Ralph Penniall

Effects of chronic ethanol treatment upon rat liver mitochondria

Abstract Liver mitochondria of male Sprague-Dawley rats chronically treated with ethanol showed a similar depression (40–50 per cent) of oxygen uptake under state 4, state 3 and DNP-uncoupled conditions with succinate as substrate. Using β-hydroxybutyrate as the substrate, respiratory rates in states 4 and 3 were depressed, but the rate under DNP-uncoupled conditions was not. ADP: 0 ratios were depressed by 16 per cent with pyruvate but not with succinate. Upon closer examination of the electron transport chain in freeze-thawed mitochondria, several heretofore undetected defects were noted. These include similar depressions (41–46 per cent) in the oxidations of NADH, β-hydroxy-butyrate, malate or glutamate with cytochrome c as electron acceptor. These results suggest that defects do not occur at the level of substrate dehydrogenation but between NADH and cytochrome c . Other defects in the electron transport chain were observed: a 30 per cent decrease in NADH:ferricyanide reductase activity; a 38 per cent decrease in ubiquinone content and a 68 per cent decrease in succinate: cytochrome c reductase activity. However, the last result may not be attributed to effects upon succinic dehydrogenase per se , because succinate: DCIP reductase activity was unaltered. A 59 per cent depression of cytochrome c oxidase activity was also noted and must reflect a 44 per cent decrease in cytochrome aa 3 content, since K m and V max values were identical for control and ethanol-treated animals.

The subunit composition of beef heart cytochrome c oxidase

Beef heart cytochrome c oxidase (EC 1.9.3.1) preparations of 10 to 12 nmol heme aa3/mg protein have previously been subjected to SDS PAGE [1-5] . Variations in both the number of bands (from four to six) and their staining intensities have been reported, leading to uncertainties concerning the subunit composition. In contrast we have found, by use of an SDS-urea system for PAGE [6], that different beef heart cytochrome c oxidase preparations contain constant amounts of eight subunits, ranging from 37 000 to 4500 daltons. Our results indicate that the failure of previous investigators to observe eight components in preparations at this level of purity was most likely due to the use of inadequate methods of electrophoresis. The increased resolution of the SDS-urea system reveals as well the presence of minor amounts of at least four and possibly five higher tool. wt components in all five oxidases. By use of the SDS-urea procedure we have established that equilibration of an oxidase preparation with an NAD÷-affinity matrix results in the complete removal of the high mol. wt components. This occurrence is accompanied by the simultaneous binding by the matrix of the several NADHor NADPHlinked reductase activities which we have reported tO be consistent contaminants of oxidase preparations [7]. Two recent reports indicate that active cytochrome c oxidase preparations may consist of as few as two or three bands in SDS PAGE [8,9]. Such preparations

Origin of mitochondrial enzymes: III. Distribution and synthesis of cytochrome c in rat liver tissue

Abstract Fractionation studies of rat liver have shown that the mitochondria thereof contain more than 90% of the total cytochrome c of the tissue upon their isolation. Reproducible small complements of the remainder of the cytochrome c were found in each of other cell fractions. The intracellular site of synthesis of cytochrome c was determined by use of 3 H-δ-aminolevulinic acid ( 3 H-ALA) as a precursor of hematoheme. In short intervals of labeling following intraperitoneal injection of 3 H-ALA into mature rats, the specific activity of the hematoheme of microsomal cytochrome c greatly exceeded that of mitochondrial cytochrome c ; after 2 hr of incorporation, the two specific activities approached a common value. When short intervals were employed for incorporation of 3 H-ALA, followed by an additional interval in the presence of a molar excess of ALA it, was possible to show a precursor-product relationship between the respective microsomal and mitochondrial complements of cytochrome c . The findings indicate that the endoplasmic reticulum catalyzes the complete assembly of cytochrome c whereupon the completed protein is then transferred to the mitochondria.

