Liver Plasma Membrane Enzyme Research Articles

Liver plasma membrane-bound enzymes and lipids in golden hamsters infected with Ancylostoma ceylanicum

Liver plasma membrane-bound enzymes and lipids in golden hamsters infected with Ancylostoma ceylanicum. International Journal for Parasitology24: 249–251. Infection of golden hamsters with Ancylostoma ceylanicum caused significant decrease in body weight, liver weight and the protein content of liver plasma membrane. Significant inhibition of liver plasma membrane enzymes activities—5'Nucleotidase, γ-glutamyl transpeptidase, NADHK3Fe (Cn)6 reductase, Na+/K+-ATPase, CA2+-ATPase and Mg2+-ATPase was observed in infected animals compared to corresponding controls. Sialic acid content and phospholipid/cholesterol ratio in liver plasma membrane of the infected group were significantly reduced. The study suggests that changes in both the structural and functional organization of membrane may be a biochemical basis of the hepatotoxic effects.

CCl4‐induced lipoperoxidation triggers a lethal defect in the liver plasma membranes

Loss of calcium regulation across the plasma membrane of hepatocytes is responsible for irreversible cell damage by CCl4. The mode of action of colchicine in CCl4 acute liver damage is not completely understood. We followed the time courses of the changes in lipoperoxidation, the activities of liver plasma membrane Ca2(+)-ATPase, gamma-glutamyl transpeptidase and alkaline phosphatase, as well as the time courses of serum markers of liver damage in rats acutely intoxicated with CCl4. We assessed the effects of colchicine in this model and evaluated the effect of this drug on liver cytochrome P-450. Increased lipoperoxidation is the earliest and shortest lasting effect of CCl4 in the liver and is followed by a decrease in the activities of plasma membrane-bound enzymes. The alterations in serum enzymes showed a slower onset and were more protracted. Colchicine pretreatment produced a small decrease in cytochrome P-450 in the liver but completely prevented most of the changes produced by CCl4 in lipoperoxidation, liver plasma membrane enzyme activities and serum enzyme activities. We conclude that CCl4 metabolites trigger lipoperoxidation and then produce a longer lasting change in the plasma membrane, which thus allows calcium accumulation. Colchicine prevents the early mechanisms of CCl4 damage, and its effect on cytochrome P-450 perhaps plays only a contributory role.

The effect of thioacetamide on rat liver plasma membrane enzymes and its potentiation by fasting

Thioacetamide, given intraperitoneally (1.4 mmol/kg body mass) to male Wistar rats 24 h before sacrifice promoted a marked elevation of serum aminotransferases, loss of microsomal cytochrome P-450 content and a significant reduction (about 50%) of the liver plasma membrane enzymatic activities (5′-nucleotidase; K +, Na +- and Mg 2+-adenosine triphosphatases; and γ-glutamyl transferase). Previous starvation for 48 h, immediately prior to thioacetamide administration, strongly potentiated the effects of thioacetamide on the serum, microsomal and liver plasma membrane parameters, while fasting itself did not affect them. The liver plasma membrane damage may be one of the reasons for the cell death in thioacetamide-intoxicated rat livers.

Effects of ethanol administration on rat liver plasma membrane-bound enzymes

Changes in the properties of rat liver plasma membranes were examined in studies designed to differentiate between direct and metabolic effects of acute and chronic ethanol ingestion. One hour after a single dose of ethanol (3 g/kg body weight) there were increases in Na +,K +-ATPase (32%) and 5′-nucleotidase (36%), and hepatic concentrations of ethanol and acetaldehyde were approximately 23 mM and 50 μM, respectively. Na +,K +-ATPase and 5′-nucleotidase activities in liver plasma membranes from control rats were not significantly changed by in vitro addition of 30 μM acetaldehyde or 50 mM ethanol. Increases in Na +,K +-ATPase (∼20%) and 5′-nucleotidase (∼30%) were also observed in liver plasma membranes isolated from rats 16 hr after feeding ethanol or sucrose supplements for 17 days. The intake of calories from dietary protein and lipid was decreased by about 25% in both the ethanol and sucrose-fed animals. Na +,K +-ATPase activities in liver plasma membranes isolated from control rats were inhibited (∼20%) by 100 mM ethanol in vitro, whereas no inhibition was observed using membrane preparations from rats fed ethanol or sucrose supplements. Our results show that changes in liver plasma membrane enzyme activities associated with a single dose of ethanol are not a direct effect correlated with blood, hepatic or plasma membrane concentrations of ethanol or acetaldehyde. Chronic ingestion of ethanol or sucrose supplements had similar effects on liver plasma membrane enzyme characteristics and parallel changes in nutrient intake maybe a more feasible explanation of these results that any analogous direct effects of the two compounds.

