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Results for “N-Acetyllactosamine Synthase”

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At least 19 recordsLinked to original sources
PubMed1975

Role of cell membrane galactosyltransferase in concanavalin A agglutination of erythrocytes.

It has been previously observed that rabbit erythrocyte cell surface galactosyltransferase appears to play a role in concanavalin A agglutination of these erythrocytes (Podolsky et al., 1974). Further, a correlation between the occurrence or level of cell surface galactosyltransferase and concanavalin A agglutinability of other cell types has also been observed. The mechanism by which rabbit erythrocyte galactosyltransferase participates in concanavalin A agglutination has now been further defined. The enzyme was solubilized and purified. Characterization of the enzyme properties has shown them to be similar to those reported for other purified galactosyltransferases. Amino acid and carbohydrate analysis showed a high asparagine content and the presence of D-mannose. Specific alpha-mannosidase treatment of the enzyme showed that some of these D-mannose residues were terminal sugars. The purified enzyme also conferred concanavalin A agglutinability to non-agglutinable human erythrocytes. However, the ability to confer concanavalin A agglutinability was unrelated to the enzyme activity per se (as measured with fetuin acceptor) but appeared to be entirely dependent on the presence of terminal alpha-linked D-mannosyl residues in the enzyme structure. These findings suggest that the presence of terminal alpha-mannosidyl residues on cell surface glycoproteins such as galactosyltransferase may be the determining factor in agglutination of cells by concanavalin A.

Asparagine↗
PubMed1979

Galactosyltransferase and membrane glycoprotein abnormality in human platelets from Tn-syndrome donors.

Early studies on the analysis of membranes isolated from the erythrocytes of Tn-patients by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a severe reduction in the staining capacity of glycophorin with the periodate-Schiff (PAS) reaction. A low sialic acid and galactose (Gal) content of the polyagglutinable red cells was confirmed while it was reported that the abnormal red cells of Tn-patients contained little or no UDPGal: GalNAc-beta-3-D-galactosyltransferase (T-transferase) activity. The glycoprotein (GP) abnormality in Tn-erythrocytes appeared to be due to incomplete synthesis of the alkali-labile oligosaccharide chaims of glycophorin. We now report studies on the membrane GP composition and the T-transferase activity of platelets isolated from there Tn-syndrome patients whose red cell membranes contain GP abnormalities which are typical of those found in this rare clinical condition.

Blood Group Antigens↗
PubMed1977

Enzymic characteristics of fat globule membranes from bovine colostrum and bovine milk.

Fat globule membranes have been isolated from bovine colostrum and bovine milk by the dispersion of the fat in sucrose solutions at 4 degrees C and fractionation by centrifugation through discontinuous sucrose gradients. The morphology and enzymic characteristics of the separated fractions were examined. Fractions comprising a large proportion of the total extracted membrane were thus obtained having high levels of the Golgi marker enzymes UDP-galactose N-acetylglucosamine beta-4-galactosyltransferase and thiamine pyrophosphatase. A membrane-derived form of the galactosyltransferase has been solubilized from fat and purified to homogeneity. This enzyme is larger in molecular weight than previously studied soluble galactosyltransferases, but resembles in size the galactosyltransferase of lactating mammary Golgi membranes. In contrast, when fat globule membranes were prepared by traditional procedures, which involved washing the fat at higher temperatures, before extraction, galactosyltransferase was not present in the membranes, having been released into supernatant fractions, When the enzyme released by this procedure was partially purified and examined by gel filtration, it was found to be of a degraded form resembling in size the soluble galactosyltransferase of milk. The release is therefore attributed to the action of proteolytic enzymes. Our observations contrast with previous biochemical studies which suggested that Golgi membranes do not contribute to the milk fat globule membrane. They are, however, consistent with electron microscope studies of the fat secretion process, which indicate that secretory vesicle membranes, derived from the Golgi apparatus, may provide a large proportion of the fat globule membrane.

Animals↗
PubMed1976

[Induction of N-acetyllactosamine synthetase activity in the mouse mammary gland by prolactin and placental lactogen hormone].

The authors show that the ovine prolactine promote induction of N. acetyl lactosamine synthetase in tissue culture of mammary glands of pregnant mice. A crude extract of human placenta has also a lactogenic activity as tested by the same method, but in this case the blank values are very high for large concentration of crude extract. The molecular forms of HCS are tested: the slow band has a lactogenic activity, the intermediate band has no activity and the rapid band seems to be inhibitory.

Animals↗
PubMed1976

The kinetic mechansim of bovine milk galactosyltransferase. The role of alpha-lactalbumin.

