Selective and continuous degradation of carbazole contained in petroleum oil by resting cells of Sphingomonas sp. CDH-7.

Microbial degradation of carbazole (CA), a model of hard-removal heterocyclic nitrogen compounds contained in petroleum oil, was examined using Sphingomonas sp. CDH-7 isolated from a soil sample by screening for CA-assimilating microorganisms. CDH-7 used CA as a sole source of carbon and nitrogen, and metabolized CA to ammonia via anthranilic acid as an intermediate product. When CDH-7 was cultivated in the medium containing CA at the concentration of 500 mg/l (2.99 mM), CA was completely degraded within 50 h. By the reaction with the resting cells of CDH-7, 500 mg/l of CA was completely degraded within 4 h, with 1.64 mM of ammonia accumulated in the reaction mixture. When CA was added at the concentration of 100 mg/l (0.599 mM) periodically to the reaction mixture ten times, 925 mg/l (5.54 mM) of CA was degraded within 48 h by the resting cells, and 4.50 mM of ammonia was accumulated in the reaction mixture with a 75.1% molar conversion yield based on total CA added. The resting cells could almost completely degrade CA in a two-liquid-phase system which consists of water and organic solvent, even in the presence of 20% (v/v) isooctane, n-hexane, cyclohexane, and kerosene as a model petroleum oil. In the presence of an organic solvent system such as 20% (v/v) pxylene, toluene, and heptanol, however, CA degradation yields decreased.     

Orthophosphate-pyrophosphate exchange catalyzed by soluble and membrane-bound inorganic pyrophosphatases. Role of H+ gradient

A comparative study of the orthophosphate-pyrophosphate exchange reaction catalyzed by the soluble pyrophosphatase from baker's yeast and by the membrane-bound pyrophosphatase of Rhodospirillum rubrum chromatophores was performed. In both systems the rate of exchange increased when the pH of the medium was raised from 6.0 to 7.8 and when the MgCl2 concentration was raised from 0.1 mM to 20 mM. For the yeast pyrophosphatase the exchange rates measured at different pH values and in the presence of 6.7 to 8.8 mM free Mg2+ superimposed as a single curve when plotted as a function of the concentrations of either HPO4(2-) or MgHPO4. This was not observed with the use of R. rubrum chromatophores. With yeast pyrophosphatase, the Km for Pi was higher than 10 mM and could not be measured when the free Mg2+ concentration in the medium was lower than 0.5 mM. There was a decrease in the Km for Pi when the free Mg2+ concentration was raised to 6.7-8.8 mM or when, in the presence of low free Mg2+, the organic solvents dimethylsulfoxide (20% v/v) or ethyleneglycol (40% v/v) were included in the assay medium. In the presence of 6.7-8.8 mM free Mg2+ the Km for total Pi was 7 mM at pH 7.0 and 12 mM at pH 7.8. For the ionic species HPO4(2-) and MgHPO4, the Km values were 5.8 mM and 4.2 mM respectively. In the presence of 0.24-0.42 mM free Mg2+ and either 20% (v/v) dimethylsulfoxide or 40% (v/v) ethyleneglycol the Km values for total Pi, HPO4(2-) and MgHPO4 were 7.6, 3.5 and 0.5 mM respectively. With R. rubrum chromatophores, the Km for Pi in the presence of 5.5-7.5 mM free Mg2+ was very high and could not be measured. In the presence of 0.24-0.45 mM free Mg2+ the ratio between the velocities of hydrolysis and synthesis of pyrophosphate measured at pH 7.8 with yeast pyrophosphatase and chromatophores of R. rubrum were practically the same. When the free Mg2+ concentration was raised to 5.5-8.8 mM this ratio decreased from 1028 to 540 when the yeast pyrophosphatase was used and from 754 to 46 when chromatophores were used.     

