Activity and characterization of sialyltransferase from serum of normal rats, of rats bearing Zajdela ascitic hepatoma, in normal host liver and in Zajdela hepatoma cells.

Comparative studies on the content of sialic acid and on the sialyltransferase activity in normal serum and in serum of rats with Zajdela ascitic hepatoma in different phases of tumor development have been conducted. Unlike the serum from animals with tumors, in which the sialic acid quantity increases in dependence of the stage of tumor development, the activity of serum sialyltransferase statistically augmented only in serum of rats at the final stage of tumor progression. The sialyltransferase activity towards asialofetuin as an acceptor in normal liver and in Zajdela hepatoma cells, was measured and a decrease in this activity in tumor cells as well as in host liver was found. When lactose was used as acceptor, again lower enzyme activity in the tumor cells in comparison with that in liver was established, but in liver and in hepatoma cells the predominant 14C-labelled product of the sialyltransferase assay was alpha (2-6) sialyllactose isomer. The results contribute to the biochemical characterization of rat Zajdela hepatoma.

Comparative studies on the uridine-5'-diphosphate-galactose: glycoprotein galactosyltransferase activity in rat liver and Zajdela ascitic hepatoma.

We report here data on the galactosyltransferase activity in serum, liver and Zajdela ascitic hempatoma cells and about the rate of galactose attachment to appropriate acceptors by beta(1-4, beta(1-3) and alpha(1-3) linkages in liver and hepatoma cell homogenates. It was established that in serum of Zajdela bearing rats, in host liver and in Zajdela ascitic cells, galactosyltransferase activity towards ovomucoid was elevated in comparison with control serum and liver. The activity towards low molecular acceptor (N-acetylglucosamine) in liver and hepatoma was nearly the same. The predominant isomer synthesized in both tissues was beta 1-4-galactose-N-acetylglucosamine. When asialofetuin was used as acceptor the activity of alpha(1-3) galactosyltransferase was measured. In Zajdela ascitic hepatoma cells alpha(1-3) galactosyltransferase activity was 3 times higher than that in liver. The elevated ability of the tumor cells to attach galactose residues with alpha(1-3) linkage to Gal beta 1-4GlcNAc-R sequence should be considered as a characteristic feature of these malignant cells.

gamma-Glutamyltranspeptidase and dipeptidyl peptidase IV activity in the serum of normal and hepatoma-bearing chickens and in the plasma membranes from liver and hepatoma Mc-29.

Investigations on the activity of gamma-glutamyltranspeptidase (GGT) and dipeptidyl peptidase IV (DPP IV) in the serum of healthy chickens and those bearing hepatoma Mc-29, and in liver and hepatoma plasma membranes were carried out. There was no difference in the serum enzyme activities of control and tumor-bearing chickens but the activity of GGT was twice higher and that of DPP IV 20 times lower in hepatoma plasma membranes than in chicken liver plasma membranes. Using thin-layer analytical isoelectric focusing in agarose gels it was established that the pI range of GGT from host serum and hepatoma plasma membranes was shifted to more acidic values. This could be interpreted as a specific feature for this enzyme considered as a tumor marker.

Pattern of sialoglycoproteins obtained by chromatofocusing of chicken liver and hepatoma Mc-29 microsomal preparations labelled in vivo with 3H-leucine and N-acetyl-14C-mannosamine.

Chromatofocusing has been used for separation of chicken liver and virus-induced hepatoma Mc-29 microsomal glycoproteins double labelled in vivo with 3H-leucine and N-acetyl-14C-mannosamine. The sialoglycoprotein profile was obtained by plotting the pH-values, as well as the values of the calculated specific activity (SA-cpm/mg protein) in each fraction, in the graphs. Different patterns for liver and hepatoma sialoglycoproteins were detected. Unlike liver microsomes in which the highest labelled compounds were registered in the alkaline zone of the pH-gradient, special feature for the hepatoma sialoglycoprotein pattern was the presence of highly labelled fraction eluted in the acidic zone of the pH-gradient. A term named "sialylation rate" of a separated sialoglycoproteins was involved. It has been found that liver sialoglycoproteins are more or less uniformly sialylated, independently of the pI-values, while those from hepatoma with acidic pI were sialylated at a higher extent in comparison to the fractions with alkaline pI.

Partial characterization of microsomal sialyltransferase from chicken liver and hepatoma Mc-29: I. Effect of nucleotides and metal ions.

