Effects of proteolytic digestion by chymotrypsin on the structure and catalytic properties of reduced nicotinamide–adenine dinucleotide–ubiquinone oxidoreductase from bovine heart mitochondria

Abstract

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.

The role of phospholipids in the reduction of ubiquinone analogues by the mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase complex

“…Obvious possibilities are that such lipid-protein interactions are required for the conformational stability of Complex I. It has been shown that lipid depletion leads to changes in the midpoint potential of the non-haem-iron centre 2 (Ohnishi et al, 1974) and to greater susceptibility of the enzyme to proteolytic digestion (Ragan, 1976b;Crowder & Ragan, 1977). The experiments with dimyristoyl-sn-glycero-3-phosphocholine did not provide any evidence that freezing the annular and extraannular lipid directly affected activity through the protein conformation.…”

Changes in NADH-Ubiquinone Reductase (Complex I) with Autolysis in the Rat Heart as Experimental Model

“…When complex I was subjected to proteolytic digestion, the NADH-ubiquinone reductase activity disappeared when peptides with molecular masses of 49,000, 42,000, 39,000, 26,000, 23,500, 18,000, and 15,500 were released [5], NADH-potassium ferricyanide reductase activity disappeared when the 53,000 molecular mass peptide was re leased [5], Loss of FMN during autolysis may be involved. Rouslin et al [33] showed that after 60 min of autolysis the acid-extractable FMN was 58% of the control values.…”

An analysis of the polypeptide composition of bovine heart mitochondrial NADH-ubiquinone oxidoreductase by two-dimensional polyacrylamide-gel electrophoresis

“…One-dimensional SDS/polyacrylamide-gel electrophoresis Separation on gels (12cmx4mm internal diam.) containing 12.5% (w/v) acrylamide and 0.34% bisacrylamide was as described by Crowder & Ragan (1977), by the procedure of Weber & Osborn (1969). Alternatively, two discontinuous systems were used.…”

“…However, this enzyme is far and away the most complicated, since purified preparations may be resolved into at least 16 components when analysed by conventional gel electrophoresis in the presence of SDS (Ragan, 1976). Analysis by discontinuous gel electrophoresis increased the number of resolvable components to 18 (Crowder & Ragan, 1977). These results are broadly in agreement with the findings of others (Hare & Crane, 1974;Dooijewaard et al, 1978a,b), although it is obvious from the literature that the number of polypeptides reported is merely a function of the resolving power of the analytical system used.…”