Adenosine triphosphatases associated with bovine brain microtubules. II. Properties of ATPases I and II

The catalytic properties of two ATPases which had been purified from bovine brain microtubules (Tominaga, S. & Kaziro, Y. (1983) J. Biochem. 93, 1085-1092) were studied. ATPase I, which had a molecular weight of 33,000, required the presence of 1.0 microM tubulin, 0.2 mM Mg2+, and 10 mM Ca2+ for maximal activity. The activation of ATPase I by tubulin was specific to the native form of tubulin, which could not be replaced by F-actin or tubulin denatured either by heat or more mildly by dialysis in the absence of glycerol. ATPase I was not specific to ATP, and GTP, and to a lesser extent, UTP and CTP were also hydrolyzed. Km for ATP of ATPase I was about 0.04 mM. ATPase I was inhibited by 5 mM Mg2+, 0.04 M K+, 10(-3) M vanadate, 10 mM N-ethylmaleimide, or 20% (v/v) glycerol. ATPase II, which was associated with membrane vesicles, required the presence of 0.2-2.0 mM Mg2+ and 20 mM KCl for activity. Tubulin stimulated the reaction of ATPase II only partially, and the addition of Ca2+ was rather inhibitory. ATPase II was specific to ATP with a Km value of 0.14 mM. It was inhibited by 1.6 mM N-ethylmaleimide and 20% (v/v) glycerol, but was not very sensitive to vanadate. Instead, ATPase II was inhibited by trifluoperazine, chlorpromazine, and nicardipin at 10(-3) M.     

Inorganic phosphate effect on alternate peripheral pathways of glucose catabolism in Pseudomonas cepacia

Abstract The effect of the inorganic phosphate concentration on the activity of the enzyme of alternate peripheral pathways of glucose catabolism was studied in Pseudomonas cepacia ATCC 17759. Growth with low glucose concentration (0.5% w/v) and 20 mM phosphate resulted in induced levels of the phosphorylative pathway enzymes when compared with the levels of these same enzymes in high glucose concentration (2% w/v). However, an expansion of the oxidative pathway was detected during growth with 0.5% (w/v) of glucose and high phosphate concentration (160 mM). Moreover, under high phosphate (160 mM) and high glucose (2% w/v) growth conditions, glucokinase activity was increased preferentially relative to levels of direct oxidative pathway enzymes.     

The synthetic rate of dipeptide catalyzed by organic solvent-stable protease from Pseudomonas aeruginosa PST-01 in the presence of water-soluble organic solvents

The initial synthetic rates of peptide Cbz-Arg-Leu-NH(2) from Cbz-Arg and Leu-NH(2) using PST-01 protease in the presence and absence of organic solvents were investigated under various conditions. The synthetic rates of Cbz-Arg-Leu-NH(2) in the presence of 50% (v/v) methanol, 50% (v/v) N,N-dimethylformamide (DMF) and 60% (v/v) dimethyl sulfoxide (DMSO) were 1.6-, 2.4-, and 5.1-times higher than that in the absence of organic solvent, respectively. The PST-01 protease was not only stable in the presence of organic solvents but also exhibited high reaction rates in the presence of methanol, DMF, and DMSO. When the Cbz-Arg concentration was lower than 60mM or the Leu-NH(2) concentration was lower than 400mM, the initial rates increased lineally with increase in their concentrations. However, the rates did not increase when the Leu-NH(2) concentration was more than 500mM. The optimum temperature and pH of the reaction were 40 degrees C and 7.0, respectively.     

Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts

Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for β-galactosidase activity, were used for the synthesis of 1-hexyl-β- -galactoside and 1-octyl-β- -galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor. The product yield was strongly influenced by the initial water content in the reaction mixture. Water content of 10% (v/v) was optimal providing 3.6–36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis. Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level. Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis. Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26%. The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50 mM lactose for at least a week.     

Effects of glutamine, proline, histidine and betaine on post-thaw motility of stallion spermatozoa

The supplementation of the freezing diluent with 3 amino acids (glutamine, proline and histidine) and 1 amino acid-related compound (betaine) in preserving stallion spermatozoa diluted in INRA82 extender containing 2.5% (v/v) glycerol and 2% (v/v) egg yolk (control extender) during freezing and thawing was studied at 0, 40, 80, 120 and 160 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 1). Glutamine and proline were studied at 0, 10, 20, 30, 40, 50, 60, 70 and 80 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 2). In each experiment, spermatozoa were evaluated after thawing by computer automated sperm analyzer. The percentage of motile spermatozoa (faster than 30 microns/sec) was assessed. In addition, the velocity of the average path (VAP), the straight line velocity (VSL), the curvilinear velocity (VCL) and the amplitude of the lateral head displacement (ALH) were also measured. In Experiment 1, only glutamine (40 mM) significantly improved sperm motility (56.0% +/- 3.0 vs 49.7% +/- 1.6; P < 0.05) compared with the control extender, while velocities were unaffected at concentrations of 40 to 120 mM. However, at 160 mM, a significant decrease in motility and velocity was observed for all amino acids. In Experiment 2, motility in glutamine (range 41.1% +/- 3.8%; 42.4% +/- 3.6) and proline (43.0% +/- 3.7; 45.6% +/- 3.8) extenders compared with the control (34.7% +/- 1.6) was improved significantly (P < 0.05). Sperm velocity was improved at concentrations higher than 40 mM glutamine and 50 mM proline.     

