Effects of membrane ribonuclease and 3'-nucleotidase on the digestion of polyuridylic acid by rat liver plasma membrane.

1. Fragments of isolated rat liver plasma membrane possess a ribonuclease activity which at pH 7.8 in the presence of 10 mM EDTA can digest polyuridylic acid (poly(U)) and polycytidylic acid (poly(C)) but not polyadenylic acid (poly(A)) and polyguanylic acid (poly(G)). Under these conditions, the membrane preparation does not degrade native or denatured DNA. 2. The products of the reaction with poly(U) (10 mM EDTA present) can be separated on DEAE-Sephadex into oligonucleotides of increasing chain length. Most of the products are di- to hexa-nucleotides which contain terminal 3'-phosphate groups. 3. When EDTA is not present (pH 7.8 or 8.8) the plasma membrane preparation degrades both poly(A) and poly(U). With poly(A) the product is all nucleoside while with poly(U) as substrate most of the product is nucleoside, but also some oligonucleotides are produced. 4. The ribonuclease releases acid soluble products very slowly from high concentrations of poly(U) (mg/ml). 5. Uridine trinucleotide with and without a terminal 3'-phosphate group is degraded by rat liver plasma membrane. The trinucleotide diphosphate is rapidly hydrolyzed to nucleoside while the trinucleotide itself is slowly digested and yields intermediate products, including nucleoside.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat.

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na+ + K+)-ATPase, leucine aminopeptidase, 5'-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na+ + K+)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48--108 h and in 5'-nucleotidase activity at 12--24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.     

Sialyl transferase activities of rat ascites hepatoma cells and rat liver

Sialyl transferase activities of the homogenate of rat ascites hepatome cells were compared with normal rat liver homogenate. The former had only 20% of the activity of the latter when an exogenous acceptor was added in the reaction mixture.Toward endogenous receptors, the activity of the hepatoma cell homogenate was 50% of that of the normal cell homogenate. A stimulation of the activity toward endogenous acceptors was observed when the homogenate of rat ascites hepatoma cells and that of rat liver were mixed. This stimulatory effect seems to be the consequence of utilization of acceptors from ascites hepatoma cells by the sialyl transferases of the rat liver.     

Gangliosides of plasma membranes from normal rat liver and Morris hepatoma.

Plasma membranes were isolated from normal rat liver and Morris hepatoma 5123tc by discontinuous sucrose gradient centrifugation. There was an average two and one-halffold enrichment of gangliosides in plasma membranes from normal liver and hepatoma as compared with their respective whole cells. The amount of total gangliosides in plasma membranes from hepatoma was eight times greater than that found in normal liver. This increase resulted from a fivefold increase in hematosides, an eightfold increase in monosialogangliosides and a twenty-twofold increase in disialogangliosides. Trisialogangliosides were present in normal liver but not in hepatoma.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na⁺ + K⁺)-ATPase, leucine aminopeptidase, 5′-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na⁺ + K⁺)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48–108 h and in 5′-nucleotidase activity at 12–24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.     

Alkaline ribonuclease and phosphodiesterase activity in rat liver plasma membranes

The ribonuclease and phosphodiesterase activities of rat liver plasma membranes, purified from the crude nuclear fraction by centrifugation in an A-XII zonal rotor and flotation, were examined and compared. The plasma membrane is responsible for between 65 and 90% of the phosphodiesterase activity of the cell and between 25 and 30% of the particulate ribonuclease activity measured at pH8.7 in the presence of 7.5mm-MgCl(2). Both enzymes were most active between pH8.5 and 8.9. Close to the pH optimum, both enzymes were more active in Tris buffer than in Bicine or glycine buffer. Both plasma-membrane phosphodiesterase and ribonuclease were strongly activated by Mg(2+), there being at least a 12-fold difference between the activity in the presence of Mg(2+) and of EDTA. There is, however, a difference in the response of the enzymes to Mg(2+) and EDTA in that the phosphodiesterase is fully activated by 1.0mm-MgCl(2) and fully inhibited by 1.0mm-EDTA, whereas the ribonuclease requires 7.5mm-MgCl(2) for full activation and 5mm-EDTA for full inhibition. Density-gradient centrifugation has indicated that on solubilization in Triton X-100 most of the ribonuclease activity is released into a small fragment of the same size as that containing the phosphodiesterase activity. The relationship between the two activities is discussed in view of these results.     

