Anticoagulant activity of low-molecular-weight sulfated derivatives of galactomannan from <Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis> (L.) seeds

Galactomannan from seeds of Cyamopsis tetragonoloba (L.) Taub. (guar) was depolymerized using immobilized enzymatic preparation celloviridin. A set of fragments whose molecular weights varied from 12.6 to 245.6 kDa was obtained. Sulfated derivatives of components of all fractions were synthesized, in which the content of HSO ₃ ^? groups was 48.05 ± 2.31%. All preparations exhibited anticoagulant activity, which was recorded in vitro in two tests—alla and aXa. The antithrombin activity (aIIa) was high (up to 65–87 U/mg) and did not depend on the molecular weight of a sulfated derivative; in the second test (aXa), the effect of molecular weight was observed. Biospecific electrophoresis allowed us to detect the ability of galactomannan sulfates to form complexes with protamine sulfate, a classic antidote to heparin.     

Anticoagulant activity of low-molecular-weight sulfated derivatives of galactomannan from Cyamopsis tetragonoloba (L.) seeds

Galactomannan from seeds of Cyamopsis tetragonoloba (L.) Taub. (guar) was depolymerized using immobilized enzymatic preparation celloviridin. A set of fragments whose molecular weights varied from 12.6 to 245.6 kDa was obtained. Sulfated derivatives of components of all fractions were synthesized, in which the content of HSO3(-) groups was 48.05% +/- 2.31. All preparations exhibited anticoagulant activity, which was recorded in vitro in two tests--aIIa and aXa. The antithrombin activity (aIIa) was high (up to 65-87 U/mg) and did not depend on the molecular weight of a sulfated derivative; in the second test (aXa), the effect of molecular weight was observed. Biospecific electrophoresis allowed us to detect the ability of galactomannan sulfates to form complexes with protamine sulfate, a classic antidote to heparin.     

Binding of heparin to antithrombin III: The use of dansyl and rhodamine labels

Low molecular weight heparin of low-anticoagulant activity and high molecular weight heparin of correspondingly high activity were prepared by chromatography on protamine-Sepharose; preparations subjected to limited N-desulfation (5–10% free amino groups) by solvolysis were labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) or rhodamine B isothiocyanate (RITC). The fluorescent heparins retained approximately 50% of the original anticoagulant activities. Dansyl-heparin on binding to antithrombin III (ATIII) exhibited a 2.5-fold enhancement of dansyl fluorescence intensity. This effect could be prevented by excess unlabeled heparin. A 7900 molecular weight dansyl-heparin preparation bound to ATIII with a stoichiometry of close to 2:1 and with an apparent association constant for binding (K_a) of 4.9 × 10⁵, m^?1, whereas a 21,600 molecular weight fraction bound at 0.7:1 with the protein and with an apparent K_a = 7.9 × 10⁵, m^?1. When ATIII reacted with a mixture of low molecular weight dansyl-heparin and low molecular weight RITC-heparin, there was enhancement of RITC fluorescence emission when excited at the dansyl excitation maximum; this effect was not observed when either of the labeled heparin species was prepared from high molecular weight material. The results are consistent with the proposal that a single molecule of high molecular weight, high-activity heparin occupies two sites when it binds to ATIII, whereas low molecular weight, low-activity heparin binds to the two sites separately.     

Engineering of class I lactate-polymerizing polyhydroxyalkanoate synthases from Ralstonia eutropha that synthesize lactate-based polyester with a block nature

