Product Entrapment Research Articles

Proteomic and biochemical evidence links the callose synthase in Nicotiana alata pollen tubes to the product of the NaGSL1 gene

The NaGSL1 gene has been proposed to encode the callose synthase (CalS) enzyme from Nicotiana alata pollen tubes based on its similarity to fungal 1,3-beta-glucan synthases and its high expression in pollen and pollen tubes. We have used a biochemical approach to link the NaGSL1 protein with CalS enzymic activity. The CalS enzyme from N. alata pollen tubes was enriched over 100-fold using membrane fractionation and product entrapment. A 220 kDa polypeptide, the correct molecular weight to be NaGSL1, was specifically detected by anti-GSL antibodies, was specifically enriched with CalS activity, and was the most abundant polypeptide in the CalS-enriched fraction. This polypeptide was positively identified as NaGSL1 using both MALDI-TOF MS and LC-ESI-MS/MS analysis of tryptic peptides. Other low-abundance polypeptides in the CalS-enriched fractions were identified by MALDI-TOF MS as deriving from a 103 kDa plasma membrane H+-ATPase and a 60 kDa beta-subunit of mitochondrial ATPase, both of which were deduced to be contaminants in the product-entrapped material. These analyses thus suggest that NaGSL1 is required for CalS activity, although other smaller (<30 kDa) or low-abundance proteins could also be involved.

Partial purification of (1,3)-beta-glucan synthase from Candida albicans.

(1,3)-beta-Glucan synthase from Candida albicans was solubilized from microsomal membranes using the detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (Chaps). Effective solubilization was dependent upon the strain and the method used to detect enzyme activity. The solubilized enzyme was purified over 765-fold using a modified product entrapment technique. Bovine serum albumin, an activator of glucan synthase, precipitated proteins during product entrapment and was replaced with BSA immobilized on agarose beads. SDS-PAGE analysis revealed a prominent 187-kDa band present in the product entrapped pellet as well as several additional polypeptides at 227, and 187, 182 and 39 kDa which were not prevalent in crude preparations.

The Correlation Between Substrate Mass Loss and Electrochemical Impedance Spectroscopy Data for a Polymer-Coated Metal

Research has been carried out to evaluate the correlation between substrate mass loss and data generated by electrochemical impedance spectroscopy (EIS). Experiments were carried out as a function of exposure time for both polymer-coated and bare samples. Mass loss was determined in situ and in real time with a vibrating sample magnetometer (VSM). In all cases, the sample was held potentiostatically at the open circuit potential (OCP) while EIS data were collected. Mass loss data calculated from electrochemical impedance (EI) spectra were then compared to equivalent data generated on the VSM. Although previous research has investigated the correlation for bare samples [W. J. Lorenz and F. Mansfeld, Corros. Sci., 21, 647 (1981)] establishing this relationship for a coated substrate is more convoluted. The difficulty results from errors associated with solution uptake within the polymer, and corrosion product entrapment. Such errors obviate the use of traditional gravimetric techniques and, until now, the relationship between substrate mass loss and EIS has not unequivocally been established for polymer-coated samples. Although the current research indicates that EIS can accurately define the mass loss for a bare sample, the correlation for coated samples has, generally, not been as good. © 2002 The Electrochemical Society. All rights reserved.

Glucan synthase complex of Aspergillus fumigatus.

The glucan synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for beta(1-3) glucans, showing that AfFks1p colocalized with the newly synthesized beta(1-3) glucans. AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the glucan synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H(+)-ATPase, and a 160-kDa protein which was labeled by an anti-beta(1-3) glucan antibody and was homologous to ABC bacterial beta(1-2) glucan transporters.

GAS Purging for Weld Root Control in Pipe Welding

For many pipe welding applications, the physical characteristics of welds may not be important. Fillet and non fully penetrating welds have inherent crack like defects but these are seldom of any consequence. In circumstances where joints have to be designed to withstand stress in sen/ice or they need to be free from the risk of product entrapment or they need to be able to offer good flow characteristics, however, special consideration needs to be given to metallurgical aspects and to weld profile. The mechanical properties of welds, particularly their fatigue properties, can be influenced significantly by their shape and composition. In particular, at the weld ischolar_main, a positive reinforcement combined with smooth transition from weld to parent material are pre-requisites if optimum mechanical strength, flow characteristics and lack of product entrapment are to be realised. Gu idance on weld ischolar_main profile control for linear butt welds is provided elsewhere. Here we examine solutions when making tubular butt welds.