Multiple nuclear protein kinase activities in rat liver and hepatoma 3924A.

Multiple protein kinase activities were isolated from nuclei of rat and hepatoma 3924A, and purified 40- to 140-fold, respectively. Hepatic protein kinase-I exhibited high activity with casein as substrate, but was relatively inactive with either liver and hepatoma chromatin or mixed histone. In contrast, hepatoma protein kinase-I showed equivalent activity with casein and liver chromatin. Protein kinase-IIA, -IIB and-IIC from both tissues were more active with liver chromatin in comparison to casein and hepatoma chromatin, and exhibited similar electrophoretic profiles of 32P-chromatin.     

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.     

Modifications of enzyme activities in rat liver plasma membrane after carbon tetrachloride poisoning

Adenylate Cyclase activity is increased in the liver of animals treated with CCl4 (250 ul/100g body wt.) after 30 min. The maximum increase is observed 2 hours after administration of the hepatotoxin. Whereas, 3',5'-nucleotidephosphodiesterase decreases significantly throughout all the experiments. Our results present evidence that there is relationship between Adenylate Cyclase and Phosphodiesterase activity and suggest that intracellular calcium ion may mediate a regulation of the synthesis and degradation of cyclic nucleotides. It is difficult to determine the exact role Ca2+ plays in regulating these two opposing reactions. Thus, in the near future the work in this laboratory will be to define carefully the effects of the CCl4 on ions on these two systems.     

Biogenesis of plasma membrane glycoproteins. Purification and properties of two rat liver plasma membrane glycoproteins

As a preliminary to a study of the biogenesis of individual plasma membrane glycoproteins, the marker enzyme nucleotide pyrophosphatase (NPPase) and a major rat liver plasma membrane sialoprotein, subsequently found to be identical with the enzyme dipeptidyl peptidase IV (DPP IV), were purified 10,000- and 2,000-fold, respectively, from rat liver. Both were amphipathic proteins which formed defined micellar complexes with detergents and aggregated in their absence. Gel filtration, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the Triton X-100 complex of NPPase to contain a single 150,000-dalton peptide, while that of DPP IV was composed of two 120,000-dalton subunits; each complex also contained about 150,000-dalton Triton X-100. Trypsin cleaved the detergent complexes with release of major hydrophilic fragments which no longer bound detergent micelles; the accompanying change in peptide size was small for NPPase and undetectable for DPP IV, which also retained the dimer structure of its native form. DPP IV was the only major glycoprotein in rat liver plasma membrane which bound strongly to wheat germ agglutinin. Monospecific rabbit antibodies against NPPase and DPP IV precipitated the antigens without affecting their enzymatic activities.     

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.     

Comparative proteomics of rat liver and Morris hepatoma 7777 plasma membranes

Plasma membranes from normal rat liver and hepatocellular carcinoma Morris hepatoma 7777 were selectively solubilized by use of different reagents. After selective solubilization, proteins were identified by nano-HPLC-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Using simple software, the patterns of proteins identified in membrane solubilizates from liver and hepatoma were compared. Proteins identified in Morris hepatoma 7777 and not in the corresponding membrane solubilizate from liver, mostly members of the annexin and heat shock protein families, are discussed as potential candidate markers for hepatocellular carcinomas.     

Cholesteryl ester hydrolytic acitivity of rat liver plasma membrane.

Cholesteryl ester hydrolyzing activity of rat liver plasma membranes was studied using acetone-dispersed [4-14-C] cholesteryl oleate as substrate. In contrast to whole liver homogenates which displayed ample activity at both acid (4.5) and neutral (6.2-7.4) pH, purified plasma membrane fractions contained little activity at neutral pH as compared to acid pH. Moreover, rate-zonal sucrose density-gradient centrifugation patterns of plasma membrane rich fractions suggested a specific association with plasma membrane only in the case of the acid activity. These findings suggest that in vivo hepatic cell surface membranes contain little or no cholesteryl ester hydrolytic activity at extracellular pH. They support the possibility that plasma lipoprotein cholesteryl esters enter hepatic parenchymal cells prior to hydrolysis.     

