Daniel M. Ziegler

Studies on the electron transport system. XLIII. The isolation of a succinic-coenzyme Q reductase from beef heart mitochondria

Abstract The isolation of the succinic-CoQ reductase from beef heart mitochondria is described. The flavine content of the preparation is 4.8 mμmoles/mg. protein, and all of the flavine is extracted by acid only after the preparation is treated with proteolytic enzymes. The preparation also contains non-heme iron, lipid, and protoheme, and the last mentioned is present in an amount equivalent to the flavine. Based on the flavine or heme content, the minimum molecular weight in terms of protein is 210,000. The heme present in the purified enzyme is not reduced by succinate, which makes its participation as an electron carrier in the reactions catalyzed by the enzyme very unlikely. A rapid spectrophotometric method for measuring the succinic-CoQ reductase activity of mitochondria and of the purified enzyme is described.

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.

Simplified methods for the estimation of iron in mitochondria and submitochondrial fractions

Abstract Two rapid and simple methods for the determination of iron in mitochondrial fractions arc described. One, which estimates total iron, is based on the quantitative extraction of both protein-bound heme and non-heme iron after treatment with mercaptoacetic acid and glacial acetic acid. The second, which estimates only non-heme iron, is based on the extractibility of non-heme iron into ethanol after reduction with sodium dithionite.

On the reduction of externally added DPN by succinate in submitochondrial particles.

Abstract It has recently been reported from several laboratories that mitochondrial DPN is reduced by succinate and that this reduction requires the addition of ATP ( Azzone, Ernster and Klingenberg, 1960 ; Chance and Hagihara, 1960 ; and Klingenberg, 1960 ). All of these studies involved intact mitochondria and bound DPN. In the present communication some experiments will be described which deal with the reduction of added DPN by a submitochondrial particle from beef heart muscle, ETPH. This particle enjoys several advantages for the study of DPN reduction; it contains little or no bound DPN, and does not have a functional Krebs cycle (which excludes any reduction of DPN by substrates other than succinate).

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

Abstract 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.

Oxidative phosphorylation by an electron transport particle from beef heart

Abstract 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 transfer system XXIX. The isolation and properties of a succinic dehydrogenase-cytochrome b complex from beef-heart mitochondria

Abstract 1. 1. The isolation and properties of a succinic dehydrogenase-cytochrome b particle from beef heart mitochondria are described. 2. 2. The relation of cytochrome b to the main chain of terminal electron transport is discussed. 3. 3. A method for assaying succini-Coenzyme Q reductase activity is described.

Studies on the mechanism of oxidative phosphorylation: II. Role of bound pyridine nucleotide in phosphorylation

Abstract The citric cycle oxidations of PETP which are induced by external DPN are not coupled to phosphorylation whereas the oxidation involving bound pyridine nucleotide is coupled. The residue particles differ from PETP in that a proportion of the DPN-induced oxidation is still coupled. With extensive washing of the residue particles the capacity to catalyze oxidation in absence of added cofactors is sharply reduced. This capacity can be restored by incubating the particles with cofactors and substrate at o° and then washing them exhaustively. Such treated particles regain both their original oxidative and phosphorylative capacity (in absence of added cofactors). DPNH oxidation is not accompanied by phosphorylation. External DPN is not reducible by the substrates of tightly coupled pyridinoprotein dehydrogenase systems.