PubMed Health⌕ Search

Biomedical subjects

L Faye

Publications and source records attributed to L Faye.

51 records · Page 3Linked to original sources
PubMed1988

Characterization of beta-fructosidase, an extracellular glycoprotein of carrot cells.

Seedlings and suspension-cultured cells of carrot (Daucus carota) contain a cell wall associated as well as a soluble form of beta-fructosidase (beta F). These two forms have different pH optima: 4.6 for cell wall beta F and 5.6 for soluble beta F. Soluble beta F is relatively more abundant in the seedlings and cell wall beta F is relatively much more abundant in the cultured cells. Protoplasts of cultured cells have only the soluble form (pH optimum 5.6) indicating that the cell wall associated form is indeed extracellular in situ. Cell wall beta F was purified to homogeneity and has an Mr = 63,000. Antibodies raised against the deglycosylated enzyme cross-reacted with two soluble enzyme forms: in cultured cells, the soluble enzyme has an Mr = 58,000 and, in seedlings, there are two forms of Mr = 58,000 and 52,000. Treatment of purified cell wall beta F with endoglycosidase H and trifluoromethanesulfonic acid (complete deglycosylation) indicated that the enzyme probably has one high mannose and two complex glycans. This was confirmed by HPLC analysis of [3H]GlcNAc- and [3H]fucose-labeled glycopeptides obtained after trypsin digestion of radioactively-labeled beta F. The amino acid composition shows that cell wall beta F has 18.6% glycine.

Cell Wall↗
PubMed1986

Characterization of the endoplasmic reticulum-associated precursor of concanavalin A. Partial amino acid sequence and lectin activity.

Concanavalin A (ConA), which is not a glycoprotein, is synthesized as a glycoprotein precursor (pro-ConA) which is post-translationally processed. This processing results in the loss of a small glycopeptide with a high mannose oligosaccharide. Carrington et al. (Carrington, D.M., Auffret, A., and Hanke, D.E. (1985) Nature 313, 64-66) determined the nucleotide sequence of a cDNA for pro-ConA, and in the derived amino acid sequence the only glycosylation site is in the middle of the molecule. Furthermore, the derived amino acid sequence of the putative precursor of ConA was found not to be colinear with that of ConA. Here we show that pro-ConA is located primarily in an endoplasmic reticulum-rich organelle fraction. Pro-ConA was purified from this fraction and subjected to amino acid sequencing. The first 12 amino acids at the N-terminal end of pro-ConA correspond to amino acids 119-130 of mature ConA, and to amino acids 30-41 of the putative pre-pro-ConA, the sequence of which was derived from the nucleotide sequence of a cDNA. Amino acid sequencing of a tryptic glycopeptide with the high mannose side chain showed that the first 17 amino acids of this peptide correspond to amino acids 154-170 of pre-pro-ConA. The last six amino acids in this series correspond to the first six amino acids of mature ConA. These data fully support the hypothesis of Carrington et al. that the biosynthesis of ConA involves a post-translational peptide cleavage, transposition, and ligation within the original polypeptide. Pro-ConA from the organelle fraction does not bind to Sephadex G-50, indicating that it has no lectin activity. The processing of pro-ConA apparently imparts biological activity to this lectin.

Amino Acid Sequence↗
PubMed1986

The position of the oligosaccharide side-chains of phytohemagglutinin and their accessibility to glycosidases determines their subsequent processing in the Golgi.