Partial purification of a GTP-insensitive (1 → 3)- β-glucan synthase from Phytophthora sojae

A (1 → 3)- β-glucan synthase activity was identified in cell membrane preparations from the oomycete Phytophthora sojae, a soybean pathogen. The activity could be solubilized using the zwitterionic detergent CHAPS at relatively low concentrations (3 mg/ml). High salt concentrations were not effective in removing the activity from the membranes. Detergent solubilization of the enzyme resulted in a six-fold increase of calculated Vmax values (2.5 vs. 0.4 nkat/mg protein) but only minor alteration of the K m (10.6 vs. 10.7 mM). Analysis of the reaction product of the solubilized enzyme by enzymatic degradation and by 2D NMR spectroscopy confirmed its identity as a linear high molecular weight (1 → 3)- β-glucan. Glucan synthase activity in both membrane and solubilized preparations was not activated by GTP or divalent cations as reported for other fungal or plant glucan synthases, The activity was inhibited, as expected, in a competitive manner by UDP with a K i of 2.9 mM. Partial purification of the enzyme was achieved by anion exchange chromatography followed by product entrapment. This procedure resulted in the selective enrichment of a protein band with apparent M r 108 000 in SDS-PAGE which was not visible in any of the steps preceding product entrapment. The glucan pellets from product entrapment contained up to 3% of the initial enzyme activity present in the fraction used for the procedure.

Membrane fractionation and enrichment of callose synthase from pollen tubes of Nicotiana alata Link et Otto.

The callose synthase (UDP-glucose: 1,3-beta-D-glucan 3-beta-D-glucosyl transferase; EC 2.4.1.34) enzyme (CalS) from pollen tubes of Nicotiana alata Link et Otto is responsible for developmentally regulated deposition of the cell wall polysaccharide callose. Membrane preparations from N. alata pollen tubes grown in liquid culture were fractionated by density-gradient centrifugation. The CalS activity sedimented to the denser regions of the gradient, approximately 1.18 g.ml-1, away from markers for Golgi, endoplasmic reticulum and mitochondria, and into fractions enriched in ATPase activity and in membranes staining with phosphotungstic acid at low pH. This suggests that pollen-tube CalS is localised in the plasma membrane. Callose synthase activity from membranes enriched by downward centrifugation was solubilised with digitonin, which gave a 3- to 4-fold increase in enzyme activity, and the solubilised activity was then enriched a further 10-fold by product entrapment. The complete procedure gave final CalS specific activities up to 1000-fold higher than those of pollen-tube homogenates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that several polypeptides co-fractionated with CalS activity through purification, with a polypeptide of 190 kDa being enriched in product-entrapment pellets.

Partial purification of (1,3)-β-glucan synthase from Candida albicans

(1,3)-β-Glucan synthase from Candida albicans was solubilized from microsomal membranes using the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (Chaps). Effective solubilization was dependent upon the strain and the method used to detect enzyme activity. The solubilized enzyme was purified over 765-fold using a modified product entrapment technique. Bovine serum albumin, an activator of glucan synthase, precipitated proteins during product entrapment and was replaced with BSA immobilized on agarose beads. SDS-PAGE analysis revealed a prominent 187-kDa band present in the product entrapped pellet as well as several additional polypeptides at 227, and 187, 182 and 39 kDa which were not prevalent in crude preparations.

In Vitro Activity of 1,3-β-D-Glucan Synthase Requires the GTP-binding Protein Rho1

