Human liver AMP-deaminase – oligomeric forms of the enzyme

AMP-deaminase (EC 3.5.4.6) is a key enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in the liver. Mechanisms regulating activity of the enzyme are not completely elucidated, till now. In this paper experimental data indicating on the potential regulatory significance of changes in oligomeric structure of the enzyme are presented. SDS-PAG electrophoresis of human liver AMP-deaminase revealed the presence of three enzyme fragments. Only largest of them (the protein fragments weighing 68 kDa) reacted immunologically with monoclonal anti- (human liver) AMP-deaminase antibodies. At physiological pH 7.0, in the absence of regulatory ligands, reaction catalysed by human liver AMP-deaminase was strongly dependent on enzyme concentration used, with half-saturation constant (S_0.5) values increasing significantly with the degree of enzyme dilution. Preincubation with activated long-chain fatty acids – substances promoting dissociation of oligomeric enzymes, inhibited the activity of AMP-deaminase studied nearly completely. Gel filtration on Sepharose CL-6B column demonstrated existence of at least three active oligomeric forms of human liver AMP-deaminase. We postulate that oligomeric structure of the enzyme is a factor determining regulatory profile of AMP-deaminase studied.     

The liver: an organ of the immune system?

The liver stands in a unique position between the gastrointestinal tract and systemic venous system. Its constant exposure to food antigens, bacterial products and potential pathogens through the mesenteric circulation, requires the liver to maintain tolerogenic capabilities while preserving the means to mount effective immune responses. The liver has the unique ability amongst solid organs, to activate na?ve CD8+ T lymphocytes in an antigen-specific manner. However, this activation can be inefficient and lead to apoptosis. This phenomenon is believed to be involved in both, the development of oral tolerance and the induction of tolerance in liver allografts. The liver is the target of both autoimmune diseases and of chronic viral infections and its unique tolerogenic environment has frequently been suggested as a factor in the development of these diseases. A better grasp of the liver's unique immunological processes would lead to a better understanding of immune tolerance mechanisms and their role in the development of autoimmune diseases and chronic viral infections.     

Nuclear protein in the liver in protein–calorie deficiency

1. The effect of protein–calorie deficiency on nuclear proteins was studied in growing and adult rats by using chemical and electrophoretic methods of fractionation. 2. After 7 days on protein-deficient diets, the amount of neutral-soluble proteins increased, and their electrophoretic pattern changed, with the appearance of a new component. 3. The histone fraction of the liver nuclei in rapidly growing rats was lower than that in either deficient or adult animals. 4. The possible role of nuclear proteins in relation to synthesis of the proteins and nucleic acids of the other parts of the cells is discussed.     

Is there a gloxylate cycle in the liver of the fetal guinea pig?

Studies on the effect of the inhibitor of fatty acid oxidation (+)-octanoylcarnitine on the perfused liver of the 48–51 days fetal guinea pig indicate that the oxidation of endogenous fatty acids is a major source of carbon for the citric acid cycle and for synthesis of hexose. Consistent with this the liver can convert isocitrate to glyoxylate and glyoxylate to malate and may therefore operate a glyoxylate cycle allowing the net production of sugars from acetyl-CoA.     

Effect of the three-dimensional organization of liver cells on the biogenesis of the γ-glutamyltransferase fraction pattern

Context Four gamma-glutamyltransferase (GGT) fractions with different molecular weights (big-, medium-, small- and free-GGT) are detectable in human plasma. Objective Verify if liver cells can release all four GGT fractions and if the spatial cell organization influences their release. Methods Hepatoma (HepG2) and melanoma (Me665/2/60) cells were cultured as monolayers or spheroids. GGT released in culture media was analysed by gel-filtration chromatography. Results HepG2 and Me665/2/60 monolayers released the b-GGT fraction, while significative levels of s-GGT and f-GGT were detectable only in media of HepG2-spheroids. Bile acids alone or in combination with papain promoted the conversion of b-GGT in s-GGT or f-GGT, respectively. Conclusions GGT is usually released as b-GGT, while s-GGT and f-GGT are likely to be produced in the liver extracellular environment by the combined action of bile acids and proteases.     

NF-κB induced the donor liver cold preservation related acute lung injury in rat liver transplantation model

Background

We have observed at our clinical work that acute lung injury (ALI) often occurs in patients transplanted with donor livers persevered for long time. So, we conducted this study to investigate the influence of cold preservation time (CPT) of donor liver on ALI induced by liver transplantation (LT), and further study the role of nuclear factor-κB (NF-κB) in the process.

