D. Rao Sanadi

Age-related changes in cytochrome concentration of myocardial mitochondria

Abstract Cytochrome c oxidase was assayed polarographically in rat tissue homogenates and mitochondria in the presence of deoxycholate. A roughly 30% decrease in the cytochrome c oxidase activity per mg protein occurred in heart muscle homogenates and in purified heart mitochondria of the old rat. An age-related decrease in cytochrome c oxidase activity was also observed in brain homogenates but not in liver and kidney homogenates. The activity of this enzyme in μatoms of oxygen consumed/min/mg protein corresponded to 3.26 ± 0.25, 2.87 ± 0.18, 2.36 ± 0.24 in heart homogenates, and 14.46 ± 0.36, 12.98 ± 0.36 and 10.60 ± 1.03 in heart mitochondria from 5-, 15- and 26- to 29-month old animals, respectively. The activity in μatoms of oxygen consumed/min/mg protein corresponded to 1.87 ± 0.07 and 1.57 ± 0.06 in brain homogenates, 1.67 ± 0.05 and 1.57 ± 0.05 in kidney homogenates and 0.38 ± 0.01 and 0.36 ± 0.02 in liver homogenates for 7- and 29-month old rats, respectively. The turnover number of this enzyme in heart mitochondria was 985 s−1 and unchanged with respect to age of the animal. The concentrations of the cytochromes in nmol/mg heart mitochondrial protein were 0.75 ± 0.040, 0.740 ± 0.060 and 0.56 ± 0.050 for c + c1; 0.447 ± 0.033, 0.466 ± 0.047 and 0.263 ± 0.075 for b + b5 and 0.520 ± 0.030, 0.480 ± 0.020 and 0.385 ± 0.024 for a + a3 for 5-, 15- and 26-month old rats, respectively. The mitochondrial protein content of the myocardium calculated from the cytochrome oxidase specific activities of the homogenate and of the isolated mitochondrria was 44 mg/g wet tissue for the 5- and 26-month old rats and 56 mg/g wet weight for the 14-month old rats. In view of these findings, it is concluded that mitochondrial membrane composition of the myocardium of the animal changes with age.

Evidence for increased degeneration of mitochondria in old rats. A brief note

An isotonic density gradient technique for separating mitochondria into two major components was applied to mitochondria isolated from young and old rats. Respiratory measuremtns indicate that the depression of state 3 respiration rates observed in the mitochondria from old rats when glutamate--malate is used as a substrate is due primarily to the faster sedimenting mitochondrial component.

Characterization of the mitochondria of the free-living nematode, Caenorhabditis elegans.

Abstract 1. 1. Conditions are discussed for obtaining population levels of 200,000/ml in 6 days using axenic cultures of the nematode, Caenorhabditis elegans. Lifespan and growth data under these conditions are presented. 2. 2. An isolation procedure which results in high quality mitochondria is presented, and metabolic data on respiaration rates, respiratory control ratios, and ADP: O ratios using various substrates are given. 3. 3. The effects of inhibitors of respiration and ATPase activity and of uncouplers of oxidative phosphorylation are presented. 4. 4. Cytochrome content of the mitochondria as determined from difference spectra is given. No indication of the large amounts of the b type pigment reported in Turbatrix aceti was found. 5. 5. The metabolism and respiratory chain of C. elegans appear to be similar to the classic mammalian systems. The only gross difference noted was the absence of ATPase stimulation by traditional uncouplers of oxidative phosphorylation. In this respect, the C. elegans mitochondria resemble more closely those of ascaris.

An aspect of translational control of protein synthesis in aging: Changes in the isoaccepting forms of tRNA in Turbatrix aceti

Abstract Transfer RNA from the nematode Turbatrix aceti was examined for age-related changes in isoaccepting forms for sixteen amino acids. This was considered to be an index for possible changes at the translational level of protein synthesis. Aged T. aceti were obtained by the use of hydroxyurea to block reproduction, and their t RNA was purified. This was compared by reverse phase chromatography with t RNA from young T.aceti exposed to hydroxyurea for a short time. Chromatography revealed single peaks for the t RNA of seven amino acids and multiple peaks for t RNA of nine. Of these, only the t RNA for arginine any tyrosine showed consistent age-related differences, which were not attributable to either any decrease in mitochondria in old worms or the presence of eggs in only the control worms. The changes were not due to a general short-term effect of hydroxyurea.

Dependence of Escherichiacoli pyridine nucleotide transhydrogenase on phospholipids and its sensitivity to N-ethylmaleimide

Abstract A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of Escherichia coli by means of deoxycholate extraction and DEAE-cellulose chromatography in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phosphatidyl ethanolamine, the major phospholipid in the outer membrane of E. coli , was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibited by N-ethylmaleimide. Protection against inhibition was achieved with NAD+ and NADP+, but NADPH served to accelerate the rate of inhibition.