In the yeast Saccharomyces cerevisiae, the family of RHO genes are implicated in the control of morphogenetic events although the molecular targets of these GTP-binding proteins remain largely unknown. The activity of 1,3-beta-D-glucan synthase, the product of which is essential for cell wall integrity, is regulated by a GTP-binding protein, which we here present evidence to be Rho1p. Rho1p was found to copurify with Fks1p, a glucan synthase subunit, in preparations of the enzyme purified by product entrapment and was also shown to be depleted by a detergent extraction procedure known to remove the GTP-binding regulatory component. Specific ADP-ribosylation of Rho1p by exoenzyme C3 inactivates glucan synthase activity specified by FKS1 and FKS2 as demonstrated in membrane preparations from fks2 and fks1 deletion strains, respectively, and in the purified enzyme containing Fks1p. Rho1p and Fks1p were co-immunoprecipitated from purified glucan synthase under conditions that maintained enzyme activity in the immunoprecipitate. Putative Rho homologs were also identified and implicated in the regulation of glucan synthase activity from Candida albicans, Aspergillus nidulans, and Cryptococcus neoformans by ribosylation studies. The regulation of 1,3-beta-D-glucan synthase activity by RHO1 is consistent with its observed role in morphogenetic control and osmotic integrity.

Characterization of chitin synthase 2 of Saccharomyces cerevisiae. II: Both full size and processed enzymes are active for chitin synthesis.

When chitin synthase 2 of Saccharomyces cerevisiae was overexpressed in yeast cells using GAL1 promoter, deletion of the N-terminal 193 amino acids significantly increased the level of the protein without affecting its characteristics. We partially purified N-terminally truncated chitin synthase 2 by product entrapment and ion exchange column chromatography, and found that it was active even without trypsin treatment when appropriate divalent cations were present in the reaction mixture. This chitin synthase activity was independent of the N-terminal 193 amino acid truncation, because partially purified full length enzyme also exhibited the activity without trypsin treatment in the presence of appropriate cations. Furthermore, the molecular weights of these two forms of chitin synthase 2 were coincident with those estimated from the deduced amino acid sequence, and most of the chitin synthase 2 in the yeast membrane was present as an unprocessed form, as judged from its molecular weight. Treatment of either full length or truncated enzyme with trypsin, however, further increased the enzyme activity by four to fivefold, and produced a 35 kDa polypeptide that specifically reacted with monoclonal antibody raised against the region containing the putative active site of chitin synthase 2. Thus, it appears that predominant native (unprocessed) chitin synthase 2 is active, but the 35 kDa region encompassing the active site is sufficient for the catalytic activity.

Signaling toward yeast 1,3-beta-glucan synthesis.

1,3-beta-glucan synthase catalyzes the synthesis of a 1,3-beta-linked glucan polymer which produces the main rigidity of the yeast cell wall. Recent success in purification of this enzyme by product entrapment (21) has provided new insights into the dynamic aspects of the cell wall. This relatively simple procedure made it possible to identify the genes encoding the catalytic subunits of glucan synthase. In addition, the involvement of a rho type GTPase in the regulation of glucan synthase was demonstrated with the purified enzyme. Based on intracellular localization of the glucan synthase subunits, we have proposed a model in which assembly of the subunits is important for the activation of glucan synthase at sites of polarized growth. In this article, we will focus on biochemistry of 1,3-beta-glucan synthase and signaling through rho type GTPase.

In vitro synthesis of a microfibrillar (1→3)‐β‐glucan by a ryegrass (Lolium multiflorum) endosperm (1→3)‐β‐glucan synthase enriched by product entrapment

A simple, rapid procedure has been developed to purify (1→3)‐β‐glucan synthase (UDP‐Glc:(1→3)‐β‐glucan 3‐β‐d‐glucosyl transferase (EC 2.4.1.34)) over 400‐fold from membrane preparations of Italian ryegrass (Lolium multiflorum) and 60‐fold from CHAPS‐extracted membranes. When a CHAPS‐extract of the membranes is treated with 8 mM CaCl2, proteinaceous material is precipitated. Although less than 10% of CHAPS‐solubilized protein is removed in this step, the total activity recovered in the supernatant increases fourfold. Thus, CaCl2 precipitation appears to be important in removing inhibitors of the (1→3)‐β‐glucan synthase. In the presence of 1 mM UDP‐glucose, the supernatant after CaCl2 treatment produces a high molecular weight, insoluble product that entraps a (1→3)‐β‐glucan synthase of high specific activity. The product‐entrapped enzyme preparation contains six major polypeptides, and comparison of the SDS—PAGE pattern of this fraction with the polypeptide profile of an immunoprecipitated (1→3)‐β‐glucan synthase preparation suggests that polypeptides at 30–31 and 55–58 kDa are the most likely candidates for participation in (1→3)‐β‐glucan synthesis. When the reaction is performed on a larger scale, milligram quantities of product can be seen precipitating from the reaction mixture within 1 h of substrate addition. This product has been characterized by methylation analysis, 1H‐ and 13C‐nmr spectroscopy, X‐ray diffraction, electron microscopy, size exclusion chromatography, UV‐induced fluorescence in the presence of the (1→3)‐β‐glucan‐specific fluorochrome from aniline blue, and enzymic hydrolysis with a specific (1→3)‐β‐glucanase. These physical, chemical and enzymic analyses clearly demonstrate that the product is a microfibrillar (1→3)‐β‐glucan with a degree of polymerization of about 1500.

