The silk protein, sericin, protects against cell death caused by acute serum deprivation in insect cell culture

Sericin is the silk protein that covers fibroin fibers and functions as a `glue' in the cocoons of silkworms, and its most abundant component, Ser1, contains repeats of Ser- and Thr-rich 38 amino acid residues. The viability of Sf9 insect cells was 20, 57 and 49% on the fifth day and 41, 91 and 70% on the ninth day after serum deprivation in the presence of no additives, 3000 g sericin hydrolysate and 350 g SerD (the peptide containing the two repetitive units) ml^–1, respectively. Thus, the sericin samples were useful in preventing cell death and promoting cellular growth after acute serum deprivation.     

Purification and characterization of trypsin from an entomopathogen,Nomuraea rileyi NRRL 13755

Nomuraea rileyi isolate NRRL-13755 produced a large amount of trypsin enzyme when cultured on basal salt medium containing 1% (w/v) gelatin. The trypsin was purified nearly 60-fold, with a recovery of about 13% of the initial activity from the culture supernatant. This protease exhibited a remarkably high specific activity of nearly 370,000 IU/mg protein. The native molecular weight was estimated by gel permeation chromatography to be 30 kDa, and the subunit molecular weight was determined to be about 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pH and temperature optima were determined to be 8.5 and 35°C, respectively. With a relative trypsin activity of 100%, this purified preparation showed about 10% chymoelastase and nearly 50% chymotrypsin activity. Metal-chelating agents such as EDTA and EGTA at 2mm inhibited the enzyme activity by 40%, whereas N-carbobenzoxy-glycyl-l-phenylalaninamide (CBZ-gly-phe-NH₂) (2mm) and DTT (2mm) had no effect on activity. Trypsin inhibitor from turkey egg-white at 100 g/ml strongly inhibited the enzyme activity.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Modulation of Rabbit Corneal Epithelial Cell Proliferation by Growth Factor-regulated K<Superscript>+</Superscript> Channel Activity

We characterized the dependence of the mitogenic response by rabbit corneal epithelial (RCE) cells to serum containing growth factors on K⁺ channel activation. Using both cell-attached and nystatin-perforated patch-clamp configurations, a K⁺ channel was identified whose current-voltage relationship is linear with a single-channel conductance of 31 pS. Its activity was barely detectable following 24 h serum starvation. Exposure of starved cells to either 10% FBS, 5 ng/ml epidermal growth factor (EGF) or 2 nM endothelin-1 (ET-1) continuously increased its activity within 30 min by 40%, 54% and 29%, respectively. EGF and ET-1 in combination had additive effects on such activity. Application of 100 µM 4-aminopyridine (4-AP), a K⁺ channel blocker, inhibited serum-stimulated K⁺ channel activity by 85%. DNA synthesis was markedly stimulated by serum, whereas incubation with either 4-AP (200 µM) or Ba²⁺ (1 mM) suppressed this increase by 51% and 23%, respectively, whereas 5 mM tetra ethyl ammonium (TEA) had no effect. Taken together, growth factor-induced increases in proliferation are dependent on K⁺ channel stimulation. As the increases in K⁺ channel activity induced by ET-1 and EGF were additive, these mitogens may stimulate K⁺ channel activity through different signaling pathways linked to their cognate receptors.     

Agaroids from New Zealand members of the Gracilariaceae (Gracilariales,Rhodophyta) — a novel dimethylated agar

Polysaccharide extracts from four New Zealand members of the Gracilariaceae have been characterized by ¹³C-NMR spectroscopy and GLC analysis of alditol acetate derivatives prepared using a new double hydrolysis-reduction procedure. All were based on variously substituted repeating disaccharide units of agarobiose and 20% of its precursor containing l-galactose-6-sulfate. Gracilaria truncata yielded a firm gelling agar with 67% methylation on the 6-position of the d-galactose residues. The other extracts belong to a new class of agar molecules having methylation on both the 6-position of the d-galactose units and the 2-position of the l-sugar units. The Curdiea coriacea polysaccharide displayed this double methylation almost completely ( 96 %); the alkali-modified polymer thus had only two free hydroxy-groups per disaccharide repeat unit, yet still gave a firm gel. The Curdiea flabellata and Melanthalia abscissa extracts had this double methylation pattern but to a lesser extent, and additional xylosyl branch units on up to 18% of the repeating disaccharide units.     