Enantioselective production of 2,2-dimethylcyclopropane carboxylic acid from 2,2-dimethylcyclopropane carbonitrile using the nitrile hydratase and amidase of Rhodococcus erythropolis ATCC 25544

The enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid was investigated in 53 Rhodococcus and Pseudomonas related strains. Rhodococcus erythropolis ATCC 25544 was selected as it showed the highest enantioselectivity. The enantioselectivity was due to the amidase activity in a two-step reaction involving nitrile hydratase. The enantiomeric excess of the amidase was highest at pH 7.0 and decreased significantly above 20 °C. For the enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid, the optimum reaction conditions of the cells were determined to be pH 7.0, 20 °C, and 10% (v/v) methanol and were the same as the optimum pH and temperature for the enantioselective conversion by the amidase. Under these conditions, the R. erythropolis ATCC 25544 cells, which harbored nitrile hydratase and amidase enzymes, produced 45 mM (S)-2,2-dimethylcyclopropane carboxylic acid from racemic 100 mM 2,2-dimethylcyclopropane carbonitrile with an 81.8% enantiomeric excess after 64 h.     

Efficient enantioselective hydrolysis of D,L-phenylglycine methyl ester catalyzed by immobilized Candida antarctica lipase B in ionic liquid containing systems

Immobilized Candida antarctica lipase B (Novozym 435)-catalyzed enantioselective hydrolysis of D,L-phenylglycine methyl ester to enatiopure D-phenylglycine was successfully conducted in the systems with ionic liquids (ILs). Novozym 435 exhibited excellent activity and enantioselectivity in the system containing the IL BMIMxBF(4) compared to several typical organic solvents tested. It has been found that the cations and, particularly, the anions of ILs have a significant effect on the reaction, and the IL BMIMxBF(4), which shows to be the most suitable for the reaction, gave the highest initial rate and enantioselectivity among various ILs examined. The reaction became much less active and enantioselective in the systems with BMIMxHSO(4). Also, it was noticed that the enzymatic hydrolysis was strongly dependent on BMIMxBF(4) content in the co-solvent systems and the favorable content of the IL was 20% (v/v). Of the assayed four co-solvents and phosphate buffer, the lowest apparent K(m) and activation energy, and the highest V(max) of the reaction were achieved using 20% (v/v) BMIMxBF(4) co-solvent with phosphate buffer. Additionally, various influential variables were investigated. The optimum pH, substrate concentration, reaction temperature and shaking rate were 8.0, 80mM, 25-30 degrees Celsius and 150rpm, respectively, under which the initial rate, the residual substrate e.e. and the enantioselectivity were 2.46mM/min, 93.8% (at substrate conversion of 53.0%) and 38, respectively. When the hydrolysis was performed under reduced pressure, the initial rate (2.64mM/min) and the enantioselectivity (E=43) were boosted.     

Unusual cell adhesion and antithrombogenic behavior of citric acid-cross-linked collagen matrices

