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.     

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.     

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.     

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.     

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.     

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.     

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%.     

Glycoprotein catabolism in rat liver. Lysosomal digestion of iodinated asialo-fetuin

(125)I-labelled asialo-fetuin, administered intravenously, rapidly accumulates in rat liver and the radioactivity is subsequently cleared from the liver within 60min. Plasma radioactivity reaches a minimum between 10 and 15 min after injection and rises slightly during the period of liver clearance. Free iodide is the only radioactive compound found in plasma during this latter period. Fractionation of rat liver at 5 and 13min after injection of (125)I-labelled asialo-fetuin supports the hypothesis that asialo-glycoprotein is taken into liver by pinocytosis after binding to the plasma membrane and is then hydrolysed by lysosomal enzymes. At 5min, radioactivity was concentrated 23-fold in a membrane fraction similarly enriched in phosphodiesterase I, a plasma-membrane marker enzyme, whereas at 13min the radioactivity appeared to be localized within lysosomes. Separation of three liver fractions (heavy mitochondrial, light mitochondrial and microsomal) on sucrose gradients revealed the presence of two populations of radioactive particles. One population banded in a region coincident with a lysosomal marker enzyme. The other, more abundant, population of radioactive particles had a density of 1.13 and contained some phosphodiesterase, but very little lysosomal enzyme. These latter particles appear to be pinocytotic vesicles produced after uptake of the asialo-fetuin bound by the plasma membrane. Lysosomal extracts extensively hydrolyse asialo-fetuin during incubation in vitro at pH4.7 and iodotyrosine is completely released from the iodinated glycoprotein. Protein digestion within lysosomes was demonstrated by incubating intact lysosomes containing (125)I-labelled asialo-fetuin in iso-osmotic sucrose, pH7.2. The radioactive hydrolysis product, iodotyrosine, readily passed through the lysosomal membrane and was found in the external medium. These results are not sufficient to account for the presence of free iodide in plasma, but this was explained by the observation that iodotyrosines are deiodinated by microsomal enzymes in the presence of NADPH.     

Purification and comparative properties of the glycoprotein nicotinamide adenine dinucleotide glycohydrolase from rat liver microsomal and plasma membranes.

NAD glycohydrolase, or NADase (NAD+ glycohydrolase, EC 3.2.2.5) was solubilized with porcine pancreatic lipase from isolated fractions of microsomes and plasma membranes obtained from rat livers. The enzyme from each organelle was further purified by DEAE-cellulose chromatography, gel filtration and isoelectric focusing. The solubilized, partially purified enzymes had similar molecular weights, pH-activity profiles and Km values. Marked charge heterogeneity was observed for the microsomal enzyme on isoelectric focusing between pH 6 and 8 with maximum activity focusing at pH 8.0. Plasma membrane NADase displayed a single peak at pH 6.7. Treatment of the partially purified microsomal or plasma membrane enzyme with neuraminidase resulted in a single peak of activity on isoelectric focusing (pH 3.5--10) with a pI of 9.2. Polyacrylamide gel electrophoresis of either NADase revealed a periodate-Schiff positive band which was coincident with enzyme activity. Compositional analyses of the microsomal enzyme focusing at pH 8.0 confirmed the presence of hexoses, hexosamines and sialic acid. Differences in carbohydrate composition might be important in determining the subcellular distribution of this enzyme.     

Characterization of the membrane proteins of rat liver lysosomes. Composition, enzyme activities and turnover.

Lysosomes prepared from the livers of untreated rats and from the livers of rats injected with either Triton WR-1339 or dextran yielded membranes that were similar in both polypeptide composition and activities of ATPase and acid 5'-nucleotidase. The administration of Triton WR-1339 (and dextran) resulted in an increase in ATPase activity of liver homogenates that was associated with a parallel increase in the ATPase activity of the lysosomal membrane. On the other hand, plasma membranes appear to be different from lysosomal membranes with respect to polypeptide composition and enzyme activities. The ATPase activity of lysosomal membranes is not affected by ouabain and suramin, inhibitors of the plasma-membrane ATPase. The plasma-membrane alkaline 5'-nucleotidase has little activity at acid pH. Pulse-labelling of lysosomal membranes with [3H]fucose and with [3H]- and [14C]-leucine occurred rapidly, faster than labelling of plasma membranes. The labelling kinetics indicate that lysosomal membranes may be assembled independently of plasma membranes. These data suggest that, in liver, little bulk transport of plasma membrane to lysosomes takes place, and lysosomal-membrane proteins may not be derived from those of plasma membranes.     

