Substrate-dependent reduction of a recombinant chromaffin granule Cyt-b561 and its R72A mutant

Mahadevan Lakshminarasimhan, A. Bérczi, Han Asard

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Cytochrome b561 (Cyt-b561) proteins constitute a family of integral membrane proteins, catalyzing ASC-driven trans-membrane electron transport. Numerous isoforms of Cyt-b561 are present in invertebrates, vertebrates, and plants. The only protein of this family, however, which has been characterized in details at both biophysical, biochemical and physiological levels so far, is the bovine chromaffin granule Cyt-b561 (CGCyt-b561). Recently, both the bovine and the mouse CGCyt-b561 has been expressed in yeast cells and the recombinant proteins were shown to have biophysical properties similar to the native bovine CGCyt-b561. We have expressed the mouse CGCyt-b561 with a His6-tag at the C terminus (CGCyt-b561(C6H)) in yeast (Saccharomyces serevisiae) cells and studied the reduction of CGCyt-b561(C6H) in the presence of different natural reducing agents. Besides the well-known natural reductant ascorbate (ASC) and the often-used artificial reductant dithionate, NADH, GSH, and dihydrolipoic acid (DHLA), also reduced the fully oxidized protein. Interestingly however, NADPH was not effective at all. When the same reductants were tested with the R72A mutant of CGCyt-b561(C6H), a mutant with impaired ASC-dependent reducibility, neither pyridine-dinucleotides could reduce the R72A mutant. DHLA-dependent and ASC-dependent reduction kinetics were very similar in case of the R72A mutant but differed in case of CGCyt-b561. These results raise the question of how many natural reductants the CGCyt-b561 may utilize in vivo.

Original languageEnglish
Pages (from-to)61-65
Number of pages5
JournalActa Biologica Szegediensis
Volume50
Issue number1-2
Publication statusPublished - 2006

Fingerprint

chromaffin granules
Chromaffin Granules
Reducing Agents
cytochromes
reducing agents
mutants
Substrates
His-His-His-His-His-His
Yeast
Proteins
NADP
Recombinant Proteins
NAD
cattle
Yeasts
Protein Isoforms
Membrane Proteins
yeasts
Cells
cytochrome b561

Keywords

  • Arginine residue
  • ASC reduction
  • Cytochrome b561
  • Dihydrolipoic acid
  • His-tagged protein
  • Reductants

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Cell Biology
  • Neuroscience(all)
  • Applied Microbiology and Biotechnology

Cite this

Substrate-dependent reduction of a recombinant chromaffin granule Cyt-b561 and its R72A mutant. / Lakshminarasimhan, Mahadevan; Bérczi, A.; Asard, Han.

In: Acta Biologica Szegediensis, Vol. 50, No. 1-2, 2006, p. 61-65.

Research output: Contribution to journalArticle

@article{1c5be9869975446baa60caf818ecee3c,
title = "Substrate-dependent reduction of a recombinant chromaffin granule Cyt-b561 and its R72A mutant",
abstract = "Cytochrome b561 (Cyt-b561) proteins constitute a family of integral membrane proteins, catalyzing ASC-driven trans-membrane electron transport. Numerous isoforms of Cyt-b561 are present in invertebrates, vertebrates, and plants. The only protein of this family, however, which has been characterized in details at both biophysical, biochemical and physiological levels so far, is the bovine chromaffin granule Cyt-b561 (CGCyt-b561). Recently, both the bovine and the mouse CGCyt-b561 has been expressed in yeast cells and the recombinant proteins were shown to have biophysical properties similar to the native bovine CGCyt-b561. We have expressed the mouse CGCyt-b561 with a His6-tag at the C terminus (CGCyt-b561(C6H)) in yeast (Saccharomyces serevisiae) cells and studied the reduction of CGCyt-b561(C6H) in the presence of different natural reducing agents. Besides the well-known natural reductant ascorbate (ASC) and the often-used artificial reductant dithionate, NADH, GSH, and dihydrolipoic acid (DHLA), also reduced the fully oxidized protein. Interestingly however, NADPH was not effective at all. When the same reductants were tested with the R72A mutant of CGCyt-b561(C6H), a mutant with impaired ASC-dependent reducibility, neither pyridine-dinucleotides could reduce the R72A mutant. DHLA-dependent and ASC-dependent reduction kinetics were very similar in case of the R72A mutant but differed in case of CGCyt-b561. These results raise the question of how many natural reductants the CGCyt-b561 may utilize in vivo.",
keywords = "Arginine residue, ASC reduction, Cytochrome b561, Dihydrolipoic acid, His-tagged protein, Reductants",
author = "Mahadevan Lakshminarasimhan and A. B{\'e}rczi and Han Asard",
year = "2006",
language = "English",
volume = "50",
pages = "61--65",
journal = "Acta Biologica Szegediensis",
issn = "1588-385X",
publisher = "University of Szeged",
number = "1-2",