Characterization and delignification activity of a thermostable α-l-arabinofuranosidase from Bacillus stearothermophilus

Bacillus stearothermophilus L1 was isolated by enrichment culture using an alkaline extract of pulp as the carbon source at 65°C and pH 9.0. The bacterium produced extracellular xylanase and -l-arabinofuranosidase (EC 3.2.1.55). The xylanase activity was high when the cells were grown in the presence of d-xylose, whereas the arabinofuranosidase activity was high when grown in media containing l-arabinose. The arabinofuranosidase was purified 59-fold with an 80% yield by DEAE Sephacel and Sephadex G-100 chromatography. The purified enzyme had an apparent molecular mass of 110 000 kDa and consisted of two subunits of 52 500 kDa and 57 500 kDa. Using p-nitrophenyl--l-arabinofuranosidase as the substrate, the enzyme had a Michaelis constant (K _m) of 2.2 × 10^–4 m, maximum reaction velocity (V_max) of 11o mol min^–1 mg^–1, temperature optimum of 70°C and pH optimum of 7.0 (50% activity at pH 8.0). The enzyme was specific for the furanoside configuration. The purified enzyme partially delignified softwood Kraft pulp. Treatment of the pulp with 38 units ml^–1 of -l-arabinofuranosidase at 65°C for 2 h at pH 8.0 and 9.0 led to lignin releases of 2.3% and 2.1%, respectively. The enzyme acted synergistically with a thermophilic xylanase in the delignification process, yielding a 19.2% release of lignin. Correspondence to: Eugene Rosenberg     

A new histochemical method for the selective periodate oxidation of total tissue sialic acids

Summary Evaluation of the intensity of the periodic acid—Schiff (PAS) staining produced following oxidation for 1 h at 4°C with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that this reagent completely oxidized all available sialic acid residues of either the sialo- or sialosulphoglycoproteins of human and rat colon or the sialoglycoproteins of rat sublingual gland. These conditions produced no visible Schiff staining of either neutral macromolecules orvicinal diols located on hexose, 6-deoxyhexose orN-acetylhexosamine residues (neutral sugars) of sialo- and sialosulphoglycoproteins. Furthermore, there was no extraction of epithelial glycoproteins or de-O-acylation of side chain substituted sialic acid residues. These data demonstrate that 0.4mm periodic acid in approximately 1m hydrochloric acid can be used as a specific reagent for the selective visualization of sialic acids in the PAS procedure.Studies of the mechanism of the oxidation of neutral sugars with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that their lack of PAS reactivity was not due to the production of Schiff unreactive hemiacetals or hemialdals. It is suggested that the selectivity of 0.4mm periodic acid in approximately 1m hydrochloric acid is a result of an increase in the rate of the oxidation of the sialic acid residues together with a decrease in the rate of oxidation of neutral sugars.     

On bacteriophage anti-Flexner

Summary 1. The Flexner strain KB ofFlu from 1921 has given rise to two strains: the Flexner strain 38 which has grown SR and the Flexner strain 39 which has grown R. The Flexner strain 39 is at this moment a spontaneously agglutinating, lyso-sensitive strain, while the Flexner strain 38 apparently has kept up its original properties and is inagglutinable and lyso-resistant. Both strains belong to the type X ofAndrewes andInman. The Flexner strain 38, however, appears to agglutinate very specifically with an X serum. This strain can also be lysed by a special group of Flexner phage and not by others.2. The Flexner strain 38, however, is probably no variation of the strain KB ofFlu.3. The results ofBurnet andMcKie as to the correlation between antigenic structure of the Flexner strains and their sensitiveness to some groups of bacteriophage anti-Flexner could be confirmed in a high measure.     

