Effects of the N-acetylgalactosaminyltransferase inhibitor on cultured cerebral cells.

The inhibitor preparation of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyltransferase (EC 2.4.1.92) (GalNAc-T) produces effects on the neurons and the glial (astrocytes) cells of the cerebrum in culture. The effect in culture is evidenced by aneuritogenesis, deficiency in the GalNAc-T activity, and decrease in the content of gangliosides, proteins, and lipids. In isolated glial cells the effect is evidenced by cytoplasm vesiculation and premature cessation of proliferation compared with control culture. The pattern of gangliosides in the inhibited culture shows a decrease in the amount of GD1a with respect to GD3; this is compatible with the notion that the effect is due to an inhibitor of the GM2 synthase. The inhibitor effects are reverted when it is eliminated after 24 or 48 hr in the culture medium.

Internalization of the inhibitor of the N-acetylgalactosaminyltransferase by chicken embryonic retina cells: reversibility of the inhibitor effects.

Retina cells from 6-day-old chicken embryos were cultured in the presence of an 125I-labeled protein inhibitor of the UDP-N-acetylgalactosamine:GM3,N-acetylgalactosaminyltransferase. The cells were labeled and did not lose the incorporated radioactivity when treated with 0.125% trypsin or 1 M NaCl at 37 degrees C for 1 hr, indicating that the iodinated inhibitor was inside the cells. Immunostaining procedures using an anti-inhibitor antibody were applied to the cells cultured in the presence of the inhibitor after permeabilization of the cells. The inhibitor was found inside the round cells virtually devoid of neurites, but not in flat glial-like cells or in process-bearing neural cells. Also found was an apparent self-recovery effect of the cells for both the anti-neuritogenic effect and the modification of the pattern of labeled gangliosides produced by the inhibitor when the agent was withdrawn from the culture medium after the initial period of 20 hr. This recovery was clearly observed 72 hr after the removal of the inhibitor.

An endogenous inhibitor of N-acetylgalactosaminyltransferase inhibits retina neuron differentiation in culture.

An inhibitor of N-acetylgalactosamine:GM3, N-acetylgalactosaminyltransferase (EC 2.4.1.92) from chicken blood serum, was tested for its activity on embryonic chicken neural retina in culture. The inhibitor did not change the cellular protein content of the cultures but produced a significant reduction of the labeling of gangliosides. The ratio of labeling of GD3 to GD1a increased from about 0.1 to about 0.8 in the cells cultured without or with the inhibitor, respectively. A striking effect of the inhibitor was seen on the morphology of the neurons, those cultured in its presence being practically devoid of neurites. Glial flat cells were apparently not affected.

Purification and characterization of an endogenous inhibitor of the sialyltransferase CMP-N-acetylneuraminate: lactosylceramide alpha 2,6-N-acetylneuraminyltransferase (EC 2.4.99.-).

The presence in the 100,000 g supernatant of rat brain homogenate of an inhibitor of the sialyltransferase has been confirmed. It is also present in chicken and bovine brain and in other rat and bovine organs. The inhibitor has been purified, a preparation with a specific activity 130-fold higher than that of the original 100,000 g supernatant of brain being obtained. It runs as a single peak in polyacrylamide-gel electrophoresis; when run in the presence of SDS, two components appeared. The apparent Mr of the components were 14,800 and 22,400. The inhibitor has been characterized as a heat-stable protein of acidic nature. It has effect on the glycolipid and the glycoprotein sialyltransferase activities but has no effect on the galactosaminyltransferase activity.

An inhibitor of the UDP-N-acetylgalactosamine:GM3, N-acetylgalactosaminyltransferase: purification and properties, and preparation of an antibody to this inhibitor.