Respiratory activity of isolated mammalian nuclei: III. The reduced nicotinamide adenine dinucleotide oxidase of rat liver nuclei and nucleoli☆

Rat liver nuclei and nucleoli have been shown to catalyze the reduction of neotetrazolium or oxygen with NADH as a substrate. Maximal NADH oxidase activity of nuclei requires the presence of exogenous cytochrome c; nucleoli require added polyethylenesulfonate as well as cytochrome c for demonstrable activity. Nuclei can also oxidize NADPH in the presence of NAD, neither nuclei nor nucleoli can oxidize succinic acid or a variety of other potential substrates, either with or without cytochrome c. Chemical and enzymic analysis as well as analysis by light and electron microscopy and low-temperature spectroscopy indicate the complete absence of contaminating organelles which could conceivably catalyze the observed activities. Isolated nuclei were consistently found to contain a complement of cytochrome c, in the absence of cytochromes a + a3. Nucleoli which were active in NADH oxidation when supplemented with cytochrome c were found to be essentially devoid of hemoproteins.

Age-associated changes in activities of rat hepatocytes. I. Protein synthesis.

Experiments involving simultaneous comparisons of cells from two animals establish an age-dependent decline in protein synthesis. Specific radioactivities of fractions from cells of aged animals (20+ months) are depressed 60% to 80% versus those from younger animals (2 to 14 months). Isotope ratio patterns derived by PAGE indicate that formation of a 20000 MW polypeptide of cytoribosomal origin is even more strongly and specifically depressed.

Origin of mitochondrial enzymes. V. The polypeptide character and the biosynthesis of rat liver cytochromec oxidase polypeptides by mitochondria

Isolated rat liver mitochondria were labeled in vitro withl-[14C]leucine. Sixty percent of the incorporated radioactivity was found to reside in subunits 1, 2, and 3 of cytochromec oxidase with apparent molecular weights of approximately 33,000, 25,000, and 20,000, respectively. The results indicate that these are the predominant products of protein synthesis under the conditions employed. The enzyme complex, as derived by immunoprecipitation, was found to contain four additional polypeptides with apparent molecular weights of 17,000, 12,500, 7000, and 3500. A comparison of electrophoretic profiles of the rat liver and beef heart enzyme reveals that the apparent molecular weights of all polypeptides are remarkably similar.

Studies on a cytochrome oxidase antibody. III. Cross reactivity

A rabbit serum antibody induced in response to injections of beef heart cytochrome oxidase has been demonstrated to be capable of the quantitative precipitation of a solubilized rat liver cytochrome oxidase.

Origin of mitochondrial enzymes. IV. On the character of the product of cytochrome c synthesis by the endoplasmic reticulum

Summary Following an interval of incorporation of radioactive δ-aminolevulinic acid, the pattern of labeling of the multiple forms of cytochrome c of rat liver tissue was determined. The results obtained indicate that the product of the synthetic activity of the endoplasmic reticulum must be a minor, nonfunctional form (Form IV) and that it is this entity which is transferred to the mitochondrion from the endoplasmic reticulum.

Origin of mitochondrial enzymes I. Cytochrome c synthesis by endoplasmic reticulum.

Considerable evidence indicates that isolated mitochondria cannot synthesize their entire complement of proteins [ 1,2] and, in vivo, the incorporation of labeled amino acids has been found detectably slower for the easily solubilized proteins of mitochondria than for structural protein and lipoproteins [3]. These observations caused us to undertake a study of the synthesis of a specific mitochondrial protein, cytochrome c. Using 3H-6-aminolevulinic acid (3HALA) as a label for hemoproteins, we have studied its incorporation into the cytochrome c of isolated subcellular fractions of liver tissue. The results of our experiments establish that the synthesis of cytochrome c occurs in the endoplasmic reticulum. Similar results, using radioactive lysine as a precursor, were recently reported by Cadavid and Campbell [4].

The effects of cationic proteins of rabbit polymorphonuclear leukocyte lysosomes on the respiratory activity of liver mitochondria

Abstract The cationic proteins of lysosomes of rabbit polymorphonuclear leukocytes exert a biphasic effect (stimulation at low concentrations; inhibition at high concentrations) on the State 4 respiration of rat liver mitochondria. Inhibition is the only effect observed on State 3 respiration. When separated into two fractions of circa 8000 and 4000 molecular weight, each containing three components, both fractions can stimulate State 4 mitochondrial respiration but neither is capable of complete inhibition of State 3 activity.