Effects of overexpression of {beta}1, 4-galactosyltransferase on glycoprotein biosynthesis in F9 embryonal carcinoma cells

ß1,4-Galactosyltransferase (GalTase) plays a centralrole in the biosynthesis of N-acetyllactosamine-containing oligo-saccharides.However, despite this seemingly important function, little isknown about how changes in the levels of GalTase affect oligosaccharidebiosynthesis. We have examined the effects of overexpressingGalTase on the glycosylation of endogenous glycoproteins inF9 mouse embryonal carcinoma cells. Cells transfected with eitherthe short form of the GalTase cDNA (encoding a protein of 386amino acids) or the long form of the GalTase cDNA (encodinga protein of 399 amino acids) had a 3-fold increase in totalGalTase activity, relative to control F9 cells. Analysis ofpronase-digested glycopeptides obtained from control and transfectedcells after metabolic labelling with [6-³H]galactose revealedno significant qualitative or quantitative differences, as assessedby Bio-Gel P-6 gel filtration chromatography and Tomato lectinaffinity chroma-tography. Furthermore, SDS-PAGE analysis ofimmuno-precipitated [³H]galactose-labelled lysosomal-associatedmembrane protein-1 (LAMP-1) glycoprotein showed no differencein amounts or mobility. Pronase digestion and subsequent analysisof the gel-fractionated LAMP-1 glycoproteins also indicatedno differences between the various cell lines. The inabilityof elevated GalTase activity to affect glycosylation was notdue to limiting levels of GalTase substrates, since an excessof substrates was detectable in lysed cells using either endogenousor exogenous GalTase and UDP-[³H]galactose. Finally, the subcellulardistribution of GalTase, as assessed by sucrose gradient fractionation,was similar between all cell types, thus suggesting that GalTasewas appropriately compartmentalized in the transfected cells.More importantly, GalTase specific activities in the Golgi membranesof the transfected cells were 3–4 times greater than incontrol cells. These results show that selectively increasingGalTase activity does not alter glycoprotein biosynthesis inF9 cells. F9 cells galactosyltransferase glycoprotein biosynthesis     

The receptor function of galactosyltransferase during cellular interactions

Summary The molecular mechanisms that underly cellular interactions during development are still poorly understood. There is reason to believe that complex glycoconjugates participate in cellular interactions by binding to specific cell surface receptors. One class of carbohydrate binding proteins that could serve as receptors during cellular interactions are the glycosyltransferases. Glycosyltransferases have been detected on a variety of cell surfaces, and evidence suggests that they may participate during cellular interactions by binding their specific carbohydrate substrates on adjacent cells or in extracellular matrix (see Refs. 1–4 for review).This review will focus on the receptor function of galactosyltransferase, in particular, during fertilization, embryonic cell adhesion and migration, limb bud morphogenesis, immune recognition and growth control. In many of these systems, the galactosyltransferase substrate has been characterized as a novel, large molecular weight glycoconjugate composed of repeating N-acetyllactosamine residues. The function of surface galactosyl-transferase during cellular interactions has been examined with genetic and biochemical probes, including the T/t-complex morphogenetic mutants, enzyme inhibitors, enzyme modifiers, and competitive substrates. Collectively, these studies suggest that in the mouse, surface galactosyltransferase is under the genetic control of the T/t-complex, and participates in multiple cellular interactions during development by binding to its specific lactosaminoglycan substrate.     

The role of phosphorylase kinase subunits in interaction with glycogen

The interaction of rabbit skeletal muscle phosphorylase kinase with CNBr-activated glycogen results in the formation of a covalent complex. The non-bound kinase was removed by chromatography on DEAE-cellulose and phenyl-Sepharose. The amount of the bound protein increased with an increase in the number of activated groups in the glycogen molecule; the enzyme activity was thereby decreased. The kinase covalently and non-covalently bound to glycogen exhibited a higher affinity for the protein substrate (phosphorylase b) as well as for Mg2+ and Ca2+ than did the kinase in the absence of glycogen. Electrophoresis performed under denaturating conditions showed that the gamma-subunit of phosphorylase kinase is responsible for the enzyme binding to CNBr-glycogen. The effect of cross-linking reagents (glutaric aldehyde, 1.5-difluoro-2.4-dinitrobenzene) on the binding of phosphorylase kinase subunits was studied. Glycogen afforded protection of the gamma-subunit from the cross-linking to other enzyme subunits. An analysis of the subunit composition of phosphorylase kinase covalently bound to CNBr-glycogen and of the enzyme treated with cross-linking reagents in the presence of glycogen-revealed that the gamma-subunit is involved in the specific binding of phosphorylase kinase to glycogen.     

Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle]

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.     

Acclimation or stress symptom? An integrated study of intraspecific variation in the clonal plant Aechmea bromeliifolia,a widespread CAM tank‐bromeliad

The clonal tank-bromeliad Aechmea bromeliifolia (Rudge) Baker was found in four different habitats in a restinga (vegetation mosaic on sandy coastal plains), of south-eastern Brazil. These habitats (swamp forest, dry forest, dry shrubland and herbaceous marsh) lie within a few hundred metres of each other along a gradient extending inland from the coast, and differ markedly in terms of light and flood regime. We compared ramet morphology, leaf anatomy and physiology, and population parameters to examine the amplitude of trait variation of this widespread species in the studied restinga. This integrated approach allowed us to examine which variation conferred acclimation and which was merely a stress symptom. A . bromeliifolia showed site-specific differences in abundance, distribution, rosette size and shape, leaf anatomical arrangement and photochemical efficiency (potential quantum yield; F _v/ F _m) during the day. Most of the variation found seemed to be related to the interaction of light and flooding. The lowest number and size of ramets at the exposed, dry shrubland was matched by a marked leaf photoinhibition, which suggested poor acclimation to local levels of light intensity and limited water supply. In the other habitats, the morpho-physiological parameters measured suggested adequate foraging behaviour and site acclimation.  © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society, 2002, 140 , 391-401.