F. Feo

Effect of cholesterol content on some physical and functional properties of mitochondria isolated from adult rat liver, fetal liver, cholesterol-enriched liver and hepatomas AH-130, 3924A and 5123

The cholesterol to phospholipid ratio in mitochondria from hepatomas AH-130, 3924A and 5123 is higher than in the particles isolated from adult or fetal rat livers. Nearly all the cholesterol of hepatoma mitochondria is located in membranes. As in liver mitochondria, in the particles isolated from hepatoma AH-130 there is more cholesterol in the outer than in the inner membrane. In mitochondria from cholesterol-enriched liver and hepatomas, there occurs a decrease in extent of hypoosmotic and phosphate-induced swelling and a decrease of conformational changes linked to energy states. The phenomenon is more marked in particles which exhibit higher cholesterol to phospholipid ratios. A statistically significant negative correlation exists between the cholesterol to phospholipid ratio and extent of volume or conformational changes. No significant modifications of these parameters were found in fetal liver mitochondria. Cholesterol content does not influence K+ uptake by cholesterol-enriched or hepatoma mitochondria. Nor does cholesterol content affect the respiratory increment related to this uptake. As a consequence of K+ uptake, total mitochondrial water exchangeable with tritiated water rises 20% while sucrose-impermeable water rises 42-48% in both adult rat liver and hepatoma AH-130 mitochondria. Absorbance changes linked to ion uptake do not correspond merely to variations in mitochondrial water content. Water content is apparently not influenced by the cholesterol to phospholipid ratio. However, the ratio is significantly correlated to both extent and initial rate of absorbance decrease of mitochondrial suspensions during K+ uptake. The higher the ratio, the lower the extent and initial rate of absorbance decrease.

Cholesterol and phospholipid composition of mitochondria and microsomes isolated from morris hepatoma 51 23 and rat liver

The major component of membranous lipids is represented by phospholipids [l] . It is well known that this class of lipids plays an important role in regulating the biochemical properties of subcellular particles [2] . Moreover, there exists a cholesterol:phospholipid ratio peculiar for several kinds of membranes [3]. Cholesterol is important in stabilizing arrays of phospholipids in cellular and cytoplasmic membranes [4,5] , and in regulating the permeability to small molecules of artificial [6,7] and natural [8] membranes. Lipid-lipid interactions play an important role in the interaction between lipid and protein [3] ; the protein conformation is greatly influenced by their association with the lipid environment [9]. Alterations in the protein components of mitochondrial membranes have been observed in hepatoma mitochondria [ 10,l l] . They could be, at least in part, explained by changes in the lipid composition of membranes. In hepatoma mitochondria, we have also found functional changes, which could be related to high fragility and to an increased resistance to deformation and stretching [ 12,131 . Similar alterations have been observed also in cholesterol-enriched mitochondria [5] . The knowledge of lipid composition of membranes isolated from tumors is lacking and, sometimes, contrasting [ 14,151 . As a first approach to this problem we have studied the phospholipid and cholesterol content of mitochondria and microsomes isolated from rat liver and Morris hepatoma 5 123. 2. Methods

Lipid phase transition and breaks in the Arrhenius plots of membrane-bound enzymes in mitochondria from normal rat liver and hepatoma AH-130

The relationships between the physical state of membrane lipids and enzymatic or transport activities have been analyzed in several publications [l-5] using various microorganisms. Discontinuities in the activation energies of enzymatic activities or transport processes were found to depend on the fatty acid composition and cholesterol content of membranes. Such discontinuities sometimes coincided with the phase transition of lipids as detected by X-ray diffraction or thermal analysis [3,5] . However, sometimes disparities between the activity transition temperatures and lipid transition temperatures were observed [ 1 ] . Analogous discrepancies have been seen in studies with isolated mitochondria. Different activities of membrane-bound enzymes of mitochondria exhibit breaks in their Arrhenius plots at 23-24°C and at lo12°C in homeothermic animals and chilling-sensitive plants, respectively [6] . Electron spin resonance experiments showed that the temperatures of the breaks in the Arrhenius plots of some enzymatic activities in rat liver mitochondria coin-

The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria

The phospholipid depletion of rat liver mitochondria, induced by acetoneextraction or by digestion with phospholipase A2 or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A2. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipid-deficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes. These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.