Class I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (PhaC_Re) was engineered so as to acquire an unusual lactate (LA)-polymerizing activity. To achieve this, the site-directed saturation mutagenesis of PhaC_Re was conducted at position 510, which corresponds to position 481 in the initially discovered class II LA-polymerizing PHA synthase (PhaC1_PsSTQK), a mutation in which (Gln481Lys) was shown to be essential to its LA-polymerizing activity (Taguchi et al., Proc Natl Acad Sci USA 105(45):17323–17327, 2008). The LA-polymerizing activity of the PhaC_ReA510X mutants was evaluated based on the incorporation of LA units into the P[3-hydroxybutyrate(3HB)] backbone in vivo using recombinant Escherichia coli LS5218. Among 19 PhaC_Re(A510X) mutants, 15 synthesized P (LA-co-3HB), indicating that the 510 residue plays a critical role in LA polymerization. The polymer synthesized by PhaC_ReA510S was fractionated using gel permeation chromatography in order to remove the low molecular weight fractions. The ¹³C and ¹H NMR analyses of the high molecular weight fraction revealed that the polymer was a P(7 mol% LA-co-3HB) copolymer with a weight-averaged molecular weight of 3.2?×?10⁵ Da. Interestingly, the polymer contained an unexpectedly high ratio of an LA-LA*-LA triad sequence, suggesting that the polymer synthesized by PhaC_Re mutant may not be a random copolymer, but presumably had a block sequence.     

Partial purification of adenosine 3',5'-cyclic monophosphate phosphodiesterases from rat pancreas in the presence of excess protease inhibitors.

(i) Three forms of cyclic AMP phosphodiesterases (3′,5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17), F1, F2-I and F2-II, were partially purified from the soluble fraction of rat pancreas in the presence of excess protease inhibitors by DEAE-cellulose column chromatography and gel filtration and were characterized. (ii) F2-II, which was purified 31-fold, exhibited a single peak of activity on both polyacrylamide-gel electrophoresis and isoelectric focusing. The enzyme had a molecular weight of about 70,000, an isoelectric point of 3.9, and an optimal pH around 8.5 and required Mg²⁺ or Mn²⁺ but not Ca²⁺ for activity. The K_m values of this enzyme for cyclic AMP and cyclic GMP were 1 and 50 μm, respectively, while V values of this enzyme for cyclic AMP and cyclic GMP were 36.1 and 12.6 nmol min^?1 (mg of protein)^?1, respectively. Cyclic GMP competitively inhibited hydrolysis of cyclic AMP by this enzyme. Ro20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone] also inhibited hydrolysis of cyclic AMP competitively, with a K_i value of 1 μm. (iii) Fraction F1, which was purified 10-fold, had a molecular weight of more than 500,000 and required Mg²⁺ for activity. Its K_m values for cyclic AMP were 1 and 5 μm. Its K_m value for cyclic GMP was 45 μm. Fraction F2-I, which was purified 26-fold, had a molecular weight of about 70,000. The ratio of the initial velocity of hydrolysis of cyclic GMP to that of cyclic AMP was 0.5 at a substrate concentration of 1 μm.     

NADP-malic enzyme from maize leaf: purification and properties.

NADP-malic enzyme (EC 1.1.1.40), which is involved in the photosynthetic C⁴ pathway, was isolated from maize leaf and purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. At the final step, chromatography on Blue-Sepharose, the enzyme had been purified approximately 80-fold from the initial crude extract and its specific activity was 101 μmol malate decarboxylated/mg protein/min at pH 8.4. The enzyme protein had a sedimentation coefficient (s_20,w) of 9.7 and molecular weight of 2.27 × 10⁵ in sucrose density gradient centrifugation, and molecular weight of 2.26 × 10⁵ calculated from sedimentation equilibrium analysis. The molecular weight of the monomeric form was determined to be 6.3 × 10⁴ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the pyruvate carboxylation reaction, HCO₃^? proved to be the active molecular species involved. With all other substrates at saturating concentration, the following kinetic constants were obtained: K_m (malate), 0.4 mm; K_m (NADP), 17.6 μm; K_m (Mg²⁺), 0.11 mm. The maize leaf malic enzyme was absolutely specific for NADP. The Arrhenius plot obtained from enzyme activity measurements was linear in a temperature range of 13 to 48 °C, and the activation energy was calculated to be 9500 cal/mol.     

Monomeric structure of glutamyl-tRNA synthetase in Escherichia coli.