Methods

Wistar rats were used as donors and recipients to establish orthotopic rat liver transplantation models. Donor livers were preserved at 4°C for different lengths of time. The effect of NF-κB inhibitor, ammonium pyrrolidinedithiocarbamate (PDTC), on ALI was detected. All samples were harvested after 3 h reperfusion. The severity of liver injury was evaluated first. The expressions of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in liver tissue and liver outflow serum were measured respectively. The severity indexes of ALI, the activity of NF-κB and inhibitor-κBα (I-κBα) in lung/liver were measured accordingly.

Results

With the prolonged liver CPT, the liver damage associated indexes and ALI-related indexes all increased significantly. TNF-α and IL-1β in liver outflow serum increased accordingly, and the activity of NF-κB in liver/lung increased correspondingly. All these ALI-associated indexes could be partially reversed by the use of PDTC.

Conclusions

Extended CPT aggravates the damage of donor liver and induces the expressions of TNF-α and IL-1β in liver. These inflammatory factors migrate to lung via liver outflow blood and activate NF-κB in lung, inducing ALI finally. NF-κB may play a critical role in LT-related ALI. Patients with or at risk of ALI may benefit from acute anti-inflammatory treatment with PDTC.     

Colorectal cancer metastases affect the biochemical characteristics of the human liver β-adrenoceptor-G-protein-adenylate cyclase system

The sympathetic-catecholamine system is involved in the regulation of hepatic metabolic pathways mainly through cAMP-linked β₂-adrenoceptors (β₂-ARs) in humans and to a lesser extent through cAMP-independent mechanisms, but no information is available about the possible biochemical changes of β₂-ARs and their signalling pathways in human colorectal cancer (CRC) and colorectal cancer hepatic metastases (CRCHM). Changes in density and distribution of β-ARs as well as in post-receptor signalling components were studied in membranes of human liver with CRCHM, and for comparison, in membranes of nonadjacent, non-metastatic human liver (NA-NM) obtained from 13 patients, using binding and competition binding studies. Studies were also carried out using normal and cancerous human colon tissues. In CRCHM, the density of β-ARs (B_max) was significantly reduced, compared to NA-NM liver tissues (40.09 ± 2.83 vs. 23.09 ± 3.24 fmol/mg protein; P < 0.001). A similar decrease in the β-AR density was observed in the colon with primary colorectal cancer compared to healthy colon (37.6 ± 2.2 vs. 23.8 ± 3.5 fmol/mg protein), whereas the affinity of ICYP binding to the receptor remained unaffected. Desensitized β-ARs were uncoupled from stimulatory G-protein (G_S), as total density of β-adrenoceptors in the high affinity state was significantly reduced. Concomitantly, CRCHM elicited decrease in the catalytic adenylate cyclase (AC) activity (cAMP formation) in response to isoproterenol plus GTP or forskolin or NaF. In NA-NM and CRCHM liver, the inhibition–concentration curves of ICI 118.551 showed the presence of a homogeneous population of the β₂-AR subtypes. Neither the binding patterns nor the inhibition constant (K_i) of ICI 118.551 were altered in CRCHM. In CRCHM, the hepatic β-AR-G-protein(s)-AC signalling system was markedly impaired, thus, these changes may well influence β-AR-mediated functions in both organs.     

Cytochrome P-450 heme and the regulation of δ-aminolevulinic acid synthetase in the liver

Hepatic δ-aminolevulinic acid synthetase was induced in rats injected with allylisopropylacetamide. The induction process was studied in relation to experimental perturbation of cytochrome P-450 in the liver. Animals were treated with either administered endotoxin or exogenous heme, both of which accelerate degradation of cytochrome P-450 heme. These manipulations were effective in blocking induction of δ-aminolevulinic acid synthetase, and the effect of each compound was proportional to its ability to stimulate degradation of cytochrome P-450 heme. The findings suggest that the heme moiety of cytochrome P-450 dissociates reversibly from its apoprotein and, prior to its degradation, mixes with endogenously synthesized heme to form a pool that regulates δ-aminolevulinic acid synthetase activity. A similar or identical heme fraction appears to mediate stimulation of heme oxygenase, which suggests that the regulation of δ-aminolevulinic acid synthetase and of heme oxygenase in the liver are closely interrelated.     

