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At least 19 recordsLinked to original sources
PubMed1978

Human serum galactosyltransferase: distinction, separation and product identification of two galactosyltransferase activities.

Two different galactosyltransferase activities have been found in normal sera from A and O donors. Galactosyltransferase A incorporated galactose from UDP-Gal into sialic-acid-free ovine submaxillary mucin (asialo-mucin), whereas galactosyltransferase B transferred galactose from UDP-Gal to free N-acetylglucosamine or N-acetylglucosamine-glycoproteins. Specificity, kinetic and stability differences permitted the distinction of the activity of galactosyltransferase A from that of galactosyltransferase B; the only substrate found for galactosyltransferase A was asialo-mucin, whereas galactosyltransferase B showed only low activity towards asialo-mucin and free N-acetyl-galactosamine, but had a main specificity for either free N-acetylglucosamine or N-acetylglucosamine-protein. Galactosyltransferase B was more stable on heat inactivation than galactosyltransferase A; galactosyltransferase B could be separated from galactosyltransferase A by affinity chromatography on N-acetylglucosamine-derivatized agarose. The products of both enzyme activities have been analyzed. The galactosyltransferase A product was cleaved from asialo-mucin by alkaline-borohydride treatment. The acceptor used to identify the galactosyltransferase B product was free N-acetylglucosamine. Periodate oxidation studies performed on the reduced disaccharides indicated the linkage type of the products. The anomeric configuration of the respective galactosyltransferase products were determined with specific galactosidases. Using these methods, galactosyltransferase A was found to form a Galbeta (1 leads to 3)GalNAc-protein linkage and galactosyltransferase B was found to form a Galbeta(1 leads to 4)GlcNAc-linkage.

Galactosyltransferases↗
PubMed1977

Incorporation of galactose into galactosyltransferase.

Bovine skim milk galactosyltransferase (EC 2.4.1.22) retained its catalytic activity after partial enzymatic removal of sialic acid and galactose. Desialylated and degalactosylated galactosyltransferase was a galactosyl acceptor in the galactosyltransferase reaction. [14C]Galactose from UDP-[14C]galactose was incorporated into the carbohydrate-depleted galactosyltransferase by native galactosyltransferase. The results suggest that galactosyltransferase participates in the biosynthesis of its glycopeptides of the sialic acid-galactose-N-acetylglucosamine type.

Animals↗
PubMed1979

The effect of wheat germ agglutinin on sialyl and galactosyltransferases of rat liver Golgi membranes.

The sialyltransferase and galactosyltransferase activities of the Golgi-rich fraction from rat liver were enhanced by the binding of wheat germ agglutinin (WGA). The sialytransferase was more sensitive than the galactosyltransferase to the WGA. Maximal stimulation of the galactosyltransferase activity resulted from the binding of 60--80 micrograms WGA to the Golgi membrane, while only 40 micrograms of WGA produced a maximal enhancement in the sialyltransferase activity. Within 5 min of WGA binding, the Golgi sialytransferase activity was doubled. After the initial binding of WGA to the Golgi fraction, the galactosyltransferase activity was decreased by 30%. However, in 15 min the activity was doubled by the binding of WGA. The activities of both enzymes were further enhanced by incubation for up to 90 min. The stimulation of both sialyltransferase and galactosyltransferase activities by WGA was reversed by N-acetyl-D-glucosamine (GlcNAc), the specific inhibitor of agglutination by WGA. Complete reversal of the enhanced activity was observed after 20--30 min in the presence of 1 micromol GlcNAc. The association constant for the binding of WGA to the Golgi membranes was calculated to be 4.16 X 10(-6) M from a Steck-Wallach plot. The 'n' value or mean binding sites was calculated as 5.26 X 10(-5) M/mg of Golgi membrane protein.

Animals↗
PubMed1977

Cancer-associated serum galactosyltransferase activity. Demonstration in an animal model system.

Two different lines of solid tumors were produced in outbred hamsters by subcutaneous injection of polyoma transformed BHK cells. Growth of the tumors correlated with the appearance in serum of an electrophoretically distinct peak of galactosyltransferase: NeuAc-, Gal-free fetuin acceptor activity on polyacrylamide gels. This slow moving peak of enzyme activity (GT-HH) was detected before solid tumors could be grossly observed and the amount of activity in this peak was also found to be linearly related with growth of the tumor. GT-IIH was not detectable in control animals and separated from a faster migrating major area of serum galactosyltransferase activity (GT-IH) found in sera of both control and tumor-bearing hamsters. These two activities were shown to maintain their respective mobilities on re-electrophoresis. Solubilized enzyme derived from excised tumors demonstrated an electrophoretic mobility on polyacrylamide gels identical to that for GT-IIH present in serum from tumor-bearing animals. In contrast, enzyme activity solubilized from livers of both control or tumor-bearing hamsters showed a mobility similar to that of the faster moving serum galactosyltransferase enzyme activity, i.e. GT-IH. In addition, medium derived from nonconfluent BHKpy cells in tissue culture contained galactosyltransferase activity which co-electrophoresed with the slower migrating characteristics of galactosyltransferase activities derived from serum (control and tumor-bearing), solid tumors, liver and BHKpy cells in tissue culture were compared. All kinetic properties were similar with the exception that the Km UDP-galactose of GT-IIH (1.0 X 10(-5) M) was half that of GT-IH (2.0 X 10(-5) M).