Hemoglobin-haptoglobin receptor in rat liver plasma membrane

The presence of a receptor specific for the hemoglobin . haptoglobin complex is demonstrated in rat liver plasma membranes. Hemoglobin . haptoglobin complex, administered intravenously to rats, was cleared from the circulation at a constant rate with exclusive incorporation of the molecule into hepatocytes. This incorporation was unaffected by the simultaneous injection of asialoglycoprotein or heme . hemopexin complex. In vitro experiments with isolated liver plasma membranes indicated the absence of competitive binding of these molecules to the membrane and suggested that this receptor might recognize an altered conformation of the haptoglobin moiety of the complex resulting from the binding with hemoglobin. These observations suggest that the mechanism of recognition and binding of hemoglobin . haptoglobin complex by the receptor is different from that of the asialoglycoprotein receptor or heme . hemopexin receptor.     

Turnover of plasma membrane proteins in rat hepatoma cells and primary cultures of rat hepatocytes

The half-lives of turnover of plasma membrane proteins in rat hepatoma tissue, culture cells, and in primary cultures of rat hepatocytes have been analyzed after resolution by two-dimensional gel electrophoresis. Cell membranes were externally labeled via iodination catalyzed by lactoperoxidase and glucose oxidase. A bimodal pattern of turnover was found for the externally oriented plasma membrane proteins of rat hepatoma cells. Three glycoproteins analyzed in these cells had an average t 1/2 of 22 h while eight proteins which did not bind to concanavalin A had an average t 1/2 of 80 h. In contrast, more heterogeneous rates of turnover were found for the externally oriented plasma membrane proteins of primary cultures of hepatocytes. Most, if not all, of the membrane proteins accessible to iodination in these cells were glycoproteins. Among the glycoproteins resolved by two-dimensional polyacrylamide electrophoresis, the receptors for asialoglycoproteins had the shortest half-lives (18 h). Other glycoproteins, mostly with higher molecular weights and different isoelectric points, showed a spectrum of half-lives ranging from 16 to 99 h. The turnover rates of membrane proteins of primary cultures of rat hepatocytes were also determined with [3H]- and [35S]methionine labeling of cells. Heterogeneous rates of turnover again were found among the labeled glycoproteins and nonglycoproteins. Among the 10 glycoproteins individually analyzed, the half-lives range from 17 to 67 h. Among the 21 proteins which do not bind to concanavalin A, the half-lives range from 18 h to more than 100 h. Three proteins analyzed showed an apparent biphasic pattern of turnover, having a fast phase with a half-life of 4-6 h and a slow phase with a half-life of 15-29 h. Several nonglycoproteins, including clathrin and actin associated with membrane vesicles had extremely long half-lives. The more than 5-fold difference in the half-life between clathrin and the receptors for asialoglycoproteins, which coexist in coated pits indicates that intrinsic proteins of the coated pits turn over at a different rate than peripheral components.     

Ontogeny of glutamine transport by rat liver plasma membrane vesicles.

Glutamine metabolism in the liver is essential for gluconeogenesis and ureagenesis. During the suckling period there is high hepatic protein accretion and the portal vein glutamine concentration is twice that in the adult, whereas hepatic vein glutamine concentration is similar between adult and suckling rats. Therefore, we hypothesized that glutamine uptake by the liver could be greater in the suckling period compared to the adult period. The present studies were, therefore, designed to investigate the transport of glutamine by plasma membranes of rat liver during maturation (suckling--2-week old, weanling--3-week old and adult--12-week old). Glutamine uptake by the plasma membranes of the liver represented transport into an osmotically sensitive space in all age groups. Inwardly directed Na+ gradient resulted in an "overshoot" phenomenon compared to K+ gradient. The magnitude of the overshoot was greater in suckling rats plasma membranes compared to adult membranes. Glutamine uptake under Na+ gradient was electrogenic and maximal at pH 7.5, whereas uptake under K+ gradient was electroneutral. Glutamine uptake with various concentrations of glutamine under Na+ gradient was saturable in all age groups with a Vmax of 1.5 +/- 0.1, 0.7 +/- 0.1 and 0.5 +/- 0.06 nmoles/mg protein/10 seconds in suckling, weanling and adult rats, respectively (P < 0.01). Km values were 0.6 +/- 0.1, 0.5 +/- 0.1 and 0.5 +/- 0.1 mM respectively. Vmax for Na(+)-independent glutamine uptake were 0.6 +/- 0.1, 0.55 +/- 0.07 and 0.54 +/- 0.06 nmoles/mg protein with Km values of 0.54 +/- 0.2, 0. +/- 0.1 and 0.5 +/- 0.2 mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)