Effect of colchicine on rat liver plasma membrane

Colchicine effect has been tested on rat liver plasma membrane-bound enzymes after in vitro or in vivo treatment. It appears that the in vitro treatment does not affect 5'-nucleotidase, Mg2+-ATPase and (Na+ + K+)-ATPase, whereas adenylate cyclase is sensitive to both in vitro and in vivo treatment, the latter condition being also effective for 5'-nucleotidase.     

Localization of phosphatidylinositol 4-kinase isoenzymes in rat liver plasma membrane domains

The presence of different isoenzymes of phosphatidylinositol 4-kinase in isolated rat liver plasma membranes and their further distribution in plasma membrane domains was examined. Both wortmannin-sensitive and -insensitive PtdIns 4-kinase activities were detected in highly purified plasma membranes obtained by aqueous two-phase affinity partitioning. The wortmannin-sensitive enzyme was identified as the 230 kDa isoform by Western blotting, whereas the 92 kDa isoform was not detected in plasma membranes. The apparent molecular weights of these isoforms were 205 and 105 kDa on SDS polyacrylamide gel electrophoresis, but approximately 500 and 230 kDa respectively on gel filtration, suggesting that both enzymes either are dimers or composed of heterologous subunits. Approximately 25% of the total 230 kDa isoenzyme present in liver, and only ca 5% of the wortmannin-insensitive one, was associated with the plasma membrane fraction. Plasma membrane domains were isolated by a combination of sucrose and Nycodenz gradient centrifugations. The 230 kDa isoform was identified in the blood sinusoidal domain, but not in the bile canalicular one, and was also found in lateral plasma membranes. The wortmannin-insensitive isoenzyme was present only in this latter material. The functional implications of this distribution of PtdIns 4-kinase isoenzymes in plasma membrane regions are discussed.     

Effect of insulin and glucagon on aldolase turnover in rat liver

The effects of exogenous and endogenous insulin and glucagon on aldolase turnover in rat liver and blood were studied. Some effects of these hormones on the biosynthesis and degradation of hepatic aldolase were specified. The rate of the "de novo" synthesis of aldolase was investigated in hepatocyte mitochondria and in blood plasma. The exogenous and endogenous hormones were shown to produce different effects on the biosynthesis and spontaneous degradation of rat liver aldolase.     

Effect of lipid composition on rat liver nuclear membrane fluidity

Nuclear membrane fluidity is measured in rat liver by use of the fluorescence anisotropy of two probes: diphenylhexatriene and its cationic derivative trimethylammonium-diphenylhexatriene. It has been shown that, in 2-month-old rat liver cells, the bilayer surface is less fluid than the hydrophobic core. The fluidity was higher in 6-day-old rat liver nuclei, in which both the amount of cholesterol and the cholesterol/phospholipid ratio decreased. The influence of the single phospholipids, and in particular of phosphatidylcholine, has been studied by increasing the phosphatidylcholine with a choline base exchange reaction in isolated nuclear membranes. After this reaction, the fluorescence anisotropy of the bilayer surface increased, whereas at the hydrophobic core it decreased. Analysis of fatty acid composition shows an increase of phosphatidylcholine unsaturated fatty acids. The results show that the fluidity of nuclear membranes changes in relation to the lipid content and to the fatty acid composition. The role of nuclear membrane fluidity in cell function is discussed. © 1997 John Wiley & Sons, Ltd.     

Preservation of hepatocyte plasma membrane domains during cell division in situ in regenerating rat liver

We have utilized antibodies against five domain-specific integral proteins of the rat hepatocyte plasma membrane to examine the fates of the plasma membrane domains during hepatocyte division in the regenerating rat liver. The proteins were quantified on immunoblots of liver homogenates prepared during the peak of hepatocyte mitotic activity, 28-30 hr after two-thirds hepatectomy. Two sinusoidal/lateral proteins, CE 9 and the asialoglycoprotein receptor, and one bile canalicular protein, dipeptidylpeptidase IV, were not changed significantly in amount; whereas one sinusoidal/lateral protein, the epidermal growth factor receptor, and one bile canalicular protein, HA 4, were reduced to less than or equal to 50% of control levels. Light microscopic examination of plastic sections of regenerating liver tissue revealed that the mitotic hepatocytes generally appeared to retain normal contacts with neighboring interphase hepatocytes. Immunofluorescence was used to localize the domain-specific proteins on mitotic hepatocytes identified in 0.5-micron frozen sections of 28- to 30-hr regenerating liver tissue. Independent of mitotic stage, the hepatocytes retained mutually exclusive bile canalicular and sinusoidal/lateral domains, as defined at the molecular level by the distributions of specific proteins, such as HA 4 and CE 9, respectively.     

Effect of insulin on the composition of chromatin phospholipid in rat liver cells

In vivo the action of insulin on rat liver chromatin phospholipid composition was investigated. It was shown that the hormone led to reliable increase (nearly 20%) of total phospholipid content. The same phenomenon was shown also in the fraction of active chromatin while the phospholipids content of non-active chromatin didn't changed. It was also shown that the content of three from six phospholipid fractions altered under the insulin action. The content of sphingomyelin and phosphatidilethanolamine increased and the quantity of phosphatidylinositol decreased under the hormone action. The decrease of monophosphoinositides content was accompanied by the reliable increase of triphosphoinositides amount. It was suggested that the fractions of chromatin polyphosphoinositides were redistributed under the action of insulin.     