Phytohemagglutinin (PHA), the glycoprotein lectin of Phaseolus vulgaris has two types of asparagine-linked oligosaccharides per polypeptide: a high-mannose chain with the formula (Man)8-9(GlcNAc)2 on Asn12 and a modified chain with fewer mannose residues and additional fucose and xylose residues on Asn60. Glycosylation of PHA is a cotranslational process, which occurs in the endoplasmic reticulum, and newly synthesized PHA has two high-mannose chains. Transport of PHA to the protein bodies via the Golgi complex is accompanied by the modification of one of the two high-mannose chains. Why is only one chain modified, while the other remains in the high-mannose configuration? By determining the effect of digestion with various glycosidases (alpha-mannosidase, endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F) on native and denatured PHA we obtained evidence consistent with the interpretation that the accessibility of oligosaccharide chains to modifying enzymes is of major importance in determining whether a high-mannose chain becomes modified or not. The high-mannose chain of mature undenatured PHA is only partially accessible to glycosidases, while PHA obtained from the endoplasmic reticulum has one high-mannose chain, which is readily accessible to alpha-mannosidase and endoglycosidases H and F. We show that this readily accessible chain is in the same position on the polypeptide (Asn60) as the modified oligosaccharide on mature PHA. Thus, accessibility of the oligosaccharide side-chains to processing enzymes in the Golgi determines whether a particular oligosaccharide side-chain is processed or not.

Concanavalin A↗
PubMed1986

Cell Wall and Cytoplasmic Isozymes of Radish beta-Fructosidase Have Different N-Linked Oligosaccharides.

When 36-hour-old dark grown radish seedlings are transferred to far-red light, there is a decrease in cytoplasmic beta-fructosidase (betaF) and an increase in cell wall betaF compared to the dark controls. Cytoplasmic and cell wall-bound beta-fructosidase are both glycoproteins and exhibit high antigenic similarities, but differ according to charge heterogeneity and carbohydrate microheterogeneity. Growth of radish seedlings in the presence of tunicamycin results in a partial inhibition of betaF glycosylation but nonglycosylated betaF still accumulates in the cell wall under far-red light. Thus, glycosylation is not necessary for intracellular transport, for correct targetting, or for wall association of an active betaF. The nonglycosylated cytoplasmic and cell wall betaF forms have the same relative molecular mass but glycosylated forms have different oligosaccharide side-chains, with respect to size and susceptibility to alpha-mannosidase and endoglycosidase D digestion. The oligosaccharides of both forms are partly removed by endoglycosidase H when betaF is denatured. Isoelectric focusing analysis of betaF shows that the cell wall-associated isozymes are more basic than the cytoplasmic isozymes, and that the charge heterogeneity also exists within a single plant. A time course of changes in betaF zymograms shows a far red light stimulation of the appearance of the basic forms of the enzyme. However, the more basic cell wall specific betaF forms are not present when N-glycosylation is prevented with tunicamycin. These results indicate that cytoplasmic and cell wall betaF probably have common precursor polypeptides and basic cell wall forms arise via processing events which are tunicamycin sensitive.

Journal Article↗
PubMed1986

Oligosaccharide Side Chains of Glycoproteins that Remain in the High-Mannose Form Are Not Accessible to Glycosidases.

Glycoproteins present in the soluble and organelle fractions of developing bean (Phaseolus vulgaris) cotyledons were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, affinoblotting, fractionation on immobilized concanavalin A (ConA), and digestion of the oligosaccharide side chains with specific glycosidases before and after protein denaturation. These studies led to the following observations. (a) Bean cotyledons contain a large variety of glycoproteins that bind to ConA. Binding to ConA can be eliminated by prior digestion of denatured proteins with alpha-mannosidase or endoglycosidase H, indicating that binding to ConA is mediated by high-mannose oligosaccharide side chains. (b) Bean cotyledons contain a large variety of fucosylated glycoproteins which bind to ConA. Because fucose-containing oligosaccharide side chains do not bind to ConA, such proteins must have both high-mannose and modified oligosaccharides. (c) For all the glycoproteins examined except one, the high-mannose oligosaccharides on the undenatured proteins are accessible to ConA and partially accessible to jack bean alpha-mannosidase. (d) Treatment of the native proteins with alpha-mannosidase removes only 1 or 2 mannose residues from the high-mannose oligosaccharides. Similar treatments of sodium dodecyl sulfate-denatured or pronase-digested glycoproteins removes all alpha-mannose residues. The results support the following conclusions: certain side chains remain unmodified as high-mannose oligosaccharides even though the proteins to which they are attached pass through the Golgi apparatus, where other oligosaccharide chains are modified. The chains remain unmodified because they are not accessible to processing enzymes such as the Golgilocalized alpha-mannosidase.

