Amino acid composition of rat and human liver microsomes in normal and pathological conditions

The amino acid composition of proteins from liver microsomes has been studied in rats and in human subjects with normal liver, with obstructive jaundice or liver cirrhosis. The pattern of the amino acid composition of microsomes appeared to be species-specific. Phenylalanine, threonine, serine, proline, histidine and [aspartic acid plus asparagine] were increased, while alanine, tyrosine, glycine and arginine were decreased in the human compared to the rat microsomes. In patients with obstructive jaundice of short duration (less than two months) only a slight decrease in leucine and phenylalanine could be noticed, while in the case of liver cirrhosis amino acid composition was markedly changed.     

Modification of the biosynthesis and composition of polyglycerophosphatides in outer and inner mitochondrial membranes by cytidine liponucleotides

The biosynthesis of [3H]polyglycerophosphatides ([3H]phosphatidylglycerophosphate and [3H]phosphatidylglycerol) in mitochondrial and submitochondrial (outer and inner) membranes isolated from guinea pig liver was examined. Experimental results have established that the amount of biosynthesized [3H]polyglycerophosphatides and the relative amounts of biosynthesized [3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate can be influenced by varying the composition of fatty acids in CDP-diglycerides and by altering the incubation time of the mixture containing CDP-diglycerides (obligatory precursor), sn-[2-3H]glycerol-3-phosphate and mitochondria or submitochondrial membranes. The changes thus obtained in respect to the amount and composition of biosynthesized [3H]polyglycerophosphatides are different in mitochondria and submitochondrial membranes. The highest amount of biosynthesized [3H]polyglycerophosphatides was obtained with CDP-didecanoin and inner mitochondrial membranes. The greatest accumulation of [3H]phosphatidylglycerol with CDP-didecanoin was obtained in mitochondria and outer mitochondrial membranes, while in inner mitochondrial membranes the amounts of [3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate accumulated were approximately the same. In general, prolongation of the incubation time decreased the relative amounts of [3H]phosphatidylglycerolphosphate and increased the amount of accumulated [3H]phosphatidylglycerol, but the absolute amounts of these [3H]polyglycerophosphatides were more dependent on fatty acid composition of CDP-diglycerides tested. The following cytidine liponucleotides were tested: CDP-didecanoin, CDP-dipalmitin, CDP-diolein, and CDP-diglycerides containing saturated and unsaturated fatty acids similar to those in egg yolk lecithin. The formation of [3H]cardiolipin from [3H]phosphatidylglycerol in the presence of CDP-didecanoin and Mn2+ was found in both the outer and inner mitochondrial membranes.     

Delta-6-desaturase activity of human liver microsomes from patients with different types of liver injury

The delta-6-desaturase (D6D) activity was evaluated in microsomes from liver fragments of cholecystectomized subjects without any liver pathology and from explanted liver of patients affected by cirrhosis of different etiologies. We observed a significant decrease in D6D activity, evaluated by a radiochemical technique using 1-[14C]-linoleic acid as substrate, in cirrhotic patients with no correlation with the etiology of the cirrhosis. The D6D activity within the pathological group was quite similar. No alteration in the 20:4/18:2 ratio obtained by gas chromatographic analysis of fatty acid methyl esters of microsomal membranes was found. Liver disease seems to be the main cause of the decreased enzyme activity independent of its etiology.     

Ultrastructural aspects and amino acid composition of the purified inner and outer membranes of human liver mitochondria as compared to rat liver mitochondria.

1. The mitochondria isolated from human or rat liver were fractionated into submitochondrial particles and purified inner and outer membrane. According to different marker enzymes the inner membranes were enriched about 5-6-fold and the outer membranes about 12-14-fold. The electron microscopical appearance of the membranes was that expected on the basis of enzymic characterization. 2. A comparison of the average amino acid composition of the membrane proteins from the two types of mitochondria has been made. In the case of submitochondrial particles there were statistically significant differences between the human and rat hydrolysates for only five amino acids. Analysing the purified mitochondrial membranes there were significant differences between the two species for nine amino acids in the case of outer membranes and for 12 amino acids in the case of inner membranes. 3. With one exception all amino acids that were increased or decreased in the outer membrane exhibited a similar trend in the inner membrane of human compared with rat liver mitochondria. It appears that liver mitochondrial membranes have a species-dependent pattern of amino acid composition of their proteins.     

