Arlene P. Martin

Aging-dependent modification of lipid composition and lipid structural order parameter of hepatic mitochondria☆

Abstract The effect of aging on the lipid composition of hepatic mitochondria has been determined using a rigorously defined group of Fischer 344 rats with known survivorship data. The age groups studied included mature adults as controls (8.5-month, 100% survivorship); an intermediate aged group (17.5-month, 90% survivorship); and an aged group (29-month, 20% survivorship). Lipid extracts of mitochondria were prepared using chloroform-methanol (2:1, by volume) and total phospholipid- P i , cholesterol (free and esterfied), and phospholipid composition were determined. In the aged animals, total phospholipid- P i decreased significantly ( P = 0.019) whereas cholesterol increased ( P = 0.048) with a progressive aging-dependent increase in the molar ratio of cholesterol/phospholipid. The lower total phospholipid content of hepatic mitochondria from the aged 29-month animals was due primarily to decreases in the major phospholipids with the most notable decrease being in cardiolipin (approximately 39%). Steady-state fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene as the probe was used to estimate the lipid structural order parameter of hepatic mitochondria. There was a highly significant ( P = 0.01) aging-dependent increase in the lipid structural order parameter which correlated well with the increased molar ratio of cholesterol/phospholipid in the hepatic mitochondria isolated from the aged animals. The data suggest alterations in mitochondrial membrane lipid-protein interactions in aging and are consistent with the hypothesis of impairment of membrane function in the aging process.

The structure of rat liver mitochondria: A reevaluation

Summary Mitochondria have been considered to have single spherical or tubular shape. Recently a new model has been proposed for the structure of yeast mitochondria which suggests that there is a single, branched, tubular mitochondrion per cell. Using the technique of serial sectioning, we present evidence showing that some, but not all mitochondria of normal adult rat liver have a branched, tubular structure. These observations should lead to a reevaluation of current concepts regarding mitochondrial function and biogenesis.

Effect of aging on the oxidative phosphorylation pathway

The proposed study was undertaken to investigate the effect of aging on control of the oxidative phosphorylation pathway. Flux control coefficients for adenine nucleotide translocase and cytochrome c oxidase were determined using the procedure of Groen et al. [J. Biol. Chem., 257 (1982) 137-144]. Hepatic mitochondrial fractions from Fischer 344 rats were isolated from control (average age 6.5 months), and aged (average age 27.3 months) groups. No aging-related changes in the extent of control of respiration by the oxidase were obtained, however, differences were observed for the translocase. For the control group of animals, the greatest regulation occurred at 80-85% maximal respiratory rates, and declined at higher rates. For the aged group, a similar flux control coefficient was obtained at 80-85% respiration, but was maintained as respiration increased to maximal rates. It is proposed that changes in the flux control coefficients at maximal respiratory rates are associated with an aging-related decrease in translocase activity. Evaluation of translocase content revealed no significant differences between the two groups supporting the concept that the decreased activity was not due to decreased content. During the course of these experiments, it also became apparent that there was a significant aging-related decrease in the rate of succinate oxidation providing an adequate supply of ADP was present. No significant changes in respiratory rates, or RCR, were evident at suboptimal concentrations of ADP as reported previously from this laboratory [Vorbeck, M.L. et al., Arch. Biochem. Biophys., 214 (1982) 67-79]. Since similar decreases in respiration were obtained upon addition of an uncoupler, the aging-related changes in respiration are attributed to differences at the level of the electron transport system, including its associated reactions. The aging-related differences in respiratory rates, and extent of control of respiration, were both observed under conditions of maximal stimulation of respiration. This suggests an inability of mitochondria from aged animals to respond to the increased demands of oxidation. Basic to these differences may be the lipid-membrane associated changes seen during aging.

Mitochondrial three-dimensional form in ascites tumor cells during changes in respiration

Serial sectioning-reconstruction techniques were used to show the presence of complex, branched mitochondrial forms in Ehrlich-Lettre ascites tumor cells. Although these were the predominant mitochondrial forms present in all cells examined, simple forms, i.e., rods and spheres, also were observed. Attempts to alter the metabolic status of the cell by changing the rate of respiration or degree of coupling of oxidation to phosphorylation did not shift the mitochondrial population from predominantly complex to simple forms. It was shown that complex, branched mitochondrial forms, as well as simple rod and sphere forms were present during coupled, uncoupled, and endogenous respiration and during anaerobiosis. Both complex as well as simple mitochondrial forms underwent transition from an orthodox to a condensed conformation during State 3 respiration. Under the present experimental conditions, differences in mitochondrial forms were not dependent on coupled versus uncoupled respiration or aerobic versus anaerobic state.

