scholarly journals Endogenous proteolysis of membrane-bound red cell cytochrome-b5 reductase in adults and newborns: its possible relevance to the generation of the soluble "methemoglobin reductase"

Blood ◽  
1983 ◽  
Vol 61 (5) ◽  
pp. 894-898 ◽  
Author(s):  
D Choury ◽  
A Reghis ◽  
AL Pichard ◽  
JC Kaplan
Keyword(s):  
Current Knowledge ◽  
Reductase Activity ◽  
Cytochrome B5 ◽  
Red Cells ◽  
Cytochrome B5 Reductase ◽  
Calcium Dependent ◽  
Membrane Bound ◽  
Relative Deficiency ◽  
Low Activity

Abstract The problem of the low activity of so-called methemoglobin reductase in red cells from newborns was reinvestigated in view of our current knowledge of this enzyme, i.e., (1) its being cytochrome-b5 reductase and (2) its presence in two forms: soluble and membrane-bound. We found that red cells from cord blood and newborns exhibited a 50% decrease of soluble cytochrome-b5 reductase activity, whereas membrane-bound activity was in the adult range. Ghosts from these cells possessed diminished ability to solubilize membrane-bound cytochrome-b5 reductase in the course of in vitro auto-incubation. This autosolubilizing ability increased with age and reached adult level concomitantly with soluble cytochrome-b5 reductase activity at 6 mo. We conclude that the relative deficiency of soluble cytochrome-b5 reductase observed at birth is due to diminished post-translational processing of the membrane-bound enzyme during erythropoiesis of fetal cells. This processing is calcium-dependent related to calmodulin.

scholarly journals Endogenous proteolysis of membrane-bound red cell cytochrome-b5 reductase in adults and newborns: its possible relevance to the generation of the soluble "methemoglobin reductase"

Blood ◽  
1983 ◽  
Vol 61 (5) ◽  
pp. 894-898
Author(s):  
D Choury ◽  
A Reghis ◽  
AL Pichard ◽  
JC Kaplan
Keyword(s):  
Current Knowledge ◽  
Reductase Activity ◽  
Cytochrome B5 ◽  
Red Cells ◽  
Cytochrome B5 Reductase ◽  
Calcium Dependent ◽  
Membrane Bound ◽  
Relative Deficiency ◽  
Low Activity

The problem of the low activity of so-called methemoglobin reductase in red cells from newborns was reinvestigated in view of our current knowledge of this enzyme, i.e., (1) its being cytochrome-b5 reductase and (2) its presence in two forms: soluble and membrane-bound. We found that red cells from cord blood and newborns exhibited a 50% decrease of soluble cytochrome-b5 reductase activity, whereas membrane-bound activity was in the adult range. Ghosts from these cells possessed diminished ability to solubilize membrane-bound cytochrome-b5 reductase in the course of in vitro auto-incubation. This autosolubilizing ability increased with age and reached adult level concomitantly with soluble cytochrome-b5 reductase activity at 6 mo. We conclude that the relative deficiency of soluble cytochrome-b5 reductase observed at birth is due to diminished post-translational processing of the membrane-bound enzyme during erythropoiesis of fetal cells. This processing is calcium-dependent related to calmodulin.

Transcriptional and translational mechanisms of cytochrome b5 reductase isoenzyme generation in humans

2001 ◽  
Vol 355 (2) ◽  
pp. 529-535 ◽  
Author(s):  
Alena LEROUX ◽  
Luisa MOTA VIEIRA ◽  
Axel KAHN
Keyword(s):  
Cytochrome B5 ◽  
Protein Isoforms ◽  
In Vitro Translation ◽  
Alu Elements ◽  
Cytochrome B5 Reductase ◽  
Dynamic Events ◽  
Membrane Bound ◽  
Essential Enzyme ◽  
Translational Mechanisms

Cytochrome b5 reductase (b5R) is an essential enzyme that exists in soluble and membrane-bound isoforms, each with specific functions. In the rat, the two forms are generated from alternative transcripts differing in the first exons. In contrast, the biogenesis of b5R isoforms in the human is not yet well understood. In the present study we have detected three novel alternative exons, designated 1S, S′ and 1B, located between the first alternative exon 1M and the common second exon in the human b5R gene. Accordingly, multiple M-type, S-type and SS′-type and B-type transcripts are generated. All types of human b5R transcript are expressed ubiquitously. An analysis of in vitro translation products demonstrated an alternative use of different AUG initiators resulting in the production of various human b5R protein isoforms. Our results indicate that the organization of the 5′ region of the b5R gene is not conserved between rodents and humans. Insertion of Alu elements into the human b5R gene, in particular just upstream of the S/S′ region, could be responsible for dynamic events of gene rearrangement during evolution.

scholarly journals Methemoglobin Reductase (Cytochrome b5 Reductase) Deficiency in Congenital Methemoglobinemia