Functional intracellular glutaminase activity in intact astrocytes

Numerous cellular metabolites such as glutamine, glutamate, phosphate, calcium, ammonia and acetyl derivatives are known to affect the phosphate-activated glutaminase activity in whole cell homogenates or extracts. Since measurements in extracts under non-physiological conditions may obscure the actual intracellular metabolic flux, the functional intracellular phosphate-activated glutaminase activity was measured by the formation of³H₂O froml-[2-³H]glutamine (Anal. Biochem. 127:134–142, 1982) in cultures of intact astrocytes, untreated and treated with dibutyryl c-AMP (DiBcAMP), in the presence of several potential effectors. These values were compared with enzyme levels determined in extracts from identical cells. The rate of¹⁴CO₂ release froml-[1-¹⁴C]glutamine was also measured in both untreated and DiBcAMP treated astrocytes. The intracellular activity of glutaminase for untreated cells assayed in MEM medium with 1mM radioactive glutamine was 88 nmol/mg protein/h and in DiBcAMP treated cells the rate was 153 nmol/mg protein/h. However, the enzymatic activity measured under optimal conditions in extracts from both untreated and treated cells was much higher, but essentially the same, about 1,750 nmol/mg protein/h. The rate of¹⁴CO₂ release froml-[1-¹⁴C]glutamine was 74 and 133 nmol/mg protein/h in untreated and DiBcAMP treated cells, respectively. This represents approximately 85% of the intracellular glutaminase activity. Furthermore, increasing the concentration of glutamine in the medium from 1 to 6.4 mM increased glutaminase intracellular activity about 3 fold in both untreated and treated cells. Addition of 250 M glutamate to the medium inhibited intracellular glutaminase activity by 70% under both treatment conditions. Deletion of glucose stimulated glutaminase activity. In contrast the removal of fetal bovine serum decreased activity by 35%. The addition of 10 mM phosphate and the alpha keto acids of isoleucine and valine marginally increased intracellular glutaminase activity. The addition of 0.4 mM ammonium chloride to the medium had no effect. An increase in media pH from 6.8 to 7.7 increased intracellular glutaminase activity almost 2 fold. These results provide evidence that phosphate-activated glutaminase activity in vivo is regulated by cellular metabolites, that its functional activity is 5–9% of the rate obtained using extracts, and this functional activity is sufficient to account for the rate of glutamine oxidation.Special issue dedicated to Dr. Elling Kvamme     

The pyridine nucleotide and non-pyridine nucleotide dependence of L-glutamate dehydrogenase in the histochemical system

Summary Under histochemical conditions (fresh frozen sections from liver, kidney and cerebellum of the rat) it was shown that the oxidation of L-glutamic acid was carried out by the NAD-dependent L-glutamate dehydrogenase (E.C. 1.4.1.2) and/or the NAD- or NADP-dependent L-glutamate dehydrogenase (E.C. 1.4.1.3) as well as by an enzyme system which is not dependent on externally added NAD, NADP, FAD, FMN or CoQ₁₀ for activity.This non-pyridine dependent activity was related to the L-glutamate dehydrogenases proper, owing to the following: a) the localization of activity noticed corresponds to that obtained with the NAD- or NADP-containing media, b) the incubation time needed for initial formation of red and blue formazans is similar to that with coenzyme-containing media, c) pre-extraction experiments reveal similarity in enzyme diffusion rates, d) the named activity is influenced by the same agents and to the same extent as the activity obtained by the inclusion of NAD or NADP (e.g. dissociation of the dehydrogenase molecule into subunits due to urea, inhibition of activity due to N-ethyl maleimide and 1.10-phenanthroline, activation due to the allosteric effect of ADP and to high substrate concentration, allosteric inhibition caused by GTP and inhibition caused by -ketoglutaric acid, no inhibitory effect of KCN), and e) the named activity was not affected by added PMS (excluding activity due to L-aminoacid oxidase).In the in situ localization of enzyme activity it was found that L-glutamate dehydrogenases E.C. 1.4.1.2 and E.C. 1.4.1.3 co-exist in the cells of kidney and cerebellum, while the L-glutamate dehydrogenase E.C. 1.4.1.3 only was present in liver cells.Finally, it was stated that incubation time should be kept as short as possible in order to avoid Nothing dehydrogenase reaction as well as inhibition due to accumulation of -ketoglutaric acid. Only gel incubation media should be applied.Recipient of a research grant from the Danish Ministry of Education     

Lectin binding by microvillous membranes and coated-pit regions of human syncytial trophoblast

Summary The distribution of saccharides on the microvillous membrane of the human syncytial trophoblast was investigated using ferritin conjugates of four lectins: concanavalin A (specific for -d-manno- and -d-glucopyranosyl residues), wheatgerm agglutinin (specific forN-acetylglucosamine),Limulus polyphemus lectin (specific forN-acetylneuraminic acid), andLotus tetragonolobus lectin (specific for -l-fucose). Concanavalin A and wheatgerm agglutinin (WGA) reacted strongly with the surface membrane and ferritin deposits were also observed in coated pit regions of the membrane. Lectins fromL. polyphemus andL. tetragonolobus, however, reacted only weakly with the microvillous border and neither reacted with coated pits.Enhanced agglutinability of trophoblast cells in comparison with other foetal cells from the same conceptus was seen with WGA. This agglutination was inhibited by addition of acetylglucosamine or by a solubilized membrane fraction which was bound by a column of WGA-Sepharose. The membrane fraction which did not bind to the column did not inhibit agglutination. Electrophoresis of the WGA-bound membrane proteins revealed six subunits, the major band having an apparent mol. wt. of 55 000. A protein of this mol. wt was also seen in coated vesicles isolated from equivalent human placentae.     

