Biosynthesis of mammalian glycoproteins. Glycosylation pathways in the synthesis of the nonreducing terminal sequences.

Six purified glycosyltransferase (a beta-galactoside alpha 2 leads to 6 sialyltransferase, a beta-galactoside alpha 2 leads to 3 sialyltransferase, an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase, a beta-galactoside alpha 1 leads to 2 fucosyltransferase, a beta-N-acetylglucosaminide alpha 1 leads to 3 fucosyltransferase, and a (fucosyl alpha 1 leads to 2) galactoside alpha 1 leads to 3 N-acetyl-galactosaminyltransferase) have been used to study the biosynthetic pathways for formation of the nonreducing terminal oligosaccharide sequences in mammalian glycoproteins. The two glycoproteins used as model acceptor substrates in this study were human asialotransferrin, which contains the nonreducing terminal oligosaccharide sequence Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man, and antifreeze glycoprotein, which contains oligosaccarides with the structure, Gal beta 1 leads to 3GalNAc alph 1 leads O-Thr. Sequential action of the six glycosyltransferases on these model substrates led to the formation of previously described oligosaccharide structures. The studies reported here indicate that the substrate specificities of the individual enzymes dictate the structures that can be synthesized and the pathways by which they may be formed. The actions of a number of the transferasesare mutually exclusive, thereby prohibiting the formation of theoretically possible oligosaccharide structures. Oligosaccharides with the terminal sequence NeuAc alpha 2 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3GalNAc and NeuAc alpha 2 leads to 6Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc cannot be formed because the prior incorporation of sialic acid by the sialyltransferases yields products that are not acceptor substrates for the fucosyltransferases, and vice versa. Synthesis of other products requires that the enzymes act sequentially in a specific order. The structures NeuAc alpha 2 leads to 6(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4GlcNAc, Fuc alpha 1 leads to 2Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc, GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4GlcNAc, and GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3GalNAc can only be synthesized if the fucosyl alpha 1 leads to 2 galactose linkage is formed first. Synthesis of the pentasaccharide sequences GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GalNAc and GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc requires that the N-acetylgalactosaminyltransferase act last on the former structure and that the alpha 1 leads to 3 fucosyltransferase act last on the latter. In those instances where a product can be formed by one of two possible pathways, the comparisons of reaction rates indicate that one pathway is usually preferred...     

C]Ethylene Metabolism during Leaf Abscission in Cotton

Changes in ¹⁴C₂H₄ metabolism in the abscission zone were monitored during cotton (cv. Deltapine 16) leaf abscission. Rates of ¹⁴C₂H₄ oxidation to ¹⁴CO₂ and tissue incorporation in abscission zone segments cut from the second true leaf of nonabscising leaves of intact plants were similar (about 200 disintegrations per minute per 0.1 gram dry weight per 5.5 hours) and relatively constant over a 5-day period. Deblading to induce abscission caused a dramatic rise in ¹⁴C₂H₄ oxidation, but tissue incorporation was not markedly affected. This rise occurred well before abscission, reaching a peak of 1,375 disintegrations per minute per 0.1 gram dry weight per 5.5 hours 2 days after deblading when abscission was 40%. The rate then gradually declined, but on day 5 when abscission reached completion, it was still nearly three times higher than in segments from nonabscising leaves. Application of 0.1 millimolar abscisic acid in lanolin to the debladed petiole ends increased the per cent abscission slightly and initially stimulated ¹⁴C₂H₄ oxidation. In contrast, naphthaleneacetic acid applied in a similar manner delayed and markedly inhibited both abscission and ¹⁴C₂H₄ oxidation.     

Endogenous lectins in chickens and slime molds: Transfer from intracellular to extracellular sites

Endogenous lectins in both cellular slime molds and chicken tissues have been localized primarily intracellularly, in contrast with the predominantly extracellular localization of the glycoproteins, glycolipids, and glycosaminoglycans with which they might interact. Here we present evidence that lectins in both of these organisms may be externalized and become associated with the cell surface and/or extracellular materials. In chicken intestine, chicken-lactose-lectin-II is shown to be localized in the secretory granules of the goblet cells, along with mucin, and to be secreted onto the intestinal surface. In embryonic muscle, chicken-lactose-lectin-I is shown to be externalized with differentiation, ultimately becoming localized on the surface of myotubes and in the extracellular spaces. In a cellular slime mold, Dictyostelium purpureum, externalization of lectin is elicited by either polyvalent glycoproteins that bind the small amount of endogenous cell surface lectin, or by slime mold or plant lectins that bind unoccupied complementary cell surface oligosaccharides. These results suggest that externalization of endogenous lectin may be a response to specific external signals. We conclude that lectins are frequently held in intracellular reserves awaiting release for specific external functions.     

Cytochemical study of different stages in the life cycle of Toxoplasma gondii. VII. Oxidation--reduction enzymes in the cyst forms]

Dehydrogenases of glycolysis, Kreb's cycle, and pentose-phosphate shunt were detected in cystozoites of Toxoplasma gondii strain SS-119 with various degrees of activity. A mixed oxidative metabolism may be postulated on this stage of the toxoplasma life cycle. Besides, the activity of cytochrome oxidase was detected in cystozoites; the addition of cytochrome c to the incubation medium significantly intensified the reaction intensity. Of interest seems the observation of a layer of higher enzymatic activity in the host brain tissue in the immediate neighbourhood with the cyst body. This may be regarded as the host cells' (or tissue') response to the presence of the parasite's alien body.     

Rapid metabolism of propylene by pea seedlings

Propylene uptake by intact pea seedlings (Pisum sativum L. cv. Alaska) was easily detected using standard gas chromatographic techniques suggesting rapid metabolism. Comparative studies with highly purified ¹⁴C₃H₆ and ¹⁴C₂H₄ under aseptic conditions verified that propylene was rapidly metabolized and indicated that some aspects of its metabolism were similar to that of ethylene since ¹⁴C₃H₆, like ¹⁴C₂H₄ (Beyer, Nature 1975, 255: 144-147), was oxidized to ¹⁴CO₂ and incorporated into water-soluble tissue metabolites. However, ¹⁴C₂H₆ was metabolized at a substantially faster rate and unlike ¹⁴C₂H₄ the rate of ¹⁴C₃H₆ tissue incorporation exceeded its rate of oxidation to ¹⁴CO₂. In addition the neutral ¹⁴C-metabolites derived from ¹⁴C₃H₆ were chromatographically distinct from those formed from ¹⁴C₂H₄.     

C(2)H(4) metabolism in morning glory flowers

Flowers of Ipomoea tricolor Cav. (cv. Heavenly Blue) were cut at various stages of development and evaluated for their ability to metabolize ethylene. Freshly cut buds or flowers were treated in glass containers for 8 hours with 6 μl/liter of highly purified ¹⁴C₂H₄. Following removal of dissolved ¹⁴C₂H₄, radioactivity was determined for the different flower tissues and trappd CO₂. ¹⁴C₂H₄ oxidation to ¹⁴CO₂ and tissue incorporation occurred at very low to nondetectable levels 2 to 3 days prior to flower opening. About 1 day prior to full bloom, just at the time when mature buds become responsive to ethylene (Kende and Hanson, Plant Physiol 1976, 57: 523-527), there was a dramatic increase in the capacity of the buds to oxidize ¹⁴C₂H₄ to ¹⁴CO₂. This activity continued to increase until the flower was fully opened reaching a peak activity of 2,500 dpm per three flowers per 8 hours. It then declined as the flower closed and rapidly senesced. A similar but smaller peak occurred in tissue incorporation and it was followed by a second peak during late flower senescence. This first peak in tissue incorporation and the dramatic peak in ethylene oxidation slightly preceded a large peak of natural ethylene production which accompanied flower senescence. The ethylene metabolism observed was clearly dependent on cellular metabolism and did not involve microorganisms since heat killing destroyed this activity and badly contaminated heat-killed flowers were unable to metabolize ethylene.     

Species-specific aggregation factor in sponges. XIII. Entire and core structure of the large circular proteid particle from Geodia cydonium

High-molecular weight particles have been isolated from the sponge Geodica cydonium. In the "native" from these particles consist of a spherical center and have 25-30 filaments attached to it. The core structure of the particles is assembled of a central circle and 25 radially-arranged filaments. The core structure is obtained from the entire structure by incubation in a medium, containing a non-ionic detergent and EDTA. The molecular weight of the enitre structure was in the range of 1.4 X 10(9) daltons or more and of the core structure 6.1 x 10(8) daltons. Two functional proteins are released from the "native" particles: the aggregation factor and the sialytransferase.     

Der Einfluss von CCC auf die Entwicklung des Roggenhalmes

Field trials on the effect of chlorocholinechloride (CCC) on rye plants of the cultivar Danae and of a selected population “WRS” proved that rye principally shows as reaction analogous to wheat. The CCC-induced decrease of stalk length is due to the reduction of elongation growth of the 4th internode. This shortening effect is mainly the result of decreased cell extension and, in the middle internode, additionally of inhibited cell division in longitudinal direction. The shape of internodes is changed under the influence of CCC. Walls of parenchyma cells of CCC-treated plants are thinner and those of sclerenchyma cells are thicker compared with cell walls of control plants.      

Molecular species of cardiolipin in relation to other mitochondrial phospholipids. Is there an acyl specificity of the interaction between cardiolipin and the ADP/ATP carrier?

Molecular species in the three major mitochondrial lipids cardiolipin, phosphatidylcholine and phosphatidylethanolamine were analysed in bovine heart and Saccharomyces cerevisiae. In both organisms cardiolipin contains mainly diacylglycerol moieties with two unsaturated chains and a significant higher proportion of C18-C18 species than phosphatidylcholine and phosphatidylethanolamine. To study whether the specific acyl composition of cardiolipin has a functional significance in lipid-protein interaction, experiments were made with the isolated ADP/ATP carrier of bovine heart mitochondria since this dimeric protein is known to be tightly associated with six molecules of cardiolipin [Beyer, K. and Klingenberg, M. (1985) Biochemistry 24, 3821-3826]. This association seems to be very strong as protein-bound cardiolipin does not exchange with soluble cardiolipin on a time scale of hours. Analysis of the species composition suggests that one carriers dimer is associated with four molecules of tetralinoleoyl cardiolipin and two molecules of trilinoleoyl-monolinolenoyl cardiolipin. Catalytic hydrogenation of the acyl chains of carrier-bound cardiolipin does not result in release of cardiolipin as judged by 31P-NMR spectroscopy. The ADP/ATP carrier was reconstituted with saturated phosphatidylcholines and spin-labelled cardiolipin whose double bonds were subsequently saturated by catalytic hydrogenation. ESR spectroscopy shows that saturation of spin-labelled cardiolipin has no significant impact on its association with the ADP/ATP carrier. However, precipitation of the detergent-solubilized ADP/ATP carrier can only be induced by addition of unsaturated but not by saturated cardiolipin. It is concluded that the specific acyl composition of cardiolipin is not a prerequisite of its high affinity for the ADP/ATP carrier, at least when the protein is reconstituted in a saturated phosphatidylcholine environment.