An investigation of the subunit structure of glutamyl-tRNA synthetase (EC 6.1.1.17) from Escherichia coli indicates that this enzyme is a monomer. The enzyme purified to apparent homogeneity is a single polypeptide chain with a molecular weight of 62,000 ± 3,000 and K^Glu_m ? 50 μM in the aminoacylation reaction. Analytical gel electrophoretic procedures were used to determine the molecular weight of species exhibiting glutamyl-tRNA synthetase activity in freshly prepared extracts of several strains of E. coli, which had been grown under various nutritional conditions and harvested at different stages of growth. In all cases, glutamyl-tRNA synthetase activity was associated with a protein having about the same molecular weight and K^Glu_m as the purified enzyme. Thus, no evidence of an oligomeric form of glutamyl-tRNA synthetase with a greater affinity for l-glutamate was obtained, in contrast to a previous report of J. Lapointe and D. Söll (J. Biol. Chem.247, 4966–4974, 1972).     

The separation and properties of two phosphoprotein phosphatases from the dimorphic fungus Mucor rouxii

Soluble preparations from mycelium of the dimorphic fungus Mucor rouxii contained detectable amounts of phosphoprotein phosphatase activity. This cytosolic phosphatase activity exhibited a molecular weight below 80,000 and could be resolved into two different forms (enzymes I and II) by chromatography on DEAE-cellulose followed by gel filtration on Sephacryl S-300. Enzyme I (M_r 64,000) was mainly a histone phosphatase activity, absolutely dependent on divalent cations, with a K_0.5 for MnCl₂ of 2 mm. Enzyme II (M_r 40,000) was active with histone and phosphorylase. Its activity was independent or slightly inhibited by Mn²⁺. This enzyme was strongly inhibited by 50 mm NaF or 1 mm ATP. When partially purified enzymes I and II were separately treated with ethanol, the catalytic properties of enzyme II were apparently not affected while those of enzyme I were drastically changed. The activity with histone, which was originally dependent on Mn²⁺, became independent or slightly inhibited by the cation. The treatment was accompanied by a notable increase in phosphorylase phosphatase activity which was strongly inhibited by Mn²⁺. Treated enzyme I eluted from DEAE-cellulose and Sephacryl S-300 columns at a position similar to that of enzyme II.     

Studies on oxidative phosphorylation: evidence for multiple forms of factor B activity.

The appearance of energy transfer factor B (Factor B) activity in discrete fractions derived from heart mitochondrial extracts has been demonstrated. Two subfractions (Fractions 50-2 and 50-10), besides Factor B (in Fraction 100-10) stimulated the activity of the ammonia-EDTA particle (AE-particle) in oxidative phosphorylation, ATP-P₁ exchange and ATP-driven NAD⁺ reduction by succinate (Succ-NAD⁺ assay). Treatment of these factor preparations with p-Chloromercuriphenylsulfonate abolished their stimulatory activity in Succ-NAD⁺, indicating the involvement of thiol groups in their function. Based on sucrose density gradient centrifugation, the molecular weight of the active component in Fraction 50-10, which had the highest Factor B activity, was 47,000. In earlier work from this laboratory, the molecular weight of Factor B was found to be 29,000. Rabbit antiserum to Factor B showed precipitin bands with and completely inhibited the stimulation of the Succ-NAD⁺ activity produced by Fractions 50-2, 50-10 and 100-2 in the presence of the AE particle.     

Purification and Properties of Oxalate Oxidase from NaCl Stressed Grain Sorghum Seedlings

The effect of NaCl stress on molecular and biochemical properties of oxalate oxidase (OXO) was studied in leaves of grain sorghum hybrid (var CSH-14) seedlings. There was no effect on molecular weight and number of subunits of the enzyme but it showed some important changes in its kinetic parameters such as K_m for oxalate and V_max. Optimum pH (5.8), activation energy (5.084 kcal mole^?1), time of incubation (6 min) and K_m for oxalate (1.21×10^t-4M) were increased, while V_max (0.182 mmole min^?1) decreased and no change in optimum temperature was observed. This showed that substrate affinity and maximum activity of the enzyme was adversely affected. The specific activity of oxalate oxidase was increased in seedlings grown in a NaCl containing medium compared to normal, which reveals the increased de novo synthesis of the enzyme to sustain oxalate degradation.     

