Electron Transport Particles Research Articles

Studies on the mechanism of oxidative phosphorylation V. The phosphorylating properties of the electron transport particle

When the heavy fraction of beef heart mitochondrial suspensions is exposed to sonic oscillation, a submitochondrial particle can be isolated which is indistinguishable from previously described ETP except that it is capable of coupling the oxidation of succinate and DPNH to phosphorylation —the maximum P/O ratios being respectively 1.2 and 2.0 ETP may be regarded as the least common denominator of both electron transport and oxidative phosphorylation. Whether phosphorylating or not, ETP is inactive in catalyzing citric cyclic oxidations. During its formation by sonication of mitochindria, some of the pyridinoprotein enzymes are completely lost while others are retained in part. The β-hydroxybutyric dehydrogenase is the only pyridinoprotein enzyme which is more concentrated in ETP than in the original mithocondrion. The presence of Mg ++ during the separation of ETP from the sonicated suspension is essential for maximal phosphorylative activity.

Studies on the electron transport system: XI. Correlation of the morphology and enzymic properties of mitochondrial and sub-mitochondrial particles

The morphological changes which mitochondria and their derivative particles undergo during isolation from beef-heart muscle have been studied by electron microscopy. Mitochondria isolated from the tissue in a 0.25 M sucrose medium are grossly modified in structure. By virtue of these modifications they can readily give rise to smaller submitochondrial particles such as ETP and PETP. Morphologically intact mitochondria can be isolated from heart tissue which is comminuted in 0.88 M sucrose but only in relatively small yield. Such mitochondria do not give rise to ETP and PETP by the methods successfully applied to the mitochondria isolated in 0.25 M sucrose. The light mitochondrial fraction contains swollen mitochondria whose outer membrane shows a marked tendency to undergo vesiculation leading to the formation of the electron transport particle. Some or all of the cristae of these swollen mitochondria can be pinched off together with a part of the outer mitochondrial membrane to form new particles which, depending upon size, conform either to the particles of the heavy mitochondrial fraction (large particle) or to PETP (small particle). When the heavy mitochondrial fraction is exposed to sonic irradiation, a smaller particle is liberated which resembles ETP closely both morphologically and chemically. It differs primarily in the respect that bound cytochrome c is readily extractable whereas the bound cytochrome c of ETP is not.

A lipid-soluble form of cytochrome c from the electron transport particle of beef-heart mitochondria

A lipid-soluble form of cytochrome c from the electron transport particle of beef-heart mitochondria

Studies on the electron transport system X. Preparation and spectral properties of a particulate DPNH and succinate cytochrome C reductase from heart muscle

1. 1. The electron transport particle (ETP) has been fractionated with a mixture of cholate and ammonium sulfate. A particulate fraction has been obtained (DPNH-DC-SDC) which catalyzes the oxidation of both DPNH and succinate by cytochrome c. At a higher concentration of ammonium sulfate, another particulate fraction (SDC) has been obtained which is active only with succinate. 2. 2. DPNH-DC-SDC contains cytochromes b and c 1 (but not c or cytochrome oxidase) in the molecular ratio 1:8:1.0. The enzyme bound hemoproteins are reduced to the same degree by either succinate or DPNH. 65% of the total cytochrome b is reducible by substrate whereas 80% of the total cytochrome c 1 is reducible by substrate. 3. 3. In aged preparations, the rates of reduction of cytochromes b, c 1 and of externally added c by DPNH are greatly decreased and the extent of reduction of cytochrome b is diminished. The reducibility of the hemes by succinate is less labile.

Studies on the terminal electron transport system VI. Fragmentation of the electron transport particle with deoxycholate

At an appropriate ratio of deoxycholate to protein the electron transport particle is fragmented into a red particle similar to the succinic dehydrogenase complex and a green particle which acts as a DPNH oxidase when supplemented with cytochrome c. The red particle contains cytochromes b and c 1 (but not a) and catalyzes the oxidation of succinate or DPNH by ferricyanide and cytochrome c but not by oxygen. The green particle contains cytochrome b, c 1 and a and catalyzes the oxidation of DPNH (but not succinate) by cytochrome c or by oxygen in presence of cytochrome c. Antimycin inhibits the oxidation of succinate by cytochrome c (catalyzed by the red particle) or oxidation of DPNH by oxygen or cytochrome c (catalyzed by the green particle).

Studies on the terminal electron transport system: V. Extraction of a soluble DPNH cytochrome C reductase from the electron transport particle

1. 1. A soluble DPNH-cytochrome- c-reductase has been isolated in high yield from the electron particle of beef heart mitochondria. 2. 2. The enzyme shows a modified flavoprotein spectrum with maxima at 270, 360, and 450 mμ. The presence of a heme component was also found which appears to be firmly attached to the flavoprotein. 3. 3. The enzyme has 2 iron atoms for each mole of flavin. The flavin prosthetic group appears not to be flavinadeninedinucleotide on the basis of enzymic and chromatographic tests. 4. 4. The properties of the enzyme extracted from the electron transport particle closely resemble those of the soluble DPNH-cytochrome- c-reductase described by Mahler et al.

Studies on the mechanism of oxidative phosphorylation: I. Preparation and properties of a phosphorylating electron transfer particle from beef heart mitochondria

Methods for the preparation of actively phosphorylating electron transport particles from beef heart mitochondrial suspensions are described. The stability of the phosphorylation process both in the beef heart mitochondria and the derivative particles is of a different order of magnitude from that of rat liver mitochondria. The maximum P/O ratios attainable with PETP are close to the highest values recorded in the literature. 2,4-Dinitrophenol abolishes oxidative phosphorylation except for substate level phosphorylation accompanying oxidation of α-ketoglutarate.

Oxidative phosphorylation by an electron transport particle from beef heart

A stable phosphorylating electron transfer particle has been isolated from beef heart mitochondria which retains the capacity for oxidatice phosphorylation almost unimpaired. Some new aspects of the mechanism of oxidative phosphorylation have been recognized.

Studies on the electron transport system: II. On the “opening” phenomenon

The preparation of two modified forms of the electron transport particle (ETP) has been described. One form (closed) can catalyze only to a slight degree the oxidation of DPNH by ferricytochrome c or the oxidation of ferrocytochrome c by oxygen except after exposure to desoxycholate. As these two activities emerge the capacity to catalyze the over-all DPNH-oxygen reaction is lost. This latter activity is restored by the addition of small amounts of cytochrome c. The second form approximates the behavior of the closed form which had been exposed to desoxycholate.

Studies on the electron transport system: III. The properties of two modified forms of ETP

Some enzymic properties and the composition of two modified forms (I and II) of the electron transport particle (ETP) have been examined. Such preparations contain per mg of protein 0.42·10 −3 μmole of flavin, 2.5·10 −3 μmoles of heme, 6.2·10 −3 μmoles of non-heme iron and 2.4·10 −3 μmoles of copper—the molecular or atomic ratios being in round numbers 1:6:15:6. The antimycin titer of the preparations was 0.5·10 −3 μmole/mg. The oxido-reduction of the prosthetic hemes in presence of DPNH and oxygen has been demonstrated. Heme reduction was partially inhibited by antimycin. The absorption spectrum of the enzyme indicates the presence of at least two groups of hemes—one group with bands at 563, 554, 535 and 525 mμ—the other with a band at 603 mμ.