Changes in tissue and mitochondrial membrane composition during rapid growth, maturation and aging in rainbow trout, Oncorhynchus mykiss

Membrane compositions, particularly of mitochondria, could be critical factors in the mechanisms of growth and aging processes, especially during phases of high oxidative stress that result in molecular damage. In the present study, liver and mitochondrial membrane phospholipid (PL) compositions were analyzed in rainbow trout during its four first years of life, a period characterized by rapid growth and high oxidative stress. Specifically, farmed fish of three ages (1-, 2- and 4-years) were studied, and PL compositions of whole liver and liver mitochondria, and fatty acid compositions of individual PL classes were determined. Liver mitochondrial membranes showed a PL composition different to that of the whole tissue suggesting adaptation of cell and subcellular membranes to specific functions. Individual PL had characteristic fatty acid compositions that were similar in whole liver and mitochondrial membranes. Whole liver and mitochondria showed increased lipid peroxidation with age along with changes in membrane PL fatty acid compositions. Most PL classes showed similar changes in fatty acid composition among the age groups, with reduced proportions of docosahexaenoic acid (DHA) and, generally, concomitantly increased levels of monounsaturated fatty acids, which together resulted in reduced peroxidation index (PIn). However, total polyunsaturated fatty acid (PUFA) content did not change significantly with age due to increased eicosapentaenoic acid, docosapentaenoic acid and, in most PL, increased n−6 PUFA. These results suggest there may be oxidation of PL DHA with compensatory mechanisms to maintain membrane fluidity and function. However, modification of fatty acid composition of specific PLs, such as cardiolipin, could affect the electron transport chain efficiency and propagate the oxidative reaction throughout the cell. In addition, both the content and fatty acid composition of sphingomyelin, which has been suggested as a possible mediator of cell dysfunction and apoptosis, changed with age differently to the other PL classes. Moreover, these changes showed different trends between mitochondria and whole liver. These data suggest there is marked oxidative stress associated with rapid growth and maturation in rainbow trout. Changes observed in membrane lipids point to their possible participation in the processes involved in this species response to oxidative stress and damage accumulation rate.     

Comparison of Cysteine and Tryptophan Content of Insoluble Proteins Derived from Wild-Type and mi-1 Strains of Neurospora crassa

The possibility of an amino acid substitution (cysteine for tryptophan) in a membrane protein of the [mi-1] strain of Neurospora crassa has been investigated in detail by using a double radioactive labeling procedure. Auxotrophic strains of Neurospora having wild-type [+] or [mi-1] cytoplasm have been grown under conditions which result in the specific labeling of protein tryptophan with (3)H and protein cysteine with (35)S. Although the least soluble 1 to 20% of the [mi-1] mitochondrial membrane protein was usually found to have a higher Cys/Trp ratio (ratio of cysteine plus half-cystine to tryptophan) than the corresponding [+] fraction, it has been shown that these differences were due mainly to the presence of differential amounts of a very insoluble, cysteine-rich (Cys-rich) material. The same Cys-rich material was found in variable amounts in both [+] and [mi-1] cultures, but the concentration was usually higher in the [mi-1] cultures. The Cys-rich material is clearly distinct from "structural protein" on the basis of amino acid composition and appears to have no direct relationship to the [mi-1] phenotype. In the absence of the Cys-rich material, no difference between the Cys/Trp ratios of corresponding [+] and [mi-1] membrane proteins could be detected. We conclude, therefore, that the previously postulated amino acid substitution of cysteine for tryptophan in [mi-1] membrane protein is incorrect.     

