Histochemische und biochemische Untersuchung der α-glucosidasen mit 2-naphthyl-α-D-glucosid

Summary Histochemical and biochemical studies yield the following method of choice for the in situ detection of neutral (microvillous) and acid (lysosomal) -glucosidases: 12 mg 2-naphthyl--D-glucoside (dissolved in 0.5 ml N,N-dimethylformamide) and 0.6–0.8 ml hexazonium-p-rosaniline in 10 ml 0.1 M citric acid phosphate buffer for aqueous or 5 ml buffer mixed with equal parts of 2% agar for incubation with semipermeable membranes, pH 5 or 6.5.With this method neutral -glucosidases can be exactly demonstrated in the brush border of the small intestine (glycoamylase, sucrase-isomaltase) and kidney of mammals, birds, fishes, amphibia and reptiles; localization of acid -glucosidases is achieved at the cellular level in many organs and tissues.Fluorometric and photometric measurements prove that 2-naphthyl--D-glucoside is superior to 6-brom-2-naphthyl--D-glucoside for the demonstration of -glucosidases in situ due to the lower Michaelis constant and higher maximal reaction velocity of the naphthol derivative. — Among the coupling reagents tested neutral -glucosidases can be localized correctly with hexazotized p-rosaniline (with and without semipermeable membranes) for simultaneous coupling. Fast Blue B delivers false positive results in the suczedaneous and simultaneous coupling procedure using aqueous incubation media; in combination with the membrane technique azo dye can not be observed in the sections. Hexazonium-p-rosaniline inhibits neutral and acid -glucosidases to nearly the same extent as Fast Blue B.Fixation of blocks of tissue in formaldehyde and glutaraldehyde suppresses -glucosidases in the intestine and epididymis. The inhibition rates amount to 50 and 70% respectively. Washing in sugar solution rises enzyme activity to 65 and 50%.Species and organ dependent activity differences of neutral and acid -glucosidases and changes of enzyme activity in the intestine and kidney after castration as well as in the course of pregnancy can be detected by means of biochemistry but not with the histochemical assay including minimal incubation. In comparison with p-nitrophenyl--D-glucoside the 2-naphthyl derivative is also the substrate of choice for the biochemical determination of -glucosidases. — Agar gel electrophoresis reveals one band in the neutral and acid pH range.

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Histochemische und biochemische Untersuchung der -glucosidasen mit 2-naphthyl--D-glucosid

Zusammenfassung Vergleichend histochemisch-biochemische Untersuchungen ergeben folgende Methode der Wahl zum in situ-Nachweis der neutralen (mikrovillären) und sauren (lysosomalen) -Glucosidasen: 12 mg 2-Naphthyl--D-glucosid (gelöst in 0,5 ml N,N-Dimethylformamid) und 0,6–0,8 ml Hexazonium-p-rosanilin in 10 ml 0,1 M Citronensäure-Phosphat-Puffer zur wäßrigen oder in 5 ml Pufferlösung 1:1 gemischt zur Inkubation mit semipermeablen Membranen, pH 5 und 6,5.Mit diesem Verfahren können die neutralen -Glucosidasen in Bürstensaum von Dünndarm (Glucoamylase, Saccharase-Isomaltase) und Niere bei Säugern, Fischen, Vögeln, Reptilien und Amphibien exakt dargestellt werden; die Lokalisation der sauren -Glucosidase gelingt auf Zellebene in zahlreichen Organen und Geweben.Fluorometrische und photometrische Messungen zeigen, daß 2-Naphthyl--D-glucosid dem Alternativsubstrat 6-Br-2-Naphthyl--glucosid zur histochemischen Untersuchung wegen seiner kleineren Michaelis-Konstante und höheren maximalen Reaktionsgeschwindigkeit überlegen ist. — Unter den geprüften Kupplungssubstanzen kann nur Hexaonium-p-rosanilin als Simultankuppler mit und ohne Membrantechnik vor allem die neutralen -Glucosidasen korrekt erfassen; Fast Blue B in wäßrigen Medien lokalisiert bei Post- und Simultankupplung falsch-positiv und liefert in Verbindung mit semipermeablen Membranen negative Resultate. Die Hemmung der -Glucosidasen durch hexazotiertes p-Rosanilin entspricht etwa der durch Fast Blue B.Stückfixation in Form- und Glutaraldehyd inhibiert die neutralen und sauren -Glucosidasen in Dünndarm und Nebenhoden zu ca. 50 bzw. 70%; nach Auswaschen liegen die Aktivitäten bei 65 bzw. 50%.Art- und organspezifische Aktivitätsdifferenzen der sauren und neutralen -Glucosidasen und Änderungen der Enzymaktivität nach Kastration sowie während der Gravidität in Dünndarm und Niere deckt zuverlässig nur die biochemische Untersuchung auf; der histochemische -Glucosidasen-Nachweis versagt hier selbst bei Minimalinkubation weitgehend. Verglichen mit p-Nitrophenyl--glucosid ist das 2-Naphthylderivat auch das biochemische Substrat der Wahl. — Mittels Agargelelektrophorese kann im alkalischen und sauren pH-Bereich 1 Bande nachgewiesen werden.