Initial rate parameters obtained with bovine galactosyltransferase at saturating Mn2+ concentrations, and a variety of acceptors including N-acetylglucosamine, glucose, ovalbumin, and di-N-acetylglucosamine are inconsistent with an ordered addition of UDP-galactose and acceptor substrates to the enzyme-Mn2+ complex. Inhibition patterns with N-acetylglucosamine or UDP-glucose as inhibitors of the galactosylation of ovalbumin indicated that either UDP-galactose or N-acetylglucosamine can bind to an enzyme-Mn2+ complex by a random equilibrium mechanism. Initial rate studies also indicate that alpha-lactalbumin may bind to either an enzyme-Mn2+-acceptor complex or an enzyme-Mn2+-UDP-galactose complex, suggesting that lactose synthesis also proceeds by a random equilibrium addition of substrates and alpha-lactalbumin. From the initial rate data assuming the random equilibrium mechanism, the dissociation constants for UDP-galactose, acceptor substrates, and alpha-lactalbumin from the appropriate complexes have been calculated. These values are in good agreement with those obtained independently by nonkinetic methods, providing additional support for the proposed random equilibrium mechanism. From similar studies with a cross-linked complex of alpha-lactalbumin and transferase, dissociation constants for UDP-galactose and acceptor substrates from the enzyme-Mn2+-alpha-lactalbumin complex were calculated. Comparison of each of the dissociation constants in the substrate addition phase shows that the binding of acceptor substrates and alpha-lactalbumin to enzyme-Mn2+ complexes is highly synergistic; the affinity of alpha-lactalbumin for the enzyme-Mn2+ acceptor complex is about 2 orders of magnitude greater than for the enzyme-Mn2+ complex. Similarly, the affinity of the acceptor for the enzyme-Mn2+-alpha-lactalbumin complex is about 2 orders of magnitude greater than the enzyme-Mn2+ complex. Synergism is also observed between alpha-lactalbumin and UDP-galactose binding but the synergism is much less than that observed with acceptor substrates and alpha-lactalbumin. Thus, the large decrease in the Michaelis constant for glucose in the presence of alpha-lactalbumin, which is observed for lactose synthesis by the galactosyltranferase, is primarily the result of the high degree of synergism in the binding of alpha-lactalbumin and glucose to enzyme-Mn2+ complexes. This synergism also accounts for the activation of N-acetyllactosamine synthesis by alpha-lactalbumin at low concentrations (less than 2 mM) of N-acetylglucosamine. An abortive enzyme-Mn2+-UDP-acceptor complex in the product release phase of the reaction appears to account for the inhibition of either lactose, or N-acetyllactosamine synthesis at a high concentration of either N-acetylglucosamine or glucose. This abortive complex is further stabilized by alpha-lactalbumin, thus the resulting substrate inhibition is observed at much lower acceptor concentrations in the presence of alpha-lactalbumin.

Acetylglucosamine↗
PubMed1978

Immobilized bovine lactose synthase. A method of topographical analysis of the active site.

Bovine galactosyltransferase (UDPgalactose: D-glucose 4beta-galactosyltransferase, EC 2.4.1.22) was covalently coupled to Sepharose 4B by reaction at pH 5.0 with the activated mixed disulfide Sepharose-glutathione-2(5-nitropyridyl)-disulfide. The Sepharose-protein conjugate was presumably coupled via the unique highly reactive cysteine of those thiols on the bovine enzyme. The gel-bound N-acetyllactosamine and lactose synthase activity of about 0.4% was consistent with the affects of diffusion and the 90% activity reduction noted upon thiol modification of the dissolved enzyme. The residual lactose biosynthetic activity of the bound enzyme appeared possible only if the reactive thiol were physically distinct from the active site since the bulky Sepharose-glutathione group must not obscure the alpha-lactalbumin binding region.

Animals↗
PubMed1978

Isolation and characterization of an enriched Golgi fraction from rat brain.

A fraction rich in membranes of the Golgi apparatus was isolated from rat brain by discontinuous density gradient centrifugation. The fraction sedimented at the characteristic Golgi density of 1.11--1.15 (g/cm3, 5 degrees C) and had specific activities of Golgi-marker enzymes (N-acetyllactosaminyl synthetase, glycoprotein (Fetuin) galactosyltransferase, thiamine pyrophosphatase), 6--7 times over those of the original homogenates. The recovery of the enzyme activities in this fraction ranged from 17 to 31 %. The incorporation [3H]fucose into glycoproteins was 3-fold higher than in homogenate. Recovery and relative specific activities of marker enzymes for other subcellular organelles were low. Electron microscopic analysis of the fraction revealed in the presence of Golgi structures, namely, large sacs or plates with attached tubules and "blebbing" of the tubules into the vesicles.

Animals↗
PubMed1975

Composition of membranes of cells transformed by tumorigenic DNA and RNA viruses.

Continuous cell lines obtained from mouse and hamster embryonic tissue and human lung contain a homologous series of acidic glycolipids known as gangliosides. These complex lipids are highly concentrated in plasma cell membranes. The ganglioside pattern becomes greatly simplified when cells are transformed by tumorigenic DNA and RNA viruses. Non-transforming viruses and lytic infection of cells by tumorigenic viruses without neoplastic tranformation do not cause changes in gangliosides. The alterations of ganglioside composition are the result of specific blocks in the biosynthesis of these lipids. The significance of these impairments of membrane glycolipid formation and the nature of the regulatory site(s) involved are considered with regard to the altered social behavior of transformed cells.

Animals↗
PubMed1978

Biochemical studies of rat liver Golgi apparatus. I. Isolation and preliminary characterization.