Swelling and shrinking phenomena in mitochondria isolated from rat liver and Yoshida ascites hepatoma

Abstract Mitochondria from the Yoshida ascites hepatoma,in the presence of different swelling conditions,swell less extensively than normal liver mitochondria.On the contrary, tumour mitochondria actively shrink under the action of ADP,DNP or Mg 2+ ;with the latter ion,shrinkage is more marked than in liver mitochondria. The inhibitor pattern with anion-promoted swelling or Mg 2+ -induced shrinkage is shown,and the possible implications of these results are discussed.

Effect of a cholesterol-rich diet on cholesterol content and phagocytic activity of rat macrophages

Treatment for 11–13 weeks with a cholesterol-rich diet induced increases in free and esterified serum cholesterol. There was also an increase in free cholesterol of peritoneal macrophages. A 2.2 times rise in the cholesterol to phospholipid ratio of plasma membranes occurred in cholesterol-enriched macrophages. No changes were observed in phagolysosomes. Cholesterol-enriched macrophages showed a 35.8% inhibition of latex particles phagocytosis. When lipid droplets were substituted for latex the inhibition was 81.7%.

The effect of 4-hydroxy-2, 3-trans-pent-en-1-al and maleylaldehyde on some mitochondrial functions

Abstract Low concentrations of HPE and MLA inhibited state 3 respiration of rat liver mitochondria in the presence of different NAD+-dependent substrates. MLA appeared to be more active than HPE. High aldehyde concentrations inhibited the state 3 respiration with succinate. The restraint of succinate oxidation by HPE and MLA and of glutamate plus malate oxidation by MLA correlated with the inhibition of succinate and glutamate dehydrogenase activites, respectively. HPE inhibited glutamate dehydrogenase at concentrations higher than those affecting glutamate oxidation. Malate dehydrogenase activity was slightly sensitive to HPE and MLA. Both aldehydes inhibited NADH oxidation by freeze-thawed mitochondria. These results suggest the existence of a site particularly sensitive to aldehydes in the electron transport chain between the specific NAD+-linked dehydrogenases and ubiquinone.

Mg2+-induced shrinkage in mitochondria isolated from yoshida ascites hepatoma

Abstract Mg2+ ions induce water extrusion paralleled by the bivalent cation uptake in metabolizing mitochondria isolated from hepatoma ascites; in the absence of metabolism relatively large amounts of Mg2+ are taken up, independently of the existence of a swollen state; in swollen mitochondria the bivalent cation induce shrinkage. The patterns of the Mg2+-induced shrinkage have been studied and the implication of the results obtained are discussed.

Effect of trypsin on morphological integrity and some functional activities of mitochondria from normal liver and AH-130 Yoshida ascites hepatoma

Abstract The effect of trypsin on morphological integrity and certain functional activities of mitochondria from normal liver and AH-130 Yoshida ascites hepatoma, has been studied. Liver mitochondria are barely attacked by trypsin which induces swelling only id added in large amounts. The swelling is preceeded by a lag phase during which liberation of little amounts of ninhydrin reacting compounds occurs. Tumour mitochondria are highly sensitive to trypsin, which induces immediate swelling even if added in amounts as little as 0.9 μg/mg protein. Evidence is provided that in tumour mitochondria trypsin induces damage of inner membrane permselectivity towards sucrose, and respiratory inhibition, also when it fails to inactivate the external membrane enzyme, NADH:cytochrome c reductase. The results are interpreted as indicative of the existence of a different structural state in membranes of normal and tumour mitochondria.