     

Motif-Guided Identification of a Glycoside Hydrolase Family 1 α-L-Arabinofuranosidase in Bifidobacterium adolescentis

Members of glycoside hydrolase family 1 (GH1) cleave glycosidic linkages with a variety of physiological roles. Here we report a unique GH1 member encoded in the genome of Bifidobacterium adolescentis ATCC 15703. This enzyme, BAD0156, was identified from over 2,000 GH1 sequences accumulated in a database by a genome mining approach based on a motif sequence. A recombinant BAD0156 protein was characterized to confirm that this enzyme alone specifically hydrolyzes p-nitrophenyl-α-L-arabinofuranoside among the 24 p-nitrophenyl-glycosides examined. Among natural glycosides, α-1,5-linked arabino-oligosaccharides served as substrates, but arabinan, debranched arabinan, arabinoxylan, and arabinogalactan did not. A time course analysis of arabino-oligosaccharide hydrolysis indicated that BAD0156 is an exo-acting enzyme. These results suggest that BAD0156 is an α-L-arabinofuranosidase. This is the first report of a GH1 enzyme that acts specifically on arabinosides, providing information on GH1 substrate specificity.     

Asymmetric acetalation of α,α-trehalose: Synthesis of α-d-galactopyranosyl α-d-glucopyranoside and 6-deoxy-6-fluoro-α-d-glucopyranosyl α-d-glucopyranoside

Selective acetalation of α,α-trehalose with ethyl or methyl isopropenyl ether and toluene-p-sulphonic acid in N,N-dimethylformamide gave the 4,6-isopropylidene acetal as the major product, isolated as its hexa-acetate 1 (38%). The gluco-galacto analogue 6 of α,α-trehalose was synthesized from 1 by the sequence: hydrolysis of the isopropylidene group with trifluoroacetic acid, mesylation of the resulting diol, benzoate displacement, and saponification of the product. Deacetylation of 1 followed by benzylation and hydrolysis of the acetal group furnished a hexa-O-benzyl derivative 9. Tosylation of the primary hydroxyl group in 9, treatment of the product with tetrabutylammonium fluoride in acetonitrile, and subsequent catalytic hydrogenolysis of the benzyl groups gave 6-deoxy-6-fluoro-α,α-trehalose (12). Compounds 6 and 12 and 6-deoxy-6-iodo-α,α-trehalose are substrates for cockchafer trehalase, but have very low V_max values.     

Detection of an alteration of the α2 gene in a patient with chronic lung disease and serum α2 deficiency

Summary ₂-Macrpglobulin (A2M) is a major human plasma protease inhibitor capable of inhibiting most endopeptidases tested so far. In the case of the other major plasma protease inhibitor, ₁-antitrypsin, genetically determined deficiency states are known to increase the risk of chronic obstructive pulmonary disease (COPD) 20- to 30-fold in affected individuals. No defects of the A2M gene have been described as yet, but A2M may play a role in the regulation of protease activity in the lung, especially with respect to those proteases not inhibited by ₁-antitrypsin. We report here the molecular genetic detection of an alteration of the A2M gene in a patient with serum A2M deficiency and chronic lung disease since childhood. The alteration involves restriction sites detected with 10 different enzymes and is most probably caused by a major deletion or rearrangement of the gene. Nine of the restriction enzymes used detected no polymorphisms in 40 healthy control subjects and 39 COPD patients. The polymorphism detected in this patient with the enzyme PvuII was different from another described previously, and was found in this patient only. The patient is heterozygous for an alteration in the A2M gene; this may be responsible for his serum A2M deficiency and may be relevant to the early onset of pulmonary disease in his case.     