Effect of cell-wall-digesting enzymes on physiological state and competence of maize coleoptile cells

Summary Protoplasts are frequently isolated from maize coleoptiles with cell-wall-degrading enzymes such as pectolyase (PEC), mazerozyme, and cellulase. Incubation of coleoptiles with these enzymes caused rapid depolarizations of the membrane voltage (V _M ). The depolarizing effect of 0.5% (w/v) mazerozyme or 1.5% (w/v) cellulase was unaffected by denaturation of the enzymes. In the case of pectolyase (0.1%, w/v), however, the active enzyme was significantly more potent than the denaturated enzyme in depolarizing coleoptile cells. Exposure to 0.1% active PEC but not to inactive PEC also caused an oxidative burst in coleoptiles and enhanced K⁺ efflux. Together this suggests that pectic breakdown products of the cell wall act as signal for wounding. Typically addition of 10 M 1-naphthylene acetic acid (NAA) to coleoptiles causes a transient depolarization followed by a slow hyperpolarization of V _M . However, in the presence of PEC, V _M only depolarized in NAA. After PEC-treated coleoptiles were washed free of the enzyme, NAA caused only small fluctuations of V _M . A similarly small V _M response to NAA appeared in coleoptiles pretreated with heatdenaturated supernatant (SUP) from a protoplast isolation buffer, the latter suspected to contain the PEC-generated wounding signal. Comparable pretreatment of coleoptiles with PEC or SUP had no significant effect on the spontaneous and NAA-evoked acidification of the incubation medium. Pretreatment with SUP also had no significant effect on the NAA-stimulated elongation of coleoptile segment. Hence, PEC treatment of coleoptile tissue affects the membrane transport properties of the cells. This effect is partly maintained after removal of the enzyme from the incubation medium, an effect not significant for NAA-generated acidification and cell elongation.Abbreviations V _M membrane voltage - V_red redox voltage - PEC pectolyase - SUP supernatant from cell wall digestion - NAA 1-naphthylene acetic acid     

Betulinic and oleanolic acids isolated from Forsythia suspensa Vahl inhibit urease activity of Helicobacter pylori

Sixteen medicinal herbs were selected from a database on traditional herbal materials as well as literature on Korean plant resources. Then ethanol (70%, v/v) extracts of these herbs were tested for inhibition of the urease activity of Helicobacter pylori. The urease activity of H. pylori was strongly (82%) inhibited by extract of Forsythia suspensa Vahl. Active compounds in extract of Forsythia suspensa Vahl were first separated by batch mode solvent extraction, followed by purification by silica gel and octadecyl silica gel column chromatography using solvents of different polarity. According to NMR analysis of the last chromatographic fraction, we identified the presence of betulinic acid and oleanolic acid, which are known to have anti-inflammatory, anti-cancer, and anti-HIV viral activities.     

Enzymatic synthesis of α-2-deoxyglucosyl derivatives catalyzed by organic solvent-resistant α-glucosidase

Organic solvent-resistant Aspergillus niger α-glucosidase (ANGase) can synthesize α-2-deoxyglucosyl derivatives (2DDs) in water-organic solvent media by a trans-addition reaction from d-glucal to various acceptors. Herein, we studied the influence of four different solvents on ANGase stability and activity. ANGase exhibited 47 or 43% residual activity following incubation in 50% (v/v) or in 70% (v/v) acetone for 4 h, respectively. When various carbohydrates were used as acceptor molecules, ANGase catalyzed the addition reaction of four different sugar alcohols, glucose, sucrose, or trehalose to d-glucal. Among the acceptor molecules tested, xylitol was the best acceptor by producing the highest yield (87% addition). The concentration of acetone/acceptor influenced the formation of 2DDs and the yields. We confirmed the molecular weight of five kinds of products by mass spectrometry and enzymatic hydrolysis. Current method is useful for the production of carbohydrates containing 2-deoxyglucose moiety.     