Journal Article↗
PubMed1985

Characterization of N-linked oligosaccharides by affinoblotting with concanavalin A-peroxidase and treatment of the blots with glycosidases.

Glycoproteins which bind concanavalin A (Con A) can be located on nitrocellulose sheets after electrophoretic transfer from slab gels, by sequential incubation of the sheets with Con A and peroxidase, and visualization of the peroxidase by an insoluble reaction product. We refer to this method as affinoblotting. Differential elution of Con A from the blots by washing the sheets with different concentrations of alpha-methylglycosides is used to demonstrate the affinity of Con A for the oligosaccharide side chains, and to differentiate between proteins with weak and those with high affinity for Con A. Concanavalin A has a high affinity for the four plant glycoproteins (phaseolin, phytohemagglutinin, jackbean alpha-mannosidase, and the glycosylated precursor of Con A) studied here. Incubation of the blots with alpha-mannosidase and endoglycosidase H (endo H) is used to demonstrate that the oligosaccharide chains can be degraded by glycosidases while the proteins are immobilized on the nitrocellulose. With this approach we show here that the four plant glycoproteins used as models in this study interact with Con A through high-mannose oligosaccharide side chains sensitive to alpha-mannosidase and endo H degradation.

Chemical Phenomena↗
PubMed1980

The effect of protein concentration on the activity of beta-fructosidase from radish seedlings.

Radish beta-fructosidase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) is inactived by diluting the enzyme solution and the activity can be restored by addition of bovine serum albumin or other proteins. The use of detergent, high molar salt solutions or silicone-coated tubes showed that decrease of specific activity upon dilution is not linked to adsorption of the enzyme on to glass walls. Albumin neither protects the enzyme from denaturation by heat nor changes its stability during conservation at room temperature. The action of added proteins is not due to removal of an inhibitor from the enzyme solutions. Some polyanions or polycations have the same effect as albumin, but dialysis or chromatography show that they do not act by reassociation of inactive products formed by dilution of the active enzyme. A molecular weight heterogeneity is observed in the enzyme population when chromatography is performed without albumin. This suggests that inactive forms, formed upon dilution, differ slightly in their molecular conformation from the active forms obtained at high protein concentration.

Adsorption↗
PubMed1979

Evidence for the glycoprotein nature of radish beta-fructosidase.

Concanavalin A (Con A) was utilized free, bound to Sepharose 4 B or cross-linked to glutaraldehyde to investigate the possibility of binding this lectin to radish beta-fructosidase (E.C.3.2.1.26). The choice of cross-linked Con A as affinoadsorbent is discussed and standard conditions for binding are defined. Specificity of precipitation of this enzyme by the lectin was especially investigated. Thus, the possibility of binding was tested in the presence of high ionic strength, ethylene glycol, alpha-methyl mannoside, alpha-methyl glucoside and during periodate oxidation of the enzyme. Based on the interactions observed between beta-fructosidase and Con A under these conditions it is concluded that the saccharide binding site of the lectin is primarily involved with a secondary contribution from the hydrophobic site. The specificity of binding and the complete precipitation of beta-fructosidase activity by the insolubilized lectin imply that all beta-fructosidase activity measured in Raphanus sativus seedling extracts is linked to (a) glycoprotein form(s) of this enzyme.

Concanavalin A↗
PubMed1977

[Photoregulation of L-phenylalanine ammia-lyase: an immunochemical approach].