Animals↗
PubMed1975

Udp-galactose: glycoprotein galactosyltransferase activity in a clonal line of rat brain.

1. UDPgalactose:glycoprotein galactosyltransferase (EC 2.4.1.-) activity was demonstrated in homogenates from whole rat brain, isolated neuromal perikarya, enriched glial cell fractions, and cultured rat glial tumor cells (clone C6). 2. Galactosyltransferase activity was enriched 3-9-fold in neuronal perikarya and 1.4--1.8-fold in the glial cell fraction over the activity in whole brains from 19- and 40-day-old rats. The activity of galactosyltransferase in neuronal perikarya decreased with age. Extensive contamination of the glial cell fraction with membranous fragments appeared to obscure the precise specific activity of this fraction. 3. The specific activity of the enzyme in glial tumor cells was 4--8-fold higher than in brain tissue when the enzyme was assayed under identical conditions using endogenous and different exogenous acceptors. 4. Galactosyltransferase activities from adult brain and glial tumor cells had similar properties. They both required Mn-2 plus and Triton, and exhibited pH optima between 5 and 7. The apparent Km of the enzyme for UDPgalactose was 1.3-10-minus 4 M for brain tissue and 2.2-10-minus 4 M for glial tumor cells. 5. The high galactosyltransferase activity in glial tumor cells and in neuronal perikarya of younger rats is compatible with the possibility of a role of this enzyme in developing brain.

Aging↗
PubMed1979

Purification by affinity chromatography and some properties of microsomal galactosyltransferase from pig thyroid.

Membrane-bound 4-beta-galactosyltransferase (lactose synthase; UDP galactose: D-glucose 4-beta-galactosyltransferase, EC 2.4.1.22) was purified 1500-fold to near homogeneity from pig thyroid microsomes with about 30% yield. The purified enzyme behaved as a lipophilic protein, rapidly losing activity and aggregating if not supplemented with either Triton X-100 or serum albumin (both of these were equally effective for long-term stabilization). The enzyme preparation showed an absolute requirement for Mn2+, which could not be replaced by other cations. Catalytic properties were very similar to those reported for soluble forms of the enzyme in biological fluids. The purified galactosyltransferase showed a major protein band of approx. 74,000 daltons on sodium dodecyl sulfate gel electrophoresis. On gel filtration, enzyme activity was eluted at approx. 70,000 daltons. It is concluded that the membrane-bound thyroid galactosyltransferase is a monomeric protein significantly larger than the soluble forms of this enzyme described earlier; but it resembles recently reported galactosyltransferases from sheep mammary Golgi membranes and liver microsomes.

Animals↗
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↗
PubMed1976

Studies on the purification and properties of UDP-galactose glycoprotein galactosyltransferase from rat liver and serum.

1. Rat liver microsomal preparations incubated with 200mM-NaCl at either 0 or 30 degrees C released about 20-30% of the membrane-bound UDP-galactose-glycoprotein galactosyl-transferase (EC 2.4.1.22) into a 'high-speed' supernatant. The 'high-speed' supernatant was designated the 'saline wash' and the galactosyltransferase released into this fraction required Triton X-100 for activation. It was purified sixfold by chromatography on Sephadex G-200, and appeared to have a higher molecular weight than the soluble serum enzyme. 2. Rat serum galactosyltransferase was purified 6000-7000-fold by an affinity-chromatographic technique using a column of activated Sepharose 4B coupled with alpha-lactalbumin. The purified enzyme ran as a single broad band on polacrylamide gels and contained no sialytransferase, N-acetylglucosaminyltransferase and UDP-galactose pyrophosphatase activities. 3. The highly purified enzyme had properties similar to those of both soluble and membrane-bound galactosyltransferase. It required 0.1% Triton X-100 for stabilization, but lost activity on freezing. The enzyme had an absolute requirement for Mn2+, not replaceable by Ca2+, Mg2+, Zn2+ or Co2+. It was active over a wide pH range (6-8) and had a pH optimum of 6.8. The apparent Km for UDP-galactose was 12.5 x 10(-6) M. Alpha-Lactalbumin had no appreciable effect on UDP-galactose-glycoprotein galactosyltransferase, but it increased the specificity for glucose rather than for N-acetylglucosamine, thus modifying the enzyme to a lactose synthetase. 4. The possibility of a conversion of higher-molecular-weight liver enzyme into soluble serum enzyme is discussed, especially in relation to the elevated activities of this and other glycosyltransferases in patients with liver diseases.

