Senescence-associated decrease of NADPH-induced lipid peroxidation in rat liver microsomes

Senescence is associated with decrease in the NADPH-induced lipid peroxidation in liver homogenate as well as rough and smooth microsomes of female rats. In the microsomal fractions, sensitivity to NADPH-induced lipid peroxidation is high in young adults (3-month-old), decreases in middle aged (12-month-old) and reaches lowest levels in senescent (30-month-old) rats. Increasing the concentration of co-factors or time of incubation does not alter this resistance observed in the senescent rats. Major factors responsible for this resistance in senescent rats seem to be low levels of substrate in the c reductase, cytochrome P-450 and high cholesterol:phospholipid ratios besides enhanced levels of superoxide dismutase, alpha-tocopherol and reduced glutathione.     

An in vitro ESR study of uncatalyzed rat liver protein-catalyzed spin-labeled phosphatidylcholine exchange

ESR spectrometry has been used to study fatty acid spin-labeled phosphatidylcholine exchange from single bilayer donor vesicles to various acceptor systems, such as intact or differently treated mitochondria, phospholipid multilamellar vesicles or single bilayer vesicles. This exchange is catalyzed by soluble non-specific rat liver protein, first investigated by Bloj and Zilversmit in 1977 (J. Biol. Chem. 252, 1613--1619). Non-catalyzed phosphatidylcholine exchange has also been studied. Full inhibition of both mechanisms occurs with lipid-depleted acceptor mitochondria, while N-ethylmaleimide-treated mitochondria behave as good acceptors during catalyzed exchange but are in no way effective during spontaneous exchange. Non-catalyzed exchange does not take place with phospholipase D-treated mitochondria as acceptors, while the pure catalyzed mechanism is inhibited by 28%. Neither multilamellar nor single bilayer phospholipid vesicles exchange spin-labeled phosphatidylcholine in the absence of protein, the former being a poorer acceptor system than the latter during catalyzed exchange, when this activity is 31 and 80%, respectively, of that of intact mitochondria. The hypothesis is made that the spontaneous mechanism is active among intact natural membranes and could be of some importance in vivo. Furthermore, the biomembrane protein moiety is assumed to be involved in the catalyzed exchange more as a phospholipid spacer than as a binder between the exchange protein and the membrane involved. Phospholipids, on the contrary, appear to be important for both functions.     

An in vitro ESR study of uncatalyzed and rat liver protein-catalyzed spin-labeled phosphatidylcholine exchange

ESR spectrometry has been used to study fatty acid spin-labeled phosphatidylcholine exchange from single bilayer donor vesicles to various acceptor systems, such as intact or differently treated mitochondria, phospholipid multilamellar vesicles or single bilayer vesicles. This exchange is catalyzed by soluble non-specific rat liver protein, first investigated by Bloj and Zilversmit in 1977 (J. Biol. Chem. 252, 1613–1619). Non-catalyzed phosphatidylcholine exchange has also been studied. Full inhibition of both mechanisms occurs with lipid-depleted acceptor mitochondria, while $\text{N-}\text{ethylmaleimide-treated}$ mitochondria behave as good acceptors during catalyzed exchange but are in no way effective during spontaneous exchange. Non-catalyzed exchange does not take place with phospholipase D-treated mitochondria as acceptors, while the pure catalyzed mechanism is inhibited by 28%. Neither multilamellar nor single bilayer phospholipid vesicles exchange spin-labeled phosphatidylcholine in the absence of protein, the former being a poorer acceptor system than the latter during catalyzed exchange, when this activity is 31 and 80%, respectively, of that of intact mitochondria. The hypothesis is made that the spontaneous mechanism is active among intact natural membranes and could be of some importance in vivo. Furthermore, the biomembrane protein moiety is assumed to be involved in the catalyzed exchange more as a phospholipid spacer than as a binder between the exchange protein and the membrane involved. Phospholipids, on the contrary, appear to be important for both functions.     

Kinetics of NADPH-induced lipid peroxidation in rat liver microsomal fractions as a function of age

The kinetics of NADPH-induced lipid peroxidation in hepatic rough and smooth microsomes have been studied in rats ranging in age from 1 day to 2 years. Apparent Km and Vmax for NADPH and the extent of lipid peroxidation show that lipid peroxidation potential is low at birth, increases during postnatal development, and decreases during senescence. Our results indicate that this trend may be due to changes in phospholipid content and NADPH cytochrome c reductase activity in microsomal fractions.     