An inhibitor of the UDP-N-acetylgalactosamine:GM3, N-acetylgalactosaminyltransferase (EC 2.4.1.92) has been purified close to 100-fold from chicken blood serum. The method of purification includes heating, dialysis, passage through a column of DEAE-Sephadex, filtration through Amicon XM 100, and passage through Sepharose 6B. The molecular weight determined by Sepharose 6B was 200,000, but on sodium dodecyl sulfate-polyacrylamide gel electrophoresis it appears as if the compound dissociated into components of 68,000. The inhibitor was not active on other glycosyl transferases and lost its inhibitory activity following treatment with pronase and trypsin. alpha-Chymotrypsin did not affect the inhibitor. An antibody to this inhibitor was prepared which decreased its inhibitory capability and precipitated with it in a radial double immunodiffusion experiment.

Synthesis and translocation of gangliosides and glycoproteins during urethane anesthesia.

In this work, we have studied (a) the contents of gangliosides, glycoproteins, and phospholipids of the vesicle and plasma membrane fractions from brains of anesthetized and control rats and chickens and (b) the labeling of gangliosides and glycoproteins in the retina ganglion cell layer and optic tectum of urethane-anesthetized and control chickens after intraocular injection of a labeled N-acetylneuraminic acid precursor and the distribution of the label after subcellular fractionation. We found an increase in the content of gangliosides relative to protein in the vesicle fraction of both anesthetized rats and chickens relative to their controls. Other values were not affected by anesthesia. These results do not reflect a faster synthesis of gangliosides stimulated by urethane, because their rate of labeling was diminished in anesthetized animals. During the 4-h period after the animals were injected intraocularly with the radioactive precursor, the highest values of ganglioside-specific radioactivity were found in the vesicle fraction of control and anesthetized animals; at longer intervals, the specific radioactivity of the vesicle and plasma membrane fractions became rather similar. These data are in accordance with previous studies from this laboratory suggesting that the synthesis of the carbohydrate chain of gangliosides is regulated by the physiological demands made by the neurotransmitting system.

Optic nerve integrity is required for light to affect retina ganglion cell gangliosides.

The labeling of retina ganglion cell and optic tectum gangliosides after an intraocular injection of N-[3H]acetylmannosamine ([3H]ManNAc) is higher in chickens exposed to light than in those maintained in darkness. In the present work we studied whether the signal for the higher labeling of ganglion cells in light originates in the photoreceptor layer or comes from the nerve terminal. For this purpose the labeling of ganglion cell gangliosides was determined in light and dark in chickens with one optic nerve severed. The results showed that the effect of light occurred only in the eye normally connected to the optic tectum. In the eye with its optic nerve severed, no difference was observed between the labeling of gangliosides in animals in light and dark, having both groups the labeling values of the normal eyes exposed to light. The results indicate that the information that decreases labeling in darkness or accelerates it in light originates in the nerve terminal.

Regulation of ganglioside and sialoglycoprotein biosynthesis-Effect of drugs affecting membrane flow.

The labelling of gangliosides and sialoglycoproteins (SGP) after injecting intraocularly 30 ?Ci of [(3)H]ManNAc was studied in chickens that had previously received, also intraocularly, 30 or 70 nmol of monensin in 10 ?l of ethanol or 24 ?g of colchicine in 10 ?l saline. Controls received ethanol or saline only. Colchicine at doses that almost completely block the axonal transport to the optic tectum does not affect the labelling of those sialocompounds. The translocation of gangliosides and SGP, measured as a percentage of accessibility to the sialidase was inhibited by colchicine, but this effect was different for gangliosides and for SGP. In contrast, monensin, also at doses that block axonal transport, inhibits the synthesis and the translocation of gangliosides and SGP. It also modifies the labelling pattern of retinal gangliosides, which with respect to the normal pattern shows an increase in the relative labelling of GM(3), GD(3), GM(1) and probably GD(1b) and a decrease in GD(1a) and GT(1b). The activity of UDP-GalNAc: GM(3), GalNAc transferase (EC 2.4.1.79) of retina membranes of chickens that received an intraocular injection of monensin 4 h before being sacrificed showed a dose-dependent decrease with respect to the controls. In experiments in vitro monensin at a concentration of 3 mM failed to inhibit this activity.