Activity of α-galactosidase in immobilized cells of Amsonia tabernaemontana Walt

Cell suspension culture Amsonia tabernaemontana Walt. were permeabilized by Tween 80 and immobilized by glutaraldehyde. The highest α-galactosidase activity was at pH 5.3 and temperature 70 °C. The hydrolysis of substrate was linear for 3 h reaching 70 - 75 % conversion. The cells characterized by high enzyme activity and stability in long-term storage showed convenient physico-mechanical properties (physical protection from shear forces and easy separation of product from biocatalysts). This revised version was published online in July 2006 with corrections to the Cover Date.     

Determination of Dopamine-β-hydroxylase Activity in Human Serum Using UHPLC-PDA Detection

Dopamine-β-hydroxylase (DBH, EC 1.14.17.1) is an enzyme with implications in various neuropsychiatric and cardiovascular diseases and is a known drug target. There is a dearth of cost effective and fast method for estimation of activity of this enzyme. A sensitive UHPLC based method for the estimation of DBH activity in human sera samples based on separation of substrate tyramine from the product octopamine in 3 min is described here. In this newly developed protocol, a Solid Phase Extraction (SPE) sample purification step prior to LC separation, selectively removes interferences from the reaction cocktail with almost no additional burden on analyte recovery. The response was found to be linear with an r²?=?0.999. The coefficient of variation for assay precision was <?10% and recovery?>?90%. As a proof of concept, DBH activity in sera from healthy human volunteers (n?=?60) and schizophrenia subjects (n?=?60) were successfully determined using this method. There was a significant decrease in sera DBH activity in subjects affected by schizophrenia (p?<?0.05) as compared to healthy volunteers. This novel assay employing SPE to separate octopamine and tyramine from the cocktail matrix may have implications for categorising subjects into various risk groups for Schizophrenia, Parkinson’s disease as well as in high throughput screening of inhibitors.     

Detection of SHV β-lactamases in Gram-negative bacilli using fluorescein-labeled antibodies

Background  

β-lactam resistance in Gram-negative bacteria is a significant clinical problem in the community, long-term care facilities, and hospitals. In these organisms, β-lactam resistance most commonly results from the production of β-lactamases. In Gram-negative bacilli, TEM-, SHV-, and CTX-M-type β-lactamases predominate. Therefore, new and accurate detection methods for these β-lactamase producing isolates are needed.     

Structure assembly of Bcl-xL through α5-α5 and α6-α6 interhelix interactions in lipid membranes

Lipid bilayer membrane is the main site where Bcl-x_L executes its anti-apoptotic function. Here we used site-directed mutagenesis and cysteine-directed cross-linking to trap the structure of Bcl-x_L upon membrane insertion. Cys151 on α5-helix and Asn185 on α6-helix of two neighboring Bcl-x_L are found in close positions, respectively. The FRET based binding assay indicated that the BH3-peptide binding pocket in Bcl-x_L is disrupted after its membrane insertion. Co-immunoprecipitation experiments showed that the membrane-bound Bcl-x_L sequestered tBid by direct interaction at physiological pH. If Bcl-x_L behaves similarly at low pH as it does at physiological pH, the membrane-bound Bcl-x_L should bind to tBid through protein regions other than the BH3 domain of tBid and the hydrophobic pocket of Bcl-x_L. Previously, a crystallography study demonstrated that Bcl-x_L formed homodimers through domain swapping in water, where Cys151 and Asn185 of two monomeric subunits are far apart from each other and the BH3-peptide binding pocket is intact. Our results indicated that Bcl-x_L dimer trapped by cross-linking in lipids is distinct from the domain swapped dimer, suggesting that Bcl-x_L transits through a structural change from the water-soluble state to the membrane-bound state and there are multiple possibilities for structural reorganization of Bcl-x_L protein.     