Identification of N5,N10-methylene tetrahydrofolate reductase as the enzyme involved in the 5-methyl tetrahydrofolate-dependent formation of a beta-carboline derivative of 5-hydroxytryptamine in human platelets.

An enzyme in human platelets or rat brain incubated with 5-methyl tetrahydrofolate (5MeH₄folate) yields formaldehyde (4, 13), which will combine with biogenic amines to form β-carbolines (5) or tetrahydroisoquinolines. This activity was purified 500-fold from human platelets which are the main storage site for 5-hydroxytryptamine in man. This enzyme was identical to N⁵, N¹⁰-methylene tetrahydrofolate (N⁵,N¹⁰-methylene H₄folate) reductase by the following criteria: (i) co-purification, (ii) heat denaturation, (iii) pH response, (iv) molecular weight, (5) cofactor requirements. A mechanism involving the enzymatic generation of formaldehyde followed by adduct formation with a biogenic amine is proposed.     

Glucose 6-phosphate dehydrogenase isozymes of maize leaves : some comparative properties

Two different forms of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been purified from etiolated and green leaves, respectively, of 6-day maize (Zea mays L. cv Fronica) seedlings. The procedure includes an ammonium sulfate step, an ion exchange chromatography, and a second gel filtration in Sephadex G-200 in the presence of NADP⁺ to take advantage of the corresponding molecular weight increase of the enzyme. The isozyme from etiolated leaves is more stable and has been purified up to 200-fold. Subunit molecular weight, measured by sodium dodecyl sulfate-gel electrophoresis, is 54,000. The active protein, under most conditions, has a molecular weight 114,000, which doubles to molecular weight 209,000 in the presence of NADP⁺. The association behavior of enzyme from green leaves is similar, and the molecular weight of the catalytically active protein is also similar to the form of etiolated leaves.

Glucose 6-phosphate dehydrogenase of dark-grown maize leaves isoelectric point (pI) 4.3 is replaced by a form with pI 4.9 during greening. The isozymes show some differences in their kinetic properties, K_m of NADP⁺ being 2.5-fold higher for pI 4.3 form. Free ATP (K_m = 0.64 millimolar) and ADP (K_m = 1.13 millimolar) act as competitive inhibitors with respect to NADP⁺ in pI 4.3 isozyme, and both behave as less effective inhibitors with pI 4.9 isozyme. Magnesium ions abolish the inhibition.

     

Comparison of free and ribosome-bound phenylalanyl-tRNA synthetase from rabbit reticulocytes.

Phenylalanyl-tRNA synthetase (l-phenylalanine:tRNA ligase [AMP], EC 6.1.1.b) from the ribosomal and the postribosomal cell supernatant fractions of rabbit reticulocytes were purified separately and characterized. Phenylalanyl-tRNA synthetase from the ribosomal fraction was purified 114-fold to a final specific activity of 1603 units/mg and is approximately 90% pure. Phenylalanyl-tRNA synthetase from the postribosomal supernatant fraction was purified 4186-fold to a final specific activity of 247 units/mg. The enzymes from the two fractions appear to be identical based on their elution from various chromatographic media, sedimentation coefficient, pH, Mg²⁺, and K⁺ optima, and heat stability. Phenylalanyl-tRNA synthetase from rabbit reticulocytes has a molecular weight of approximately 245,000 with an α₂β₂ subunit structure. The molecular weights of the subunits are 57,000 and 67,200.     