The photoregulation of L-Phenylalanine ammonia-lyase (PAL) is studied by immunochemical methods. We used a partly purified L-Phenylalanine ammonia-lyase: F1 light fraction, the corresponding inactive one provided from dark-grown cotyledons: F1 dark fraction and the antisera specific of these two fractions. The complete absorption of PAL activity from F1 light fraction with the anti-F1 light immune serum shows the antigenicity of PAL and the specificity of this serum for all forms of PAL present in F1 light fraction. The presence of an inactive L-Phenylalanine ammonia-lyase in the 36 h dark-grown cotyledons suggested by preliminary results of absorption is conformed by showing that less PAL activity is precipitated from the fraction F1 light by a same amount of IgG anti-F1 light when F1 dark fraction is added. This result is explained by a competition between active and inactive forms of PAL for the IgG extracted from an immune serum specific for F1 light fraction. By measuring the absorption of PAL activity from FO fraction (crude extract) obtained from 18 h, 36 h and 48 h light-grown cotyledons when increasing amounts of IgG anti-F1 36 h light are added, we demonstrate the presence of at least two isozymes A and B, the synthesis of B being shifted in time in comparison to A.

Ammonia-Lyases↗
PubMedDate not supplied

Electrophoretic analysis of plant cysteine and serine proteinases using gelatin-containing polyacrylamide gels and class-specific proteinase inhibitors.

Inclusion of gelatin in polyacrylamide gels provides a sensitive way of detecting multiple proteolytic activities in crude extracts from any source. The present study describes a method allowing discrimination between cysteine and serine proteinases in plant extracts, using gelatin-containing gels in combination with class-specific proteinase inhibitors. Preincubation of extracts with 4 mM phenylmethylsulfonyl fluoride, a serine proteinase inhibitor, or with 25 microM L-trans-epoxysuccinyl-L-leucylamido(4-guanidino) butane, a cysteine proteinase inhibitor, allowed the identification of enzymes from both classes in extracts of tomato fruit and papaya latex. The efficiency of the two low molecular weight inhibitors used was very high, and the irreversibility of the inhibiting effect was maintained during electrophoresis conducted in the presence of sodium dodecyl sulfate. The analytic procedure described here, with a detection threshold of less than 100 pg enzyme, is the first that allows quick and accurate discrimination of plant cysteine and serine proteinases separated in electrophoretic gels. This simple and rapid technique could be of interest for studying the evolution of class-specific proteinases in plant extracts during various developmental, physiological, and pathogenic processes. It is also potentially applicable to the majority of eucaryotic and procaryotic systems.

Cysteine Endopeptidases↗
PubMedDate not supplied

Functional conservation of calreticulin in Euglena gracilis.

Calreticulin is the major high capacity, low affinity Ca2+ binding protein localized within the endoplasmic reticulum. It functions as a reservoir for triggered release of Ca2+ by the endoplasmic reticulum and is thus integral to eukaryotic signal transduction pathways involving Ca2+ as a second messenger. The early branching photosynthetic protist Euglena gracilis is shown to possess calreticulin as its major high capacity Ca2+ binding protein. The protein was purified, microsequenced and cloned. Like its homologues from higher eukaryotes, calreticulin from Euglena possesses a short signal peptide for endoplasmic reticulum import and the C-terminal retention signal KDEL, indicating that these components of the eukaryotic protein routing apparatus were functional in their present form prior to divergence of the euglenozoan lineage. A gene phylogeny for calreticulin and calnexin sequences in the context of eukaryotic homologues indicates i) that these Ca2+ binding endoplasmic reticulum proteins descend from a gene duplication that occurred in the earliest stages of eukaryotic evolution and furthermore ii) that Euglenozoa express the calreticulin protein of the kinetoplastid (trypanosomes and their relatives) lineage, rather than that of the eukaryotic chlorophyte which gave rise to Euglena's plastids. Evidence for conservation of endoplasmic reticulum routing and Ca2+ binding function of calreticulin from Euglena traces the functional history of Ca2+ second messenger signal transduction pathways deep into eukaryotic evolution.

Amino Acid Sequence↗