Blood ◽  
1974 ◽  
Vol 44 (6) ◽  
pp. 879-884 ◽  
Author(s):  
Takeshi Kitao ◽  
Yoshiki Sugita ◽  
Yoshimasa Yoneyama ◽  
Kenichi Hattori
Keyword(s):  
Reductase Activity ◽  
Cytochrome B5 ◽  
Electrophoretic Pattern ◽  
Heat Stability ◽  
Red Cells ◽  
Reductase Deficiency ◽  
Cytochrome B5 Reductase ◽  
Diaphorase Activity ◽  
Enzymatic Reduction ◽  
Normal Enzyme

Abstract Two NADH diaphorases, diaphorase I and II, were isolated from normal red cells and congenital methemoglobinemic red cells by CM-cellulose and DE 32 column chromatography. For methemoglobinemic sample, activities of diaphorase I and diaphorase II were 80% and less than 5% of those for the normal red cells, respectively. Only diaphorase II showed cytochrome b5 reductase activity. The cytochrome b5 reductase deficiency seems to manifest methemoglobinemia through the decrease in the enzymatic reduction of cytochrome b5 and subsequent nonenzymatic reduction of methemoglobin by the reduced cytochrome b5. The methemoglobinemic diaphorase II was found similar to the normal enzyme with respect to Kms for the dye and NADH, heat stability, effect of pH, and electrophoretic pattern. The ratio of the diaphorase activity to the cytochrome b5 reductase activity was the same for both enzymes. Although the production of an abnormal enzyme molecule can not be excluded, it is possible that in this case the rate of enzyme formation is decreased.

scholarly journals Properties of NADH-cytochrome-b5 reductase from human neutrophils

Blood ◽  
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.

scholarly journals Properties of NADH-cytochrome-b5 reductase from human neutrophils

Blood ◽  
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

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.

Abstract 15696: Cytochrome B5 Reductase 3 Sensitizes Soluble Guanylate Cyclase to Nitric Oxide

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Anh T Nguyen ◽  
Mizanur M Rahaman ◽  
Stephanie M Mutchler ◽  
Megan Miller ◽  
Josef T Prchal ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Blood Pressure ◽  
Soluble Guanylate Cyclase ◽  
Cytochrome B5 ◽  
Heme Iron ◽  
Proximity Ligation Assay ◽  
Cytochrome B5 Reductase ◽  
Cgmp Production ◽  
Iron Reductase

Impaired soluble guanylyl cyclase (sGC)-dependent nitric oxide (NO) signaling has been linked to numerous cardiovascular diseases (CVD) such as hypertension, myocardial infarction and atherosclerosis. Despite emerging evidence indicating the importance of sGC function within the cardiovascular system, the basic mechanisms that regulate sGC activity remain incompletely understood. Herein, we provide in vitro and in vivo evidence that cytochrome b5 reductase 3 (Cyb5R3) is an sGC heme iron reductase and regulates downstream cGMP signaling. Of major significance, we also demonstrate that a Cyb5R3 T116S polymorphism with allele frequency of 0.23 in African Americans associates with increase blood pressure and is incapable of reducing sGC. Proximity ligation assay (PLA) experiments show that endogenous Cyb5R3 and oxidized sGC associate. Knockdown of Cyb5R3 results in reduced cGMP production and downstream signaling in rat aortic smooth muscle cells (SMC). Overexpression of Cyb5R3 not only rescues cGMP production but also increases baseline cGMP, whereas T116S mutant does not. Finally, inhibition of Cyb5R3 in mice significantly increases systemic blood pressure. Our studies are the first to identify an sGC heme iron reductase, provide evidence for Cyb5R3 as a key biological regulator of sGC activity and vascular tone in SMC, and link a human polymorphism of Cyb5R3 to increased blood pressure; all of which may lead to the development of novel therapeutics targeting Cyb5R3 for the treatment of CVD. Importantly, the co-expression of Cyb5R3 and sGC in multiple cells types suggests that this regulation of sGC activity may have broad applications for multiple physiological and pathophysiological processes. Results: Conclusions:

scholarly journals Resolution of Distinct Membrane-Bound Enzymes from Enterobacter cloacae SLD1a-1 That Are Responsible for Selective Reduction of Nitrate and Selenate Oxyanions

2006 ◽  
Vol 72 (8) ◽  
pp. 5173-5180 ◽  
Author(s):  
Helen Ridley ◽  
Carys A. Watts ◽  
David J. Richardson ◽  
Clive S. Butler
Keyword(s):  
Nitrate Reductase ◽  
Reductase Activity ◽  
Selective Reduction ◽  
Enterobacter Cloacae ◽  
Anaerobic Growth ◽  
Elemental Selenium ◽  
Membrane Bound ◽  
Membrane Bound Enzymes ◽  
Distinct Membrane

ABSTRACT Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with α and β subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (αβγ) complex with an apparent molecular mass of ∼600 kDa. The individual subunit sizes are ∼100 kDa (α), ∼55 kDa (β), and ∼36 kDa (γ), with a predicted overall subunit composition of α3β3γ3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be ∼2 mM, with an observed V max of 500 nmol SeO4 2− min−1 mg−1 (k cat, ∼5.0 s−1). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.

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