Inhibition by polyamines of lipid peroxide formation in rat liver microsomes.

Both NADPH- and ascorbic acid-dependent lipid peroxidations were inhibited by spermine, the degree of inhibition being greater with the former peroxidation. The effective concentration of spermine required for inhibition was higher when larger amounts of microsomes were used. However, the activities of NADPH-cytochrome c reductase and NADPH-peroxidase were not influenced by spermine. These results suggest that spermine inhibits lipid peroxidation by binding to phospholipids in the microsomes.     

NADPH-dependent inhibition of lipid peroxidation in rat liver microsomes.

Microsomal NADPH-driven electron transport is known to initiate lipid peroxidation by activating oxygen in the presence of iron. This pro-oxidant effect can mask an antioxidant function of NADPH-driven electron transport in microsomes via vitamin E recycling from its phenoxyl radicals formed in the course of peroxidation. To test this hypothesis we studied the effects of NADPH on the endogenous vitamin E content and lipid peroxidation induced in liver microsomes by an oxidation system independent of iron: an azo-initiator of peroxyl radicals, 2,2'-azobis (2,4-dimethylvaleronitrile), (AMVN), in the presence of an iron chelator deferoxamine. We found that under conditions NADPH: (i) inhibited lipid peroxidation; (ii) this inhibitory effect was less pronounced in microsomes from vitamin E-deficient rats than in microsomes from normal rats; (iii) protected vitamin E from oxidative destruction; (iv) reduced chromanoxyl radicals of vitamin E homologue with a 6-carbon side-chain, chromanol-alpha-C-6. Thus NADPH-driven electron transport may function both to initiate and/or inhibit lipid peroxidation in microsomes depending on the availability of transition metal catalysts.     

Stimulation of acyl-CoA:cholesterol acyltransferase activity in rat liver microsomes by phosphatidylcholine

Acyl-CoA:cholesterol acyltransferase (ACCAT) activity of rat liver microsomes was stimulated by phosphatidylcholine. The stimulatory effect varied with the composition of the phosphatide: dimyristyl-, dipalmityl-, distearyl- and dioleylphosphatidylcholine were stimulatory, whereas dicaproyl- and dilinoleylphosphatidylcholine were not. The results suggest that increased fluidity of the membrane induced by phosphatide is probably not involved in the stimulation of cholesterol esterification. Phosphatide exerted its effect directly on the microsomes and did not extract cholesterol or ACCAT from the microsomes to an appreciable extent.Hydrolysis of microsomal phosphatide suppressed ACCAT activity. Enztme activity was restored with the addition of phosphatidylcholine. The results suggest that phosphatide may be required for cholesterol esterification.     

Dihydroxyfumaric acid induced lipid peroxidation in rat liver microsomes.

Dihydroxyfumaric acid induced lipid peroxidation in rat liver microsomes. This reaction was heat-insensitive contrary to the mitochondrial peroxidation reported in the previous paper, and was enhanced by p-chloromercuribenzoate. Additions of Fe²⁺ and Fe³⁺ stimulated both the lipid peroxidation and the disappearance of dihydroxyfumaric acid. On the other hand, addition of Mn²⁺ or Cu²⁺, which stimulated the disappearance of dihydroxyfumaric acid, inhibited the lipid peroxidation. Hydroxyl radical scavengers, superoxide dismutase and catalase had no effect on this lipid peroxidation and dihydroxyfumaric acid disappearance. The cytochrome p-450 content decreased about 70 % in parallel with the lipid peroxidation.     

Formation of lipid peroxides in isolated rat liver microsomes by singlet molecular oxygen.

Rat liver microsomes were incubated in neutral aqueous solution of potassium peroxychromate, a system which generates singlet molecular oxygen. Such incubation resulted both in a rapid decline in NADPH-cytochrome c reductase activity, and in an increase in formation of lipid peroxides. These reactions were not inhibited by either superoxide dismutase (SOD) or mannitol, nor were they entirely duplicated by incubating microsomes with hydrogen peroxide. However, a high concentration of 1,4-diazabicyclo-[2,2,2]octane (DABCO), a known scavenger of singlet oxygen, prevented both decline in reductase activity and formation of lipid peroxides. These results suggest that the observed effects are, in fact, attributable to singlet oxygen, and not to hydrogen peroxide, superoxide radical, or hydroxyl radical.     

Asymmetry of the site of choline incorporation into phosphatidylcholine of rat liver microsomes.

[14C]Choline was incorporated into microsomal membranes in vivo, and from CDP-[14C]choline in vitro, and the site of incorporation determined by hydrolysis of the outer leaflet of the membrane bilayer using phospholipase C from Clostridium welchii. Labelled phosphatidylcholine was found to be concentrated in the outer leaflet of the membrane bilayer with a specific activity approximately three times that of the inner leaflet. During incorporation of CDP-choline and treatment with phospholipase C the vesicles retained labelled-protein contents indicating that they remained intact. When the microsomes were opened with taurocholate after incorporation of [14C]choline in vivo, the labelled phosphatidylcholine behaved as a single pool. Selective hydrolysis of labelled phosphatidylcholine in intact vesicles is not, therefore, a consequence of specificity of phospholipase C. These results indicate that the phosphatidylcholine of the outer leaflet of the microsomal membrane bilayer is preferentially labelled by the choline-phosphotransferase pathway and that this pool of phospholipid does not equilibrate with that of the inner leaflet.     

Enhancement of hydroperoxide-dependent lipid peroxidation in rat liver microsomes by ascorbic acid

Simultaneous addition of ascorbic acid and organic hydroperoxides to rat liver microsomes resulted in enhanced lipid peroxidation (approximately threefold) relative to incubation of organic hydroperoxides with microsomes alone. No lipid peroxidation was evident in incubations of ascorbate alone with microsomes. The stimulatory effect of ascorbate on linoleic acid hydroperoxide (LAHP)-dependent peroxidation was evident at all times whereas stimulation of cumene hydroperoxide (CHP)-dependent peroxidation occurred after a lag phase of up to 20 min. EDTA did not inhibit CHP-dependent lipid peroxidation but completely abolished ascorbate enhancement of lipid peroxidation. Likewise, EDTA did not significantly inhibit peroxidation by LAHP but dramatically reduced ascorbate enhancement of lipid peroxidation. The results reveal a synergistic prooxidant effect of ascorbic acid on hydroperoxide-dependent lipid peroxidation. The inhibitory effect of EDTA on enhanced peroxidation suggests a possible role for endogenous metals mobilized by hydroperoxide-dependent oxidations of microsomal components.     

Inhibition of protein carbonyl formation and lipid peroxidation by glutathione in rat liver microsomes.

The peroxidation of rat liver microsomal lipids is stimulated in the presence of iron by the addition of NADPH or ascorbate and is inhibited by the addition of glutathione (GSH). The fate of GSH and the oxidative modification of proteins under these conditions have not been well studied. Rat liver microsomes were incubated at 37 degrees C under 95% O2:5% CO2 in the presence of 10 microM ferric chloride, 400 microM ADP, and either 450 microM ascorbic acid or 400 microM NADPH. Lipid peroxidation was assessed in the presence 0, 0.2, 0.5, 1, or 5 mM GSH by measuring thiobarbituric acid reactive substance (TBARS) and oxidative modification of proteins by measuring protein thiol and carbonyl groups. GSH inhibited TBARS and protein carbonyl group formation in both ascorbate and NADPH systems in a dose-dependent manner. Heat denaturing of microsomes or treatment with trypsin resulted in the loss of this protection. The formation of protein carbonyl groups could be duplicated by incubating microsomes with 4-hydroxynonenal. Ascorbate-dependent peroxidation caused a loss of protein thiol groups which was diminished by GSH only in fresh microsomes. Both boiling and trypsin treatment significantly decreased the basal protein thiol content of microsomes and enhanced ascorbate-stimulated lipid peroxidation. Protection against protein carbonyl group formation by GSH correlated with the inhibition of lipid peroxidation and appeared not to be due to the formation of the GSH conjugate of 4-hydroxynonenal as only trace amounts of this conjugate were detected. Ninety percent of the GSH lost after 60 min of peroxidation was recoverable as borohydride reducible material in the supernatant fraction. The remaining 10% could be accounted for as GSH-bound protein mixed disulfides. However, only 75% of the GSH lost during peroxidation appeared as glutathione disulfide, suggesting that some was converted to other soluble borohydride reducible forms. These data support a role for protein thiol groups in the GSH-mediated protection of microsomes against lipid peroxidation.     

Topography of phosphatidylcholine,phosphatidylethanolamine and triacylglycerol biosynthetic enzymes in rat liver microsomes

The topography of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol biosynthetic enzymes within the transverse plane of rat liver microsomes was investigated using two impermeant inhibitors, mercury-dextran and dextran-maleimide. Between 70 and 98% of the activities of fatty acid : CoA ligase (EC 6.2.1.3), $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were inactivated by mercury-dextran. Dextran-maleimide caused 52% inactivation of the $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase. Inactivation of each of these activities except fatty acid : CoA ligase occurred in microsomal vesicles which remained intact as evidenced by the maintenance of highly latent mannose-6-phosphatase activity (EC 3.1.3.9). These glycerolipid biosynthetic activities were not latent, indicating that substrates have free access to the active sites. Moreover, ATP, CDP-choline and CMP appeared unable to penetrate the microsome membrane. These data indicate that the active sites of these enzymes are located on the external surface of microsomal vesicles.It is concluded that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.     

Topography of phosphatidylcholine, phosphatidylethanolamine and triacylgycerol biosynthetic enzymes in rat liver microsomes

The topography of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol biosynthetic enzymes within the transverse plane of rat liver microsomes was investigated using two impermeant inhibitors, mercury-dextran and dextran-maleimide. Between 70 and 98% of the activities of fatty acid : CoA ligase (EC 6.2.1.3), sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were inactivated by mercury-dextran. Dextran-maleimide caused 52% inactivation of the sn-glycerol-3-phosphate acyltransferase. Inactivation of each of these activities except fatty acid : CoA ligase occurred in microsomal vesicles which remained intact as evidenced by the maintenance of highly latent mannose-6-phosphatase activity (EC 3.1.3.9). These glycerolipid biosynthetic activities were not latent, indicating that substrates have free access to the active sites. Moreover, ATP, CDP-choline and CMP appeared unable to penetrate the microsome membrane. These data indicate that the active sites of thease enzymes are located on the external surface of microsomal vesicles. It is concluded that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.     

Effect of thiols on lipid peroxidation in rat liver microsomes

The stimulatory or inhibitory effects of various thiol compounds on in vitro lipid peroxidation by iron-ascorbate in rat liver microsomes were determined. Glutathione had no measurable pro-oxidant capacity, in contrast, it protected against lipid peroxidation. N-Acetyl l-cysteine and S-methyl-glutathione had no effect on in vitro lipid peroxidation. l-Cysteine stimulated lipid peroxidation and also of d-penicillamine and dl-dithiothreitol the pre-oxidant capacity predominated the anti-oxidant capacity. Cysteamine afforded a pronounced protection against in vitro lipid peroxidation. In contrast to the labile character of the glutathione dependent protection, the protection by cysteamine was not affected by heat-pretreatment of the liver microsomes or alkylating protein sulfhydryl groups by N-ethyl maleimide. Again in contrast to glutathione, the protection against in vitro microsomal lipid peroxidation by cysteamine was not reduced after in vivo lipid peroxidation induced by CC14. This suggests that even after the process of lipid peroxidation has been started, administration of cysteamine might still be beneficial.     

Ultrasonic studies of lipid bilayer. Phase transition in synthetic phosphatidylcholine liposomes

The ultrasonic velocity at 3 MHz and the density in the nonsonicated and sonicated liposomes of dipalmitoylphosphatidylcholine have been measured in the temperature range from 0 degrees C to 55 degrees C. The results indicate that nonsonicated multilamellar vesicles undergo a weak first order transition which is analogous to the nematic-isotropic transition of liquid crystals. A sharp change in the ultrasonic velocity associated with the first order transition disappears when the multilamellar vesicles are sonicated. The bulk modulus of the lipid bilayer calculated from the ultrasonic velocity and the density of sonicated liposomes has a value of 3.0 X 10(10) dyne/cm2 at 20 degrees C, reaches a minimum value of 2.1 X 10(10) dyne/cm2 at its transition temperature and increases slightly to 2.2 X 10(10) dyne/cm2 at 50 degrees C.     

Stimulation of lipid peroxidation in rat liver microsomes by tetraphenylporphine and its metallocomplexes

It is shown that tetraphenylporphyrin (TPP) and its complexes with metals decrease the rate of the diene conjugate formation. The above compounds increase the malonic dialdehyde accumulation. The effect of TPP and its complexes with metals is connected with stimulation of lipid peroxidation in biomembranes.     

Relation between lipid polymorphism and transbilayer movement of lipid in rat liver microsomes

We have studied the effects of trinitrophenylation on the transbilayer movement of phosphatidylcholine and the macroscopic lipid structure in rat liver microsomal membranes. The transbilayer movement of phosphatidylcholine was investigated using the PC-specific transfer protein. ³¹P-NMR was employed to monitor the phospholipid organization in intact microsomal vesicles. The results indicate that modification of microsomes with trinitrobenzenesulfonic acid enhances the transbilayer movement of phosphatidylcholine at 4°C. Furthermore, phosphatidylethanolamine headgroup trinitrophenylation in microsomes increases the isotropic component in the ³¹P-NMR spectra even at 4°C, possibly representing the appearance of intermediate non-bilayer lipid structures. The observed parallel between these data suggests that phosphatidylethanolamine molecules in the microsomal membrane, probably in combination with a protein component, are able to destabilize the bilayer organization, thereby facilitating the transmembrane movement of phospholipids.     

Cytosol-stimulated remodeling of phosphatidylcholine in rat lung microsomes

When 600 × g supernatants of 10% (w/v) rat lung homogenates were incubated with CDP[Me-¹⁴C]choline, both saturated and unsaturated species of phosphatidylcholine were formed from endogenous diacylglycerols. The percentage radioactivity in the disaturated species of total phosphatidylcholine increased with time from 12% after 5 min to 30% after 60 min incubation. In similar experiments with 20000 × g supernatants, the increase in the disaturated species of microsomal phosphatidylcholine was from 25 to 37% over the same time period. In incubations of isolated microsomes in buffer, the percent of ¹⁴C label in disaturated phosphatidylcholine remained constant at a level of 25%. To investigate a possible role of cytosolic factor(s) in the increase in the percentage of disaturated phosphatidylcholine with time, microsomes were prelabeled by incubation in buffer with CDP[Me-¹⁴C]choline to give a fixed ratio of radioactive saturated and unsaturated phosphatidylcholine species. When the reisolated microsomes were incubated in buffer, the distribution of radioactivity over saturated and unsaturated species remained constant. In contrast, incubation of prelabeled microsomes in the presence of cytosol caused an increase in the percent radioactivity in saturated phosphatidylcholines from a starting value of 18 to 30% after 60 min incubation, while leaving total phosphatidylcholine radioactivity unaffected. These results indicate a remodeling of phosphatidylcholine under the influence of a cytosolic factor(s). Evidence is presented that suggests that Ca²⁺-independent cytosolic phospholipase A₂ activity as well as a microsomal ATP-independent CoA-mediated acyltransferase activity might contribute to this remodeling. The cytosol donates the necessary CoA for this acyl transfer as well as saturated acyl-CoA for the reacylation of lysophosphatidylcholine.     

The composition of rat liver microsomes. The structural proteins of rat liver microsomes

1. The structural-protein component of microsomal membranes was isolated by three separate methods. Analysis by polyacrylamide-gel electrophoresis indicated that the microsomal structural component is made up of a heterogeneous group of proteins. These proteins were further characterized by their phospholipid-binding capacity. The electrophoretic patterns of microsomal structural proteins were found to differ significantly from those of mitochondrial structural proteins. 2. The reticulosomal fraction was also characterized by electrophoresis with reference to total microsomal proteins, microsomal structural proteins and ribosomal proteins. The reticulosomes gave an electrophoretic pattern significantly different from those of the other three preparations examined. It is suggested that reticulosomes consist largely of enzymic proteins of the endoplasmic reticulum.