Effects of ranolazine on fatty acid transformation in the isolated perfused rat liver

It has been proposed that in the heart, ranolazine shifts the energy source from fatty acids to glucose oxidation by inhibiting fatty acid oxidation. Up to now no mechanism for this inhibition has been proposed. The purpose of this study was to investigate if ranolazine also affects hepatic fatty acid oxidation, with especial emphasis on cell membrane permeation based on the observations that the compound interacts with biological membranes. The isolated perfused rat liver was used, and [1-¹⁴C]oleate transport was measured by means of the multiple-indicator dilution technique. Ranolazine inhibited net uptake of [1-¹⁴C]-oleate by impairing transport of this fatty acid. The compound also diminished the extra oxygen consumption and ketogenesis driven by oleate and the mitochondrial NADH/NAD⁺ ratio, but stimulated ¹⁴CO₂ production. These effects were already significant at 20 μM ranolazine. Ranolazine also inhibited both oxygen consumption and ketogenesis driven by endogenous fatty acids in substrate-free perfused livers. In isolated mitochondria ranolazine inhibited acyl-CoA oxidation and β-hydroxybutyrate or α-ketoglutarate oxidation coupled to ADP phosphorylation. It was concluded that ranolazine inhibits fatty acid uptake and oxidation in the liver by at least two mechanisms: inhibition of cell membrane permeation and by an inhibition of the mitochondrial electron transfer via pyridine nucleotides.     

Synthesis of biologically active spin-labelled radioactive cytidine diphosphodiglyceride, a novel probe for biological membranes.

A versatile synthesis of spin-labelled radioactive cytidine diphospho-sn-1,2-diacylglycerol (CDP-diglyceride) has been developed based on the combination of the enzymatic acylation of radioactive sn-glycero-3-phosphate with 12-doxyl stearic acid and the chemical conversion of the thus obtained spin-labelled radioactive phosphatidic acid with cytidine monophosphomorpholi-date into spin-labelled radioactive CDP-diglyceride. The method for the isolation and purification of the latter compound was described. This obtained CDP-[2-3H]diglyceride contained 10% of fatty acids of paramagnetic nature, presumably present as a covalently bound 12-doxyl stearic acid esters. The biological activity was tested by using the synthesized compound as a substrate in the mitochondrial biosynthesis of phosphatidylglycerol. It was found that spin-labelled CDP-[2-3H]diglyceride prepared as described can be converted in the presence of sn-[2-14C]-glycero-3-phosphate into a spin-labelled [2-3H, 2'-14C]phosphatidylglycerol with isolated rat liver mitochondria, establishing therefore that the site of its utilization is identical with the site of phosphatidylglycerol synthesis in isolated mitochondria, i.e. inner mitochondrial membrane. Results described demonstrate that the synthesized spin-labelled CDP-diglyceride can be used as a specific probe for the spin- and radioactive covalent labelling of polyglycerophosphatides of mitochondrial membranes. Some implications and further possibilities in the study of biological membranes using the spin-labelled radioactive CDP-diglyceride are discussed.     

Both the outer mitochondrial membrane and the microsomal forms of cytochrome b5 reductase contain covalently bound myristic acid. Quantitative analysis on the polyvinylidene difluoride-immobilized proteins.

NADH-cytochrome b5 reductase is known to be located on two distinct membranes, i.e. endoplasmic reticulum and outer mitochondrial membranes. The endoplasmic-reticulum-associated form of the enzyme contains myristic acid in an amide linkage to its N-terminal glycine [Ozols, Carr & Strittmatter (1984) J. Biol. Chem. 259, 13349-13354]. To investigate whether the dual subcellular localization of the reductase corresponds to a difference in fatty acylation, the enzyme was purified from well-characterized rat liver microsomal and mitochondrial fractions and analysed by a new quantitative analytical procedure. The purified reductases were run on SDS/polyacrylamide gels and blotted on to polyvinylidene difluoride membranes. The reductase-containing bands were treated with hydroxylamine, and amide-linked fatty acids were then detached by acid hydrolysis. The detached fatty acids were extracted, derivatized and analysed as phenylacyl esters by reverse-phase h.p.l.c., and the protein content of the samples was determined by amino acid analysis of the acid hydrolysates. Myristic acid was found in both the microsomal and mitochondrial reductases in a molar ratio of 1:1 with protein. These results demonstrate for the first time the presence of a myristylated protein on outer mitochondrial membranes, and show that the microsomal and mitochondrial reductases are also identical in their fatty acylation.     