α-l-Rhamnosidase Activity in Culture Filtrate of Corticium rolfsii Enzymic Activity at Low pH

A phosphorylation system for formation of ATP from AMP by Zymolyase-treated cells of Candida boidinii (Kloeckera sp.) No. 2201 was developed as an ATP production process. This system was shown to be an energy conversion system, from a reduced C₁ -compound to ATP through reduction of NAD⁺ and oxidative phosphorylation but not substrate level phosphorylation, together with phosphorylation of AMP to ADP.

Reaction conditions for the ATP production were optimized in respect of substrate and coenzyme concentrations, pH and temperature, osmotic pressure, and oxygen supply. Under the optimal conditions, 26 mM (13 g/liter) and 8.5 dim (4g/liter) of ATP were produced with methanol and formate as C₁ -substrate, respectively.     

Norepinephrine Drives Persistent Activity in Prefrontal Cortex via Synergistic α1 and α2 Adrenoceptors

Optimal norepinephrine levels in the prefrontal cortex (PFC) increase delay-related firing and enhance working memory, whereas stress-related or pathologically high levels of norepinephrine are believed to inhibit working memory via α1 adrenoceptors. However, it has been shown that activation of Gq-coupled and phospholipase C-linked receptors can induce persistent firing, a cellular correlate of working memory, in cortical pyramidal neurons. Therefore, despite its importance in stress and cognition, the exact role of norepinephrine in modulating PFC activity remains elusive. Using electrophysiology and optogenetics, we report here that norepinephrine induces persistent firing in pyramidal neurons of the PFC independent of recurrent fast synaptic excitation. This persistent excitatory effect involves presynaptic α1 adrenoceptors facilitating glutamate release and subsequent activation of postsynaptic mGluR5 receptors, and is enhanced by postsynaptic α2 adrenoceptors inhibiting HCN channel activity. Activation of α2 adrenoceptors or inhibition of HCN channels also enhances cholinergic persistent responses in pyramidal neurons, providing a mechanism of crosstalk between noradrenergic and cholinergic inputs. The present study describes a novel cellular basis for the noradrenergic control of cortical information processing and supports a synergistic combination of intrinsic and network mechanisms for the expression of mnemonic properties in pyramidal neurons.     

Detection of a new hybrid α2 globin gene among American Blacks

Summary We have examined the globin gene complex for 49 individuals with -thalassemia-2 (–^3.7). Crossovers resulting in -thalassemia-2 (type I) were observed in all 57 chromosomes with the –^3.7 defect. Except for one -thalassemia-2 chromosome, all were linked to the absence of an Rsa I restriction site located 0.7 kb 5 to the 2-globin gene; this polymorphic site was observed for 10 of 38 non--thalassemia chromosomes from Black Americans. In four Black families with a heterozygous -thalassemia-2 [–^3.7 (I)], an Apa I restriction site has been identified in the IVS-2 of the 2 gene of the normal chromosome (labeled the ^*2 gene). The ^*2 gene of one Black subject was cloned and a segment located 5 to the Cap site as well as the IVS-2, exon 3, and a 3 segment were sequenced. The data show that the ^*2 gene is an 2 gene except for a segment between nucleotides (nts) 580–81 and nt 509 (Cap site=nt 1), and perhaps as far upstream as nt-634, which has an 1 sequence. This ^*2 hybrid gene probably originated through a double crossover; the structural identity of its IVS-2 with that of the 1 gene adequately explains the presence of the Apa I restriction site.     

