Limited proteolysis of the nitrate reductase from spinach leaves.
The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.
Kubo
Y
Y
Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University, Japan.
Ogura
N
N
Nakagawa
H
H
D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D009565:Nitrate Reductases / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D009565:Nitrate Reductases; D008058:Dihydrolipoamide Dehydrogenase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin