title: Methylenetetrahydrofolate reductase. Evidence for spatially distinct subunit domains obtained by scanning transmission electron microscopy and limited proteolysis.
abstract: Scanning transmission electron microscopy of individual unfixed molecules of methylenetetrahydrofolate reductase has been used to determine the molecular mass distribution of the protein. Methylenetetrahydrofolate reductase, which has a subunit molecular mass of 77 kilodaltons, was found to exist predominantly as a dimer with an apparent molecular mass of 136 +/- 29 kilodaltons. The mass distribution of the enzyme molecules was unchanged in the presence of the allosteric inhibitor S-adenosylmethionine. Examination of negatively stained protein molecules suggested that each subunit of the dimer consists of two globular domains of approximately equal size. Limited proteolysis of the enzyme by trypsin gave results which were entirely consistent with the presence of two domains per subunit. In the presence of 1% trypsin, the enzyme was cleaved into two fragments. The masses of these fragments were 39 and 36 kilodaltons as assessed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Tryptic cleavage did not lead to loss of NADPH-menadione or NADPH-methylenetetrahydrofolate oxidoreductase activity, and the flavin prosthetic group remained bound to the protein. However, the cleaved protein was completely desensitized with respect to inhibition by S-adenosylmethionine. These results suggest that each subunit of methylenetetrahydrofolate reductase contains two domains and that allosteric inhibition requires specific interactions between these domains. The region between these two domains appears to be very sensitive to proteolysis, while the domains themselves are relatively resistant to further degradation.
mesh_terms: D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2); D000429:Alcohol Oxidoreductases / Q000032:analysis*; D000818:Animals; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008855:Microscopy, Electron, Scanning; D008970:Molecular Weight; D012436:S-Adenosylmethionine / Q000494:pharmacology; D013552:Swine; D014357:Trypsin / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D012436:S-Adenosylmethionine; D000429:Alcohol Oxidoreductases; D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2); D014357:Trypsin