Inhibition of Vesicular Monoamine Transporter-2 Activity in α-Synuclein Stably Transfected SH-SY5Y Cells

α-Synuclein plays a key role in the pathological neurodegeneration in Parkinson’s disease. Although its contribution to normal physiology remains elusive, the selective degeneration of α-synuclein-containing dopaminergic neurons in Parkinson’s disease may be linked to abnormal α-synuclein induced toxicity. In the present study, a complex of α-synuclein and vesicular monoamine transporter-2 was identified by GST-Pull Down experiment. In wild-type α-synuclein stably transfected SH-SY5Y cell lines, the activity of vesicular monoamine transporter-2 decreased by 31% as determined by [³H] dopamine uptake, and its expression also decreased in both protein and mRNA levels using western and northern blot analysis. Overexpression of wild-type α-synuclein did not induce cell death or apoptosis, but significantly enhanced the intracellular reactive oxygen species level as assayed by flow cytometry. These data suggest that Up-regulated α-synuclein expression inhibits the activity of vesicular monoamine transporter-2, thereby interrupting dopamine homeostasis and resulting in dopaminergic neuron injury in Parkinson’s disease.     

Simple Detection of Xylosidase Activity in Single Colonies,Using 1-Naphthyl-β-d-xylopyranoside

Since the xylosidase of Bacillus pumilus hydrolyzed 1-naphthyl-β-d-xylopyranoside (naphthyl-X) to produce xylose and 1-naphthol and a chromogenic azo compound is produced by coupling 1-naphthol and Fast Blue Salt B, a simple method for detection of xylosidase activity in single colonies was studied. Escherichia coli JM109 carrying the xylosidase gene of B. pumilus was cultivated at 37°C for 18 h on an LB plate containing 0.5 mg/ml naphthyl-X, and then the plate was overlaid with 3 ml of a top layer containing 24 mg of agar and 6 mg of Fast Blue Salt B. After incubation of the plate at 37°C for 1 h, each colony became reddish-brown. Even a small colony with the xylosidase on the plate was easily distinguished from colonies without the enzyme.     

Effects of Calcium Ion on the Catalytic Activity of α-Chymotrypsin in Organic Solvents

Addition of small amounts of calcium ion markedly accelerated the transesterification of N-acetyl-l-tyrosine methyl ester to its ethyl ester by the catalysis of α-chymotrypsin in organic solvents. Maximum increase of the reaction rate was about 12-fold in the presence of 25 μm of calcium ion in ethanol. The rate increase was strongly dependent on calcium ion concentration and nature of organic solvents. Esterification of N-acetyl-l-tyrosine and hydrolysis of N-acetyl-l-tyrosine ethyl ester by α-chymotrypsin in organic solvents were also accelerated by calcium ion. The reactions obeyed Michaelis–Menten kinetics, and the acceleration of the reactions was due to the increase in k_cat.     

Production of α-linolenic acid in Yarrowia lipolytica using low-temperature fermentation

α-Linolenic acid (ALA) is an essential ω-3 fatty with reported health benefits. However, this molecule is naturally found in plants such as flaxseed and canola which currently limits production. Here, we demonstrate the potential to sustainably produce ALA using the oleaginous yeast Yarrowia lipolytica. Through the use of a recently identified Δ12–15 desaturase (Rk Δ12–15), we were able to enable production in Y. lipolytica. When combined with a previously engineered lipid-overproducing strain with high precursor availability, further improvements of ALA production were achieved. Finally, the cultivation of this strain at lower temperatures significantly increased ALA content, with cells fermented at 20 °C accumulating nearly 30% ALA of the total lipids in this cell. This low-temperature fermentation represents improved ALA titer up to 3.2-fold compared to standard growth conditions. Scale-up into a fed-batch bioreactor produced ALA at 1.4 g/L, representing the highest published titer of this ω-3 fatty acid in a yeast host.

     

Differential involvement of α4β2, α7 and α9α10 nicotinic acetylcholine receptors in B lymphocyte activation in vitro