We have developed a novel biodegradable matrix that has unusual cell adhesion and antithrombogenic properties. The prepared biodegradable matrix was alkali-treated collagen (AlCol) cross-linked with citric acid derivative (CAD), named as AlCol-CAD. The swelling ratio of AlCol-CAD decreased with increasing CAD concentration, but with a further increase of the CAD concentration, the swelling ratio of AlCol-CAD also began to increase; this behavior showed a point where the swelling ratio reached a minimum value before increasing. The highest shrinkage states of 7.5%, 15%, and 30% (w/v) in AlCol-CAD were observed at CAD concentrations of 10, 20, and 40 mM, respectively, and moreover, the residual amino groups in AlCol-CAD were found to decrease with increasing CAD concentration. On the other hand, increases in carboxyl groups of 7.5% and 15% (w/v) in AlCol-CAD were found at CAD concentrations higher than 10 and 20 mM, respectively, whereas, at CAD concentrations from 10 to 40 mM, no significant change of the carboxyl groups was observed in 30% (w/v) AlCol-CAD. Human umbilical vein endothelial cell (HUVEC) adhesion on 15% (w/v) AlCol-CAD increased with increasing CAD concentration up to 20 mM and then slightly decreased. In the case of 30% (w/v) AlCol-CAD, the number of adhered HUVECs on AlCol-CAD increased with increasing CAD concentration. Furthermore, it was observed that HUVECs had excellent cell proliferation on 15% (w/v) AlCol-CAD at CAD concentrations of 20 and 40 mM, after incubation for 7 days. No thrombus formation was observed on 15% (w/v) AlCol-CAD at CAD concentrations above 20 mM. These results suggested that the 15% (w/v) AlCol-CAD at CAD concentrations above 20 mM has both HUVEC adhesion and antithrombogenic properties.     

Enzymatic production of pyruvate from fumarate — an application of microbial cyclic-imide-transforming pathway

For the purpose of producing pyruvate from fumarate through microbial cyclic-imide-transforming pathway, various cyclic-imide-utilizing microorganisms were isolated from soil. Among them, strain g31 was the best producer and was identified as Pseudomonas sp. With the resting cells of the strain, the conditions were optimized for pyruvate production from fumarate. The cells cultivated in the medium containing 2% (w/v) of fumarate showed the highest production with sufficient yield. The optimized wet-cell concentration, pH and temperature of the reaction were 1% (w/v), pH 6 to 7, and 30°C, respectively. Aeration was found to be an effective factor, and vigorous shaking during the reaction mixture resulted in higher production. Under the optimized reaction conditions, 100 mM of fumarate was almost stoichiometrically converted into pyruvate (94 mM) in 24 h.     

Enzymatic enantioselective transcyanation of silicon-containing aliphatic ketone with (S)-hydroxynitrile lyase from Manihot esculenta

(S)-Hydroxynitrile lyase from Manihot esculenta (MeHNL) was shown for the first time to be able to catalyze the enantioselective transcyanation of acetyltrimethylsilane (ATMS) with acetone cyanohydrin to form (S)-2-trimethylsilyl-2-hydroxyl-propionitrile in an aqueous/organic biphasic system. To better understand the reaction, various influential variables were examined. The most suitable organic phase, optimal buffer pH, aqueous phase content, shaking rate, temperature, concentration of ATMS, acetone cyanohydrin and crude enzyme were diisopropyl ether (DIPE), 5.4, 13% (v/v), 190 rpm, 40°C, 10 mM, 20 mM, and 35 U/ml, respectively, under which the initial reaction rate, substrate conversion and product enantiomeric excess (e.e.) were 19.5 mM/h, 99.0% and 93.5%, respectively. A comparative study demonstrated that silicon atoms in the substrate had a great effect on the reaction, and that ATMS was a much better substrate for MeHNL than its carbon analogue 3,3-dimethyl-2-butanone (DMBO) with respect to the initial reaction rate, substrate conversion and product e.e. MeHNL has greater affinity towards ATMS than its carbon analogue as indicated by the much lower K_m. The activation energy of MeHNL-catalyzed transcyanation of ATMS was also markedly lower than that of DMBO. The silicon effect on the reaction was rationalized on the basis of the special characteristics of silicon atoms and the catalytic mechanism of MeHNL.     

Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts

Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for beta-galactosidase activity, were used for the synthesis of 1-hexyl-beta-d-galactoside and 1-octyl-beta-d-galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor. The product yield was strongly influenced by the initial water content in the reaction mixture. Water content of 10% (v/v) was optimal providing 3.6-36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis. Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level. Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis. Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26%. The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50mM lactose for at least a week.     

