Neuraminidase in Calf Retinal Outer Segment Membranes

Abstract: An enzyme catalyzing the hydrolysis of sialic acid ( N -acetylneuraminic acid: NeuNAc)-containing glycoconjugates has been found in bovine retinal rod outer segment (ROS) membranes. The enzymatic activity is optimal at pH 4.0 and is stimulated by 0.15% Triton X-100. Total activity was determined by the release of NeuNAc from endogenous and exogenous substrates (GDla). The ROS enzyme preferentially hydrolyses the ROS gangliosides, possibly because they are more accessible than the glycoproteins as substrates for the neuraminidase. Release of NeuNAc from gangliosides leads to important changes in the ganglioside patterns; whereas the amounts of GM1 increased throughout the incubation, the levels of polysialogangliosides GTlb and GD3 diminished owing to their rapid hydrolysis. The finding that gangliosides are hydrolysed more extensively than glycoproteins suggests that endogenous ROS gangliosides may be the principal source of metabolically available sialic acid in ROS. It was also observed that the activity of ROS neuraminidase is not affected by illumination of the membranes.     

Trypanosoma cruzi glycoprotein of Mr 56000: characterization and assessment of its potential to protect against fatal parasite infections

A ∼ 56 000 Da membrane glycoprotein purified from epimastigotes of Trypanosoma cruzi was characterized biochemically and tested for its efficacy to induce protection in mice from a lethal challenge with this protozoan parasite. Immunofluorescence assays with live and formalin-fixed epimastigotes and trypomastigotes localized the glycoprotein to the flagellum, the body of the parasite, and the cell membrane. Immunoblotting demonstrated the glyco-protein's presence in nearly equal amounts in all developmental stages of several T. cruzi isolates. Mice immunized with the purified glycoprotein and challenged with 10000 infectious trypomastigote forms of isolate Y survived the controls by up to four days. This significant protection makes this antigen a potential candidate for a multi-subunit vaccine against 7. cruzi.     

Sulpholipid metabolism in developing chicken retina.

Glycolipid analysis of chicken retina and brain indicated the presence of cerebroside, cerebroside 3-sulphate and sulphogalactosylglycerolipid In retina, the ratio of cerebroside to cerebroside 3-sulphate was approximately half compared to brain. During chicken retina ontogenesis the ratio of cerebroside 3-sulphate to sulphogalactosylglycerolipid increased rapidly and in the adult animal, the amount of cerebroside 3-sulphate was 14 times higher than that of sulphogalactosylglycerolipid. The activity of PAPS: cerebroside sulphotransferase and arylsulphatase A in developing chicken retina indicated that the general ontogenic profiles of retinal PAPS: cerebroside sulphotransferase and arylsulphatase A were similar to those obtained for the brain. Both the enzymes showed the highest activity just before hatching. The significance of occurrence of sulpholipids in retina is discussed.     

ENZYMATIC BIOSYNTHESIS OF ETHANOLAMINE- AND CHOLINE-PHOSPHOGLYCERIDES IN RETINA DURING DEVELOPMENT. EFFECT OF LIGHT STIMULATION

Choline- and ethanolamine-phosphoglycerides (CPG and EPG) are the most abundant phospholipids of retinal membranes. We have investigated some regulatory mechanisms involved in the final steps of their biosynthesis, namely those catalysed by CDP-choline 1,2 diradyl-sn-glycerol choline phosphotransferase (CPT) and CDP-ethanolamine 1,2 diradyl-sn-glycerol ethanolamine phosphotransferase (EPT). We have studied both enzymes in the retina which offers an excellent model for the investigation of the molecular basis of the effect of its physiological stimulus, the light. In chick retina. the specific activity (SA) of EPT reached a maximum at the 18th day of embryonic life and decreased thereafter. In the case of CPT, a similar peak of SA was observed at hatching. The time of maximum SA of EPT and CPT corresponded to the period during which retinal rod outer segments are formed. The apparent K_m values of EPT and CPT determined with whole retinal homogenates for CDP-bases showed different profiles. The apparent K_m of EPT decreased during embryonic life and increased thereafter whereas the apparent K_m of CPT did not change during ontogenesis. Light stimulation of calf retinal homogenates had different effects on phosphotransferase activities. In the presence of only endogenous diacylglycerol (DAG) the SA of CPT was 2-fold higher for dark-adapted retinas, whereas no differences in EPT activities were observed. After addition of exogenous DAG (4mM) to the incubation medium, light stimulation of the retina led to a 50% increase of EPT activity whereas no effect was observed for CPT. These different effects could be related to the cyclic nucleotides present in retina before and after light stimulation. In addition all the data presented in this study indicate that, as in brain, CPT and EPT in retina are two different enzymes.     