Cellular localization and substrate specificity of isoelectric forms of human liver neuraminidase activity.

The four major isoelectric forms of human liver neuraminidase (with pI values between 3.4 and 4.8) have been isolated by preparative isoelectric focusing and characterized with regard to their substrate specificity using glycoprotein, glycopeptide, oligosaccharide and ganglioside natural substrates. All forms exhibited a rather broad linkage specificity and were capable of hydrolyzing sialic acid glycosidically linked alpha 2-3, alpha 2-6 and alpha 2-8, although differential rates of hydrolysis of the substrates were found for each form. The most acidic form 1 (pI 3.4) was most active on sialyl-lactose, whereas form 2 (pI 3.9) and 3 (pI 4.4) were most active on the more hydrophobic ganglioside substrates. Form 4 (pI 4.8) was most active on the low-Mr hydrophilic substrates (fetuin glycopeptide, sialyl-lactose). Each form was less active on the glycoprotein fetuin than on a glycopeptide derived from fetuin. Organelle-enriched fractions were prepared from fresh human liver tissue and neuraminidase activity on 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid was recovered in plasma membrane, microsomal, lysosomal and cytosolic preparations. Isoelectric focusing of the neuraminidase activity recovered in each of these preparations resulted in significantly different isoelectric profiles (number, relative amounts and pI values of forms) for each preparation. The differential substrate specificity of the isoelectric forms and the different isoelectric focusing profiles of neuraminidase activity recovered in subcellular-enriched fractions suggest that specific isoelectric forms with broad but defined substrate specificity are enriched at separate sites within the cell.     

Distribution and partial purification of a liver membrane protein capable of inactivating cytosol enzymes.

1. The inactivation of cytosol enzymes in liver extracts was carried out by several subcellular fractions, with plasma membranes having the highest specific activity. Rough and smooth microsomal fractions were both active, whereas lysosmal inactivation capacity appeared to be derived entirely from contaminating plasma-membrane fragments. 2. Inactivation capacity in liver fractions was derived from parenchymal cells. Of the non-liver cells tested, plasma membranes from H35 hepatoma cells were able to inactivate glucose 6-phosphate dehydrogenase (EC 1.1.1.49), adipocyte "ghosts" showed slight activity and erythrocyte and reticulocyte "ghosts" were inactive. 3. Liposomes prepared from pure lipids with net negative, positive or neutral charge did not possess inactivation capacity. 4. Liver plasma-membrane inactivation capacity was destroyed by heating at 50 degrees C. 5. Inactivation factor solubilized from membranes by trypsin plus Triton X-100 treatment was partially purified by (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and hydroxyapatite chromatography. 6. Partially purified inactivation factor analysed by gel electrophoresis gave a major protein band that co-migrated with capacity for inactivation of glucose 6-phosphate dehydrogenase. 7. It is concluded that inactivation factor is a membrane protein whose intracellular distribution and other properties are consistent with a possible role for this activity in the initial step of protein degradation.     

Comparison of ornithine decarboxylase from rat liver, rat hepatoma and mouse kidney.

Comparisons were made of ornithine decarboxylase isolated from Morris hepatoma 7777, thioacetamide-treated rat liver and androgen-stimulated mouse kidney. The enzymes from each source were purified in parallel and their size, isoelectric point, interaction with a monoclonal antibody or a monospecific rabbit antiserum to ornithine decarboxylase, and rates of inactivation in vitro, were studied. Mouse kidney, which is a particularly rich source of ornithine decarboxylase after androgen induction, contained two distinct forms of the enzyme which differed slightly in isoelectric point, but not in Mr. Both forms had a rapid rate of turnover, and virtually all immunoreactive ornithine decarboxylase protein was lost within 4h after protein synthesis was inhibited. Only one form of ornithine decarboxylase was found in thioacetamide-treated rat liver and Morris hepatoma 7777. No differences between the rat liver and hepatoma ornithine decarboxylase protein were found, but the rat ornithine decarboxylase could be separated from the mouse kidney ornithine decarboxylase by two-dimensional gel electrophoresis. The rat protein was slightly smaller and had a slightly more acid isoelectric point. Studies of the inactivation of ornithine decarboxylase in vitro in a microsomal system [Zuretti & Gravela (1983) Biochim. Biophys. Acta 742, 269-277] showed that the enzymes from rat liver and hepatoma 7777 and mouse kidney were inactivated at the same rate. This inactivation was not due to degradation of the enzyme protein, but was probably related to the formation of inactive forms owing to the absence of thiol-reducing agents. Treatment with 1,3-diaminopropane, which is known to cause an increase in the rate of degradation of ornithine decarboxylase in vivo [Seely & Pegg (1983) Biochem. J. 216, 701-717] did not stimulate inactivation by microsomal extracts, indicating that this system does not correspond to the rate-limiting step of enzyme breakdown in vivo.     

Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. I. Biochemical determination

The distribution of adenylate cyclase (AC) in Golgi and other cell fractions from rat liver was studied using the Golgi isolation procedure of Ehrenreich et al. In liver homogenate the AC activity was found to decay with time, but addition of 1 mM EGTA reduced the rate of enzyme loss. The incorporation of 1 mM EGTA into the sucrose medium used in the initial two centrifugal steps of the Golgi isolation method stabilized the enzyme activity throughout the entire procedure and resulted in good enzyme recovery. In such preparations, AC activity was demonstrated to be associated not only with plasma membranes but also with Golgi membranes and smooth microsomal membranes as well. Furthermore, under the conditions used, enzyme activity was also associated with the 105,000 g x 90 min supernatant fraction. The specific activity of the liver homogenate was found to be 2.9 pmol-mg protein-1-min-1, the nonsedimentabel and microsomal activity was of the same order of magnitude, but the Golgi and plasma membrane activities were much higher. The specific activity of plasma membrane AC was 29 pmol-mg proten-1-min-1. The Golgi activity varied in the three fractions, with the highest activity (14 pmol) in GF1 lowest activity (1.8) in GF2, and intermediate activity (5.5) in GF3, when the Golgi activity was corrected for the presence of content protein, the activity in GF1 became much higher (9 x) than that of the plasma membrane while the activities in GF2 and GF3 were comparable to that of plasma membrane. In all locations studied, the AC was sensitive to NaF stimulation, especially the enzyme associated with Golgi membranes. The activities in plasma and microsomal membranes were stimulated by glucagon, whereas the Golgi and nonsedimentable AC were not.     

pH-dependent conversion of liver-membranous alkaline phosphatase to a serum-soluble form by n-butanol extraction

Alkaline phosphatase released from rat liver plasma membrane under usual conditions was electrophoretically not identical with a soluble form in serum which was derived from the liver. The liver-membranous alkaline phosphatase, however, was converted to the serum-soluble form when the liver plasma membrane was treated with n-butanol under the acidic conditions lower than pH 6.5. Such pH-dependent conversion of the enzyme was not observed in plasma membrane of rat ascites hepatoma AH-130 cells. The converting activity for alkaline phosphatase was detected not only in plasma membrane but also in lysosomal membrane of rat liver.     

Arylamidases of rat liver and chemically induced hepatomas 1. Subcellular distribution of l-leucine 2. Naphthylamidase-active antigens

The subcellular distribution of arylamidase-active antigens in rat liver and in two chemically induced hepatomas (D23 and D33) was investigated. Soluble antigens or detergent-solubilized membrane antigens from isolated subcellular fractions were tested in fused rocket immunoelectrophoresis against antisera prepared against each of the fractions. The arylamidase active antigens were identified by means of a zymogram technique using l-leucine 2-naphthylamide as substrate.Two arylamidase-active antigens were shown to be shared between plasma membranes, microsomes, lysosomal membranes and lysosomal content of the hepatocytes. One of these occurred predominantly in the plasma membranes (the plasma membrane arylamidase) while the other was preferentially found in the lysosomal content (the lysosomal content arylamidase). Also a third arylamidase-active antigen was identified and was shown to be restricted to the microsomes and the lysosomal membranes (the microsomal/lysosomal arylamidase).The rat liver plasma membrane arylamidase-active antigen was also present in plasma membrane, microsomal an cell-sap fractions of both the hepatomas. However, in the hepatomas this antigen occurred predominantly in the microsomal fraction. The plasma membrane arylamidase was the only arylamidase-active antigen found in the hepatoma D33 while the plasma membrane and microsomal fractions of hepatoma D23 also contained another antigen with this activity. Neither the lysosomal content arylamidase nor the microsomal/lysosomal arylamidase could be detected in any of the hepatoma fractions.     

