) in unfractionated membranes, along with action of inhibitors (e.g., ADP, p-chloromercuribenzoate) and the pH optimum, indicate that virtually all of the membrane-bound ferricyanide reductase activity in these cells is NADH-cytochrome-b5 reductase. This reductase, however, is only slightly solubilized (ca. 10%) by a phosphate buffer extraction procedure that is effective with the liver enzyme.

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