An assay method for ganglioside synthase using anion-exchange chromatography

A rapid procedure which is based on combined ion-exchange chromatography and solubility was established for determination of the activity of ganglioside synthases and cerebroside sulfotransferase. The procedure consists of selective elution of radiolabeled reaction products (acidic glycolipids) freed from labeled precursors and breakdown products on a DEAE-Sephadex column and of direct radioassay of the products in the eluate. Monosialogangliosides were eluted from the column with 40 mM ammonium acetate (AcONH4) in methanol, cerebroside sulfate with 90 mM AcONH4 in methanol, and disialogangliosides with 40 mM AcONH4 in isopropanol/n-hexane/water (55/20/19, v/v/v). The established procedure is simple, reproducible, and economical. Using rat Golgi membrane as enzyme source the recovery rate of the products was over 95%.     

Novel reversible indole-3-carboxylate decarboxylase catalyzing nonoxidative decarboxylation

After enrichment culture with indole-3-carboxylate in static culture, a novel reversible decarboxylase, indole-3-carboxylate decarboxylase, was found in Arthrobacter nicotianae FI1612 and several molds. The enzyme reaction was examined in resting-cell reactions with A. nicotianae FI1612. The enzyme activity was induced specifically by indole-3-carboxylate, but not by indole. The indole-3-carboxylate decarboxylase of A. nicotianae FI1612 catalyzed the nonoxidative decarboxylation of indole-3-carboxylate into indole, and efficiently carboxylated indole and 2-methylindole by the reverse reaction. In the presence of 1 mM dithiothreitol, 50 mM Na2 S2O3, and 20% (v/v) glycerol, indole-3-carboxylate decarboxylase was partially purified from A. nicotianae FI1612. The purified enzyme had a molecular mass of approximately 258 kDa. The enzyme did not need any cofactor for the decarboxylating and carboxylating reactions.     

The role of hydration in enzyme activity and stability: 1. Water adsorption by alcohol dehydrogenase in a continuous gas phase reactor

The enzymatic synthesis of the tripeptide derivative Z-Gly-Trp-Met-OEt is reported. This tripeptide is a fragment of the cholecystokinin C-terminal octapeptide CCK-8. Studies on the alpha-chymotrypsin catalyzed coupling reaction between Z-Gly-Trp-R(1) and Met-R(2) have focused on low water content media, using deposited enzyme on inert supports such as Celite and polyamide. The effect of additives (polar organic solvents), the acyl-donor ester structure, the C-alpha protecting group of the nucleophile, enzyme loading, and substrate concentration were tested. The best reaction medium found was acetonitrile containing buffer (0.5%, v/v) and triethylamine (0.5%, v/v) using the enzyme deposited on Celite as catalyst (8 mg of alpha-chymotrypsin/g of Celite). A reaction yield of 81% was obtained with Z-Gly-Trp-OCam as acyl donor, at an initial concentration of 80 mM. The tripeptide synthesis was scaled up to the production of 2 g of pure tripeptide with an overall yield of 71%, including reaction and purification steps. (c) 1996 John Wiley & Sons, Inc.     

Improved HPLC method for the simultaneous determination of tramadol and O-desmethyltramadol in human plasma

This paper describes an HPLC method for the determination of tramadol and its major active metabolite, O-desmethyltramadol (ODT), in human plasma. Sample preparation involved liquid-liquid extraction with diethyl ether-dichloromethane-butanol (5:3:2, v/v/v) and back extraction with sulphuric acid. Tramadol, ODT and the internal standard, sotalol, were separated by reversed phase HPLC using 35% acetonitrile and an aqueous solution containing 20 mM sodium phosphate buffer, 30 mM sodium dodecyl sulphate and 15 mM tetraethylammonium bromide pH 3.9. Detection was by fluorescence with excitation and emission wavelengths of 275 and 300 nm, respectively. The method was linear for tramadol (3-768 ng/ml) and ODT (1.5-384 ng/ml) with mean recoveries of 87.2% and 89.8%, respectively. Intra- and inter-day precisions were 10.34% and 8.43% for tramadol and 9.43% and 8.75% for ODT at the respective limits of quantitation (3 and 1.5 ng/ml). Accuracy for tramadol ranged from 96.2% to 105.3%. The method was applied to a pharmacokinetic study of tramadol in human volunteers.     

