Protein synthesis by microsomal particles from regenerating rat liver

1. Washed microsome particles from regenerating liver were shown to incorporate [(14)C]leucine into protein more actively than similar preparations from normal liver. 2. The total incorporation in the preparations from regenerating liver increased linearly with the amount of protein incubated, whereas this was not so with preparations from normal liver. 3. The greater activity of regenerating-liver microsomes appeared to be associated with the bound polysomes. 4. The size distribution of polysomes obtained after removal of membrane with deoxycholate was the same in normal and regenerating liver. 5. In general the activity of polysome preparations from normal and regenerating liver was similar. 6. It is concluded that the greater activity of the particles in the microsome fraction from regenerating liver is to be attributed to the ribosomes bound to membrane and that their activity is controlled by factors present in the membrane.     

Increased acidic phospholipids in rat brain membranes after chronic ethanol administration

Two types of plasma membranes isolated from rat brain cortex were used to study the membrane-perturbing properties of ethanol. Rats administered ethanol in the form of a liquid diet showed an increase in levels of phosphatidylserines, phosphatidylinositols and phosphatidic acids as compared to controls. The results present evidence that chronic ethanol treatment results in an increase in the acidic phospholipids in brain membranes. This type of membrane modification may have important implications for the function of membrane transport enzymes such as (Na+, K+)-ATPase, which also increases in activity upon chronic ethanol administration.     

Changes in activities of some ammonia-metabolizing enzymes in the rat liver and the brain after chronic ethanol administration

The changes in the activities of ammonia-metabolizing enzymes in liver and brain after ethanol intoxication has been investigated in rats. After administration of ethanol 30% (w/v) 6g kg-1 for 4 weeks we found an increase in liver glutamate dehydrogenase and glutaminase activity. In brain tissue the glutaminase activity was significantly higher and glutamate dehydrogenase was significantly lower. Glutamine synthetase activity in liver and brain was practically unchanged. The reasons for these changes in the activities of some ammonia-metabolizing enzymes in liver and brain after ethanol ingestion have been discussed.     

Enzymic synthesis of ether types of choline and ethanolamine phosphoglycerides by microsomal fractions from rat brain and liver.

The formation of product by ethanolamine phosphotransferases (EC 2.7.8.1) and cholinephosphotransferases (EC 2.7.8.2) in microsomal fractions from brains and livers of mature rats is increased several fold by 1,2-diacyl-sn-glycerols. With the addition of 1-alkyl-2-acyl-sn-glycerols, we have found an 11-fold increase with brain microsomes and a 20-fold increase with lvier microsomes in the synthesis of choline ether lipids (1-alkyl-2-acyl- and 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylcholines). For the synthesis of ethanolamine ether lipids (1-alkyl-2-acyl and 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylethanolamines), the stimulation of alkylacylglycerols was 7-fold for brain microsomes and 18-fold for liver microsomes. The alkylacyl glycerols (8 mM) also inhibited the synthesis of diacyl phosphoglycerides by 44 to 65%, indicating that the same ethanolaminephosphotransferases and cholinephosphotransferases are utilized for the synthesis of alkylacyl phosphoglycerides and diacyl phosphoglycerides. A desaturation of the alkyl groups may take place in the same reaction mixture. The rate of incorporation of phosphorylcholine into alkenylacyl glycerophosphorylcholines (choline plasmalogens) with alkylacylglycerols, cytidine diphosphate choline, and liver microsomes was 15 nmoles per mg protein per hour. The in vitro synthesis of choline plasmalogens with alkylacylglycerols had not been observed previously. The corresponding rate of incorporation of phosphorylethanolamine into ethanolamine plasmalogens was 10 nmoles per mg protein per hour, a value greater than any of the previously reported values for ethanolamine plasmalogen formation from alkylacyl glycerophosphorylethanolamines.     

Purification of membrane-bound galactosyltransferase from rat liver microsomal fractions