Effect of dieldrin on rat liver plasma membrane enzymes

Dieldrin, an organochlorine insecticide, when administered orally to male albino rats at a dose of 5 mg/kg body weight/day for 15 days inhibits the enzyme Mg 2+ -ATPase and stimulates the activity of 5 ' nucleotidase and NADH-dehydrogenase in liver plasma membrane. l-ascorbic acid supplementation offers some protection in relation to the inhibition of Mg 2+ -ATPase.

Effect of chronic alcohol consumption on the activities of liver plasma membrane enzymes: Gamma-glutamyltransferase, alkaline phosphatase and 5'-nucleotidase

To study the effect of chronic alcohol administration on the activities of liver plasma membrane enzymes such as gamma-glutamyltransferase, alkaline phosphatase and 5'-nucleotidase, female rats were pair-fed for 6 weeks nutritionally adequate liquid diets containing either ethanol or isocaloric carbohydrates as controls. Compared to the control diet, chronic alcohol administration resulted in a significant enhancement of serum activities of gamma-glutamyltransferase, alkaline phosphatase and 5'-nucleotidase by 91% (P < 0.005), 80% (P < 0.001) and 65% (P < 0.01), respectively. Concomitantly, chronic alcohol intake led to a striking increase of gamma-glutamyltransferase activities in liver homogenates by 68% (P < 0.001), in liver plasma membranes rich in bile canaliculi by 80% (P < 0.025), and in liver plasma membranes free of bile canaliculi by 24% (P < 0.02). However, chronic ethanol consumption had no effect on alkaline phosphatase activities in liver homogenates and liver plasma membranes but significantly suppressed 5'-nucleotidase activities. These results therefore show that chronic intake of ethanol increases serum activities of enzymes originating from liver plasma membranes but has different effects on the enzyme activity in liver plasma membranes itself, suggesting that the alcohol-mediated increase of serum activities of various enzymes originating from liver plasma membranes might be due to different mechanisms.

Alteration of rat liver plasma membrane fluidity and ATPase activity by chlorpromazine hydrochloride and its metabolites

Chlorpromazine therapy frequently is complicated by asymptomatic alkaline phosphatase elevation and occasionally by cholestatic jaundice. To investigate the mechanism of this toxicity, we studied the effect of Chlorpromazine and several of its metabolites in vitro on rat liver plasma membrane ATPase activity and membrane fluidity, two factors of potential importance for normal bile formation. Liver plasma membranes were isolated by discontinuous sucrose ultracentrifugation and were enriched, compared with homogenate, 53-fold in NaK-ATPase, 23-fold in Mg-ATPase, and 28-fold in alkaline phosphatase, suggesting the presence of sinusoidal and lateral as well as canalicular hepatocyte membrane surfaces. Chlorpromazine and its metabolites caused an immediate, dose-dependent and reversible inhibition of NaK-ATPase activity over a 2–200 μM concentration range. Compared with the parent compound, relative inhibition of NaK-ATPase activity by Chlorpromazine metabolites varied—chlorpromazine sulfoxide was half as potent, 7-hydroxychlorpromazine equally potent, and 7,8-dihydroxychlorpromazine and 7,8-dioxochlorpromazine five times more active. Mg-ATPase activity was inhibited by Chlorpromazine and the 7-hydroxy and sulfoxide metabolites, but not by the 7,8-dihydroxy or 7,8-dioxo metabolites. The selective nature of this inhibition was further evidenced by the failure of chlorpromazine or its metabolites to inhibit the activity of 5′-nucleotidase, another liver plasma membrane enzyme. ATPase inhibition by 100 μM Chlorpromazine appeared to be noncompetitive. Chlorpromazine and each of its metabolites also altered liver plasma membrane lipid fluidity, as determined by fluorescence polarization, and the potency with which CPZ and its metabolites altered ATPase activity was closely related to their ability to alter lipid fluidity. These studies indicate that chlorpromazine-induced cholestasis may result from alteration of both physical and enzymatic properties of liver plasma membranes and further suggest that formation of more toxic metabolites in some individuals might account for their susceptibility to Chlorpromazine hepatotoxicity.