Arylamidases of rat liver and chemically induced hepatomas. 1. Subcellular distribution of L-leucine. 2. Naphthylamidase-active antigens.

The subcellular distribution of arylamidase-active antigens in rat liver and in two chemically induced hepatomas (D23 and D33) was investigated. Soluble antigens or detergent-solubilized membrane antigens from isolated subcellular fractions were tested in fused rocket immunoelectrophoresis against antisera prepared against each of the fractions. The arylamidase active antigens were identified by means of a zymogram technique using L-leucine 2-naphthylamide as substrate. Two arylamidase-active antigens were shown to be shared between plasma membranes, microsomes, lysosomal membranes and lysosomal content of the hepatocytes. One of these occurred predominantly in the plasma membranes (the plasma membrane arylamidase) while the other was preferentially found in the lysosomal content (the lysosomal content arylamidase). Also a third arylamidase-active antigen was identified and was shown to be restricted to the microsomes and the lysosomal membranes (the microsomal/lysosomal arylamidase). The rat liver plasma membrane arylamidase-active antigen was also present in plasma membrane, microsomal and cell-sap fractions of both the hepatomas. However, in the hepatomas this antigen occurred predominantly in the microsomal fraction. The plasma membrane arylamidase was the only arylamidase-active antigen found in the hepatoma D33 while the plasma membrane and microsomal fractions of hepatoma D23 also contained another antigen with this activity. Neither the lysosomal content arylamidase nor the microsomal/lysosomal arylamidase could be detected in any of the hepatoma fractions.     

Purification and properties of a mouse liver plasma-membrane glycoprotein hydrolysing nucleotide pyrophosphate and phosphodiester bonds

1. A mouse liver plasma-membrane preparation was solubilized in an N-dodecylsarcosinate-Tris buffer, pH7.8, and the proteins and glycoproteins were separated by a rate-zonal centrifugation in sucrose-detergent gradients. 2. A peak of alkaline phosphodiesterase activity which sedimented ahead of the 5'-nucleotidase peak was associated with a major glycoprotein component of the plasma membrane. 3. The phosphodiesterase activity was then purified further by gel filtration and gave a single glycoprotein band after electrophoresis on polyacrylamide gels. The apparent molecular weight of the polypeptide at pH7.4 and 8.9 was 128000-130000 and was independent of the polyacrylamide concentration. Electrophoresis in gels containing deoxycholate showed that the protein band was coincident with phosphodiesterase activity. 4. After two-dimensional immunoelectrophoresis, with agarose containing rabbit anti-(mouse plasma-membrane) antiserum as second dimension, the enzyme showed one component which was also coincident with the phosphodiesterase activity. 5. An amino acid composition of the glycoprotein is presented. Carbohydrate analysis indicated the presence of glucosamine, neutral sugars and sialic acid. 6. The enzyme was also a nucleotide pyrophosphatase, as shown by a similar enrichment during purification of activity towards ATP, NAD(+), UDP-galactose and UDP-N-acetylglucosamine. The phosphodiesterase activity, measured by using dTMP p-nitrophenyl ester as substrate, was competitively inhibited by nucleotide pyrophosphate substrates. The enzyme showed little or no activity towards RNA, cyclic AMP, AMP, ADP and glycerylphosphorylcholine. 7. The significance of this enzyme activity in the plasma membrane is discussed.     

Characterization of different molecular forms of 5''-nucleotidase in normal serum and in serum from cholestatic patients and bile-duct-ligated rats.

Serum 5'-nucleotidase in rat and man is derived from the plasma membrane rather than the cytosol by the criteria of inhibition with [alpha beta-methylene]ADP and antisera. In individuals with cholestasis the serum enzyme is mainly present as a high-Mr form that in the presence of the zwitterionic detergent Sulphobetaine 14 has the electrophoretic characteristics of liver plasma-membrane ectoenzyme. A minor form of 5'-nucleotidase in cholestatic serum and all the enzyme in normal serum appears to be half the molecular size of the liver plasma-membrane ectoenzyme. 5'-Nucleotidase from both normal and cholestatic rat serum was found to contain a polypeptide chain of apparent Mr 70 000 by immunoblotting techniques. It is suggested that the major form of 5'-nucleotidase in cholestatic serum is an ectoenzyme dimer derived from liver plasma membrane. All of the enzyme in normal serum and some of the enzyme in cholestatic serum is present as an active monomer derived from the ectoenzyme dimer.