CMP-N-acetylneuraminic acid: glycoprotein sialyltransferase activities were assayed in microsomal fractions from chicken liver and hepatoma, induced by the leukosis virus strain Mc-29, using asialofetuin as the substrate acceptor of N-acetylneuraminic acid. The effect of some nucleotides and metal ions on the enzyme activity was investigated. Kinetic studies revealed that the Km values toward asialofetuin at a saturation concentrations of CMP-N-acetylneuraminic acid for both liver and hepatoma enzymes are very closed, while V value was lower for the tumor enzyme. The liver and hepatoma enzymes have no exogenous Mn cations requirement and are inhibited by CTP, CMP and ATP. CMP was shown to act as a competitive inhibitor with an apparent Ki of 0.24 mM for the liver and 0.16 mM for hepatoma enzyme, respectively.

Partial characterization of microsomal sialyltransferase from chicken liver and hepatoma Mc-29: II. Measurement of enzyme activities utilizing microsomal glycoproteins as exogenous acceptors.

Microsomal sialyltransferase was assayed in chicken liver and hepatoma Mc-29 utilizing liver and hepatoma microsomal glycoprotein fractions, treated with Triton X-100, as exogenous acceptors. In a homologous assay system containing enzyme and acceptor from one and the same tissue no quantitative dependence of enzyme activity was revealed with increasing amount of the acceptor. In mixed experiments in which liver enzyme activity was tested towards hepatoma acceptor glycoproteins, a gradual drop in sialyltransferase activity occurred with increasing quantities of the acceptor. This effect seems to be a consequence of the presence of some inhibitor in the microsomal fractions from the hepatoma cells.

A comparison of galactosyltransferase activity in mouse ascites lymphoma cells (Ly/Ya), in cultured lymphoma cells, in ascitic fluid and culture medium.

Comparative studies were carried out on the galactosyltransferase activity in ascites lymphoma cells isolated from mouse with ascitic lymphoma Ly/Ya, in these cells grown in vitro (24 hrs culture), in ascitic fluid and culture medium. The effect of varying amounts of UDP-galactose on transfer rate of galactose to ovomucoid by the cell enzyme (ascitic and cultured lymphoma cells) and by the soluble enzyme (ascitic fluid and culture medium) was studied. The activity of the enzyme in the cell culture medium was 2.5-fold higher than that in ascitic fluid. The apparent Km values for UDP-galactose of the enzyme from both kinds of cells and from the two fluids was 7.14 x 10(-7) M. At saturating concentrations of donor substrate, V values for the cells and culture medium was 765 pmoles/10(6) cells/h and 180 pmoles/10(6) cells/h for the ascitic fluid.

Effect of hydrocortisone on sialyltransferase activity in the rat small intestine during maturation. Changes along the villus-crypt axis and in fetal organ culture.

Sialyltransferase activity was assayed in rat intestinal cells isolated as fractions reflecting the villus-crypt axis of differentiation. In 13-day-old rats both endo- and exogenous sialyltransferase activity reached their maximum in undifferentiated crypt cells and their peaks overlapped. In contrast, sialyltransferase of the adult intestine was 4-fold lower than that of sucklings in the crypts, with slight tendency to be transferred to the villus cells. Hydrocortisone applied to 10-day-old rats caused three days later a precocious drop of sialyltransferase activity in the crypt cells. Unlike in vivo, glucocorticoid responsiveness was accompanied by increased sialyltransferase activity in fetal small intestine cultivated for 17 days.

Effect of fluororotic acid on the sialylation of the chicken liver, hepatoma MC-29 and serum glycoconjugates.

Fluororotic acid, an orotic acid analogue which interferes with the nucleotide and RNA biosynthesis, was tested to determine its effect on: a) 14C-glucosamine and 14C-N-acetylmannosamine incorporation into acid-soluble nucleotide-sugars and into b) glycoconjugates from chicken liver and hepatoma induced by the avian leukosis strain Mc-29. In vivo metabolism studies indicated that this agent alters the nucleotide biosynthesis in both tissues investigated and causes a decrease in the sialylation rate of liver, hepatoma and serum glycolipids and glycoproteins.

Isolation and characterization of chicken liver and hepatoma Mc-29 endoplasmic reticulum and Golgi apparatus membranes and biosynthesis of their glycoconjugates.

Rough and smooth endoplasmic reticulum (ER) membranes and trans-Golgi apparatus (GA) fraction, intermediate GA fraction, and cis-GA fraction from White Leghorn chicken liver and hepatoma Mc-29 were isolated. Their purity was checked by electron microscopy and by determination of the activity of the marker enzymes. Rough and smooth membranes were also identified by calculation of the ratio between the content of RNA and the total phospholipids. It was found that the membrane fractions were pure enough. The tumor exhibited a decreased amount of ER and a small GA when compared when the ER and GA in the liver. The biogenesis of membrane glycoconjugates was followed by in vivo experiments either with [14C]glucosamine or by double labeling with [14C]N-acetylmannosamine and [3H]leucine. The radioisotope studies indicated that the synthesis rate of protein in liver and hepatoma rough ER was nearly the same, but in the tumor cells the glycosylation of the nascent polypeptide chain and the formation of glycolipids were significantly decreased.