}

TY - JOUR

T1 - Substrate-dependent reduction of a recombinant chromaffin granule Cyt-b561 and its R72A mutant

AU - Lakshminarasimhan, Mahadevan

AU - Bérczi, A.

AU - Asard, Han

PY - 2006

Y1 - 2006

N2 - Cytochrome b561 (Cyt-b561) proteins constitute a family of integral membrane proteins, catalyzing ASC-driven trans-membrane electron transport. Numerous isoforms of Cyt-b561 are present in invertebrates, vertebrates, and plants. The only protein of this family, however, which has been characterized in details at both biophysical, biochemical and physiological levels so far, is the bovine chromaffin granule Cyt-b561 (CGCyt-b561). Recently, both the bovine and the mouse CGCyt-b561 has been expressed in yeast cells and the recombinant proteins were shown to have biophysical properties similar to the native bovine CGCyt-b561. We have expressed the mouse CGCyt-b561 with a His6-tag at the C terminus (CGCyt-b561(C6H)) in yeast (Saccharomyces serevisiae) cells and studied the reduction of CGCyt-b561(C6H) in the presence of different natural reducing agents. Besides the well-known natural reductant ascorbate (ASC) and the often-used artificial reductant dithionate, NADH, GSH, and dihydrolipoic acid (DHLA), also reduced the fully oxidized protein. Interestingly however, NADPH was not effective at all. When the same reductants were tested with the R72A mutant of CGCyt-b561(C6H), a mutant with impaired ASC-dependent reducibility, neither pyridine-dinucleotides could reduce the R72A mutant. DHLA-dependent and ASC-dependent reduction kinetics were very similar in case of the R72A mutant but differed in case of CGCyt-b561. These results raise the question of how many natural reductants the CGCyt-b561 may utilize in vivo.

AB - Cytochrome b561 (Cyt-b561) proteins constitute a family of integral membrane proteins, catalyzing ASC-driven trans-membrane electron transport. Numerous isoforms of Cyt-b561 are present in invertebrates, vertebrates, and plants. The only protein of this family, however, which has been characterized in details at both biophysical, biochemical and physiological levels so far, is the bovine chromaffin granule Cyt-b561 (CGCyt-b561). Recently, both the bovine and the mouse CGCyt-b561 has been expressed in yeast cells and the recombinant proteins were shown to have biophysical properties similar to the native bovine CGCyt-b561. We have expressed the mouse CGCyt-b561 with a His6-tag at the C terminus (CGCyt-b561(C6H)) in yeast (Saccharomyces serevisiae) cells and studied the reduction of CGCyt-b561(C6H) in the presence of different natural reducing agents. Besides the well-known natural reductant ascorbate (ASC) and the often-used artificial reductant dithionate, NADH, GSH, and dihydrolipoic acid (DHLA), also reduced the fully oxidized protein. Interestingly however, NADPH was not effective at all. When the same reductants were tested with the R72A mutant of CGCyt-b561(C6H), a mutant with impaired ASC-dependent reducibility, neither pyridine-dinucleotides could reduce the R72A mutant. DHLA-dependent and ASC-dependent reduction kinetics were very similar in case of the R72A mutant but differed in case of CGCyt-b561. These results raise the question of how many natural reductants the CGCyt-b561 may utilize in vivo.

KW - Arginine residue

KW - ASC reduction

KW - Cytochrome b561

KW - Dihydrolipoic acid

KW - His-tagged protein

KW - Reductants

UR - http://www.scopus.com/inward/record.url?scp=33846404094&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33846404094&partnerID=8YFLogxK

M3 - Article

VL - 50

SP - 61

EP - 65

JO - Acta Biologica Szegediensis

JF - Acta Biologica Szegediensis

SN - 1588-385X

IS - 1-2

ER -