Mechanism of Bilirubin Diglucuronide Formation in Intact Rats

Although it is well established that bilirubin monoglucuronide is formed in the liver from bilirubin by a microsomal bilirubin uridine diphosphate (UDP)-glucuronosyltransferase, the subcellular site of conversion of monoglucuronide to diglucuronide and the molecular mechanism involved in diglucuronide synthesis have not been identified. Based on in vitro studies, it has been proposed that two fundamentally different enzyme systems may be involved in diglucuronide synthesis in rat liver: (a) a microsomal UDP-glucuronosyltransferase system requiring UDP-glucuronic acid as sugar donor or (b) a transglucuronidation mechanism that involves transfer of a glucuronosyl residue from one monoglucuronide molecule to another, catalyzed by a liver plasma membrane enzyme. To clarify the mechanism by which bilirubin monoglucuronide is converted in vivo to diglucuronide, three different experimental approaches were used. First, normal rats were injected with either equal amounts of bilirubin-IIIalpha [(14)C]monoglucuronide and unlabeled bilirubin-XIIIalpha monoglucuronide, or bilirubin-XIIIalpha [(14)C]monoglucuronide and unlabeled bilirubin-IIIalpha monoglucuronide. Analysis of radiolabeled diglucuronide excreted in bile showed that [(14)C]glucuronosyl residues were not transferred between monoglucuronide molecules. Second, in normal rats infused intravenously with dual-labeled [(3)H]bilirubin [(14)C]monoglucuronide, no transfer or exchange of the [(14)C]glucuronosyl group between injected and endogenously produced bilirubin monoglucuronide could be detected in the excreted bilirubin diglucuronide. Third, in homozygous Gunn rats, injected (14)C-labeled or unlabeled bilirubin mono- or diglucuronides were excreted in bile unchanged (except that diglucuronide was hydrolyzed to a minor degree). This indicates that Gunn rats, which lack bilirubin UDP-glucuronosyltransferase activity, are unable to convert injected monoglucuronide to diglucuronide. Collectively, these findings establish that a transglucuronidation mechanism is not operational in vivo and support the concept that bilirubin diglucuronide is formed by a microsomal UDP-glucuronosyltransferase system.

A histochemical study about changes in rat liver plasma membrane enzyme activities after galactosamine administration

In rats changes in plasma membrane enzyme activities due to Gal-N intoxication were studied by enzymehistochemical methods. The bile canalicular 5'-nucleotidase and nucleoside polyphosphatase activities decreased; the sinusoidal 5'-nucleotidase remained unchanged. The bile canalicular leucyl-beta-naphthyl-amidase showed an increase in activity; the alkaline phosphatase activity remained unchanged. In contrast to the spotty necrosis, changes in plasma membrane enzyme activities were seen in all liver cells, suggesting that changes of these activities, occurring after Gal-N treatment, do not correlate with cell death. The conclusion was drawn that the deviations of the enzyme activities might be due to changes in the lipid environment of the enzyme proteins in the membrane. With the exception of alkaline phosphatase, partial hepatectomy caused the same changes in enzyme activities as did Gal-N intoxication. Nevertheless Gal-N administration to partial hepatectomized rats did not lead to hepatic necrosis. Galactose given simultaneously or within two hours after Gal-N prevented both changes in plasma membrane enzyme activities and hepatocellular damage. This suggests an important role of galactolipids and galactoproteins in the plasma membrane alterations.

Changes in the form of Arrhenius plots of the activity of glucagon-stimulated adenylate cyclase and other hamster liver plasma-membrane enzymes occurring on hibernation.

1. Arrhenius plots of the glucagon-stimulated adenylate cyclase, 5'-nucleotidase, (Na+ + K+)-stimulated adenosine triphosphatase and Mg2+-dependent adenosine triphosphatase activities of control hamster liver plasma membranes exhibited two break points at around 25 and 13 degrees C, whereas Arrhenius plots of their activities in hibernating hamster liver plasma membranes exhibited two break points at around 25 and 4 degrees C. 2. A single break occurring between 25 and 26 degrees C was observed in Arrhenius plots of the activities of fluoride-stimulated adenylate cyclase, basal adenylate cyclase and cyclic AMP phosphodiesterase of liver plasma membranes from both control and hibernating animals. 3. Arrhenius plots of phosphodiesterase I activity showed a single break at 13 degrees C for membranes from control animals, and a single break at around 4 degrees C for liver plasma membranes from hibernating animals. 4. The temperature at which break points occurred in Arrhenius plots of glucagon- and fluoride-stimulated adenylate cyclase activity were decreased by about 7--8 degrees C by addition of 40 mm-benzyl alcohol to the assays. 5. Discontinuities in the Arrhenius plots of 4-anilinonaphthalene-1-sulphonic acid fluorescence occurred at around 24 and 13 degrees C for liver plasma membranes from control animals, and at around 25 and 4 degrees C for membranes from hibernating animals. 6. We suggest that in hamster liver plasma membranes from control animals a lipid phase separation occurs at around 25 degrees C in the inner half of the bilayer and at around 13 degrees C in the outer half of the bilayer. On hibernation a change in bilayer asymmetry occurs, which is expressed by a decrease in the temperature at which the lipid phase separation occurs in the outer half of the bilayer to around 4 degrees C. The assumption made is that enzymes expressing both lipid phase separations penetrate both halves of the bilayer, whereas those experiencing a single break penetrate one half of the bilayer only.