Isoenzymes of glucose 6-phosphate dehydrogenase from the plant fraction of soybean nodules

Two isoenzymes of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been separated from the plant fraction of soybean (Glycine max L. Merr. cv Williams) nodules by a procedure involving (NH₄)₂SO₄ gradient fractionation, gel chromatography, chromatofocusing, and affinity chromatography. The isoenzymes, which have been termed glucose 6-phosphate dehydrogenases I and II, were specific for NADP⁺ and glucose 6-phosphate and had optimum activity at pH 8.5 and pH 8.1, respectively. Both isoenzymes were labile in the absence of NADP⁺. The apparent molecular weight of glucose 6-phosphate dehydrogenases I and II at pH 8.3 was estimated by gel chromatography to be approximately 110,000 in the absence of NADP⁺ and double this size in the presence of NADP⁺. The apparent molecular weight did not increase when glucose 6-phosphate was added with NADP⁺ at pH 8.3. Both isoenzymes had very similar kinetic properties, displaying positive cooperativity in their interaction with NADP⁺ and negative cooperativity with glucose 6-phosphate. The isoenzymes had half-maximal activity at approximately 10 micromolar NADP⁺ and 70 to 100 micromolar glucose 6-phosphate. NADPH was a potent inhibitor of both of the soybean nodule glucose 6-phosphate dehydrogenases.     

Partial purification and characterization of endoproteinases from senescing barley leaves

Two major endoproteinases were purified from senescing primary barley leaves. The major enzyme (EP₁) appeared to be a thiol proteinase and accounted for about 85% of the total proteolytic activity measured in vitro. This proteinase was purified 5,800-fold and had a molecular weight of 28,300. It was highly unstable in the absence of dithiothreitol or at a pH greater than 7.5. Leupeptin, at a concentration of 10 micromolar, inhibited this enzyme 100%. A second proteinase (EP₂) was purified approximately 50-fold and had a molecular weight of 67,000. It was inhibited 20% by 1 millimolar dithiothreitol and 50% by 1 millimolar phenylmethyl sulfonylfluoride. EP₂ contributed about 15% of the total proteolytic activity measured in vitro. Both proteinases hydrolyzed a variety of artificial and protein substrates, and both had pH optima of 5.5 to 5.7 when either azocasein or [¹⁴C]ribulose-1,5-bisphosphate carboxylase ([¹⁴C]RuBPCase) was the substrate. The thiol endoproteinase hydrolyzed azocasein linearly but hydrolyzed [¹⁴C]RuBPCase biphasically. A third endoproteinase (EP₃), not detected by standard proteolytic assays, was observed when [¹⁴C]RuBPCase was the substrate.     

Purification and properties of a highly active catalase from cabbage loopers,Trichoplusia ni

Using ethanol-chloroform fractionation in conjunction with standard column chromatography techniques catalase has been purified to electrophoretic homogeneity from mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni. The specific activity of purified catalase was 2.2 × 10⁵ units (IU = 1 μmol H₂O₂ decomposed mg protein⁻¹ min⁻¹). The purified enzyme's native molecular weight was in the 247,000–259,000 Da range and was tetrameric with an apparent molecular weight of 63,000 Da for each subunit. In addition, biochemical properties of the enzyme were studied with emphasis on substrate specificity, kinetics, and the mechanism of inactivation by the irreversible inhibitor 3-amino-1,2,4-triazole (AT). The apparent K_m of the purified catalase for H₂O₂ was 54.2 mM and 50% of the maximal rate occurred at 16 mM H₂O₂. Purified catalase was ineffective in metabolizing organic hydroperoxides and, unlike other catalases, lacked peroxidase activity. Lastly, AT in the presence and absence of H₂O₂ was an effective inhibitor of catalase activity (I₅₀ = 100 mM) suggesting that a portion of the purified catalase was complexed with hydrogen peroxide in a compound 1 configuration.     