Hepatic stem cells: from inside and outside the liver?

The liver is normally proliferatively quiescent, but hepatocyte loss through partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response from all cell types in the liver to perfectly restore liver mass. Moreover, hepatocyte transplants in animals have shown that a certain proportion of hepatocytes in foetal and adult liver can clonally expand, suggesting that hepatoblasts/hepatocytes are themselves the functional stem cells of the liver. More severe liver injury can activate a potential stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of bipotential transit amplifying cells (oval cells), that can ultimately differentiate into hepatocytes and biliary epithelial cells. A third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells may contribute to the albeit low renewal rate of hepatocytes, but can make a more significant contribution to regeneration under a very strong positive selection pressure. In such instances, cell fusion rather than transdifferentiation appears to be the underlying mechanism by which the haematopoietic genome becomes reprogrammed.     

Control of the redox state of the nicotinamide–adenine dinucleotide couple in rat liver cytoplasm

1. A study has been made of the ability of rat liver in vivo to maintain equilibrium in the combined glyceraldehyde 3-phosphate dehydrogenase, 3-phosphoglycerate kinase and lactate dehydrogenase reactions, i.e. in the system: [Formula: see text] Attempts were made to upset equilibrium. The [lactate]/[pyruvate] ratio was rapidly changed by injection of ethanol or crotyl alcohol, and the value of [ATP]/[ADP][HPO(4) (2-)] was rapidly changed by injection of ethionine or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone. 2. The concentrations of the metabolites occurring in the above equation were measured in freeze-clamped liver. 3. Although the injected agents caused large changes in the concentrations of the individual components, near-equilibrium in the system was maintained, as indicated by the fact that the value of [ATP]/[ADP][HPO(4) (2-)], referred to as the phosphorylation state of the adenine nucleotides, measured directly agreed with the value calculated for equilibrium conditions from the above equation. 4. The results are discussed and taken to confirm that the order of magnitude of the value of the redox state of the cytoplasmic NAD couple in rat liver is controlled by the phosphorylation state of the adenine nucleotide system.     

Studies on the mechanism of the conversion of cytosol δ-aminolevulinic acid synthase to the mitochondrial enzyme in the liver of the allylisopropylacetamide-treated rat

δ-Aminolevulinic acid (ALA) synthase was partially purified from liver cytosol fraction of rats treated with allylisopropylacetamide (AIA). The cytosol ALA synthase showed an apparent molecular weight of 320,000. The cytosol ALA synthase of this size dissociates into at least three protein components when subjected to sucrose density gradient centrifugation in the presence of 0.25 m NaCl: one is the catalytically active protein with an s value of about 6.4 or a molecular weight of 110,000, and the other two are catalytically inactive binding proteins showing s values of about 4 and 8, respectively. Recombination of the 6.4 S protein and the 4 S protein yielded a protein complex with an apparent molecular weight of 170,000 and recombination of all three protein components resulted in formation of the original cytosol ALA synthase. The cytosol ALA synthase also loses its binding proteins when treated with various proteases; thus, the enzyme-active protein obtained after papain digestion was very similar, if not identical, to mitochondrial ALA synthase. When treated with trypsin, however, the cytosol ALA synthase was converted to an enzyme showing an apparent molecular weight of 170,000, which probably represents the complex of the mitochondria-type enzyme and the 4 S binding protein. The cytosol ALA synthase tends to aggregate to form a dimer with an apparent molecular weight of 650,000–700,000. The aggregated form of the cytosol ALA synthase was less susceptible to trypsin digestion. Hemin strongly stimulated dimer formation of the cytosol ALA synthase and the aggregate produced by contact with hemin was very tight and did not easily dissociate into its respective protein components by sucrose gradient centrifugation or even after treatment with trypsin. The possible mechanisms of the conversion of cytosol ALA synthase to the mitochondrial enzyme and also of the inhibition by hemin of the intracellular translocation of ALA synthase are discussed.     