Glycerophosphatide biogenesis: I. Subcellular localization of cytidine triphosphate: Phosphatidic acid cytidyl transferase

Abstract The subcellular localization of cytidine triphosphate: phosphatidic acid cytidyl transferase (CTP: PA cytidyl transferase) has been investigated. Rat liver and bovine heart homogenates were fractionated by differential centrifugation and the composition of the fractions evaluated by marker enzymes and electron microscopy. In both rat liver and bovine heart preparations, the highest relative specific activity was associated with the mitochondrial fraction and closely paralleled the distribution pattern for succinoxidase, a mitochondrial marker.

Aging-related decrease in hepatic cytochrome oxidase of the Fischer 344 rat.

Abstract The effect of aging on the hepatic mitochondrial population has been determined using a rigorously defined group of Fischer 344 rats with known survivorship data. The age groups studied included mature adult controls (8.5 months; 100% survivorship), an intermediate aged group (17.5 months; 90% survivorship), and an aged group (29 months; 20% survivorship). Cytochrome oxidase activity and content were determined in homogenates and mitochondrial fractions. The mitochondrial fractions were characterized by determination of respiratory activity, and monoamine oxidase activity as well as evaluation of the polypeptide composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The yield of protein in the isolated mitochondrial fraction as well as the mitochondrial specific content decreased significantly as a function of aging. Mitochondrial specific content was determined from the specific activities of cytochrome oxidase in the homogenate (per gram liver) and in the isolated mitochondrial fraction (per mg protein). Specific activity of hepatic cytochrome oxidase decreased approximately 15% (P = 0.035) in homogenates from the 17.5-month animals with a further, highly significant (P = 0.0002) decrease (29%) in the 29-month animals. In contrast, there was no statistically significant difference among the age groups in the cytochrome oxidase specific activity in the isolated hepatic mitochondrial fractions. However, the percentage of the total homogenate cytochrome oxidase activity recovered in the isolated mitochondrial fraction decreased significantly in the 29-month animals (P = 0.0063 vs the 8.5-month controls; P = 0.022 vs the 17.5-month group). Cytochrome aa3 content of total liver homogenates from aged animals decreased (P = 0.00064) which is in agreement with the decline in cytochrome oxidase specific activity in this age group. In the mitochondrial fraction from the aged animals, cytochrome aa3 content was essentially unchanged which is consistent with the lack of aging-related change in mitochondrial cytochrome oxidase specific activity. In freshly isolated mitochondrial fractions, no aging-related alterations were observed in respiratory control and ADP O ratios. The addition of exogenous NADH and cytochrome c did not change significantly the respiratory rate of hepatic mitochondria from control or aged animals. These results demonstrate the integrity of freshly isolated mitochondrial preparations from both control and aged Fischer 344 rats. In addition, there was no aging-related alteration in either monoamine oxidase specific activity or polypeptide composition. The similarities observed in the specific activities of cytochrome oxidase and monoamine oxidase, as well as in the cytochrome aa3 content and polypeptide composition of the isolated mitochondrial fraction, suggest a generalized decrease in hepatic mitochondrial content as a function of aging rather than a selective loss of mitochondrial components.

Lipid-protein interactions of erythrocyte membranes: Comparison of normal O,Rh(D) positive with the rare O,Rhnull

Abstract Phospholipase A 2 modification of lipid-protein interactions of normal O,Rh(D) positive erythrocyte membranes increased the fluorescence intensity of the membrane bound probe, 1-anilinonaphthalene-8-sulfonate (ANS) and increased the N-1-[ 14 C]-ethyl maleimide ([ 14 C]-NEM) labeling of sulfhydryl groups in two proteins of molecular weight >200,000. In marked contrast, phospholipase A 2 modification of the rare phenotype O,Rh null membranes resulted in no significant increase in ANS fluorescence or labeling of sulfhydryl groups by [ 14 C] NEM. Since the O,Rh null erythrocytes demonstrated an increased osmotic fragility and decreased survival time, the fluorescence and sulfhydryl labeling data support the conclusion that hydrophobic bonding between β-fatty acid side chains and non-polar regions of asymmetric proteins is necessary for maintaining the native structure of the O,Rh(D) positive membrane. Comparative studies with phospholipase C or D implied that ionic bonding played a similar though less important structural role in both membranes.

