Giorgio Lenaz

Organic structural specificity and sites of coenzyme Q in succinoxidase and DPNH-oxidase systems.

In succinoxidase, CoQ1 had low activity, and CoQ2 through CoQ10 had comparable activities in both the acetone- and pentane-extracted systems. In the DPNH-oxidase system, CoQ1 had low activity; CoQ2, CoQ3, and CoQ4 had about one-fourth the activity; and CoQ5 and CoQ6 had about one-half the activity of CoQ10. CoQ7, CoQ8, and CoQ9 had activity comparable with that of CoQ10. Hexahydro- and octahydrocoenzyme Q4 had activities comparable with that of CoQ4 and CoQ10 for succinoxidase, but about 10–15% that of CoQ10 for DPNH-oxidase. The double bond in the isoprenoid unit adjacent to the nucleus, and possible cyclization, are not necessary for activity. 2,3-Dimethoxy-5-methyl-6-heptadecyl-l,4-benzoquinone shows succinoxidase and DPNH-oxidase activities comparable with that of hexahydrocoeuzyme Q4. The 5-methyl group is not essential for succinoxidase activity, but has some significance for DPNH-oxidase. A tertiary hydroxyl group in the isoprenoid unit adjacent to the nucleus abolishes activity for succinoxidase but not completely for DPNH-oxidase. Rhodoquinone had no activity in the succinoxidase system and 25% that of CoQ10 in the DPNH-oxidase system. Representatives of biosynthetic precursors, 2-methoxy-5-methyl-6-phytyl-1,4-benzoquinone and 2-methoxy-3-hydroxy-5-methyl-6-phytyl-1,4-benzoquinone, had no activity in either the succinoxidase or DPNH-oxidase systems. Four compounds having a mechanistic relationship between CoQ and vitamin E had no activity. These data support the concept of two sites for the electron transfer of CoQ. The site for CoQ in succinoxidase is not very specific in the structural and steric requirement for the isoprenoid side chain, and has an electron potentiality that is not met by rhodoquinone. The site for CoQ in DPNH-oxidase has a rather specific requirement for a given length of side chain, and an electron potentiality that is significantly met by rhodoquinone.

Mitochondrial structural protein I: Methods of preparation and purification: Characterization by gel electrophoresis

Abstract Structural protein prepared from heavy beef heart mitochondria by a variety of methods, was shown to be heterogeneous by polyacrylamide gel electrophoresis. Procedures are described which have been found to be effective in purifying structural protein to a stage at which it shows one band when electrophoresed on polyacrylamide gel. When structural protein so purified was oxidized with performic acid, and subjected to electrophoretic analysis, four major protein bands were visualized. A possible explanation of this observation is discussed. The solubility properties of structural protein varied according to the method of preparation.

Stoichiometric aspects of the uncoupling of oxidative phosphorylation by carbonyl cyanide phenylhydrazones

Abstract Heavy beef heart mitochondria (HBHM) were titrated with carbonyl cyanide m-chlorophenylhydrazone and with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (pF3 CO-CCP) under a variety of conditions. For 100% uncoupling as little as one molecule of uncoupler sufficed for as many as 27 potential coupling sites. From a determination of the maximal experimental respiratory rate of the HBHM it was possible to calculate the “concentration of active chains” in each experiment. Significantly, an almost exact stoichiometry was found between the uncoupler and the number of “active phosphorylation sites” (“active chains” × control P O ). The possible constancy of this stoichiometric relationship was further tested by studying the degree of uncoupling resulting from the addition of varying levels of uncouplers to mitochondria exhibiting varying initial P O ratios and respiring at rates that were varied by use of the respiratory inhibitors rotenone, malonate, and cyanide. All the data were consistent with the concept that the uncouplers act preferentially on “active phosphorylation sites,” and that this affinity relates to the existence of a high-energy intermediate rather than to the state of reduction of these sites. The “stoichiometric efficiency” (i.e., the degree of uncoupling per molecule of uncoupler per active phosphorylation site) was determined over a wide range of respiratory rates (maximal obtainable respiratory rate/observed rate in the range between 3 and 40 or more). Over this entire range there was only a marginal overall decline in the former parameter; over 80% of the observed points fell between 0.7 and 1.4. The maintenance of a constant stoichiometry supports the concept that uncouplers such as pF3 CO-CCP are highly mobile within the mitochondrial membrane and have a very high affinity only for actively functioning coupling sites. The amount of uncoupler required to produce a given degree of uncoupling is usually dependent only upon the rate of generation of high-energy intermediates.

The membrane systems of the mitochondrion: V. The inner membrane of beef heart mitochondria☆

Abstract A method is described, involving digestion of mitochondria with phospholipase, which leads to a clean separation of the two mitochondrial membrane systems. An inner membrane fraction, R 2 , can be prepared which is essentially free from the activities known to be associated with the outer membrane, but is enriched in components of the inner membrane and in some of its main activities (electron transfer, and ATPases stimulated by divalent cations). Electron microscopy shows that the R 2 fraction is an essentially complete, unruptured inner membrane. Ca ++ ions translocated into the mitochondrion are completely recovered in the R 2 fraction; this has been taken as support for the premise that the R 2 particle is a morphologically intact inner membrane. The fact that sequences which depend upon energy transformations (oxidative phosphorylation and energized translocation) are partially lost in the inner membrane fraction, R 2 , suggests that, although morphologically intact, the functionality of the inner membrane has been somewhat damaged by the digestive procedure.

Studies on mitochondrial structural protein: III. Physical characterization of the structural proteins of beef heart and beef liver mitochondria

Abstract The several species of purified structural proteins isolated from beef heart and beef liver mitochondria have been shown to have similar solubility properties, similar composition of amino acids, similar peptide maps after tryptic digestion, and similar if not identical molecular weights, and to contain either aspartate or alanine as N -terminal amino acid. These similarities and identities provide the experimental foundation for considering the various species of structural protein as members of a general class of noncatalytic proteins, all of which represent the predominant protein species (some 50%, of the total protein) in the detachable sectors of membrane repeating units.

Mitochondrial structural protein: II. Determination of the molecular weight of structural proteins by sedimentation analysis

Abstract The molecular weight of structural protein isolated from beef heart or beef liver mitochondria was determined. Samples of these proteins were purified to the point of electrophoretic homogeneity. The proteins were depolymerized by several different procedures. The molecular weight was determined by the methods of synthetic boundary sedimentation velocity, analytical zone sedimentation velocity and sedimentation equilibrium. The values for the molecular weight of structural protein clustered in the range of 55,000–70,000.

Studies on ultrastructural dislocations in mitochondria: II. On the dislocation induced by lyophilization and the mechanism of uncoupling

Abstract Lyophilization has been shown to uncouple bovine heart mitochondria; however, well-coupled particles may be derived by sonication of the mitochondria uncoupled in this way. An ultrastructural dislocation has been demonstrated by electron microscopy in the cristael membrane of the lyophilized particles. This dislocation is believed to be the morphological basis of uncoupling in lyophilized mitochondria. A mechanism is proposed whereby coupled submitochondrial particles may be derived by sonic irradiation of uncoupled mitochondria.

Studies on ultrastructural dislocations in mitochondria: I. Reconstitution of oxidative phosphorylation in lyophilized mitochondria by sonic irradiation

Abstract Exposure of mitochondria to lyophilization, freezing and thawing, Ca++, or distilled water reduces or eliminates the capability for coupled electron transfer. Restoration of coupling capability can be achieved by sonic irradiation of the treated mitochondrial suspensions.