Studies on the binding of d-α-tocopherol to rat liver nuclei

The present study describes the nature and characteristics of the intranuclear binding sites of [³H]d-α-tocopherol in rat liver. When radioactively labeled d-α-tocopherol was intravenously administered to rats, approximately 55% of the nuclear radioactivity was associated with an intranuclear nucleoprotein complex. This complex, which was extractable by high concentrations of NaCl, was characterized by equilibrium density ultracentrifugation on a 30 to 60% linear sucrose gradient. About 50% of the high-salt-extracted radioactivity was coprecipitable with macromolecules by 10% ice-cold trichloroacetic acid (TCA). This TCA-precipitable radioactivity was completely ethanol soluble. Alkaline conditions favored the solubilization of the vitamin-receptor complex. Among various enzymes tested, only Pronase and trypsin were capable of dissociating the vitamin-receptor complex. Both ionic (sodium dodecyl sulfate) and nonionic (Triton X-100) detergents solubilized α-tocopherol from the nuclei and concomitantly released some of the associated macromolecules. In addition, treatment of nuclei with low concentrations of Triton X-100 showed that about 30% of the nuclear bound α-tocopherol is associated with inner core sites in the nucleoprotein complex with very high affinity for the vitamin. Dissociation of the nucleoprotein complex (chromatin) by high-salt solubilization and subsequent partial reassociation of the components by salting out procedures revealed the high affinity association of α-tocopherol with the reconstituted DNA-protein complex. Subfractionation of this complex further revealed that α-tocopherol is predominantly associated with the fraction containing phenol-soluble nonhistone proteins having a high affinity for DNA. In vitro binding studies also showed that there are specific saturable binding sites for d-α-tocopherol in rat liver nuclei.     

Interaction of pyridoxal 5′-phosphate with the liver glucocorticoid receptor-DNA complex

The rat liver glucocorticoid receptor has been eluted from DNA-cellulose with pyridoxal 5′-phosphate at low ionic strength. This elution is concentration dependent with 80–90% of the receptor eluted in 30 rain at 0 °C when the concentration of pyridoxal 5′-phosphate is 10 mm. This elution is specific for the 4′-aldehyde group of pyridoxal 5′-phosphate since vitamin B₆ analogs lacking this group are inactive in eluting the steroid-receptor complex from DNA-cellulose. Receptor has also been eluted from rat liver nuclei with similar results. The receptor eluted with pyridoxal 5′-phosphate has been compared with the receptor eluted with 0.45 m NaCl. Both methods of elution yield a steroid-receptor complex which sediments at about 3.7 S. The pyridoxal 5′-phosphate-eluted receptor however, is less prone to aggregation at low ionic strength and more stable with respect to steroid binding than the 0.45 m NaCl-eluted steroid-receptor complex. The complement of proteins eluted from DNA-cellulose with pyridoxal 5′-phosphate is very similar to that eluted with NaCl as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Insulin resistance in the liver: Deficiency or excess of insulin?

In insulin-resistant states (obesity, pre-diabetes, and type 2 diabetes), hepatic production of glucose and lipid synthesis are heightened in concert, implying that insulin deficiency and insulin excess coexists in this setting. The fact that insulin may be inadequate or excessive at any one point in differing organs and tissues has many biologic ramifications. In this context the concept of metabolic compartmentalization in the liver is offered herein as one perspective of this paradox. In particular, we focus on the hypothesis that insulin resistance accentuates differences in periportal and perivenous hepatocytes, namely periportal glucose production and perivenous lipid synthesis. Subsequently, excessive production of glucose and accumulation of lipids could be expected in the livers of patients with obesity and insulin resistance. Overall, in this review, we provide our integrative perspective regarding how excessive production of glucose in periportal hepatocytes and accumulation of lipids in perivenous hepatocytes interact in insulin resistant states.     

Structural analysis of the carbohydrate moieties of α-l-fucosidase from human liver

Acid -l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kD_a and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHz¹H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 31. While the oligomannoside-type chains show some heterogeneity in size (Man_5–8GlcNAc₂), theN-acetyllactosaminetype chains are exclusively bi-(2–6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of -l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339–50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, -N-acetylhexosaminidase, -l-fucosidase and -galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified.     

In vitro study of alginate–chitosan microcapsules: an alternative to liver cell transplants for the treatment of liver failure

The application of alginate–chitosan (AC) microcapsules to liver cell transplantation has not been previously investigated. In the current in vitro study, we have investigated the potential of AC microcapsules for the encapsulation of liver cells and show that the AC membrane supports the survival, proliferation and protein secretion by entrapped hepatocytes. The AC membrane provides cell immuno-isolation and has the potential for cell cryopreservation. The AC microcapsule has several advantages compared to more widely used alginate–poly-L-lysine (APA) microcapsules for the application of cell therapy.