Posttranslational incorporation of [14C]arginine into rat brain proteins. Acceptor changes during development.

Many of the cytosolic proteins of the rat brain appear to have the capacity to incorporate L-[14C]arginine posttranslationally. Scanning of the electrophoretic pattern of the labeled proteins showed two main radioactive peaks: peak A, found in the region of proteins of MW above 200 kD, and peak B, found in the region of 33 kD. The ratio of peaks A/B tends to decrease with the age of the rats. Another zone of radioactivity has an apparent MW similar to that of albumin (approximately 66 kD). No differences were found between the effects of ionic strength and of inhibitors on the arginyl transferase of brain and those described for the transferases of other organs.

Inhibition of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyl transferase by gangliosides.

Gangliosides in the range of 0.1-0.4 mM inhibited the UDP-N-acetylgalactosamine:GM3, N-acetylgalactosaminyl transferase (EC 2.4.1.79) of chicken retina. Other lipids such as phosphatidylethanolamine, sphingomyelin, sulfatides, and phosphatidic acid in concentrations similar to those of gangliosides did not affect the enzyme activity significantly. GM3 has an inhibition capability slightly less than that of gangliosides with two or three sialyl groups in their molecules, while asialo-GM1 is clearly less inhibitory. The inhibitory effect of a constant amount of GT1 ganglioside was higher at low concentrations of membrane preparation, but the inhibition was similar at different concentrations of the substrates GM3 or UDP-N-acetylgalactosamine and at all incubation times studied. The added gangliosides were found attached to the membranes. In this attached state they may act either as substrate or inhibitor. The inhibitory effect of gangliosides was not apparent when a mixture of Triton CF 54-Tween 80 was added to the incubation medium at concentrations greater than 0.33%.

Inhibition of the chicken retinal UDP-GaINAc:GM3, N-acetylgalactosaminyl-transferase by blood serum and by pineal gland extracts.

The effects of the pineal gland extract and blood serum on the activity of the UDP-GalNAc:GM3, N-acetylgalactosaminyltransferase (GM3:GalNAc-T) from chicken retina were studied. Both preparations have inhibitory capability on the enzyme activity. Two types of inhibitory capabilities were found: one is heat labile and decomposes UDP-GalNAc and another is heat stable. When the pineal gland extracts were prepared from light-exposed chickens, the inhibitory capability increased with respect to the extracts from dark-maintained animals; vice versa, blood serum from dark-maintained animals had higher heat-labile and heat-stable capabilities than that from light-exposed chickens. In in vitro experiments, no difference was found in the inhibitory capability of blood serum extracts from pinealectomized animals compared to control animals. In vivo labeling experiments with pinealectomized animals in either light or dark showed similar differences in the labeling of the optic tectum gangliosides as the normal animals.

The labeling of the retina and optic tectum gangliosides and glycoproteins of chickens in darkness or exposed to light.

Chickens that received an intraocular injection of 3H-ManNAc and were exposed to light had more labeled gangliosides in the retina ganglion cell layer and in the contralateral optic tectum than similarly treated animals that remained in darkness. The effect is not due to the turning on or off of the light. The sialyl groups of sialoglycoproteins showed similar effect but the labeling of proteins in chickens that received 3H-proline did not show significant differences. So far the effect has been obtained only with retina linked to the optic tectum through the optic nerve. If the nerve is severed the effect disappears. The gangliosides GD1a and GT1 are powerful inhibitors of the GM3-N-acetylgalactosaminyl transferase. The main effect of those gangliosides is expressed when they are linked to the membranes containing the enzyme in such a form that they are not released by washing with water. The hypothesis is advanced that the utilization of gangliosides in the nerve ending during the interneuronal transmission produces a small decrease in their concentration that in turn is transmitted backwards to the neuronal perikarya where it accelerates the synthesis of new gangliosides.

Inhibition of brain tubulinyl-tyrosine carboxypeptidase by endogenous proteins.