Monoclonal antibodies to mitochondrial coupling factor B

Two monoclonal antibodies (MAb 1 and IV) have been prepared which showed high and specific reactions towards bovine heart mitochondrial coupling factor B (FB). Both have been identified as sub‐type IgG1 of mouse immunoglobulins. MAb I reacts with purified and functionally active fB, alkylated or oxidized forms of FB and even with peptides formed on digestion of FB with trypsin. When used together, MAb I and IV reacted with FB in immunoblots of normal and urea treated samples of mitochondria, submitochondrial particles, ammonia‐EDTA extracted particles, and H+‐ATPase. Both MAbs inhibited FB‐stimulated ATP‐dependent reverse electron flow activity when fB was incubated with the antibody either before or after its addition to FB‐deficient AE‐particles. Reactivity of MAb I towards fB declined upon exposure of fb to guanidine HCl while reactivity of MAb IV remained unaltered.

Amino-terminal amino acid sequence of beef heart mitochondrial coupling factor B

Bovine heart mitochondrial coupling factor B was isolated and purified to homogeneity in its active form. The amino‐terminal amino acid sequence of the alkylated protein was determined. Two chains with exactly the same sequence except for the presence of an additional Phe at the aminoterminus on one of them were obtained. The 55 amino acid sequence appears to be largely hydrophilic with several charged amino acid residues. This sequence showed no homology with the E. coli unc operon, oligomycin sensitivity conferring protein, or coupling factor 6 or any protein in the data base.

Diamide blocks H+ conductance in mitochondrial H+-ATPase by oxidizing FB dithiol

Effects of diamide on proton conductance of electron transport particles (ETPH), purified H+‐ATPase (F1–F0), F0 of the H+‐ATPase from beef heart mitochondria and binding of cadmium (109Cd) to the H+‐ATPase have been examined in the present paper. When ETPH and purified H+‐ATPase are treated with 1 mM diamide, ATP‐dependent generation of membrane potential, monitored by the absorbance change produced by the redistribution of oxonol VI, is consistantly inhibited. Diamide also blocks passive H+ conductance driven by a K+ diffusion potential in the membrane sector, F0 of H+‐ATPase. Furthermore, diamide treatment drastically reduces the binding of 109Cd2+ to H+‐ATPase, showing competition for the FB dithiol group.

Further observations on coupling factor A

The isolation, purification and characterization of coupling factor A from beef heart mitochondria has been reported [l-3]. Its subunit composition is similar to that of Fr-ATPase [4]. These studies revealed that factor A is a different form of mitochondrial Fr-ATPase [5]. It stimulates the activity of urea-depleted sub~tochond~al particles in oxidative phospho~lation coupled to NADH or succinate oxidation, ATP-supported reversed electron flow, and Pi-ATP exchange. It differs from Fi-ATPase in that it is cold-stable, and the ATPase specific activity is latent, although it can be enhanced to match Fr -ATPase activity by exposing it to elevated temperatures. It has been suggested in the past that the lower ATPase activity of factor A may result from the presence of mitochondrial ATPase inhibitor [6] in amounts sufficient to mask most of the enzyme activity [7,8]. Analysis of factor A and Fr-ATPase for in~bitor content showed that both had roughly the same ~~bitor content (0.4 mol1340 000 g ATPase protein) but only Fr had high ATPase activity [3]. However, there remained the possibility that the inhibitor was bound to a non-specific, non-functional site in Fr , but it was located at its proper site in factor A leading to masking of the ATPase activity. The only way to overcome this criticism is to prepare factor A in a form that had zero inhibitor content. It was found [9] that membrane bound, as well as soluble Fr and Fi-X [IO] preparations form a fluorescent complex with the antibiotic inhibitor, aurove~in. They reported that a factor A preparation did not form such a complex with aurovertin and further proposed that it was either dissociated into

Comparative studies on mitochondrial coupling factor B and FB

Abstract Beef heart mitochondrial protein factor FB [Higashiyama et al , Biochemistry 14 , 4117–4121 (1975)] was purified and its properties were compared with those of coupling factor B. Both proteins stimulated ATP-driven NAD+ reduction in ammonia and EDTA-treated (AE-) submitochondrial particles, but the extent of stimulation (maximum activity of particles) was very low with FB. FB was found to be ineffective in stimulating Pi-ATP exchange in either AE-particles or reconstituted oligomycin-sensitive ATPase vesicles. Furthermore, FB failed to stimulate ATP-driven NAD+ reduction activity of AE-particles in the presence of saturating amounts of dithiothreitol (DTT). DTT alone stimulates the particle activity extensively as reported earlier. Rabbit antiserum to FB did not show a precipitin band with purified Factor B, nor did the antibody inhibit Factor B stimulated activity of the AE-particles. The data suggest that FB and Factor B are two different molecular species with different functions and fail to provide evidence that FB is a coupling factor.