Galactosyltransferase: multiple forms in serum of normal and hepatoma Mc-29 bearing chickens and from liver and hepatoma microsomal and plasma membrane preparations.

The multiple forms of galactosyltransferase in chicken serum and in microsomal and plasma membrane preparations from liver and viral induced hepatoma Mc-29 have been studied by isoelectric focussing. An elevation of the hepatoma plasma membrane enzyme activity was described and in the pattern of the multiple forms of the enzyme two forms were found (pI-5.34 and 8.22) which were similar to those described in the serum of hepatoma bearing chickens (pI-5.36 and 8.24). A conclusion is drawn that these enzyme forms are apparently present to a greater extent in the hepatoma plasma membrane enriched fractions than in liver membranes and are probably shed into the serum of the tumor bearing animals.

Characterization of sialyltransferases from serum of normal and hepatoma Mc-29 bearing chickens.

Sialyltransferase was measured in serum of normal and hepatoma Mc-29 bearing chickens. By preparative isoelectric focusing the multiple forms of sialyltransferase from both kind of serums was studied as well. By using influenza virus neuraminidase an attempt was made for partial structural characterization of the sialylation sites in asialofetuin applied as exogenous acceptor for sialyltransferase determination. It was established an elevated serum sialyltransferase activity in tumor bearing chickens with tumor an enzyme form was detected with pI-4.99 identical with an enzyme form described previously in solubilized plasma membrane preparations from hepatoma Mc-29. Monitoring of multiple forms of serum glycosyltransferases may be of value in answering the problem concerning the tissue origin of serum enzymes.

Glycosidases in normal and regenerating chicken liver, hepatoma Mc-29, Rous sarcoma, in turkey poult liver and hemocytoblastomes, provoked by the leukosis virus strain Mc-31.

Four glycosidases (beta-galactosidase, alpha-mannosidase, alpha-fucosidase and beta-N-acetylglucosaminidase) were studied in chicken normal and regenerating liver, in turkey poult liver and in virus induced avian tumors--chicken hepatoma (strain Mc-29), Rous sarcoma (strain Schmidt-Ruppin) and turkey poult hemocytoblastoma nodules (strain Mc-31). The multiple forms of beta-N-acetylglucosaminidase were assayed as well. A particular enzyme pattern was found in the tumor lines under investigation. A characteristic property of hepatoma cells was the elevation of beta-galactosidase activity and of the former enzyme and that of beta-N-acetylglucosaminidase for the hemocytoblastoma. In Rous sarcoma the glycosidase activities (except that of alpha-fucosidase) were much lower, compared to the other two solid tumors. All enzyme activities were compared with those in the normal liver of the corresponding avian species, and with the liver of tumor bearing fowls and with regenerating chicken liver. Unlike the rat liver in the avian normal and tumor tissues the percentual ratio between the multiple forms A and B of beta-N-acetylglucosaminidase was found to be 30:70%.

Studies on N-acetylneuraminic acid biosynthesis in chicken liver and hepatoma Mc-29 by using [14C]N-acetylmannosamine and [14C]glucosamine.

The biosynthesis of free N-acetylneuraminic (sialic) acid (N-acetylneuraminic + CMP-N-acetylneuraminic acid) in chicken liver and hepatoma Mc-29 by using [14C]N-acetylmannosamine and [14C]glucosamine was studied in vivo. The specific activity (SA) of hepatoma N-acetylneuraminic acid labelled with [14C]glucosamine is lower than that of liver, showing that the rate of conversion of UDP-N-acetylglucosamine to N-acetylneuraminic acid is reduced in tumor cells. The biosynthesis rate of sialic acid in hepatoma cells is higher when [14C]N-acetylmannosamine was applied. The UDP-N-acetylglucosamine-2'-epimerase activity was nearly 3-fold lower in hepatoma compared to that in liver. The time course of [14C]N-acetylmannosamine incorporation into free and protein bound sialic acid in hepatoma and liver was also showed. The SA of hepatoma protein bound sialic acid remained lower in all time points investigated. The results agree with the assumption that the metabolic pathways leading to sialic acid synthesis and to sialylation of tumor glycoconjugates are altered after malignant transformation.

Multiple forms of chicken liver and hepatoma Mc-29 microsomal and plasma-membrane sialyl and fucosyltransferases.

