The effects of proteolytic digestion by trypsin on the structure and catalytic properties of reduced nicotinamide-adenine dinucleotide dehydrogenase from bovine heart mitochondria.
1. At 21 degrees C incubation of NADH-ubiquinone-1 reductase (Complex 1) with trypsin caused selective inhibition of nicotinamide nucleotide transhydrogenase activity. The reduction of K3Fe(CN)6 by NADH or NADPH was unaffected, but a slow decrease in the rate of reduction of ubiquinone-1 by NADH was observed. 2. The pH-dependence of nicotinamide nucleotide transhydrogenase activity differed in Complex I and trypsin-treated Complex I. The trypsin-labile activity had a pH optimum of approx. 6.5, whereas the trypsin-resistant activity had a pH optimum of approx. 5.5 or less. 3. The trypsinlabile transhydrogenase activity was specifically inhibited by butanedione or phenylglyoxal and was identified with the enzyme catalysing energy-linked transhydrogenase activity in submitochondrial particles. 4. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate revealed that trypsin caused degradation of a polypeptide of mol.wt 20500 in parallel with the loss of transhydrogenase activity. 5. At 30 degrees C and higher trypsin concentrations, the rate of reduction of K3Fe(CN)6 by NADH or NADPH slowly decreased. Increased lability of NADH-K3Fe(CN)6 reductase activity to trypsin was observed when the endogenous phospholipid of Complex I was depleted by detergent or phospholipase A treatment. 6. Polyacrylamide-gel electrophoresis indicated that removal of phospholipid allowed much more extensive degradation of constituent polypeptides by trypsin. The subunits of the low-molecular-weight (type II) dehydrogenase (53000 and 26000 mol.wt.) were, however, relatively resistant to trypsin even in phospholipid-depleted preparations.
D000818:Animals; D002384:Catalysis; D002417:Cattle; D008931:Mitochondria, Muscle / Q000201:enzymology*; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D009243:NAD; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis / Q000037:antagonists & inhibitors; D009249:NADP; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
D010455:Peptides; D009243:NAD; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; D014357:Trypsin