Acetylglucosamine↗
PubMed1976

Uridine 5'-diphosphate-galactose:glycoprotein galactosyltransferase activity in the ovarian cancer patient.

Uridine 5'-diphosphate-galactose:glycoprotein galactosyltransferase activity was demonstrated in homogenates of normal ovary and ovarian epithelial adenocarcinomas. The specific activity of the enzyme in ovarian tumors was 3 to 5 times higher than in normal ovaries when the enzyme was assayed under identical conditions. The glycoprotein fetuin, from which terminal sialic acid and penultimate galactose were removed (fetuin minus N-acetylneuraminis acid and galactose), acted as an excellent exogenous acceptor. Galactosyltransferase from normal ovary and ovarian tumor cells had similar properties. Both required Mn2+ and Triton X-100 and had broad pH optima between 5.5 and 7. Galactosyltransferase activity was also measured in serum samples from ovarian cancer patients and normal healthy individuals in the presence of fetuin minus N-acetylneuraminic acid and galactose as exogenous acceptor. The enzyme levels were significantly elevated in the sera of ovarian cancer patients as compared to normal controls. The differences in the levels of this enzyme in the tissues and sera of normal individuals and ovarian cancer patients were not due to differential levels of the degrading enzymes such as uridine 5'-diphosphate-galactose pyrophosphatase or beta-D-galactosidase. Serial determinations were carried out on the sera of 5 ovarian cancer patients over a long period of time. The serum level of galactosyltransferase activity appeared to correlate with tumor volume as well as with the clinical status of the patient, which suggests possible leakage of the tumor enzyme into the host sera. Serial determination of this enzyme level in ovarian cancer patients seems promising in measuring tumor progression or success of therapeutic approaches.

Adenocarcinoma↗
PubMed1977

Galactosyltransferase activity of the microvillous surface of human placental syncytial trophoblast.

The syncytial trophoblast has previously been shown to have minimal intrasyncytial galactosyltransferase activity at term. The biochemical and autoradiographic study reported here shows that the microvillous surface of term human placental syncytial trophoblast has a galactosyltransferase activity capable of transferring 3H-galactose from uridine diphosphate-D-galactose-1-3H to trichloroacetic-acid precipitable, endogenous acceptors. This capability of resynthesizing cleaved galactose moieties in the glycocalyx, without dependence on cytoplasmically located galactosyltransferases, would allow for reinstatement of the original surface molecular configuration without requiring synthesis and insertion of a completely new membrane molecule. It is suggested that the surface galactosyltransferase might function to repair damage to syncytial trophoblast glycocalyx induced by the enzymes in maternal blood.

Autoradiography↗
PubMed1975

Purification and properties of galactosyltransferase from human colostrum.

The galactosyltransferase (Uridine diphosphate-D-galactose: D-glucose 1-galactosyltransferase EC 2.4.1.22) was purified from human colostrum by chromatography on DEAE-cellulose, cellulose phosphate, sephadex G-100, and hydroxylapatite after removal of caseins by centrifugation. The final preparation showed two forms of protein on polyacrylamide disc gel electrophoresis, and both of them exhibited galactosyltransferase activity. The molecular weights of the two forms of the protein were estimated as 44,000 to 45,000 and 55,000 to 57,000 by polyacrylamide disc gel electrophoresis containing sodium dodecyl sulfate. General properties of galactosyltransferase were investigated.

Chromatography↗
PubMed1977

Age-related changes in human skin collagen galactosyltransferase and collagen glucosyltransferase activities.

Collagen galactosyltransferase and collagen glucosyltransferase activities were assayed in human skin specimens of about 100 mg wet weight. The assay of the glucosyltransferase activity was found to be highly specific. The assay of the galactosyltransferase activity was somewhat less specific, but there was no difference in specificity between the foetal and adult human skin samples. The activities of the two collagen glycosyltransferases in human skin extract were found to vary with age, being highest in foetal skin, and higher in the skin of young children that in that of adults. The galactosyltransferase and glucosyltransferase activities in foetal skin were respectively about 4 times and 6 times those in adult skin. The magnitudes of the changes with age in the two collagen glycosyltransferase activities were smaller than those occurring in the activities of the two other intracellular enzymes of collagen biosynthesis namely prolyl and lysyl hydroxylase. This difference suggests that the four intracellular enzyme activities of collagen biosynthesis are not regulated in an identical manner.