Comparison of cytidinediphospho-sn-1,2-diglyceride transfer from microsomal and liposomal to mitochondrial membranes

Transfer of [3H]CDP-diglycerides from isolated guinea pig liver microsomal and liposomal membranes to guinea pig mitochondrial membranes was studied by incubating microsomal or liposomal membranes carrying [3H]CDP-diglycerides with mitochondrial membranes and determining the CDP-diglyceride-dependent incorporation of sn-3-[14C]glycerolphosphate into mitochondrial [14C]polyglycerophosphatides. A significant difference in the amount of transferred [3H]CDP-diglycerides and the composition of mitochondrial [14C]polyglycerophosphatides was found depending on whether [3H]CDP-diglycerides were transferred from microsomal or liposomal membranes. This amount was around 12% when [3H]CDP-diglycerides were transferred from the microsomal membranes and around 4.6% when they were transferred from the liposomal membranes. Furthermore, about 60% of [14C]phosphatidylglycerol and 35% of [14C]phosphatidylglycerophosphate were found in the microsomes-mitochondria system and about 9% of [14C]phosphatidylglycerol and 79% of [14C]phosphatidylglycerophosphate were found in the liposomes-mitochondria system, establishing an important role for the membrane donor in the transfer of [3H]CDP-diglycerides to mitochondria. Furthermore, if the transfer of [3H]CDP-diglycerides from the microsomal to the mitochondrial membranes was assayed by the determination of [3H]CDP-diglycerides in reisolated mitochondrial membranes without further incorporation into mitochondrial polyglycerophosphates, it amounted to about 38%.     

Glucosyltransferase activity in mitochondria. Biosynthesis of glucosyl-phosphoryl-dolichol in inner mitochondrial membranes

1. Inner mitochondrial membranes are able to transfer [14C]glucose from UDP-[14C]glucose onto dolichylmonophosphate. 2. Synthesis of dolichyl-phosphoryl-glucose takes place only in the presence of exogenous dolichyl-monophosphate loaded into phospholipid vesicles. 3. Neutral phospholipids interact preferentially with the membrane-bound enzyme. The effect of phospholipids is not related to the length of fatty acid chains but a correlation between the activation and the degree of unsaturation of fatty acid chains has been found. 4. This enzyme required divalent cations for activity. Such a requirement might be related to lipid-protein interactions which favour a suitable conformation of glycosyltransferase.     

Fatty acid composition of phospholipids in mitochondria and microsomes during diethylnitrosamine carcinogenesis in rat liver

Changes in lipid composition and function of subcellular organelles have been described in transplanted and primary tumours. We examine here the fatty acid composition of individual phospholipids (PL) in hyperplastic nodules and primary hepatoma induced by diethylnitrosamine (DEN), compared to that of normal liver and of transplantable Yoshida AH-130 hepatoma. Phosphatidylcholine and phosphatidylethanolamine fatty acid composition in mitochondria and microsomes from primary hepatoma were markedly different from normal liver; C18:0/C18:1 ratio was lower and the ratio between monosaturated and polyunsaturated fatty acids was higher. Linoleic acid content of mitochondrial cardiolipin, usually very high in normal rat liver, was notably lower in primary hepatoma. Cholesterol/phospholipid ratio in both microsomes and mitochondria from DEN-induced hepatoma was higher than in normal liver. Hyperplastic nodules showed no changes in cholesterol content whereas modifications in fatty acid composition were already observable. These modifications of membrane structure may be related to the functional changes found in nodular cells. Changes in fatty acid composition of membrane phospholipids, occurring in both primary hepatoma and preneoplastic nodules, might be one of the causes for decreased rate of lipid peroxidation peculiar to these tissues.     

Occurrence of 9- and 13-keto-octadecadienoic acid in biological membranes oxygenated by the reticulocyte lipoxygenase

Membranes of intact rabbit reticulocytes and rat liver mitochondrial membranes oxygenated by the pure reticulocyte lipoxygenase contain 13-keto-9Z,11E-octadecadienoic acid and 9-keto-10E,12Z-octadecadienoic acid. In mitochondrial membranes not treated with lipoxygenase and in rabbit erythrocyte membranes these products were not detected. The chemical structure of the compounds has been identified by cochromatography with authentic standards on various types of HPLC columns, by uv and ir spectroscopy and GC/MS. In the membranes of rabbit reticulocytes up to 2% of the linoleate residues are present as its 9- and 13-keto derivatives. Most of the keto compounds (up to 90%) are esterified in the membrane ester lipids, only about 10% were found in the free fatty acid fraction. It is proposed that the keto dienoic fatty acids are formed via decomposition of hydroperoxy polyenoic fatty acids originating from the oxygenation of the membrane lipids by the reticulocyte lipoxygenase.     

Dietary fatty acids modulate liver mitochondrial cardiolipin content and its fatty acid composition in rats with non alcoholic fatty liver disease

No data are reported on changes in mitochondrial membrane phospholipids in non-alcoholic fatty liver disease. We determined the content of mitochondrial membrane phospholipids from rats with non alcoholic liver steatosis, with a particular attention for cardiolipin (CL) content and its fatty acid composition, and their relation with the activity of the mitochondrial respiratory chain complexes. Different dietary fatty acid patterns leading to steatosis were explored. With high-fat diet, moderate macrosteatosis was observed and the liver mitochondrial phospholipid class distribution and CL fatty acids composition were modified. Indeed, both CL content and its C18:2n-6 content were increased with liver steatosis. Moreover, mitochondrial ATP synthase activity was positively correlated to the total CL content in liver phospholipid and to CL C18:2n-6 content while other complexes activity were negatively correlated to total CL content and/or CL C18:2n-6 content of liver mitochondria. The lard-rich diet increased liver CL synthase gene expression while the fish oil-rich diet increased the (n-3) polyunsaturated fatty acids content in CL. Thus, the diet may be a significant determinant of both the phospholipid class content and the fatty acid composition of liver mitochondrial membrane, and the activities of some of the respiratory chain complex enzymes may be influenced by dietary lipid amount in particular via modification of the CL content and fatty acid composition in phospholipid.     

Homeoviscous adaptation under pressure. III. The fatty acid composition of liver mitochondrial phospholipids of deep-sea fish

Homeoviscous adaptation of biological membranes to high hydrostatic pressure has been investigated by determining the differences in lipid composition of membranes from fish obtained from depths between 200 and 4000 m. The fatty acid composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine/inositol and cardiolipin from a liver mitochondrial fraction was analysed by capillary gas-liquid chromatography. The ratio of saturated to unsaturated fatty acids significantly and negatively related to depth in PC and PE as predicted by homeoviscous adaptation to pressure. Thus, deep sea species possess greater proportions of unsaturated fatty acids than shallow species. Cardiolipin showed the opposite trend. An unsaturation index was not significantly related to depth in any phospholipid fraction.     

Effects of fasting on oxidative stress in rat liver mitochondria

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O₂ consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N^epsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.     