Synthesis and decomposition of alcohol esters of 5′-AMP by rat liver plasma membrane

1. 1. Recent data in the literature indicated that particulate fractions prepared from rat liver and adipose tissue were capable of synthesizing an unknown nucleotide from ATP and glycerol. In the present study, we provide direct evidence that the enzyme responsible for this activity is present in purified plasma membranes from rat liver, and that the nucleotide can be characterized as glycerol ester of adenosine 5-monophosphate. The synthetic activity was of similar extent with intact hepatocytes and with broken hepatocyte cell preparations, indicating an external location of alcohol-AMP-synthesizing enzyme in plasma membrane. 2. 2. Glycerol-AMP synthesis by rat liver plasma membranes was inhibited by EDTA; among the divalent cations tested, only Zn 2+ could effectively restore its synthesis. 3. 3. The enzymic reaction, studied with both ATP and glycerol as varying substrates, followed a sequential mechanism. The competitive inhibition of ATP by pyrophosphate was consistent with this mechanism. 4. 4. The K m values for ATP and glycerol were 0.30–0.80 mM and 5 M, respectively, at pH 9.5, with different preparations of plasma membrane. 5. 5. The synthesis of glycerol-AMP was competitively inhibited by 5′-AMP, but non-competitively (partial competition) by NADH. A number of substrates of the nucleotide pyrophosphatase were also inhibitory. Unlike 5′-AMP, the inhibitory effect of NADH was dependent on the concentration of glycerol. These data suggest that the catalytic site of nucleotide pyrophosphatase (EC 3.6.1.9) was not involved in the synthesis of glycerol AMP. 6. 6. Glycerol-AMP was decomposed by a liver plasma membrane enzyme, resulting in the formation of 5′-AMP. Such hydrolytic activity was inhibited by the presence of 5′-AMP.

Liver plasma membrane enzyme activities following glutaraldehyde fixation

The Wachstein-Meisel ATPase histochemical method has been previously used to demonstrate the ultrastructural localization of this enzyme in both whole liver and isolated plasma membranes following fixation in glutaraldehyde. In the present study biochemical assay, of liver plasma membrane enzymes following fixation in cold 2.5% glutaraldehyde showed that approximately 40% of Mg2+-ATPase, but only 4% of (Na+-K+)-ATPase activity remained in membranes from either control or ANIT-treated rats. In addition, 5'-nucleotidase activity was almost abolished by fixation. The present results indicate that the Wachstein-Meisel method, when applied to biliary canaliculi, can reliably be used to demonstrate the ultrastructural, histochemical localization of Mg2+-ATPase but not that of (NA+-K+)-ATPase. Furthermore, the method permits a valid comparison to be made of the relative Mg2+-ATPase activity in normal and chemically damaged biliary canaliculi.

Effect of essential fatty acid deficiency on activity of liver plasma membrane enzymes in the rat

Liver plasma membranes (LPM) were isolated from rats fed an essential fatty acid-supplemented diet (+EFA) or from rats fed an essential fatty acid-deficient diet (-EFA). The proportions of linoleate and arachidonate in membrane total fatty acids in the -EFA preparations were one-half or less than the values for the +EFA preparations. Basal, F-, or glucagon-stimulated adenylate cyclase activities were significantly lower in EFA-deficient livers than in nondeficient ones. Addition of GTP significantly enhanced glucagon-stimulated adrenylate cyclase in both groups, but extent of stimulation above basal was greater in EFA-deficient livers. Portal vein injection of glucagon in vivo resulted in significantly higher cAMP formation in +EFA livers than in -EFA livers. When glucagon was used in vitro at 1-1,000 nM, stimulation of adenylate cyclase remained lower in EFA-deficient membranes, but extent of stimulation above basal activity was larger in -EFA membranes than in +EFA. Total Na+, K+ (Mg2+)-ATPase from EFA-depleted LPM exhibited significantly higher values of apparent Km and Vmax-5'-Nucleotidase activity, in contrast, was considerably decreased in EFA-deficient rats. These findings show that, in animals, changes in unsaturated fatty acid composition can affect the properties of membrane-bound enzymes. These alterations could be due to changes in membrane physical properties and/or prostaglandin formation.