When a 25-50% ammonium-sulphate-insoluble fraction from a bovine brain preparation was chromatographed on a cellulose phosphate column, several protein fractions which inhibit the activity of tubulinyl-tyrosine carboxypeptidase were obtained. One of these fractions exhibited activity of fructose-bisphosphate aldolase (EC 4.1.2.13) and the enzyme accounted for more than 95% of the protein of this fraction as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The inhibitory activities of the two protein fractions which had the highest activity per mg of protein were practically abolished by pretreatment with pronase; preincubation with trypsin, on the other hand, caused only a partial inactivation of the inhibitors. The inhibitory activities were little affected by heating at 90 degrees C for 5 min. Preincubation with purified tubulinyl-tyrosine carboxypeptidase caused a great decrease of the inhibitory activities of these two fractions, leaving open the possibility that these inhibitors act as substrates of the carboxypeptidase.

Short term labeling of proteins, gangliosides and glycoproteins of the optic tract of chickens exposed to light or darkness.

In contrast with previous findings of the labeling of the glycosidic moieties of the gangliosides and glycoproteins in chickens injected with N-[ (3)H ] acetylmannosamine , the labeling of the ganglion cell layer and optic tectum proteins of chicks exposed to light after an intraocular injection of [(3)H]proline showed no differences with those of their counterpart chickens that remained in darkness. The same failure in finding a difference was met when the cytosolic or the particulate proteins or the acid soluble fraction in the retina were compared. Cycloheximide and puromycin inhibited the labeling of retina and optic tectum proteins, gangliosides and glycoproteins in both illumination conditions. Since the labelings in the optic tectum appeared more inhibited than those in the retina ganglion cell layer it was concluded that cycloheximide and puromycin, besides the synthesis of those compounds, also inhibit their axonal transport. On the basis of these contrasting results the working hypothesis is advanced that light stimulation enhances the activity of the Golgi apparatus but not (or less) that of the polyribosomes.

Ganglioside glycosyltransferase activities in the cerebral hemispheres from developing rat embryos.

The developmental patterns of three ganglioside glycosyltransferases were determined in the embryonic rat cerebral hemispheres from day 14 of gestation until birth. Considering the values of day 14 of gestation as 100%, the activity per µg of DNA at birth of the CMP-NeuAc:GM3 sialosyltransferase decreased to 40%, that of UDP-GalNAc:GM3 N-acetylgalactosaminyltransferase increased to 230% and that of UDP-Gal:GM2 galactosyltransferase showed minor variations. The changes in the activities of these enzymes correlated with the changes occurring in this embryonic period in the complexity of the oligosaccharide chain of gangliosides which result in a relative increase of gangliosides having the gangliotetraosyl backbone.

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study.

On t.l.c. plates 125I-cholera toxin binds to a disialoganglioside tentatively identified as GD1b with about 10 times less capacity then to ganglioside GM1. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM1 and GD1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.

The effect of light exposure following an intraocular injection of [3H]N-acetylmannosamine on the labeling of gangliosides and glycoproteins of retina ganglion cells and optic tectum of singly caged chickens.

Ten-day-old chickens that after a 2-day-period of adaptation to dark received an intraocular injection of [3H]N-acetylamannosamine ([3H]ManNAc) and were exposed, individually housed, to light, have more labeling in the gangliosides and glycoproteins of the ganglion cell layer of retina and in the contralateral optic tectum compared to their counterparts that remained in darkness. No differences were found in the labeling of the acid soluble fraction of the ganglion cell layer between the animals in dark and light at 0.5 and 5 h after the injection of [3H]ManNAc. No differences could be observed in the quality or storage of the gangliosides labeled in light with respect to those labeled in dark, but those labeled in light had a higher percent of labeling released by neuraminidase at 5 h after the intraocular injection of the labelled precursor. In animals exposed to intermittent light, the increased labeling with respect to dark was smaller than that found in animals exposed continuously to light.