Characterization and Gene Cloning of 1,3-beta-D-Glucan Synthase from Saccharomyces Cerevisiae

1,3-beta-D-Glucan synthase of Saccharomyces cerevisiae was solubilized and purified up to 700-fold by product entrapment. The specific activity of the partially purified enzyme was around 4 mumol glucose incorporated.min-1.mg protein-1. In SDS/PAGE, enrichment of a 200-kDa protein was clearly observed in parallel with the increase in specific activity. mAbs that could immunoprecipitate the 1,3-beta-D-glucan synthase activity were isolated, and some of them also recognized this 200-kDa protein in the Western blot. Internal amino acid sequences of this 200-kDa protein were determined after lysyl endopeptidase digestion. With the information of these amino acid sequences, we cloned two genes, GSC1 and GSC2 (glucan synthase of S. cerevisiae 1 and 2), which are very similar to each other (88% at the amino acid level); hydropathy profiles of both proteins suggest that these genes encode integral membrane proteins which can be assumed to have approximately 16 transmembrane domains. Disruption of each gene was not lethal, but disruption of both genes was lethal. The 1,3-beta-D-glucan synthase activities of membrane and partially purified enzyme of gsc1::URA3 cells were significantly lower than those of the wild-type and gsc2::LEU2 cells.

Characterization and gene cloning of 1,3-beta-D-glucan synthase from Saccharomyces cerevisiae.

1,3-beta-D-Glucan synthase of Saccharomyces cerevisiae was solubilized and purified up to 700-fold by product entrapment. The specific activity of the partially purified enzyme was around 4 mumol glucose incorporated.min-1.mg protein-1. In SDS/PAGE, enrichment of a 200-kDa protein was clearly observed in parallel with the increase in specific activity. mAbs that could immunoprecipitate the 1,3-beta-D-glucan synthase activity were isolated, and some of them also recognized this 200-kDa protein in the Western blot. Internal amino acid sequences of this 200-kDa protein were determined after lysyl endopeptidase digestion. With the information of these amino acid sequences, we cloned two genes, GSC1 and GSC2 (glucan synthase of S. cerevisiae 1 and 2), which are very similar to each other (88% at the amino acid level); hydropathy profiles of both proteins suggest that these genes encode integral membrane proteins which can be assumed to have approximately 16 transmembrane domains. Disruption of each gene was not lethal, but disruption of both genes was lethal. The 1,3-beta-D-glucan synthase activities of membrane and partially purified enzyme of gsc1::URA3 cells were significantly lower than those of the wild-type and gsc2::LEU2 cells.

(1,3)β-Glucan synthase activity of Neurospora crassa: identification of a substrate-binding protein

(1,3)β-Glucan synthase activity from the filamentous Ascomyceet Neurospora crassa was purified 1300-fold to a specific activitiy of 14 000 nmol glucose incorporated/min per mg protein. Hyphae were disrupted and crude membrane fractions obtained by high-speed centrifugation. Membrane fractions with Tergitol NP-40 and a second high-speed particulate fraction was obtained. Enzyme activity was solubilized with (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate and octyl-β- d-glucoside from Tergitol-extracted membrane preprations. Solubilized enzyme activity was purified by product entrapment and recovered by low-speed centrifugation through a layer of sucrose. SDS-PAGE analysis revealed 2 proteins of 165 and 100 kDa as likely candidates for subunits of the (1,3)β0-glucan synthase enzyme complex. 5-Azido-[ 32P]-UDP-glucose was photo-crosslinked to UDP-glucose-binding proteins in each fraction of the purification procedure. Autoradiograms of SDS-PAGE gels revealed a single protein of 165 kDa enriching with enzyme activity and labeling with the substrate analog. Photoincorporation of 5-azido-[ 32P]UDP-glucose by the 165 kDa protein was competed by 0.25 mM UDP-glucose (80%) and TDP-glucose (65%) while ADP-glucose (27%), CDP-glucose (36%), and GDP-glucose (8%) where less effective. These results were similar to in vitro inhibition of enzyme activity by the same compounds. These data strongly suggest that the 165 kDa protein is a substrate-binding subunit of (1,3)β-glucan synthase.