An ELISA for screening hybridoma cultures for monoclonal antibodies against a detergent solubilized integral membrane protein

A method is described for the binding of a detergent solubilized integral membrane protein to polystyrene immunoassay plates. Addition of Bouins fluid, a histochemical fixative, to wells of plates containing the detergent solubilized antigen, followed by low speed centrifugation, is sufficient to promote binding of antigen in the presence of Triton X-100 concentrations as high as 1.75%. The binding of antigen is rapid and the entire binding procedure, including removal of fixative and washing of the plates, can be accomplished in less than 15 min. Immunological specificity of the bound antigen is retained. This method has been used to effectively screen hybridoma cultures for specific antibodies.

Mitochondrial ultrastructure and volume during swelling.

The ultrastructure of hepatic mitochondria was studied during energy-linked swelling and compared with overall mitochondrial volume and optical density measurements. Total mitochondrial volume change during swelling was determined directly by resistive particle counting using a pulse height analyzer in conjunction with a Coulter counter Model ZB. The data presented provide direct evidence that the optical density decrease during swelling is separate and distinct from the actual increase in overall mitochondrial volume. Although 84% of the total swelling, as determined by optical density measurements, was complete within 3 min, overall mitochondrial volume, as determined with the Coulter counter, increased only 20%. The Coulter counter system provides the first direct evidence that, as suggested by earlier gravimetric and ultrastructural studies, optical density decrease during swelling does not reflect an increase in total mitochondrial volume but rather is associated with alterations to the inner membrane-matrix space. Only following sequential changes to the inner membrane-matrix space does mitochondrial size increase as reflected by the increase in overall volume measured by the Coulter counter.

Phosphate-dependent, trifluoperazine-sensitive Ca2+ efflux from rat liver mitochondria. Modulation by a cytosol factor.

Mitochondria possess separate pathways for the uptake and release of Ca2+: an electrophoretic uniporter for Ca2+ influx, and an independent electroneutral efflux mechanism operational when a high membrane potential is retained (reviews [ 1,2]). Ca2+ efflux from heart and brain mitochondria appears to be directly coupled to Na’ entry [3,4] ; in liver mitochondria, however, the mechanism(s) which regulates the release (or retention) of Ca2+ is unclear. The role of Pi in this process is uncertain as, under diverse experimental conditions, Pi has been reported to stimulate [5], inhibit [6] or efflux in parallel with Ca2+ efflux [7]. Hepes and ruthenium red were obtained from Sigma. Ruthenium red was further purified according to [8]. Calmodulin was obtained from Calbiochem-Behring Corp., and trifluoperazine was a gift from Smith Kline and French (Philadelphia PA). Sprague-Dawley rats (175-250 g) were used without fasting. Unless noted otherwise, all procedures were carried out at 4°C. Livers were homogenized as in [9] in 70 mM sucrose, 220 mM mannitol containing 10 mM Hepes (pH 7.4) (SMH). The mitochondrial fraction was isolated by differential centrifugation [9] and washed twice by resuspension in SMH and centrifugation at 95 000 X g. The cytosol was obtained by centrifugation of the post-mitochondrial supernate at 1OOOOOXgfor 1 h. These apparently conflicting reports regarding Pi prompted us to re-evaluate its effect on Ca2+ efflux in liver mitochondria. In our hands, Pi stimulated mitochondrial Ca2+ efflux. We have discovered a heatstable factor in liver cytosol which significantly enhanced Pi-dependent Ca2+ efflux. This enhanced Ca2+ effIux was effectively blocked by the phenothiazine antipsychotic drug, TFP. These results suggest involvement of a cytosol factor in the modulation of Ca” release from rat liver mitochondria.