Multiple forms of microsomal and plasma membrane sialyl and fucosyltransferases from chicken liver and transplantable hepatoma Mc-29 have been separated by means of isoelectric focusing. A net different pattern was distinguished between liver and hepatoma microsomal and plasma-membrane associated transferases. Microsomal sialyltransferase from hepatoma Mc-29 has typical forms with pI = 5.69, 7.43, 8.05 and 8.56, while in plasma membrane, enzymes with pI = 5.00 and 8.70 occur. The presence of 9 forms of fucosyltransferase within the pH range 3.46-9.57 for hepatoma microsomes and within pH 4.52-9.60 for plasma membranes was detected. Forms with pI 5.10, 5.75 and 7.87 could be considered specific for the hepatoma microsomal enzyme, and forms with pI 4.52, 4.85 and 5.20 for the plasma-membrane associated enzyme.

Comparative study on fucosylation of chicken liver and virus-induced hepatoma Mc-29.

Comparative studies on fucoprotein metabolism of chicken liver and hepatoma Mc-29 have been carried out and the following parameters were determined: the incorporation rate of [14C]fucose into hepatoma and liver total tissue homogenate, acid-soluble and acid-insoluble fractions, acid-soluble nucleotide fraction and into plasma-membrane acid-precipitable fraction; the activity of microsomal and plasma-membrane fucosyltransferase; the electrophoretic pattern of hepatoma and liver plasma-membrane proteins and the incorporation of [14C]fucose into the glycoprotein fractions in both plasma-membrane preparations. It was found that the labelling of hepatoma tissue homogenate and plasma membranes was higher than that of the same liver preparations 3 hr after the [14C]fucose injection. This finding was supported by a considerably elevated hepatoma fucosyltransferase activity. The labelling rate of numerous fucoproteins from hepatoma plasma membranes was greatly increased and some of the individual glycoprotein bands were labelled to a higher extent compared with liver. The data presented show specific alterations of fucose and fucoprotein metabolism which could be considered as a characteristic feature of chicken viral-induced hepatoma Mc-29.

Effect of silymarin (Carsil) on the microsomal glycoprotein and protein biosynthesis in liver of rats with experimental galactosamine hepatitis.

The effect of the hepatoprotective drug silymarin (Carsil) on the incorporation rate of 14C-leucine into total proteins and on the biosynthesis of UDP-N-acetylhexosamine and microsomal glycoproteins using 14C-glucose of rat liver with D-galactosamine hepatitis was studied. It was found that i.p. treatment with Carsil in a dose of 140 mg/kg applied for 4 consecutive days partly abolishes the inhibitory effect of galactosamine on protein and glycoprotein biosynthesis. The specific activity of 14C-labelled UDP-N-acetylhexosamine is higher in the liver of D-galactosamine treated rats, compared with the specific activity of the nucleotide from liver pretreated with Carsil and then injected with galactosamine. This fact supports the suggestion that Carsil probably activates the metabolic conversion of UDP-hexosamine to the acetylated metabolite-UDP-N-acetylhexosamine, which is the normal liver cell metabolite, in liver cells.

Effect of D-glucosamine on the content and synthesis of UDP-sugars and plasma membrane associated carbohydrates in chicken liver and hepatoma Mc-29.

1. Comparative studies on the effect of D-glucosamine on the synthesis of UDP-sugars and on the incorporation rate of [14C]glucose into neutral sugars, sialic acid and glucosamine isolated from chicken liver and hepatoma Mc-29 plasma membranes have been carried out. 2. The influence of D-glucosamine on the activity of microsomal UDP-N-acetylglucosamine: glycoprotein N-acetylglucosaminyltransferase to lipid (dolichol monophosphate) and to protein acceptor was studied too. 3. It has been shown that D-glucosamine provokes alterations in the content and synthesis of UDP-sugars and it is an inhibitor of the glycosylation processes. An inhibitory effect of this sugar analog on the N-acetylglycosaminyltransferase has been established.

Phospholipid composition of subcellular fractions and phospholipid-exchange activity in chicken liver and MC-29 hepatoma.

The phospholipid composition of mitochondria, microsomes and plasma membranes from liver and MC-29 hepatoma from White Leghorn chickens has been investigated. It was established that all mitochondria and microsome phospholipid fractions obtained from MC-29 hepatoma are increased strongly compared to those from liver. The sphingomyelin augmentation was particularly great. In hepatoma plasma membranes only the sphingomyelin quantity was increased. Sphingomyelin- and phosphatidylcholine-exchange activities were observed in avian liver for the first time. These two activities were increased in MC-29 hepatoma cells. Three phospholipid-exchange proteins have been established in chicken liver 105000 X g supernatant. One of them specifically transports phosphatidylcholine, the second one is non-specific for phosphatidylcholine and sphingomyelin, and the third one is specific only for sphingomyelin. In hepatoma cells only a non-specific phosphatidylcholine- and sphingomyelin-exchange protein was found.