α-Naphthyl-isothiocyanate-induced cholestasis in the rat: studies of liver plasma membrane enzymes

Oral administration of a single dose of alpha-naphthyl-isothiocyanate (ANIT) to rats produced a conjugated hyperbilirubinaemia, maximal at 2 days and which subsided by 7. The activities of 3 liver plasma membrane enzymes, Mg-2+-ATPase, (Na-+-K-+)-ATPase and 5-nucleotidase, and serum bilirubin levels were studied for up to 7 days after treatment. Activities of the 3 enzymes were significantly decreased at 2 days after treatment and returned to normal by 7, thus varying inversely with the degree of hyperbilirubinaemia. Enzyme histochemistry used to demonstrate canalicular localization of Mg-2+-ATPase in sections of whole liver and of isolated plasma membrane pellets showed that the reduction in activity was not a uniform partial loss, but represented a range of reductions in most canaliculi with a few retaining normal staining intensity. The results suggest that after ANIT intoxication there is a membrane lesion which may be responsible for the observed hyperbilirubinaemia due to the failure of secretion of biliary constituents into the canaliculus. However, more direct studies are necessary to determine whether any one of these enzymes is directly involved in the transport of biliary constituents across the bile canalicular membrane.

Studies on plasma membranes XXI. Inhibition of liverplasma membrane enzymes by tumourpromoting phorbolester, mitotic inhibitors and cytochalasin B

The tumour promotor tetradecanoyl phorbol acetate (TPA) inhibited the Mg 2+-, Ca 2+- and (Na +-K +)ATPases of rat-liver plasma membranes. A nonpromoting phorbolester derivative was without effect. Colchicine and/or vinblastine inhibited the (Na +-K +)ATPase, glucagon-stimulated adenylate cyclase, and cyclic adenosine-3, 5-monophosphate (c-AMP) phosphodiesterase, but were without significant effect on the Mg 2+- or Ca 2+-ATPase. Cytochalasin B inhibited the (Na +-K +)ATPase. The results furnish the first direct evidence that these drugs may interact with plasma membranes. The mechanism of the enzyme inhibitions is briefly discussed.

Modification by bovine growth hormone of liver plasma membrane enzymes, phospholipids, and circular dichroism

Liver plasma membrane phospholipid distribution, protein conformation, and 5′-nucleotidase, Mg 2+-adenosine triphosphatase and (Na + + K +)-adenosine triphosphatase specific activities, were shown to depend on pituitary status and treatment with bovine growth hormone. In whole liver homogenates, hypophysectomy produced a decrease in the proportion of phosphatidyl serine, lysophosphatidyl choline, and phosphatidic acid and diphosphatidyl glycerol and an increased proportion of phosphatidyl ethanolamine. The phospholipid distribution in liver plasma membranes was the same for normal and hypophysectomized rats. Plasma membranes obtained from bovine growth hormone-treated hypophysectomized rats had approximately 50%, more phosphatidyl serine than membranes obtained from untreated hypophysectomized or normal rats. Plasma membranes from hypophysectomized rats had 75% of the 5′-nucleotidase, the same level of (Na + + K +)-adenosine triphosphatase, and twice the Mg 2+-adenosine triphosphatase of membranes from normal rats. Twelve hours after administration of bovine growth hormone to hypophysectomized rats, (Na + + K +)-adenosine triphosphatase had almost doubled and Mg 2+-adenosine triphosphatase decreased by 50%. 5′-Nucleotidase remained unchanged. Twenty-four hours after bovine growth hormone administration, both (Na + + K +)-adenosine triphosphatase and 5′-nucleotidase had increased. Mg 2+-adenosine triphosphatase was 23% of the baseline level of untreated hypophysectomized rats. Treatment for 3 days or 5 days increased the 5′-nucleotidase 2-fold. Circular dichroism spectra of liver plasma membranes isolated from hypophysectomized rats consistently showed greater negative ellipticity in the far ultraviolet range (250-190 nm) than those from normal rats or rats treated with bovine growth hormone.