Studies on some aspects of peroxidase from submaxillary gland

A peroxidase has been purified 25- to 30-fold over crude homogenate from goat submaxillary gland, which shows a single band of protein on polyacrylamide gel electrophoresis at four different pH values (4.6–10.0). A molecular weight (M_r) of approximately 2 × 10⁴ per heme binding site has been found. The molecular weight of the enzyme determined by Sephadex-gel filtration method, appeared to be 4 × 10⁴. The sedimentation pattern of the purified enzyme shows a symmetrical peak, although there was evidence of some small heterogenous material near the meniscus. The sedimentation coefficient of the enzyme (s^o 20^wat 0.4% of the enzyme concentration) was found to be 4.18, which indicates the molecular weight of the enzyme to be approximately 6 × 10⁴.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A₄ hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A₄ to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B₄ was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and apparent K_m for leukotriene A₄ between 2 · 10^?5 and 3 · 10^?5 M. The purified leukotriene A₄ hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A₄ hydrolase from previously purified epoxide hydrolases.     

Evidence for Chloroplastic Localization of an Ammonium-Inducible Glutamate Dehydrogenase and Synthesis of Its Subunit from a Cytosolic Precursor-Protein in Chlorella sorokiniana

Chlorella sorokiniana cells, cultured for 12 hours in 30 millimolar ammonium medium, contained an ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzyme with subunits having a molecular weight of 53,000. In vitro translation of total cellular poly(A)⁺ RNA, isolated from fully induced cells, resulted in synthesis of an NADP-GDH antigen with a molecular weight of 58,500. The 58,500 dalton antigen was processed in vitro, with a 100,000g supernatant prepared from broken fully induced Chlorella cells, to a protein with a molecular weight of 53,000. These data support the inference that the NADP-GDH subunit (M_r = 53,000) is initially synthesized as a larger precursor protein (M_r = 58,500). By use of a cytochemical staining procedure, dependent upon NADP-GDH catalytic activity, the holoenzyme was shown to be chloroplast-localized. An immunoelectron microscopy procedure, employing anti-NADP-GDH immunoglobulin G and Protein A-gold complex, showed that NADP-GDH antigen was absent from the nucleus but present in both the chloroplast and cytosol. Since synthesis of the enzyme can be inhibited by cycloheximide, the detection of NADP-GDH antigen in the cytosol was probably due to binding of the NADP-GDH antibody to nascent polypeptide chains of the precursor-protein being synthesized on cytosolic 80S ribosomes.     

Organization of the glycolytic enzymes in Escherichia coli

Differential centrifugation of osmotically lysed lysozyme-EDTA spheroplasts from Escherichia coli sedimented 50–70% of the glycolytic activities examined in a low speed pellet; the remaining activity, occurring in a high speed supernatant, contained the soluble enzymes of the cell. The distribution pattern of the enzymes could be altered by extrusion of the spheroplasts through the French Press or by lysis at different pH values. Electron micrographs of the pellet fraction revealed lysed spheroplasts mostly devoid of cellular constituents but consisting of cytoplasmic membranes surrounded by partially degraded cell wall fragments. Washing of the pellet showed that the enzymes were not all bound to the same degree to the membrane fraction. Throughput activity of the glycolytic pathway was demonstrated for the membrane fraction, but none was observed for the soluble fraction of the cell (i.e. for enzymes present in the supernatants) unless these were first concentrated by ultrafiltration. The supernatant from the lysed spheroplasts, together with a further supernatant obtained by washing the membrane pellet, was concentrated by ultrafiltration and chromatographed on a Bio-Gel column. The eluate contained glycolytic activities both in fractions corresponding to relatively high and relatively low molecular weight material The high molecular weight species, containing a proportion of all the enzymes studied, had a molecular weight of at least 1.2 × 10⁶. A multienzyme aggregate containing one each of the glycolytic enzymes would have a molecular weight of ～ 1.3 × 10⁶. The specific rate of pyruvate formation from glucose by the high molecular weight species was similar to that obtained from a preparation in which the fractions containing all the low molecular weight material enzyme activities were pooled and concentrated by ultrafiltration. Using the high molecular weight material, studies were made of the ability of added unlabelled glycolytic intermediates to compete for catalytic sites with intermediates produced endogenously from [¹⁴C₆] glucose. The relatively weak competition observed indicated a high degree of protection afforded the labelled intermediates derived from [¹⁴C₆] glucose.