Biosynthesis of glycosylated human lysozyme mutants.

Complementary DNA encoding human lysozyme was subjected to oligonucleotide-directed mutagenesis. At one of three selected positions, amino acid residues 22, 68, or 118, the signal for N-linked glycosylation was created. The mutant DNAs were inserted into a eucaryotic vector and transfected into cultured hamster cells. The three mutant cDNAs directed synthesis of lysozyme mutants, which were named LI, LII, and LIII. The mutant lysozymes LI and LII comprised mixtures of glycosylated and nonglycosylated forms. The glycosylated and nonglycosylated forms of mutant LI were found to have an enzymatic activity similar to normal human milk lysozyme. The usage of the glycosylation sites in the mutants was similar in Chinese hamster ovary (CHO) and baby hamster kidney cells. Approximately two of every three molecules in mutant LI, approximately one of every eight molecules in mutant LII, and practically no molecules in mutant LIII became glycosylated. In CHO cells, the processing of the oligosaccharide side chains yielded several larger products than in baby hamster kidney cells. This size variability of glycosylated lysozyme from CHO cells may be explained by the presence of biantennary and triantennary endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides with N-acetyllactosamine repeats of variable length and by the presence of hybrid oligosaccharides, as suggested by affinity to several lectins and sensitivity to endo-beta-galactosidase. In both cell types, the majority of the glycosylated forms were secreted and thus behaved similarly to nonglycosylated lysozyme. A small proportion of mutant LI lysozyme remained associated with the cells. The retained lysozyme was recruited predominantly from the molecules bearing high mannose oligosaccharides. These molecules were targeted to lysosomes, and their carbohydrate was trimmed to an endo-beta-N-acetylglucosaminidase H-resistant form. Owing to the small size of mutant LI lysozyme, minor changes in the size of its carbohydrate moiety result in detectable changes in the electrophoretic mobility of the whole glycoprotein. We suggest that this novel glycoprotein could be used as a reporter in studies on processing and segregation of glycoproteins.     

Gangliosides in various brain areas of three inbred strains of mice

The ganglioside patterns of cerebellum, cortex, pons-medulla, hypothalamus, hippocampus and caudate nucleus of three inbred strains of mice (C57BL/6J, DBA/2J and BALB/cJ) have been analysed. All brain areas contained both the simple and complex species of gangliosides. GD1a was the major ganglioside in cortex, hippocampus and caudate nucleus whereas GT1b was the major species in cerebellum, hypothalamus and pons-medulla. In hippocampus, the percentages of GT1b and GD1a were quite similar. Pons and medulla exhibited the highest levels of GM1 (which approaches the value of GT1b) and the lowest values of GD1a. A ganglioside, termed here GT1L, was located between GD1b and GT1b. This ganglioside, which was present in highest amounts in cerebellum disappeared after alkali treatment. Highly significant differences were observed in the amounts and patterns of gangliosides among brain areas of the three strains. Highly significant differences (p<0.001) were also found in the ganglioside distribution of various brain areas among the strains, especially for tri-and tetrasialogangliosides between Balb and DBA. A significant difference of GM1 was observed in the cerebellum when comparing DBA with the two other strains. It is likely that the differences might be related to their relative abundances in certain cell types and for defining synaptic circuits in brain areas of some strains.     

Magnesium binding in the digesta of the chicken.

Two experiments involving laying hens and mature roosters were carried out to determine the effect of pH on the ultrafilterable fraction of Mg((Mg)u) present in the digesta along the gastrointestinal tract. Concurrently, the approximate molecular weight of the (Mg)u was determined. The average pH's for laying hens were: crop 4.88, proventriculus 5.27, ventriculus 4.77, duodenum 5.57, jejunum 6.15, ileum 7.82, and colon 6.65. Similar pH values were obtained for mature roosters. The (Mg)u fraction decreased significantly in the ileum where the pH was greater than 7.00. However, treating the ileal digesta with an acid pH buffer released about 70% of the nonfilterable Mg. The molecular weight of the (Mg)u was found to be less than 700, suggesting that the Mg was present in digesta as an inorganic salt or as an organic complex of low molecular weight.