1. Rat liver microsomal preparations incubated in 1% Triton X-100 at 37°C for 1h released about 60% of the membrane-bound UDP-galactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. The supernatant galactosyltransferase which was solubilized but not purified by this treatment had a higher molecular weight than the serum enzyme as shown by Sephadex G-100 column chromatography. 2. The galactosyltransferase present in the high-speed supernatant was purified 680-fold by an affinity-column-chromatographic technique by using a column of activated Sepharose 4B coupled with α-lactalbumin. The galactosyltransferase ran as a single band on polyacrylamide gels and contained no sialyltransferase, N-acetylglucosaminyltransferase or UDP-galactose pyrophosphatase activities. 3. The purified membrane enzyme had properties similar to serum galactosyltransferase. It had an absolute requirement for Mn²⁺ that could not be replaced by Ca²⁺, Mg²⁺, Zn²⁺ or Co²⁺, and was active over a wide pH range (6–8) with a pH optimum of 6.5. The apparent K_m for UDP-galactose was 10.8μm. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose. 4. The molecular weight of the membrane enzyme was 65000–70000, similar to that of the purified serum enzyme. Amino acid analyses of the two proteins were similar but not identical. 5. Sephadex G-100 column chromatography of the purified membrane enzyme showed a small peak (2–5%) of higher molecular weight than the purified serum enzyme. Inclusion of 1mm-ε-aminohexanoic acid in the isolation procedures increased this peak to as much as 30% of the total enzyme recovered. Increasing the ε-aminohexanoic acid concentration to 100mm resulted in no further increase in this high-molecular-weight fraction.     

Characterization of CDPdiacylglycerol hydrolase in mitochondrial and microsomal fractions from rat lung

CDPdiacylglycerol pyrophosphatase (E.C. 3.6.1.26) activity has been examined in rat lung mitochondrial and microsomal fractions. While the mitochondrial hydrolase exhibited a broad pH optimum from pH 6-8, the microsomal activity decreased rapidly above pH 6.5. Apparent Km values of 36.2 and 23.6 microM and Vmax values of 311 and 197 pmol.min-1.mg protein-1 were observed for the mitochondrial and microsomal preparations, respectively. Addition of parachloromercuriphenylsulphonic acid led to a marked inhibition of the microsomal fraction but slightly stimulated the mitochondrial activity at low concentrations. Mercuric ions were inhibitory with both fractions. Although biosynthetic reactions utilizing CDPdiacylglycerol require divalent cations, addition of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+ all inhibited the catabolic CDPdiacylglycerol hydrolase activity in both fractions. EDTA and EGTA also produced an inhibitory effect, especially with the mitochondrial fraction. Although addition of either adenine or cytidine nucleotides led to a decrease in activity with both fractions, the marked susceptibility to AMP previously reported for this enzyme in Escherichia coli membranes, guinea pig brain lysosomes, and pig liver mitochondria was not observed. These results indicate that rat lung mitochondria and microsomes contain specific CDPdiacylglycerol hydrolase activities, which could influence the rate of formation of phosphatidylinositol and phosphatidylglycerol for pulmonary surfactant.     

Retinol and retinyl esters in parenchymal and nonparenchymal rat liver cell fractions after long-term administration of ethanol

Chronic ethanol consumption reduces the liver retinoid store in man and rat. We have studied the effect of ethanol on some aspects of retinoid metabolism in parenchymal and nonparenchymal liver cells. Rats fed 36% of total energy intake as ethanol for 5-6 weeks had the liver retinoid concentration reduced to about one-third, as compared to pair-fed controls. The reduction in liver retinoid affected both the parenchymal and the nonparenchymal cell fractions. Plasma retinol level was normal. Liver uptake of injected chylomicron [3H]retinyl ester was similar in the experimental and control group. The transport of retinoid from the parenchymal to the nonparenchymal cells was not found to be significantly retarded in the ethanol-fed rats. Despite the reduction in total retinoid level in liver, the concentrations of unesterified retinol and retinyl oleate were increased in the ethanol fed rats. Hepatic retinol esterification was not significantly affected in the ethanol-fed rats. Since our study has demonstrated that liver uptake of chylomicron retinyl ester is not impaired in the ethanol-fed rat, we suggest that liver retinoid metabolism may be increased.     

Effects of acute and chronic administration of ethanol on rat brain arylsulphatase A and B

Acute (4g/kg i.p.) and chronic (Sustacal^TM diet containing 10% ethanol for 20 days) administration of ethanol to male Sprague-Dawley rats produced no change in the content or enzyme activity of brain arylsulphatase A. In contrast to the lack of effect on arylsulphatase A, the acute and chronic administration of ethanol resulted in an increase in the activity of brain arylsulphatase B (15.8% and 18.4%, respectively). However, the enhancement of the activity of arylsulphatase B was observed only in the brain homogenates which were subjected to osmotic shock. No enhancement of the arylsulphatase B activity was found in the supernatant soluble fraction after the acute and chronic administration of ethanol. Furthermore, acute and chronic ethanol administration did not alter the activities of arylsulphatase A and B in microsomes which have been suggested as sites of the synthesis of lysosomal hydrolases. In addition, 80 mM ethanol, in vitro, did not affect the activity of arylsulphatase A and B. The results of the present study suggest that the acute or chronic administration of ethanol might enhance the activity of lysosomal membrane bound arylsulphatase B via altering the lipid metabolism of lysosomal membranes.     