Mouse B lymphocytes express several nicotinic acetylcholine receptor (nAChR) subtypes, their exact functions being not clearly understood. Here we show that α7 nAChR was present in about 60%, while α4β2 and α9(α10) nAChRs in about 10% and 20% of mouse spleen B lymphocytes, respectively; Balb/c and C57Bl/6 mice possessed different relative amounts of these nAChR subtypes. α4β2 and α7, but not α9(α10) nAChRs, were up-regulated upon B lymphocyte activation in vitro. Flow cytometry and sandwich ELISA studies demonstrated that α7 and α9(α10) nAChRs are coupled to CD40, whereas α4β2 nAChR is coupled to IgM. B lymphocytes of both α7(-/-) and β2(-/-) mice responded to anti-CD40 stronger than those of the wild-type mice, whereas the cells of β2(-/-) mice responded to anti-IgM worse than those of the wild-type or α7(-/-) mice. Inhibition of α7 and α9(α10) nAChRs with methyllicaconitine resulted in considerable augmentation of CD40-mediated B lymphocyte proliferation in cells of all genotypes; stimulation of α4β2 nAChRs with epibatidine increased the IgM-mediated proliferation of the wild-type and α7(-/-), but not β2(-/-) cells. Inhibition of α9(α10) nAChRs with α-conotoxin PeAI exerted weak stimulating effect on CD40-mediated proliferation. This nAChR subtype was up-regulated in α7(-/-) B-cells. α7 nAChRs were found recruited to immune synapses between human T and B lymphocytes, both of which produced acetylcholine. It is concluded that α7 nAChR fulfills inhibitory CD40-related mitogenic function, α4β2 nAChR produces a stimulatory IgM-related effect, while α9α10 nAChR is a "reserve" receptor, which partly compensates the absence of α7 nAChR in α7(-/-) cells. Acetylcholine is an additional mediator to modulate activation of interacting T and B lymphocytes.     

Lack of α-Xylosidase Activity in Arabidopsis Alters Xyloglucan Composition and Results in Growth Defects

Xyloglucan is the main hemicellulose in the primary cell walls of most seed plants and is thought to play a role in regulating the separation of cellulose microfibrils during growth. Xylose side chains block the degradation of the backbone, and α-xylosidase activity is necessary to remove them. Two Arabidopsis (Arabidopsis thaliana) mutant lines with insertions in the α-xylosidase gene AtXYL1 were characterized in this work. Both lines showed a reduction to undetectable levels of α-xylosidase activity against xyloglucan oligosaccharides. This reduction resulted in the accumulation of XXXG and XXLG in the liquid growth medium of Atxyl1 seedlings. The presence of XXLG suggests that it is a poor substrate for xyloglucan β-galactosidase. In addition, the polymeric xyloglucan of Atxyl1 lines was found to be enriched in XXLG subunits, with a concomitant decrease in XXFG and XLFG. This change can be explained by extensive exoglycosidase activity at the nonreducing ends of xyloglucan chains. These enzymes could thus have a larger role than previously thought in the metabolism of xyloglucan. Finally, Atxyl1 lines showed a reduced ability to control the anisotropic growth pattern of different organs, pointing to the importance of xyloglucan in this process. The promoter of AtXYL1 was shown to direct expression to many different organs and cell types undergoing cell wall modifications, including trichomes, vasculature, stomata, and elongating anther filaments.The primary wall that surrounds the growing cells of plants has to be able to extend in response to turgor pressure. This process needs to be tightly regulated to avoid a mechanical failure of the wall. The direction of expansion also needs to be controlled so that different cell types can develop their particular morphology. In addition, the growth of the different tissues in an organ has to be tightly coordinated so that it can achieve its final shape (Baskin, 2005). The mechanical behavior of the expanding cell wall has been likened to a fiber-reinforced composite, with crystalline cellulose microfibrils embedded in an amorphous matrix of hemicellulose and pectin. How this works at the molecular level is still the subject of much research and speculation (Geitmann and Ortega, 2009).Xyloglucan is the main hemicellulose in the primary cell walls of gymnosperms and most angiosperm families and is present in all extant groups of land plants, although with some differences in structure (Peña et al., 2008; Scheller and Ulvskov, 2010). All these xyloglucans have a backbone of (1→4)-linked β-d-glucopyranosyl residues, many of which are substituted with α-d-xylopyranosyl residues at O6. In many vascular plants, including Arabidopsis (Arabidopsis thaliana), every fourth glucosyl residue of the xyloglucan backbone is unsubstituted (Vincken et al., 1997). In the standard nomenclature for xyloglucan structures, these residues are represented by G, while X, L, and F indicate Glc residues substituted, respectively, with α-d-Xylp, β-d-Galp-(1→2)-α-d-Xylp, and α-l-Fucp-(1→2)-β-d-Galp-(1→2)-α-d-Xylp side chains (Fry et al., 1993). Conventionally, the reducing end of the molecule is positioned to the right. Treatment of Arabidopsis xyloglucan with an endoglucanase that attacks unsubstituted residues results in oligosaccharide mixtures that include XXG, GXXG, XXXG, XXLG, XLXG, XLLG, XXFG, and XLFG, with some of the Gal residues O-acetylated (Madson et al., 2003; Obel et al., 2009).Although the detailed arrangement and possible connections of the different components of primary cell walls are still unclear, xyloglucan chains are long enough to attach simultaneously to neighboring microfibrils and thus could generate resistance to cell wall extension (Obel et al., 2007). There is also considerable evidence for the covalent linkage of xyloglucan to the pectic polysaccharide rhamnogalacturonan I (Popper and Fry, 2008). The attachment of xyloglucan to cellulose microfibrils is based on hydrogen bonds, and it might be controlled by expansin proteins (Cosgrove, 2005). Xyloglucan connections between microfibrils could also be broken or created by enzymes in the xyloglucan transglycosylase/hydrolase (XTH) family (Nishitani and Vissenberg, 2007). These enzymes cleave xyloglucan chains in front of unsubstituted Glc residues and stay covalently bound to this residue, forming an enzyme-donor complex (Johansson et al., 2004). They can later attach the Glc residue to the nonreducing end of another xyloglucan molecule, acting as xyloglucan endotransglucosylases (XETs). A group of XTHs can also use water as an acceptor, acting as xyloglucan endohydrolases, but they seem to be a minority (Baumann et al., 2007; Eklöf and Brumer, 2010). It is unclear at the moment if endoglucanases from other families are involved in xyloglucan metabolism (Lopez-Casado et al., 2008).The importance of xyloglucan as a regulator of cell wall extension has been thrown into doubt by the identification of an Arabidopsis mutant that has no detectable xyloglucan but still manages to develop normally (Cavalier et al., 2008). Apart from being slightly smaller, this mutant has defective root hairs, but it seems clear that Arabidopsis must have alternative ways of regulating the separation of cellulose microfibrils. It is interesting nonetheless that microfibrils seem to be more irregularly spaced in the xyloglucan-deficient mutant (Anderson et al., 2010).The end result of endoglucanase activity on xyloglucan is the release of oligosaccharides with an unsubstituted Glc at the reducing end. Specific exoglycosidase activities are then necessary to release each type of residue (Iglesias et al., 2006). α-Xylosidase activities in both pea (Pisum sativum) and Tropaeolum majus can only remove unsubstituted Xyl residues from the nonreducing end of the molecule (O’Neill et al., 1989; Fanutti et al., 1991). A β-glucosidase is then required to remove the unsubstituted Glc before α-xylosidase can act again (Crombie et al., 1998). β-Galactosidase and α-fucosidase activities are also required for the complete disassembly of the different Arabidopsis oligosaccharides (Edwards et al., 1988; Léonard et al., 2008). There is currently no information on the enzymes that might be involved in xyloglucan deacetylation or on how the presence of acetyl residues affects exoglycosidases.The Arabidopsis gene AtXYL1 (At1g68560) was identified as coding for an α-xylosidase activity against xyloglucan oligosaccharides by the similarity of its product to purified cabbage (Brassica oleracea var capitata) α-xylosidase (Sampedro et al., 2001). The identification was confirmed through heterologous expression in yeast. According to the Carbohydrate Active Enzymes database (http://www.cazy.org/), AtXYL1 is a member of glycosyde hydrolase family 31, which includes mainly α-glucosidases and α-xylosidases(Cantarel et al., 2009). A reduction of up to 70% of α-xylosidase activity was reported in antisense lines where AtXYL1 was silenced, but this reduction did not cause changes in morphology (Monroe et al., 2003). This article presents the characterization of two independent insertional mutants in AtXYL1 that have no detectable α-xylosidase activity and show remarkable changes in xyloglucan composition along with alterations in the growth pattern.