Isolation and characterization of [3H]fucose-labeled glycoproteins from the serum of normal rats and rats bearing Zajdela hepatoma.

Specific [3H]fucose-labeled glycoproteins were found in the serum of rats bearing Zajdela hepatoma, which possess different isoelectric points in comparison with serum glycoproteins from normal rats. The electrophoretic profile of the serum glycoproteins is significantly altered. There is approximately a 2.5 times increase of [3H]fucose incorporation into serum glycoproteins from rats with an ascitic form of hepatoma, compared with normal rats and to animals bearing a solid form of the tumor (which have a considerably greater survival). Serum fucoproteins identical for the two forms of hepatoma were isolated, as well as glycoproteins strongly specific for each of the forms.

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.

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.

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%.

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 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.

Adenylate cyclase activity in plasma membranes of chicken liver and of Mc-29 virus induced hepatoma.

1. Adenylate cyclase activity in homogenate and plasma membrane preparations from chicken liver and from Mc-29 virus induced transplantable hepatoma has been investigated. The basal enzyme activity from both liver and hepatoma homogenates and plasma membranes was similar. 2. NaF stimulated the enzyme activity in both liver and hepatoma plasma membranes, while only liver adenylate cyclase was activated by glucagon and epinephrine. 3. No inhibitory effect of insulin was established on liver and hepatoma enzyme activity. 4. Adenylate cyclase activity in liver and hepatoma plasma membranes was inhibited by colchicine at the same degree.

Isolation and partial characterization of plasma membranes from chicken liver and from Mc-29 virus induced transplantable hepatoma.

Plasma membranes have been isolated from chicken liver and from Mc-29 virus induced transplantable hepatoma. The purity of membrane preparations has been checked by electron microscopy and by determination of the activity of some enzymes: 5'-nucleotidase, Na+, K+-ATP-ase, Mg2+-ATP-ase, alkaline beta-glycerophosphatase and glucose-6-phosphatase. In hepatoma membranes the activity of 5'-nucleotidase, Na+, K+-ATP-ase and Mg2+-ATP-ase was lower, that of alkaline phosphatase higher, than in liver membrane preparation. The incorporation rate of glucosamine-14C into UDP-N-acetylglucosamine and into plasma membrane glucosamine have been studied as well. The rate of synthesis of UDP-N-acetylglucosamine was faster in liver than in tumor cells. The labeling of hepatoma plasma membranes with glucosamine-14C occurred more slowly than that of liver ones. The rate of transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to membrane-bound glucosamine is lower in hepatoma, than in liver cells.