Na+/K+/Cl− cotransport in cultured vascular smooth muscle cells: Stimulation by angiotensin II and calcium ionophores,inhibition by cyclic AMP and calmodulin antagonists

Summary The specific activity of the Na⁺/K⁺/Cl^– cotransporter was assayed by measuring the initial rates of furosemide-inhibitable⁸⁶Rb⁺ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 m, respectively. The dependence of furosemide-inhibitable⁸⁶Rb⁺ influx on external Na⁺ and K⁺ was hyperbolic with apparentK _m values of 46 and 4mm, respectively. The dependence on Cl^– was sigmoidal. Assuming a stoichiometry of 112 for Na⁺/K⁺/Cl^–, aK _m of 78mm was obtained for Cl^–. In quiescent smooth muscle cells cotransport activity was approximately equal to Na⁺ pump activity with each pathway accounting for 30% of total⁸⁶Rb⁺ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na⁺ pump activity was not significantly different in the gorwing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores, A23187 and ionomycin. The removal of external Ca²⁺ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited⁸⁶Rb⁺ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.     

Regulation of aspartokinase activity in non-fermentative,marine eubacteria

Summary Cell-free extracts of gram-negative, non-fermentative, marine eubacteria were assayed for aspartokinase activity. The organisms tested included polarly flagellated species and groups which had GC contents in their DNAs of 46 to 64 moles % (Alteromonas, Pseudomonas) as well as species which had peritrichous flagellation and moles % GC contents of 53 to 68 (Alcaligenes). The results of these studies suggested that in all the strains tested, aspartokinase activity was catalyzed by a single enzyme. On the basis of the effect ofl-threonine,l-lysine,l-methionine, andl-isoleucine on activity, five different types of aspartokinases (designated I through V) were delineated. In aspartokinase types I through IV,l-threonine andl-lysine inhibited activity by means of a concerted feedback inhibition; in type V, activity was inhibited byl-threonine but unaffected byl-lysine. In types I, III, and IV,l-threonine andl-lysine alone were inhibitory, while in type II these effectors had virtually no effect on activity when tested singly. Three distinct responses were observed in the presence of two other end products of the aspartate pathway,l-methionine andl-isoleucine. In types I and II, these two amino acids usually stimulated activity and overcame the inhibition byl-threonine andl-lysine; in types IV and V,l-methionine andl-isoleucine had no effect; and in type III these amino acids inhibited activity. The results of this study indicate that the aspartokinases of a number of species and groups of marine bacteria have similarities and differences which should be of use in making future taxonomic groupings.     

Arabitol accumulation in Geotrichum candidum

Culture conditions which lead to the intracellular accumulation of arabitol and mannitol in Geotrichum candidum were investigated. The accumulation of arabitol was dependent on the concentrations of metabolizable hexoses, the non-metabolizable disaccharide sucrose, NaCl and KCl in the growth medium. In media containing 2% (w/v) glucose, fructose or l-sorbose cultures contained only mannitol after 48 h or 72 h growth. In media containing 10% (w/v) to 30% (w/v) glucose, or 25% (w/v) fructose or l-sorbose there was an increase in the total concentration of intracellular polyol due to the accumulation of arabitol. This pentitol was also found to accumulate intracellularly when the organism was grown in medium containing 34% (w/v) sucrose, 0.7 M NaCl or 0.7 M KCl in addition to 2% (w/v) glucose. Under the conditions tested no change in the accumulation of mannitol or ethanol-soluble carbohydrate, believed to be primarily composed of trehalose, was evident.Intracellular polyol was released during incubation of arthrospores obtained from media containing 25% or 10% glucose, in distilled water at 25° C, but no polyol was released under these conditions from arthrospores obtained from growth in 2% glucose medium.     

Gene cloning and overproduction of low-specificity d-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for parkinsonism drug

The dtaAX gene encoding a pyridoxal 5′-phosphate (pyridoxal-P)-dependent low-specificity d-threonine aldolase was cloned from the chromosomal DNA of Alcaligenes xylosoxidans IFO 12669. It contains an open reading frame consisting of 1,134 nucleotides corresponding to 377 amino acid residues. The predicted amino acid sequence displayed 54% identity with that of d-threonine aldolase from gram-positive bacteria Arthrobacter sp. DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes. This gram-negative bacterial enzyme was highly overproduced in recombinant Escherichia coli cells, and the specific activity of the enzyme in the cell extract was as high as 18 U/mg (purified enzyme 38.6 U/mg), which was 6,000 times higher than that from the wild-type Alcaligenes cell extract. The recombinant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography steps. The recombinant low-specificity d-threonine aldolase was shown to be an efficient biocatalyst for resolution of l-β-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinson's disease. Received: 9 September 1999 / Received revision: 1 November 1999 / Accepted: 12 November 1999     