Study of insulin-receptor interactions in plasma membrane of rat liver using antibodies against insulin

Preincubation of rat liver cell plasma membranes with antiinsulin serum (AIS) results in a 3-fold increase of specific binding of [125I]insulin added to AIS-free membranes. The degree of such increase depends on preincubation time and AIS concentration; at a rise of both parameters the dependence curve shown a plateau. The Scatchard plots suggest that preincubation with AIS increases the high and low affinity of binding sites; their number remains thereby unchanged. Using several types of immune and non-immune sera for preincubation the specificity of this effect was established. It was assumed that the antibodies initiate the immunological reaction of the membrane. Study of AIS effect on dissociation of membrane-bound labelled insulins differing in their affinities for the given AIS revealed that the antibodies can interact with the newly formed hormone-receptor complex but cannot affect the receptor within insulin. It is concluded that the intact hepatocyte membrane contains a receptor-bound insulin which is a target of AIS action. The antigen-antibody interaction changes the functional state of occupied and free receptors which bind [125I]insulin. The latter becomes repeatedly involved in this process as a result of destabilization of site-site interactions provoked by antibodies against the hormones.     

Effect of calorie restriction on skeletal muscle and liver insulin binding in growing rat

The effect of specific calorie deprivation was studied in meal-fed growing rats. It resulted in a 50% decrease in growth rate. Blood glucose and most non-essential blood free amino acid levels were depressed. Postprandial plasma insulin was decreased. With insulin ranging from 0.01 to 100 nM, insulin binding to crude Triton X-100 solubilized membranes from liver was higher in calorie restricted rats when compared with control rats. Wheat germ agglutinin (WGA) purified receptor preparations also exhibited higher insulin binding in liver from experimental group but the significance (P less than 0.05) was only visible with low insulin levels; both basal and insulin-stimulated tyrosine-kinase activity were left unchanged. In contrast, whatever the skeletal muscle insulin receptor preparation (enriched plasma membranes, crude Triton X-100 solubilized or wheat-germ agglutinin purified extracts) insulin binding was similar in control and calorie-restricted rats.     

The rat liver insulin receptor

Using insulin affinity chromatography, we have isolated highly purified insulin receptor from rat liver. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the rat liver receptor contained the Mr 125,000 alpha-subunit, the Mr 90,000 beta-subunit, and varying proportions of the Mr 45,000 beta'-subunit. The specific insulin binding of the purified receptor was 25-30 micrograms of 125I-insulin/mg of protein, and the receptor underwent insulin-dependent autophosphorylation. Rat liver and human placental receptors differ from each other in several functional aspects: (1) the adsorption-desorption behavior from four insulin affinity columns indicated that the rat liver receptor binds less firmly to immobilized ligands; (2) the 125I-insulin binding affinity of the rat liver receptor is lower than that of the placental receptor; (3) partial reduction of the rat liver receptor with dithiothreitol increases its insulin binding affinity whereas the binding affinity of the placental receptor is unchanged; (4) at optimal insulin concentration, rat liver receptor autophosphorylation is stimulated 25-50-fold whereas the placental receptor is stimulated only 4-6-fold. Conversion of the beta-subunit to beta' by proteolysis is a major problem that occurs during exposure of the receptor to the pH 5.0 buffer used to elute the insulin affinity column. The rat receptor is particularly subject to destruction. Frequently, we have obtained receptor preparations that did not contain intact beta-subunit. These preparations failed to undergo autophosphorylation, but their insulin binding capacity and binding isotherms were identical with those of receptor containing beta-subunit. Proteolytic destruction and the accompanying loss of insulin-dependent autophosphorylation can be substantially reduced by proteolysis inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of streptozotocin-induced diabetes on epidermal growth factor receptors in rat liver plasma membrane

The effect of streptozotocin-induced diabetes on 125I-labeled epidermal growth factor (EGF) binding was studied in microsomal membranes from rat liver. The binding of EGF in membranes from diabetic animals was significantly low, the value being about 60% of the control level. Scatchard analysis of the binding data clearly showed that the decrease in EGF binding was due to a decrease in the number of receptors. Treatment of diabetic animals with insulin restored EGF receptors to control levels, whereas the treatment with triiodothyronine had no effect. Serum EGF concentrations measured were almost the same among the control, diabetic, and insulin-treated diabetic groups. These results suggest that insulin deficiency in vivo causes a decrease in hepatic EGF receptors.     

Effect of detergents on the enzyme activities of the rat liver plasma membrane

The anionic detergents sodium dodecyl sulfate (SDS) and Alipal CO-433 and the non-ionic detergent Trition X-100 at concentrations of 0.02–0.10% cause a more rapid solubilization of phospholipid than proteins in isolated rat liver plasma membranes. All three detergents cause an increase in membrane turbidity at low detergent concentration (0.01–0.04%) but then decrease the turbidity at higher detergent concentration (0.04–0.10%). Each detergent gives a characteristic turbidity-detergent concentration profile which is pH dependent.The activities of the membrane-bound enzymes Mg²⁺ ATPase, 5′-nucleotidase and acid and aklaline phosphatase were influenced by each detergent to a different extent. Each enzyme gave a characteristic activity-detergent concentration profile. Mg²⁺ ATPase was inhibited by all detergents. 5′-Nucleotidase was stimulated by Triton and Alipal but inhibited by SDS. Alkaline phosphatase was stimulated by Alipal and SDS and not influenced by Triton. Acid phosphatase was stimulated by Triton and inhibited by Alipal and SDS. 56% of the total membrane-bound alkaline phosphatase and 23% of the total membrane-bound 5′-nucleotidase was solubilized in an active form by 0.06% and 0.05% SDS respectively.