Heterologous expression and site-directed mutagenesis of an ascorbate-reducible cytochrome b561

Bérczi, Alajos; Su, Dan; Lakshminarasimhan, Mahadevan; Vargas, Amy; Asard, Han

doi:10.1016/j.abb.2005.09.006

Cited by 32 publications

(62 citation statements)

References 53 publications

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“…Reduction by Asc (25 mM) reached 79.56 Ϯ 3.97% of the reduction level obtained with dithionite (2 mM) (n ϭ 3), as is also observed for most other Cyts b 561 (30,33). As reported previously (30), no such characteristic peak was detected in stripped membrane fractions from yeast cells transformed with the empty pESC-His vector (data not shown).…”

Section: Evidence That Dcytb Is An Mdha Reductase In Humanmentioning

confidence: 46%

“…Cells were washed with ice-cold homogenization buffer (50 mM Mops-KOH, pH 7.0, 5 mM EDTA, 100 mM KCl, and 100 mM sucrose) and broken in a bead beater (Biospec Products, Bartlesville, OK). Yeast protein extraction and membrane preparation were done as reported previously (30).…”

Section: Methodsmentioning

confidence: 99%

“…DCytb was also heterologously expressed in yeast cells and HEK-293 cells, although in these cells another band of ϳ52 kDa was observed. In fact, a band of 50 -55 kDa is often observed for each Cyt b 561 and is believed to represent an aggregation product produced by freezing and thawing of samples (30,38).…”

Section: Dcytb Expression In Erythrocytementioning

confidence: 99%

“…It has been observed that recombinant and native plant and mammalian Cyts b 561 have characteristic Asc-reducible spectra (30,33,(41)(42)(43)(44). To confirm this biochemical characteristic for DCytb, the absorption spectra were measured using stripped membrane fractions from yeast cells expressing mouse DCytb (Fig.…”

Section: Evidence That Dcytb Is An Mdha Reductase In Humanmentioning

confidence: 99%

“…Protein Gels and Western Blot-Proteins were resolved by SDS-PAGE using 12% acrylamide gels. Samples were not heated or boiled before loading on the gels, because this caused Cyt b 561 aggregation and prevented them from penetrating the gel (30,33). After SDS-PAGE, proteins were transferred onto a polyvinylidene difluoride membrane with a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad).…”

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confidence: 99%

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Human Erythrocyte Membranes Contain a Cytochrome b561 That May Be Involved in Extracellular Ascorbate Recycling

May

Koury

et al. 2006

Journal of Biological Chemistry

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Human erythrocytes contain an unidentified plasma membrane redox system that can reduce extracellular monodehydroascorbate by using intracellular ascorbate (Asc) as an electron donor. Here we show that human erythrocyte membranes contain a cytochrome b 561 (Cyt b 561 ) and hypothesize that it may be responsible for this activity. Of three evolutionarily closely related Cyts b 561 , immunoblots of human erythrocyte membranes showed only the duodenal cytochrome b 561 (DCytb) isoform. DCytb was also found in guinea pig erythrocyte membranes but not in erythrocyte membranes from the mouse or rat. Mouse erythrocytes lost a majority of the DCytb in the late erythroblast stage during erythropoiesis. Absorption spectroscopy showed that human erythrocyte membranes contain an Asc-reducible b-type Cyt having the same spectral characteristics as recombinant DCytb and biphasic reduction kinetics, similar to those of the chromaffin granule Cyt b 561 . In contrast, mouse erythrocytes did not exhibit Asc-reducible b-type Cyt activity. Furthermore, in contrast to mouse erythrocytes, human erythrocytes much more effectively preserved extracellular Asc and transferred electrons from intracellular Asc to extracellular ferricyanide. These results suggest that the DCytb present in human erythrocytes may contribute to their ability to reduce extracellular monodehydroascorbate.

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Section: Evidence That Dcytb Is An Mdha Reductase In Humanmentioning

confidence: 46%

Section: Methodsmentioning

confidence: 99%

Section: Dcytb Expression In Erythrocytementioning

confidence: 99%

Section: Evidence That Dcytb Is An Mdha Reductase In Humanmentioning

confidence: 99%

mentioning

confidence: 99%

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Human Erythrocyte Membranes Contain a Cytochrome b561 That May Be Involved in Extracellular Ascorbate Recycling

May

Koury

et al. 2006

Journal of Biological Chemistry

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Spectral characterization of the recombinant mouse tumor suppressor 101F6 protein

et al. 2009

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Tumor suppressor protein 101F6, a gene product of the 3p21.3 (human) and 9F1 (mouse) chromosomal region, has recently been identified as a member of the cytochrome b561 (Cyt-b561) protein family by sequence homology. The His(6)-tagged recombinant mouse tumor suppressor Cyt-b561 protein (TSCytb) was recently expressed in yeast and purified, and the ascorbate reducibility was determined. TSCytb is auto-oxidizable and has two distinct heme b centers with redox potentials of approximately 40 and approximately 140 mV. Its split alpha-band in the dithionite-reduced spectrum at both 295 and 77 K is well resolved, and the separation between the two alpha-peaks is approximately 7 nm (approximately 222 cm(-1)). Singular value decomposition analysis of the split alpha-band in the ascorbate-reduced spectra revealed the presence of two major spectral components, each of them with split alpha-band but with different peak separations (6 and 8 nm). Similar minor differences in peak separation were obtained when the split alpha-bands in ascorbate-reduced difference spectra at low (<1 mM) and high (>10 mM) ascorbate concentrations were analysed. According to low-temperature electron paramagnetic resonance (EPR) spectroscopy, the two heme b centers are in the low-spin ferric state with maximum principal g values of 3.61 and 2.96, respectively. These values differ from the ones observed for other members of the Cyt-b561 family. According to resonance Raman spectroscopy, the porphyrin rings are in a relaxed state. The spectroscopic results are only partially in agreement with those obtained earlier for the native chromaffin granule Cyt-b561.

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Dihydrolipoic acid reduces cytochrome b561 proteins

2012

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Cytochrome b561 (Cyt-b561) proteins constitute a family of trans-membrane proteins which are present in a wide variety of organisms. Two of their characteristic properties are the reducibility by ascorbate (ASC) and the presence of two distinct b-type hemes localized on two opposite sides of the membrane. Here we show that the tonoplast-localized (TCB) and the putative tumour suppressor (TSCB) Cyt-b561 proteins can be reduced by other reductants than ASC and dithionite. A detailed spectral analysis of the ASC-dependent and dihydrolipoic acid (DHLA)-dependent reduction of these two Cyt-b561 proteins are also presented. Our results are discussed in relation to the known antioxidant capability of DHLA as well as its role in the regeneration of other antioxidant compounds of cells. These results allow us to speculate on new biological functions for the trans-membrane Cyt-b561 proteins.

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Heterologous expression and site-directed mutagenesis of an ascorbate-reducible cytochrome b561

Cited by 32 publications

References 53 publications

Human Erythrocyte Membranes Contain a Cytochrome b561 That May Be Involved in Extracellular Ascorbate Recycling

Human Erythrocyte Membranes Contain a Cytochrome b561 That May Be Involved in Extracellular Ascorbate Recycling

Spectral characterization of the recombinant mouse tumor suppressor 101F6 protein

Dihydrolipoic acid reduces cytochrome b561 proteins

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