Adolescent↗
PubMed1978

Further characterization of collagen galactosyltransferase from chick embryos.

Optimum extraction of collagen galactosyltransferase activity from chick embryos required relatively high concentrations of detergent and salt. The activity was inhibited by concanavalin A, and the enzyme had a high affinity for columns of this lectin coupled to agarose; these results suggest the presence of carbohydrate units in the enzyme molecule. Collagen galactosyltransferase was highly labile, and only 1% of the originally bound enzyme activity could be eluted from the concanavalin A-agarose column with a buffer containing methyl glucoside and ethylene glycol. The purification of the activity over the original supernatant of chick embryo homogenate was 250-300-fold, with the optimum reaction conditions for the purified transferase differing somewhat from those for crude enzyme preparations. The reaction was inhibited by glucose-free basement-membrane collagen, UDP and galactosylhydroxylsine, and also by Co2+ and a number of compounds resembling UDP-galactose. Hydroxylysine was also a weak inhibitor. Immobilized hydroxylysine and UDP-glucuronic acid did not bind the collagen galactosyltransferase, but the enzyme was retarded in a column of UDP-galacturonic acid linked to agarose.

Animals↗
PubMed1979

The charge heterogeneity of soluble human galactosyltransferases isolated from milk, amniotic fluid and malignant ascites.

UDP-galactose: N-acetylglucosamine galactosyltransferase was isolated from pooled human milk, pooled amniotic fluid and from two different individual samples of malignant ascites. The purification procedure involving two successive affinity chromatography steps on N-acetylglucosamine--agarose and alpha-lactalbumin--agarose yielded an enzyme preparation homogeneous by size. Under non-denaturing conditions the ascites and amniotic fluid enzymes had identical electrophoretic mobility, but they moved faster than the milk enzyme. Isoelectric analysis in the presence and absence of urea resolved the milk enzyme into at least 13 different forms, nine of which had the same isoelectric points after refocusing. All enzyme forms showed similar activity when free N-acetylglucosamine, ovalbumin, sialic-acid-free ovine submaxillary mucin and glucose, in the presence of alpha-lactalbumin, were used as acceptor substrates. Comparative isoelectric focusing of the three galactosyltransferases revealed identical patterns of the amniotic and ascites enzymes, but only partial overlap with the milk enzyme, which was less negatively charged. Neuraminidase treatment of ascites and milk galactosyltransferases produced very similar focusing patterns. The possible structural basis for this charge heterogeneity is briefly discussed.

Amino Acids↗
PubMed1978

Galactosyltransferase in fetal, neonatal, and adult colon: relationship to differentiation.

Microsomal galactosyltransferase activity of fetal rat colon increased fourfold between 18 and 22 days of gestation and then more slowly during neonatal life reaching adult levels after 14 days. The Km for uridinediphosphate- (UDP) galactose, pH optimum, cation, and detergent requirements were identical in fetal and adult galactosyltransferase. Cytidine 5'-diphosphate-choline stimulated the adult but not fetal colonic galactosyltrasferase activity by inhibition of UDP-galactose pyrophosphatase. The increase in colonic galactosyltransferase in late fetal development is correlated with our previous observation that incorporation of [3H]galactose is markedly increased during differentiation of the fetal colon.

Aging↗
PubMed1976

[Biosynthesis of glycoproteins in pulmonary parenchyma. II. Galactosyltransferase activity in pneumocyte subcellular fractions].

A soluble galactosyltransferase exists in the cell sap of the alveolar cells of the lung. The optimal conditions of galactosyltransferase are: pH 6, 20 DEGREES C, 10 mM Mn2+, 1 mM Mg2+. Michaelis constant for substrate UDP-galactose is 390 nM. By electrofocusing on Ampholine column, galactosyltransferase appears in a unique peak, at pHi 4.8. The parameters of the purified enzyme are the same as that of the crude enzyme. UDP is a noncompetitive inhibitor for the purified enzyme.

Animals↗
PubMed1978

Correlation of UDP-galactose glycoprotein:galactosyltransferase levels in the sera with the clinical status of ovarian cancer patients.

Uridine diphosphate-galactose : glycoprotein galactosyltransferase (EC 2.4.1.22) was measured serially prior to and after surgery in 4 patients with ovarian epithelial cancer. The levels of this enzyme in the sera correlated well with the clinical status of the patients. In 2 other patients, the follow-up was designed to detect recurrence, and the enzyme assay was started when the patients were clinically disease free. Elevation of galactosyltransferase preceded the clinical appearance of disease by 3-7 months. Serial determination of glycoprotein galactosyltransferase in serum may be useful for evaluating the effectiveness of therapeutic programs.

Adenocarcinoma↗