Flavin reductase: Structural studies and tissue distribution.
Quandt, Kimberly Sue
1993
Abstract
Flavin reductase is a soluble enzyme that catalyzes electron transfer from pyridine nucleotides to flavins and a variety of other electron acceptors including methylene blue, 2,6-dichlorophenolindophenol, and pyrroloquinoline quinone. This catalysis is the basis of the therapeutic use of riboflavin and methylene blue in the treatment of congenital and acquired methemoglobinemia. Although a clinical role of flavin reductase has been established, the physiological role of the enzyme is not known. To obtain a better understanding of the structure and function of flavin reductase, studies were undertaken to determine its primary structure and tissue distribution. The protein component of bovine erythrocyte flavin reductase was found to be identical to that of bovine erythrocyte green heme-binding protein based on amino acid analysis and N-terminal amino acid sequence analysis. Moreover, the two proteins were immunochemically cross-reactive and were indistinguishable when compared by sodium dodecylsulfate-polyacrylamide gel electrophoresis and by immuno-double diffusion. Flavin reductase was immunochemically detected in the soluble fractions of bovine, rat, rabbit and human erythrocytes, in cytosolic fractions of bovine, rat and rabbit liver, and in cytosolic fractions of rat and rabbit heart. By immunoblot analysis, the concentration of flavin reductase in bovine erythrocyte was estimated to be 10 $\mu$M. Two peptides derived from a partial acid hydrolysis of bovine erythrocyte flavin reductase, and three peptides derived from a Lys-C digest were isolated and partially or completely sequenced. Degenerate oligonucleotides, based on amino acid sequences of erythrocyte reductase peptides, were used as primers in PCR to selectively amplify a partial cDNA that encodes the reductase. A PCR product was used as a specific probe to screen a bovine liver cDNA library. The sequence determined from two overlapping clones contains an open reading frame of 621 nucleotides and encodes 206 amino acids. The amino acid sequence deduced from the bovine liver flavin reductase cDNA matches the amino acid sequences determined for erythrocyte reductase-derived peptides, and the predicted molecular mass of 22,001 daltons for the liver reductase agrees well with the molecular mass of 21,994 daltons determined for the erythrocyte reductase by electrospray mass spectrometry. The amino acid sequence at the N-terminus of the reductase has homology to sequences of pyridine nucleotide-dependent enzymes, and the predicted secondary structure, $\beta\alpha\beta$, resembles the common nucleotide-binding structural motif. RNA blot analysis detected a single 1-kilobase reductase transcript in human heart, kidney, liver, lung, pancreas, placenta and skeletal muscle, indicating that flavin reductase is expressed in a wide variety of tissues.Other Identifiers
(UMI)AAI9409788
Subjects
Biology, Molecular Health Sciences, Pharmacology Chemistry, Biochemistry
Types
Thesis
Metadata
Show full item recordCollections
Remediation of Harmful Language
The University of Michigan Library aims to describe library materials in a way that respects the people and communities who create, use, and are represented in our collections. Report harmful or offensive language in catalog records, finding aids, or elsewhere in our collections anonymously through our metadata feedback form. More information at Remediation of Harmful Language.
Accessibility
If you are unable to use this file in its current format, please select the Contact Us link and we can modify it to make it more accessible to you.