Purification of 1,3-beta-D-glucan synthase activity from pea tissue. Two polypeptides of 55 kDa and 70 kDa copurify with enzyme activity.

From pea plasma membranes isolated by aqueous polymer two-phase partitioning we have purified 1,3-beta-D-glucan synthase [glucan synthase-II (GS-II) or callose synthase], an enzyme that several reports have suggested consists of between six and nine different subunits. The procedure involves (a) preliminary removal of peripheral proteins by 0.1% digitonin; (b) solubilization of GS-II with 0.5% digitonin; (c) precipitation of activity-irrelevant proteins from the digitonin extract by Ca2+, spermine and cellobiose, which are GS-II effectors needed in step (d); (d) product entrapment by formation of 1,3-beta-D-glucan from UDP-Glc by GS-II in the presence of the mentioned effectors, followed by centrifugal sedimentation of product micelles and elution of proteins therefrom with buffer; (e) preparative isoelectric focusing (IEF) of product-entrapped proteins; and (f) glycerol gradient centrifugation of the fractions of peak GS-II activity from IEF. The procedure yields 300-fold enrichment of GS-II specific activity over that in isolated plasma membranes, and 5500-fold over that in the original homogenate. Out of approximately six principal polypeptides that occur after the product entrapment step, the glycerol gradient GS-II activity peak contains only two major polypeptides, one of 55 kDa and another of 70 kDa, plus minor amounts of one or two others whose distribution and occurrence indicate are not responsible for GS-II activity. Antisera against either the 55-kDa or the 70-kDa polypeptide adsorb more than 60% of the GS-II activity from a product-entrapped preparation. After native gel electrophoresis, GS-II activity is associated with a single protein band of very large molecular mass, whose principal components are the 55-kDa and 70-kDa polypeptides, accompanied by minor amounts of a few other polypeptides most of which do not occur in enzyme preparations purified by the previously described procedure. The 55-kDa but not the 70-kDa component can be labeled by ultraviolet irradiation of the plasma membranes in the presence of [alpha-32P]UDP-Glc under GS-II assay conditions. It seems likely, therefore, that the 55-kDa and 70-kDa polypeptides form a large catalytic complex of which the 55-kDa component is the UDP-Glc-binding subunit.

Limited proteolysis of (1,3)-beta-glucan (callose) synthase from Beta vulgaris L: topology of protease-sensitive sites and polypeptide identification using Pronase E.

Plasma membrane (PM) vesicles of defined sidedness were obtained from Beta vulgaris L. and subjected to limited proteolysis to investigate the topology and subunit composition of UDP-glucose: (1,3)-beta-glucan (callose) synthase (CalS). Latency experiments demonstrated that protease-sensitive sites on the CalS complex are located primarily at the cytoplasmic face of the PM, with little or no CalS inactivation occurring as the result of proteolysis at the apoplastic face. In the PM-bound form, CalS activity was resistant to inactivation by Pronase E, however at least four polypeptides previously implicated as possible CalS components (92, 83, 57 and 43 kDa) were extensively hydrolyzed. Polypeptides of 31, 29 and 27 kDa resisted Pronase E hydrolysis and were also enriched in CalS fractions purified by glycerol gradient centrifugation and product entrapment. In contrast to PM-bound CalS, purified CalS was rapidly hydrolyzed by Pronase E, indicating that most Pronase E-sensitive sites are deeply embedded within the PM. This study provides direct biochemical evidence that hydrophobic integral membrane proteins oriented primarily towards the cytoplasmic face of the PM are important for callose biosynthesis in Beta. Furthermore, these results form the basis of a biochemically derived working model largely consistent with morphologically derived models proposed for intramembrane PM-bound, microfibril-synthesizing complexes in higher plants.