Sidedness of ceramide-phosphoethanolamine synthesis on rat liver and brain microsomal membranes

Phosphatidylethanolamine:ceramide-ethanolamine-phosphotransferase catalyzes the synthesis of ceramide-phosphoethanolamine, a sphingomyelin analogue. Its localization was studied in rat liver and brain microsomes. After testing the integrity and the sidedness of microsomal vesicles, trypsin treatment of intact or deoxycholate-disrupted microsomes made it possible to conclude that both the transferase and the ceramide-phosphoethanolamine are located in the cisternal leaflet of the membrane bilayer. Using trinitrobenzenesulfonic acid as a probe, no trace of newly synthesized ceramide-phosphoethanolamine was detectable on the cytoplasmic side of the microsomes.     

Protein synthesis rates in rat brain regions and subcellular fractions during aging

In vivo protein synthesis rates in various brain regions (cerebral cortex, cerebellum, hippocampus, hypothalamus, and striatum) of 4-, 12-, and 24-month-old rats were examined after injection of a flooding dose of labeled valine. The incorporation of labeled valine into proteins of mitochondrial, microsomal, and cytosolic fractions from cerebral cortex and cerebellum was also measured. At all ages examined, the incorporation rate was 0.5% per hour in cerebral cortex, cerebellum, hippocampus, and hypothalamus and 0.4% per hour in striatum. Of the subcellular fractions examined, the microsomal proteins were synthesized at the highest rate, followed by cytosolic and mitochondrial proteins. The results obtained indicate that the average synthesis rate of proteins in the various brain regions and subcellular fractions examined is fairly constant and is not significantly altered in the 4 to 24-month period of life of rats.A preliminary report of these results was previously presented at: WFN-ESN Joint Meeting on: Cerebral Metabolism in Aging and Neurological Disorders, Baden, August 28–31, 1986.     

Effects of acute and chronic ethanol administration on thromboxane and prostacyclin levels and release in rat brain cortex

The effects of acute (3 g/kg i.p. two jours before sacrifice) and chronic (6% in drinking water and libitum for 15 days) ethanol administration to male rats (200 g body weight) on basal levels and release of TxB_2n2 and 6-keto-PGF_1α in brain cortex were studied. Also the effects of chronic ethanol (30 days) on the fatty acid composition of brain cortical tissue and liver phospholipids were investigated. Acute treatment reduced basal levels of 6-keto-PGF_1α in brain cortical tissue (rats sacrificed by microwave radiation) and decreased the accumulation of 6-keto-PGF_1α in brain cortex after post-decapitation ischemia (PDI). Basal TxB₂ levels were also reduced in brain cortex, but TxB₂ release during PDI was enhanced. Chronic treatment (15 days) induced changes of TxB₂ and 6-ketoPGF_1α levels and release during PDI in brain cortex less pronounced than those observed after acute treatment. The reduced effectiveness of chronic ethanol on brain vasoactive eicosanoids suggest adaptation processes. After chronic treatment (30 days), the fatty acid composition of brain cortex total phospholipids were not significantly modified. Changes of eicosanoid production after ethanol were thus independent from modifications of the fatty acid precursor pool(s). Ethanol-induced changes in the production of vascular eicosanoids in the CNS may be of relevance to the action of the compound on the CNS and may also have implications for the clinic.     

Effects of acute and chronic ethanol administration on thromboxane and prostacyclin levels and release in rat brain cortex

The effects of acute (3 g/kg i.p. two hours before sacrifice) and chronic (6% in drinking water and libitum for 15 days) ethanol administration to male rats (200 g body weight) on basal levels and release of TxB2 and 6-keto-PGF1 alpha in brain cortex were studied. Also the effects of chronic ethanol (30 days) on the fatty acid composition of brain cortical tissue and liver phospholipids were investigated. Acute treatment reduced basal levels of 6-keto- PGF1 alpha in brain cortical tissue (rats sacrificed by microwave radiation) and decreased the accumulation of 6-keto-PGF1 alpha in brain cortex after post-decapitation ischemia (PDI). Basal TxB2 levels were also reduced in brain cortex, but TxB2 release during PDI was enhanced. Chronic treatment (15 days) induced changes of TxB2 and 6-keto-PGF1 alpha levels and release during PDI in brain cortex less pronounced than those observed after acute treatment. The reduced effectiveness of chronic ethanol on brain vasoactive eicosanoids suggest adaptation processes. After chronic treatment (30 days), the fatty acid composition of brain cortex total phospholipids were not significantly modified. Changes of eicosanoid production after ethanol were thus independent from modifications of the fatty acid precursor pool(s). Ethanol-induced changes in the production of vascular eicosanoids in the CNS may be of relevance to the action of the compound on the CNS and may also have implications for the clinic.     