A method is described for the isolation of morphologically well-preserved Golgi apparatus from rat liver. The method is essentially the same as that of Morré et al. (Morré, D.J., Hamilton, R.L., Mollenhauser, H.H., Mahley, R.W., Cunningham, W.P., Cheetham, R.D., & Lequire, V.S. (1970) J. Cell Biol. 44, 484-491) except that mild cell disruption is achieved by means of a stainless-steel sieve. The average recoveries of protein and galactosyltransferase in the isolated fraction are about 6 mg from 10 g of perfused liver and about 35% from the homogenate, respectively. The preparation is virtually free from succinate-cytochrome c reductase, glucose-6-phosphatase, acid phosphatase, and 5'-nucleotidase. The Golgi fraction as well as its vesicular fragments is homogeneous upon isopycnic centrifugation in both sucrose and dextran density gradients. Their buoyant densities in sucrose are significantly higher than those in dextran, indicating that both forms of the organelle are closed systems which are impermeable to macromolecules. The galactosyltransferase activity of a freshly prepared Golgi fraction, measured with ovalbumin as galactosyl acceptor, is activated 26-fold by the addition of Triton X-100, whereas those of homogenized, sonicated, and aged preparations are only activated 2- to 4-fold.

Animals↗
PubMed1978

Biochemical studies on rat liver Golgi apparatus. II. Further characterization of isolated Golgi fraction.

Although the preparation of rat liver Golgi apparatus isolated by our method contains appreciable activities of NADH- and NADPH-cytochrome c reductases and glucose-6-phosphatase, these enzymes as well as thiamine pyrophosphatase of the extensively fragmented Golgi fraction are partitioned in aqueous polymer two-phase systems quite differently from those associated with microsomes. Similarly, the partition patterns of acid phosphatase and 5'-nucleotidase of the Golgi fragments differ from those of homogenized lysosomes and plasma membrane, respectively. It is concluded that most, if not all, of these marker enzymes in the Golgi fraction cannot be ascribed to contamination by the non-Golgi organelles. In sucrose density gradient centrifugation the NADH- and NADPH-cytochrome c reductase activities of the Golgi fraction behave identically with galactosyltransferase but differently from the reductase activities of microsomes, again indicating that the reductases are inherently associated with the Golgi apparatus. NADPH-cytochrome c reductase of the Golgi preparation is immunologically identical with that of microsomes. The marker enzymes mentioned above and galactosyltransferase behave differently from one another when the Golgi fragments are subjected to partitioning in aqueous polymer two-phase systems, suggesting that these enzymes are not uniformly distributed in the Golgi apparatus structure.

Acid Phosphatase↗
PubMed1975

Cross-linking of the components of lactose synthetase with dimethylpimelimidate.

The cross-linking of the two components of lactose synthetase, alpha-lactalbumin and a galactosyltransferase, with dimethylpimelimidate was examined. The extent of the cross-linking at pH 8.1 was found to be dependent upon the presence of substrates or inhibitors for the galactosyltransferase. N-acetylglucosamine and mixtures of either N-acetylglucosamine, Mn-2+ and UDP, or UDP-galactose and Mn-2+ promoted the formation of cross-linked species. Glucose or a mixture of UDP and Mn-2+ were much less effective in promoting cross-linking. Two types of intermolecularly cross-linked species of alpha-lactalbumin and the galactosyltransferase were obtained. Each was a 1:1 cross-linked complex of alpha-lactalbumin and either of the two forms of the transferase with molecular weights of about 42,000 and 48,000, respectively. Cross-linked complexes were not observed with more than 1 molecule each of alpha-lactalbumin and the transferase. The cross-linked complexes were obtained in homogeneous form by gel filtration on Sephadex and absorption of uncross-linked enzyme by affinity chromatography on alpha-lactalbumin-Sepharose in the presence of N-acetylglucosamine. They migrated on gel electrophoresis in sodium dodecyl sulfate with mobilities in accord with their predicted molecular weights as 1:1 complexes of alpha-lactalbumin and the transferase. The amino acid composition of the cross-linked complex was in reasonable agreement with the expected composition of a 1:1 mixture of alpha-lactalbumin and galactosyltransferase. The enzymic properties of the cross-linked and uncross-linked enzymes were compared. The cross-linked complex had a much higher intrinsic lactose synthetase activity than did uncross-linked enzyme although only about 1% of the potential activity of uncross-linked enzyme in the presence of optimal concentrations of alpha-lactalbumin. The lactose synthetase activity of the cross-linked complex, however, was unaffected by exogenous alpha-lactalbumin. In addition, the complex readily catalyzed the transfer of galactose from UDP-galactose to xylose in the absence of exogenous alpha-lactalbumin. The N-acetyllactosamine synthetase activity of the complex was low compared to its activity with other monosaccharides. Ovalbumin, which is a good acceptor for the uncross-linked transferase, was not an acceptor for the cross-linked complex. Kinetic studies of the complex suggest that its modified catalytic activity is not the result of the modification by dimethylpimelimidate but reflects the expected effects of is provided, and that

Amino Acids↗