Enhancement of adenosine triphosphatase activity of purified chloroplast coupling factor 1 in aqueous organic solvent

The ATPase activity of purified coupling factor 1 (CF1) of spinach chloroplasts [EC 3.6.1.3] was reversibly enhanced in some aqueous organic solvents, notably methanol, ethanol, and acetone. Pretreatment of CF1 with 20% (v/v) methanol did not affect the subsequent activity. The activity depended entirely on the final concentration of methanol in the reaction mixture. In the presence of 20% methanol, the Km of Ca2+-ATPase from ATP was lowered from 0.4 mM to 0.2 mM. Not only Ca2+, but also Cd2+, Mg2+, Mn2+, and Zn2+ supported the ATPase activity at rates of higher than 7 mumol.mg protein-1 . min-1. Co2+, Ni2+, and Pb2+ supported the activity at rates of 0.5-1.0 mumol.mg protein-1 . min-1. The activities supported by the following cations, if any, were less than 0.2 mumol.mg protein-1 . min-1; Ba2+, Cu2+, Fe2+, Hg2+, Sn2+, and Sr2+. The optimum concentration of methanol for Ca2+-ATPase and Mg2+-ATPase activities was about 30% (v/v). The optimum pH values for Ca2+-ATPase and Mg2+-ATPase activities were about 8.0 and 8.8, respectively. The enhancing effect of organic solvents appears to be associated with their relative lipophilic character as defined by the octanol-water partition coefficient. The Ca2+-ATPase activities of th trypsin-activated and the heat-activated CF1 were inhibited and their Mg2+-ATPase activities were enhanced by the presence of methanol in the reaction mixture.     

Ribulose bisphosphate carboxylase/oxygenase from Thiocapsa roseopersicina

d-Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified 80-fold from malate-grown Thiocapsa roseopersicina by salting out the enzyme from the high-speed supernatant between 68–95% saturation with respect to (NH₄)₂SO₄, gelfiltration through Sephadex G-100, and DEAE-cellulose chromatography followed by sedimentation into a 14–34% glycerol gradient. The specific activity of enzyme for the carboxylase reaction was 2.45 mol RuBP-dependent CO₂ fixed/min · mg protein (at pH 8.0 and 30° C) and for the oxygenase reaction was 0.23 mol RuBP-dependent O₂ consumed/min · mg protein (at pH 8.6, and 25° C). The enzyme, which was ultracentrifugally homogeneous in the presence of 4 and 10% v/v glycerol, was stable for at least one year at-80° C in the presence of 10% glycerol. S_{20, w} values obtained in the presence of 4 and 10% glycerol were 19.3 and 16.2, respectively. The enzyme contained both large (53,000-daltons) and mixed small subunits (15,000- and 13,500-daltons).Borate-dependent inactivation of the enzyme by 2,3-butadione, which was greatly reduced in the presence of the product 3-phosphoglycerate, suggested that one or more arginines are at the active site.Abbreviations DTT dithiotreitol - RuBP d-ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate - TCA trichloroacetic acid - TEMBDG buffer (pH 8.0 at 25°C) containing 20 mM Tris, 1 mM disodium EDTA · 2 H₂O, 10 mM MgCl₂·6 H₂O, 50 mM NaHCO₃, 0.1 mM DTT and 10% glycerol (v/v)     

Isolation of lectins from hemolymph of decapod crustaceans by adsorption on formalinized erythrocytes

Hemolymph of decapod crustaceans contains lectins of important specificity. An isolation procedure, based on adsorption of hemolymph lectins on red blood cells (RBC) fixed with formaldehyde, is described. Hemolymph is let to clot for 3 h at 22-28 degrees C (RT) and for 24 h at 5 degrees C; centrifuged at 13000 g for 30 min; filtered through 5-microm filters; diluted with an equal volume of 50 mM NaCl, 100 mM CaCl(2); supplemented with protease as well as phenoloxidase inhibitors; centrifuged at 13000 g for 20 min. Formalinized RBC (FRBC) are mixed with diluted hemolymph to a suspension of about 20% v/v FRBC. After incubation for 30 min at RT, FRBC are washed five times with 150 mM NaCl, 10 mM CaCl(2). The lectins adsorbed on FRBC are desorbed using either 100-500 mM of carbohydrate solutions in 0.9% NaCl or 50 mM Tris-HCl buffer, pH 8.0 containing 100 mM NaCl and 20 mM entylenediaminetetraacetate (EDTA). The procedure is efficient in isolating the hemolymph lectins of the decapods Liocarcinus depurator and Potamon potamios.