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

1986 ◽  
Vol 239 (2) ◽  
pp. 393-403 ◽  
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.

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

Blood ◽  
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

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.

Phenobarbital-induced increase of NADH-cytochrome b5 reductase activity in rat liver microsonies

1978 ◽  
Vol 27 (3) ◽  
pp. 367-368 ◽  
Author(s):  
Ademar Vieira De Barros ◽  
Jean-Claude Kaplan ◽  
Philippe Duvaldestin ◽  
Pierre Berthelot
Keyword(s):  
Rat Liver ◽  
Reductase Activity ◽  
Cytochrome B5 ◽  
Cytochrome B5 Reductase ◽  
Nadh Cytochrome

scholarly journals Analysis of mutant NADH-cytochrome b5 reductase: apparent "type III" methemoglobinemia can be explained as type I with an unstable reductase

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 808-814 ◽  
Author(s):  
T Nagai ◽  
K Shirabe ◽  
T Yubisui ◽  
M Takeshita
Keyword(s):  
Enzyme Activity ◽  
Cytochrome B5 ◽  
Base Change ◽  
Mutant Enzyme ◽  
Type I ◽  
Wild Type ◽  
Cytochrome B5 Reductase ◽  
Type Iii ◽  
Patient Reported ◽  
Nadh Cytochrome

Abstract A patient in Kurobe, Japan, was previously reported to have a new class of hereditary methemoglobinemia, type III. In this patient, NADH cytochrome b5 reductase deficiency was observed in lymphocytes and platelets as well as in erythrocytes, but this was not associated with mental retardation. A base change was identified in the gene for NADH cytochrome b5 reductase, causing an amino acid substitution from Leu- 148 to Pro. In the present study, the mutant enzyme (Leu-148-->Pro) was expressed in Escherichia coli, purified, and characterized. The mutant enzyme retained about 60% of the catalytic activity of the wild type, but was remarkably heat unstable. By incubating the mutant enzyme at 42 degrees C for 10 minutes, 80% of the enzyme activity was lost, whereas the wild-type enzyme lost < 20% activity after incubation at 50 degrees C for 30 minutes. Another mutant in which Leu-148 was replaced by Ala was prepared to establish the role of the residue. This mutant was apparently less heat stable than the wild type, implying a structural role for Leu-148. Reinvestigation of the enzyme activity in the blood cells and fibroblasts of the type III Kurobe patient, revealed that about 40% of the normal activity was detected in these cells, in contrast to the previous report. Thus, this patient reported previously as having hereditary meth-hemoglobinemia type III was shown to have type I.

scholarly journals Localization and biosynthesis of NADH-cytochrome b5 reductase, an integral membrane protein, in rat liver cells. I. Distribution of the enzyme activity in microsomes, mitochondria, and golgi complex.

1980 ◽  
Vol 85 (3) ◽  
pp. 501-515 ◽  
Author(s):  
N Borgese ◽  
J Meldolesi
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Golgi Complex ◽  
Cytochrome B5 ◽  
Liver Cells ◽  
Cytochrome C Reductase ◽  
Cytochrome B5 Reductase ◽  
Galactosyl Transferase ◽  
Rat Liver Cells ◽  
Nadh Cytochrome

The subcellular distribution of NADH-cytochrome b5 reductase in rat liver cells was reinvestigated. In fresh heavy and light Golgi fractions (GF3 and GF1 + 2) and in mitochondria, the specific activity of rotenone-insensitive NADH-cytochrome c reductase was approximately 100, 60, and 30%, respectively, of the value found in microsomes. However, the Golgi enzyme was unstable inasmuch as pelleting and resuspending the fresh fractions resulted in a considerable inactivation (40--60%), which was further increased with subsequent storage at 4 degrees C. A similar inactivation was observed using cytochrome b5 but not ferricyanide as electron acceptor. The inactivation of Golgi NADH-cytochrome c reductase activity was independent of the protein concentration of the fractions during storage, was unaffected by the addition of the antioxidant butylated hydroxytoluene, but was partly prevented by buffering the fractions at neutral pH and by storage at--20 degrees C. A total Golgi fraction was analyzed by density equilibration on continuous sucrose gradients after exposure to digitonin. As expected, the distribution of both protein and galactosyl transferase were shifted to higher densities by this treatment. However, not all galactosyl transferase-bearing elements were shifted to the same extent by exposure to the detergent, suggesting a biochemical heterogeneity of the Golgi complex. In contrast to their behavior in microsomes, the distribution of NADH-cytochrome c reductase and cytochrome b5 of Golgi fractions was shifted by digitonin, although to a lesser extent than that of galactosyl transferase. These results indicate that NADH-cytochrome b5 reductase is an authentic component of Golgi membranes, as well as of microsomes and of mitochondria. The conflicting results reported in the past on the Golgi localization of the enzyme could be due, on the one hand, to the differential lability of the activity in its various subcellular locations and, on the other, to the heterogeneity of the Golgi complex in terms of both cholesterol and enzyme distribution.

scholarly journals Analysis of mutant NADH-cytochrome b5 reductase: apparent "type III" methemoglobinemia can be explained as type I with an unstable reductase

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 808-814 ◽  
Author(s):  
T Nagai ◽  
K Shirabe ◽  
T Yubisui ◽  
M Takeshita
Keyword(s):  
Enzyme Activity ◽  
Cytochrome B5 ◽  
Base Change ◽  
Mutant Enzyme ◽  
Type I ◽  
Wild Type ◽  
Cytochrome B5 Reductase ◽  
Type Iii ◽  
Patient Reported ◽  
Nadh Cytochrome

A patient in Kurobe, Japan, was previously reported to have a new class of hereditary methemoglobinemia, type III. In this patient, NADH cytochrome b5 reductase deficiency was observed in lymphocytes and platelets as well as in erythrocytes, but this was not associated with mental retardation. A base change was identified in the gene for NADH cytochrome b5 reductase, causing an amino acid substitution from Leu- 148 to Pro. In the present study, the mutant enzyme (Leu-148-->Pro) was expressed in Escherichia coli, purified, and characterized. The mutant enzyme retained about 60% of the catalytic activity of the wild type, but was remarkably heat unstable. By incubating the mutant enzyme at 42 degrees C for 10 minutes, 80% of the enzyme activity was lost, whereas the wild-type enzyme lost < 20% activity after incubation at 50 degrees C for 30 minutes. Another mutant in which Leu-148 was replaced by Ala was prepared to establish the role of the residue. This mutant was apparently less heat stable than the wild type, implying a structural role for Leu-148. Reinvestigation of the enzyme activity in the blood cells and fibroblasts of the type III Kurobe patient, revealed that about 40% of the normal activity was detected in these cells, in contrast to the previous report. Thus, this patient reported previously as having hereditary meth-hemoglobinemia type III was shown to have type I.

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

Blood ◽  
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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