Protein and lipid disturbances in rat liver microsomal membranes after bile duct ligation

An analysis of proteins, phospholipids and cholesterol from liver microsomal membranes was performed in normal and post-cholestatic rats. Bile duct ligated rats showed a progressive decrease of these membrane constituents. Minor changes in peptide analysis, a marked decrease of phosphatidylcholine and phosphatidylinositol, disappearance of phosphatidylethanolamine and sphingomyelin, and a clear increment of phosphatidylserine was observed in post-cholestatic as compared to normal group. It was concluded that extra-hepatic cholestasis produces structural changes on the liver microsomes, particularly on phospholipid profile.     

Caspase-3 and calpain activities after acute and repeated ethanol administration during the rat brain growth spurt

Ethanol administration during the rat brain growth spurt triggers apoptotic neurodegeneration that appears to be mediated by caspase-3 activation. In order to gain more insight on the role of this caspase in ethanol-induced developmental neurotoxicity, we studied its expression and activity under different conditions of ethanol exposure during development. Furthermore, because of the cross-talk between caspase-3 and calpain we extended our study also at this protease. Ethanol was administered by gavage to rat pups as a single-day exposure on postnatal day (PN) 7 or from PN4 to PN10. Cleaved caspase-3 expression peaked in the cerebral cortex 12 h after ethanol treatment and returned to control values at 24 h. An identical pattern was found for caspase-3-like activity, that was increased only with the highest dose of ethanol tested (5 g/kg) and mostly in PN4. Repeated ethanol exposure, at a dose that was previously found to induce microencephaly, did not increase caspase-3 expression and activity although it decreased procaspase-3 expression and released mitochondrial cytochrome c. Repeated ethanol administration also increased calpain activity. These data show that acute and repeated ethanol administration differentially affect caspase-3 and calpain activity, suggesting that calpain activation may play a role in developmental neurotoxicity of ethanol.     

Butylated hydroxyanisole-stimulated NADPH oxidase activity in rat liver microsomal fractions

NADPH-dependent oxygen utilization by liver microsomal fractions was stimulated by the addition of increasing concentrations of butylated hydroxyanisole concomitant with the inhibition of benzphetamine N-demethylase activity. The apparent conversion of monooxygenase activity to an oxidase-like activity in the presence of the antioxidant was correlated with the partial recovery of the reducing equivalents from NADPH in the form of increased hydrogen peroxide production. The progress curve of liver microsomal NADPH oxidase activity in the presence of butylated hydroxyanisole displayed a lag phase indicative of the formation of a metabolite capable of uncoupling the monooxygenase activity. Ethyl acetate extracts of microsomal reaction mixtures obtained in the presence of butylated hydroxyanisole, oxygen, and NADPH stimulated the NADPH oxidase activity of either liver microsomes or purified NADPH-cytochrome c (P-450) reductase. Using high performance liquid chromatography, gas chromatography, and mass spectrometry techniques, two metabolites of butylated hydroxyanisole, namely t-butylhydroquinone and t-butylquinone, were identified. The quinone metabolite and/or its 1-electron reduction product interact with the flavoprotein reductase to directly link the enzyme to the reduction of oxygen which results in an inhibition of the catalytic activity of the cytochrome P-450-dependent monooxygenase.     

Effect of chronic ethanol treatment on lipid peroxidation in rat liver homogenate and subcellular fractions

1. The effect of chronic ethanol treatment on the level of lipid peroxidation in rat liver homogenate and subcellular fractions was measured using chemiluminescence technique and malondialdehyde formation. 2. It was shown that after chronic ethanol treatment the level of Fe/ADP-ascorbate-induced lipid peroxidation was decreased in the whole and "postnuclear" liver homogenates. Dilution of the homogenates prevented depressive effect of ethanol on lipid peroxidation. 3. Chronic ethanol treatment did not affect the intensity of the Fe/ADP-ascorbate-induced process in rat liver mitochondria and microsomes. 4. Peroxidative alteration of the liver lipids in vivo was evaluated by measurement of conjugated dienes (absorbance at 233 nm). It was shown that ethanol did not increase the level of u.v. absorption of lipids from mitochondria and microsomes. Chronic alcohol treatment did not influence the steady-state concentration of malonic dialdehyde in the whole liver homogenate. 5. The data obtained indicate that cytosol from the ethanol treated rat liver contains a factor(s) which prevents Fe/ADP-ascorbate-dependent lipid peroxidation in biological membranes.