Purification of 1,3-β-Glucan Synthase from Neurospora crassa by Product Entrapment

Awald, P., Zugel, M., Monks, C., Frost, D., and Selitrennikoff, C. P. 1993. Purification of 1,3-β-glucan synthase from Neurospora crassa by product entrapment. Experimental Mycology, 17, 130-141. 1,3-β-Glucan synthase activity of the ascomycete Neurospora crassa was purified ∼700-fold from hyphae. Hyphae were disrupted by bead-beating, and membrane-enriched fractions were obtained by high-speed centrifugation. Membranes were treated with (3-[(3-cholamidopropyl)dimethyl-ammoniol]I-propanesulfonate) and octyl-β-D-glucoside to solubilize enzyme activity. Soluble glucan synthase activity was incubated with substrate (UDP-glucose) and purified by centrifugation of enzyme associated with glucan (product entrapment). Purification was specific for UDP-glucose, the optimal concentration being 0.25 mM; no other nucleotide diphosphate sugar was able to significantly product-entrap enzyme activity. Partially purified enzyme activity formed β(1,3)-linked glucan, had a mean specific activity of 1900 nmol glucose incorporated/min/mg protein, a Km,app of 0.7 mM, and a Vmax of 0.5 nmol glucose incorporated/min. Separation of partially purified enzyme activity by SDS-PAGE showed a number of proteins copurifying with enzyme activity; computer analysis of digitized gel images revealed that proteins of 21, 25, 28, 45, 53, and 78 kDa were enriched. These results reinforce the view that 1,3-β-glucan synthase activity of fungi is a multimeric enzyme.

Beta]-Glucan Synthesis in the Cotton Fiber (III. Identification of UDP-Glucose-Binding Subunits of [beta]-Glucan Synthases by Photoaffinity Labeling with [[beta]-32P]5[prime]-N3-UDP-Glucose.

Using differential product entrapment and photolabeling under specifying conditions, we identifIed a 37-kD polypeptide as the best candidate among the UDP-glucose-binding polypeptides for the catalytic subunit of cotton (Gossypium hirsutum) cellulose synthase. This polypeptide is enriched by entrapment under conditions favoring [beta]-1,4-glucan synthesis, and it is magnesium dependent and sensitive to unlabeled UDP-glucose. A 52-kD polypeptide was identified as the most likely candidate for the catalytic subunit of [beta]-1,3-glucan synthase because this polypeptide is the most abundant protein in the entrapment fraction obtained under conditions favoring [beta]-1,3-glucan synthesis, is coincident with [beta]-1,3-glucan synthase activity, and is calcium dependent. The possible involvement of other polypeptides in the synthesis of [beta]-1,3-glucan is discussed.

Characterization and partial purification of β-1,3- d-glucan (callose) synthase from barley ( Hordeum vulgare) leaves

The plasma membrane bound β-1,3- d-glucan (callose) synthase, assumed to be involved in the resistance to the powdery mildew fungus ( Erysiphe graminis f.sp. hordei), was partially purified from a microsomal fraction of green barley leaves ( Hordeum vulgare L.). Plasma membranes were enriched by aqueous polymer two-phase partitioning of the microsomal fraction in a polyethylene glycol 3350/Dextran T-500 system. The plasma membrane bound callose synthase was dependent on uridine 5′diphosphate (UDP)-glucose ( K m 0.39 mM) and was activated by Ca 2+, digitonin, cellobiose and polyamines. The enzyme was inhibited by UDP and uridine 5′triphosphate (UTP). Glucanase digestion of the in vitro product showed that it was a β-1,3-linked polysaccharide. Two different procedures were used for further enrichment of polypeptides involved in callose synthase activity. Sucrose gradient centrifugation with concomitant product entrapment showed enrichment of four polypeptides with relative molecular masses ( M rs) of 36, 52, 66 and 170 kDa. Non-denaturing polyacrylamide gel electrophoresis (PAGE) separated the callose synthase from most of the other plasma membrane proteins. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the proteins in the callose activity stained zone revealed six dominant polypeptides with M rs of 36, 52, 54, 60, 70 an d94 kDa. The 36 and 52 kDa polypeptides were found by both methods suggesting that they could constitute true components of the barley leaf callose synthase. These results are in accordance with previous attempts to isolate the polypeptides involved in callose synthesis from dicot plants.