Kearney 1960 J Biol Chem
| Kearney EB (1960) Studies on succinic dehydrogenase. XII. Flavin component of the mammalian enzyme. J Biol Chem 235:865-77 |
Kearney EB (1960) J Biol Chem
Abstract: 1. Unlike other known flavoproteins, in which the flavin is relatively loosely bound and is easily liberated by suitable methods of denaturation, in succinic dehydrogenase from beef heart the flavin component is so tightly held that neither treatment with strong acids nor thermal denaturation separates it from the protein.
2. Extensive digestion of the purified dehydrogenase with suitable proteolytic enzymes liberates the flavin in an acid-soluble form, which is not, however, identical with known derivatives of riboflavin. The flavin appears in the digest in several chromatographically distinct forms, which may be separated from each other by purification on ion exchange resins or by chromatography on filter paper.
3. The main flavin components have been extensively purified and degraded to the mononucleotide and dephosphorylated flavin levels. The dinucleotide contains 1 mole of 5’-adenylic acid, 2 atoms of phosphorus bound in pyrophosphate linkage and 1 mole of ribose. It differs from authentic flavin adenine dinucleotide (FAD) in numerous regards, including its inactivity in the n-amino acid oxidase test, shifted absorption spectrum, shifted pH-fluorescence curve, and in the presence of cationic group(s). After degradation to the mononucleotide and dephosphorylated flavin level, similar differences exist between the resulting compounds and authentic riboflavin 5’-phosphate and riboflavin, respectively. Irradiation in alkali degrades the flavin further, but the resulting compound is not identical with lumiflavin.
4. These differences and the greater water solubility of the unphosphorylated compound as compared with riboflavin are best explained by the hypothesis that the flavin in the dehydrogenase is held to a peptide chain by a covalent linkage which survives proteolytic digestion. The compounds in the digest, therefore, would be peptides of FAD, representing fragments of the original enzyme.
5. Evidence for the flavin peptide hypothesis has come from the finding that throughout very extensive purification by a variety of methods the flavin is always accompanied by peptide material. In the most purified fraction, believed to be free of contaminating peptides, alanine, serine, threonine, glutamic acid, and valine were present in molar ratio to the flavin and an additional mole of serine was present as N-terminal group. Similar amino acid compositions were found in 2 other samples, purified by different procedures.
6. Evidence pertaining to the flavin peptide hypothesis and the possible structure of the flavin is discussed.
• Bioblast editor: Gnaiger E
Selected quotes
- The flavoprotein nature of mammalian succinic dehydrogenase was suggested by several early observations. For example, Keilin and Hartree (2) in 1939 observed a reduction in absorption at 455 nm when succinate was added to heart muscle preparations, and Axelrod et al. (3) noted a relationship between riboflavin deficiency and the succinic dehydrogenase activity of liver. With purified succinic dehydrogenase, the first direct indication of the presence of flavin came from the appearance of a difference spectrum with a maximum at 460 nm when the enzyme was treated with succinate or hydrosulfite (4-6). Attempts to demonstrate the presence of flavin in the purified enzyme by conventional methods (release of flavin by cold acid or by thermal denaturation) were uniformly unsuccessful, however.
- It was subsequently found by Green et al. (7) that tryptic digestion of their particulate preparation, the “succinic dehydrogenase complex,” liberated appreciable amounts of flavin-type material which had not been extractable by acidification or boiling. This proved to be true also with the soluble, purified enzyme from beef heart, but the flavin liberated was not identifiable as FAD or FMN’ (8, 9). Similar results have been reported by Wang et al. for the dehydrogenase isolated from pig heart (10). Independently of these observations, Boukine (11) noted the presence of bound flavins, liberated only by proteolytic digestion, in a variety of plant and animal tissues, particularly in those rich in succinic dehydrogenase.
- In preliminary reports (8, 12, 13), we have suggested that succinic dehydrogenase, in contrast to other flavoproteins, contains FAD, held by covalent linkage, and that the unusual properties of the flavin in proteolytic digests may be a consequence of being in combination with a peptide chain. The present paper includes a detailed report of our findings and a discussion of the current status of this problem.
- The experiments described establish that the prosthetic group of mammalian succinic dehydrogenase is a flavin dinucleotid .
Labels:
Preparation: Enzyme
Enzyme: Complex II;succinate dehydrogenase