A new arabinofuranohydrolase from Bifidobacterium adolescentis able to remove arabinosyl residues from double-substituted xylose units in arabinoxylan

An arabinofuranohydrolase (AXH-d3) was purified from a cell-free extract of Bifidobacterium adolescentis DSM 20083. The enzyme had a molecular mass of approximately 100 kDa as determined by gel filtration. It displayed maximum activity at pH 6 and 30 °C. Using an arabinoxylan-derived oligosaccharide containing double-substituted xylopyranosyl residues established that the enzyme specifically released terminal arabinofuranosyl residues linked to C-3 of double-substituted xylopyranosyl residues. In addition, this arabinofuranohydrolase released arabinosyl groups from wheat flour arabinoxylan polymer but showed no activity towards p-nitrophenyl α-l-arabinofuranoside or towards sugar-beet arabinan, soy arabinogalactan, arabino-oligosaccharides and arabinogalacto-oligosaccharides. Received: 15 July 1996 / Received revision: 18 October 1996 / Accepted: 18 October 1996     

α-d-Galactosylation of surface fucoglycoconjugate(s) upon stimulation/activation of murine peritoneal macrophages

Murine resident macrophages express, on their surface, carbohydrate epitopes which undergo changes during their stimulation/activation as monitored by binding of¹²⁵I labelledEvonymus europaea andGriffonia simplicifolia I-B₄ lectins. Treatment of the stimulated macrophages with coffee bean -galactosidase abolished binding of the GS I-B₄ isolectin and changed the binding pattern of theEvonymus lectin. The affinity (K _a) ofEvonymus lectin for -galactosidase-treated macrophages decreased approximately 23-fold, from 1.25×10⁸ M^–1 to 5.5×10⁶ M^–1. Subsequent digestion of -galactosidase-treated macrophages with -l-fucosidase fromTrichomonas foetus, further reduced binding ofEvonymus lectin. Resident macrophages showed the same pattern ofEvonymus lectin binding, with the same affinity, as -galactosidase-treated, stimulated macrophages. These results, together with a consideration of the carbohydrate binding specificity of theEvonymus lectin which, in the absence of -d-galactosyl groups, requires -l-fucosyl groups for binding, indicate the presence, on resident macrophages, of glycoconjugates with terminal -l-fucosyl residues. It is also concluded that during macrophage stimulation/activation -d-galactosyl residues are added to this glycoconjugate and that they form part of the receptor forEvonymus lectin. The same glycoconjugate(s) is/are also expressed on the activated macrophage IC-21 cell line which exhibits the same characteristics as that of stimulated peritoneal macrophages, i.e., it contains -d-galactosyl end groups and is resistant to the action of trypsin. Both lectins were also specifically bound toCorynaebacterium parvum activated macrophages.Abbreviations BSA bovine serum albumin - GS I-B₄ Griffonia simplicifolia I-B₄ isolectin - PBS 0.01m phosphate buffer (pH 7.1) with 0.15m NaCl (unless stated otherwise this buffer contained 3mm azide and was free of divalent cations) - PMSF phenyl methane sulfonyl fluoride - TG thioglycollate brewers medium.     

Purification and characterization of sialidase fromClostridium sordellii G12

Sialidase secreted by the urease-positiveClostridium sordellii strain G12 was isolated from culture medium and purified to apparent homogeneity as estimated by Fast Protein Liquid Chromatography (FPLC) and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). For this purpose, ion-exchange chromatography, gel filtration, isoelectric focusing, and FPLC on ion-exchange resin and gel filtration materials were used. The sialidase was purified 159 300-fold from 5 l of culture medium, yielding 9 g of enzyme protein with a specific activity of 480 U/mg. For the denatured (SDS-PAGE) and native (FPLC) sialidase relative molecular masses of 40 000 and 38 500 Da, respectively, were estimated. The substrate specificity, kinetic data, and pH-optimum of the enzyme are similar to those of other bacterial sialidases. The influences of salt or serum proteins on enzyme activity are of interest.Abbreviations MU-Neu5Ac 4-methylumbelliferyl -d-N-acetylneuraminic acid - Ganglioside GD1a IV³NeuAc, ll³NeuAc-GgOse₄Cer - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid