Effects of proteolytic digestion by chymotrypsin on the structure and catalytic properties of reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase from bovine heart mitochondria.
1. Incubation of NADH-ubiquinone oxidoreductase (Complex I) with chymotrypsin caused loss of rotenone-sensitive ubiquinone-1 reduction and an increase in rotenone-insensitive ubiquinone reduction. 2. Within the same time-course, NADH-K(3)Fe(CN)(6) oxidoreductase activity was unaffected. 3. Mixing of chymotrypsin-treated Complex I with Complex III did not give rise to NADH-cytochrome c oxidoreductase activity. 4. Gel electrophoresis in the presence of sodium dodecyl sulphate revealed selective degradation of several constituent polypeptides by chymotrypsin. 5. With higher chymotrypsin concentrations and longer incubation times, a decrease in NADH-K(3)Fe(CN)(6) oxidoreductase was observed. The kinetics of this decrease correlated with solubilization of the low-molecular-weight type-II NADH dehydrogenase (subunit mol.wts. 53000 and 27000) and with degradation of a polypeptide of mol.wt. 30000. 6. Phospholipid-depleted Complex I was more rapidly degraded by chymotrypsin. Specifically, a subunit of mol.wt. 75000, resistant to chymotrypsin in untreated Complex I, was degraded in phospholipid-depleted Complex I. In addition, the 30000-mol.wt. polypeptide was also more rapidly digested, correlating with an increased rate of transformation to type II NADH dehydrogenase.
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010455:Peptides / Q000032:analysis; D010743:Phospholipids / Q000378:metabolism; D011808:Quinone Reductases / Q000378:metabolism*; D012402:Rotenone / Q000378:metabolism
D010455:Peptides; D010743:Phospholipids; D012402:Rotenone; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D011808:Quinone Reductases; D002918:Chymotrypsin