Total Synthesis of Destruxin B

Rats were divided into low casein diet and high casein diet fed groups, and the fatty acid composition of liver mitochondrial phosphatides was analyzed. It was found that in liver mitochondrial phosphatides the value of arachidonic acid decreased slightly, and that of the polyenoic fatty acid which was infered as 4, 7, 10, 13, 16-docosapentaenoic acid increased by low casein diet. The analysis was performed also on whole liver phosphatides. There was no great difference between the fatty acid composition of whole liver and liver mitochondrial phosphatides     

Biochemical studies on abnormal erythrocyte membranes protein abnormality of erythrocyte membrane in biliary obstruction

Biochemical studies on erythrocyte membranes from eleven obstructive jaundice patients (due to various disorders) have been undertaken. By scanning electron microscopic observation these erythrocytes were spur and target in appearance. The lipid composition showed a marked increase in both cholesterol and phosphatidylcholine. In addition to these changes, it was unexpectedly demonstrated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate that a specific membrane protein component 4.2 was reduced or absent in all cases tested. This membrane protein abnormality was identical with that of hereditary spherocytosis erythrocyte membranes. It is of particular interest to note that after surgical relief of biliary obstruction in a typical case of common duct cholelithiasis, the disc electrophoretic pattern of erythrocyte membranes became normal and both lipid composition and red cell morphology returned to normal.     

Molecular species of phosphatidylcholine and phosphatidylethanolamine in subcellular membranes from rat liver.

Four subfractions of phosphatidycholine and phosphyatidylethanolamine according to the degree of unsaturation of their fatty acids have been separated from lipid extracts of microsomes, and inner and outer mitochondrial membranes. The predominant species found in the three membranes contained one saturated and one unsaturated fatty acid. In microsomes completely saturated species of both phosphatidylcholine and phosphatideylethanolamine were practically nonexistent. In outer mitochondrial membranes species with two unsaturated fatty acids were absent. In the inner mitochondrial membranes, however, disaturated species and those with two unsaturated fatty acids were found.     

Effect of polyunsaturated fatty acids and phospholipids on [3H]-vitamin E incorporation into pulmonary artery endothelial cell membranes

Vitamin E, a dietary antioxidant, is presumed to be incorporated into the lipid bilayer of biological membranes to an extent proportional to the amount of polyunsaturated fatty acids or phospholipids in the membrane. In the present study we evaluated the distribution of incorporated polyunsaturated fatty acids (PUFA) and phosphatidylethanolamine (PE) in various membranes of pulmonary artery endothelial cells. We also studied whether incorporation of PUFA or PE is responsible for increased incorporation of [3H]-vitamin E into the membranes of these cells. Following a 24-hr incubation with linoleic acid (18:2), 18:2 was increased by 6.9-, 9.2-, and 13.2-fold in plasma, mitochondrial, and microsomal membranes, respectively. Incorporation of 18:2 caused significant increases in the unsaturation indexes of mitochondrial and microsomal polyunsaturated fatty acyl chains (P less than .01 versus control in both membranes). Incubation with arachidonic acid (20:4) for 24 hr resulted in 1.5-, 2.3-, and 2.4-fold increases in 20:4 in plasma, mitochondrial, and microsomal membranes, respectively. The unsaturation indexes of polyunsaturated fatty acyl chains of mitochondrial and microsomal membranes also increased (P less than .01 versus control in both membranes). Although incubations with 18:2 or 20:4 resulted in several-fold increases in membrane 18:2 or 20:4 fatty acids, incorporation of [3H]-vitamin E into these membranes was similar to that in controls. Following a 24-hr incubation with PE, membrane PE content was significantly increased, and [3H]-vitamin E incorporation was also increased to a comparable degree, i.e., plasma membrane greater than mitochondria greater than microsomes. Endogenous vitamin E content of the cells was not altered because of increased incorporation of PE and [3H]-vitamin E. When [3H]-vitamin E was incorporated into lipid vesicles prepared from the total lipid extracts of endothelial cells and varying amounts of exogenous PE, vitamin E content was directly related to PE content. These results demonstrate that PUFA and PE distribute in all pulmonary artery endothelial cell membranes. However, only increases in PE were associated with increased incorporation of [3H]-vitamin E in membranes of these cells.