Calcium Phosphate Gel Research Articles

Inflammation et résistance antitumorale III. — effets précoces d'une réaction inflammatoire sur lévolution de la tumeur de lewis

Following the inflammatory reaction induced in mice by subcutaneous implantation into the dorsal area of talc embedded in a calcium phosphate gel, one day after the intradermal inoculation of Lewis carcinoma cells in the sternal area, the tumour growth was delayed. In contrast with a decreased mitotic index and a tumour cell injury, no differences were found between the controls and the treated animals concerning the average number of vessels or the number of leukocytes infiltrating the tumour.

47] d-Galactonate dehydratase

This chapter describes the purification and properties of the enzyme galactonate dehydratase that functions in the metabolism of D-galactose and D-galactonic acid in a pseudomonad designated “MSU-1.” The α-keto acid formed during the reaction is measured as its semicarbazone by the procedure of MacGee and Doudoroff. The purification process of the enzyme involves growth of organism, preparation of cell extracts, protamine sulfate treatment, ammonium sulfate fractionation, Sephadex G-200, calcium phosphate gel, and purification by diethylaminoethyl (DEAE)-cellulose. Studies indicate that the enzyme is specific for D-galactonate, and the optimal pH is 7.1 in glycylglycine buffer. The enzyme is quite stable to thermal treatment and possesses a half-life of over 5 minutes at 90 ° C. The native protein has a molecular weight of 240,000 to 245,000 as shown by gel filtration and sedimentation on sucrose density gradients. The enzyme does not possess 2-keto-3-deoxygalactonate aldolase, 2-ketodeoxygluconate aldolase, 2-keto-3-deoxygalactonate kinase, or 2-keto-3-deoxy-6-phosphogalactonate aldolase activity.

Isolation of Salmonella wien heat-labile enterotoxin : Alfredo CAPRIOLI 1, Giuliano D'AGNOLO 2, Vincenzo FALBO 1, L.Giorgio RODA 3 and Maurizio TOMASI 2 Laboratorio di Malattie Batteriche e Virali 1, Laboratorio di Biologia Cellulare e Immunologia 2, Istituto Superiore di Sanità, Viale Regina Elena 299 - 00161 Rome; Laboratorio di Farmacologia 3, Università di Ancona, Italy

An enterotoxic activity has been identified in culture filtrates of Salmonella wien. The enterotoxin causes fluid accumulation in rabbit ligated ileal loops, firm induration and erythema in rabbit skin and morphological alteration in chinese hamster ovary (CHO) cell cultures; it was revealed by treatment with calcium phosphate gel, and purified on DEAE-Sephadex A-50 and BioGel A-1.5 m. The enterotoxic activity was eluted from the BioGel column in two peaks. Approximately 50-70% of the enterotoxic activity of the first peak, corresponding to the excluded volume, was resistant to heating at 75 degrees C for 30 min, while the activity of the second peak was completely destroyed by this treatment. From the heat-labile peak a protein, in homogeneous form, was isolated exploiting its affinity towards agarose gel filtration media. This protein, with enterotoxic activity was also present as shown by SDS-PAGE, in the first peak, eluted from the Bio-Gel column, where it appears to be closely associated with cell wall or membrane components and thus protected from heat denaturation. The isolated enterotoxin is stable in alkaline conditions but it is sensitive to acidic pH values; moreover, it stimulates adenylate cyclase in cell culture systems. Thus, it appears to possess properties similar to both cholera toxin and the heat-labile enterotoxin of Escherichia coli. These results indicate that the enterotoxin is a protein in nature and it is postulated that it may participate in the pathogenesis of S. wien infection.

Incidence d'une réaction inflammatoire sur la résistance des souris envers Plasmodium berghei

An inflammatory reaction induced in mice by a subcutaneous injection of magnesium silicate embedded in a calcium phosphate gel is followed by an increased resistance against Plasmodium berghei. The occurrence of this increased resistance is related to the time elapsed between the induction of the inflammatory process and the infection. The delayed mortality is correlated with a slowed development of parasitemia. It is hypothesized that the protective effect may be related to the capacity of the inflammatory reaction to promote in mice both specific and non-specific antiparisitic immune responses.

Glutamine Synthetase of Germinating Peanuts

Glutamine synthetase activity, extracted from an acetone powder of 7-day germinated peanuts (Arachis hypogaea L.), was precipitated by ammonium sulfate (40-60% saturation) and further purified by gel filtration and calcium phosphate gel treatment. When it was adsorbed to and subsequently eluted from a column of diethylaminoethyl-cellulose, two peaks of activity (designated glutamine synthetase 1 and 2) were obtained which were enriched 150- and 20-fold, respectively, over the initial extract. Glutamine synthetase 1 was present in ungerminated seeds and in the cotyledons during germination; glutamine synthetase 2 appeared during germination and was found largely in the developing plant. Compared with glutamine synthetase 2, glutamine synthetase 1 appeared to have a slightly smaller molecular weight and was more stable to heat and storage. The catalytic properties of the two forms were essentially the same. Whereas neither form catalyzed gamma-glutamyltransferase activity with 4-methyleneglutamine, both glutamine synthetases 1 and 2 catalyzed an ATP- and NH(4) (+)-dependent conversion of [(14)C]-4-methyleneglutamic acid to [(14)C]-4-methyleneglutamine, but the K(m) value for 4-methyleneglutamic acid was 10-fold greater and the V(max) only one-fourth that measured with l-glutamic acid. This is the first report of glutamine synthetase activity with 4-methyleneglutamic acid as substrate, although the level of this activity does not appear adequate to account for the rapid synthesis of 4-methyleneglutamine observed in germinating peanuts.

51] 3-Hexulose-6-phosphate synthase from Methylomonas (Methylococcus) capsulatus

This chapter describes two assay methods (method I and II) and the purification procedure for 3-hexulose-6-phosphate synthase from Methylomonas (Methylococcus) capsulatus . The enzyme catalyzes a key step in the assimilation of carbon during growth of some bacteria on reduced C 1 compounds. Method 1 involves discontinuous colorimetric measurement of the rate of ribulose 5-phosphate-dependent removal of formaldehyde. Method 2 involves continuous measurement of the rate of ribulose 5-phosphate- and formaldehyde-dependent formation of hexose phosphate. Method 2 is more convenient to use but it depends on having previously purified phosphohexuloisomerase as a coupling enzyme. The purification procedure involves preparation of cell-free extract, preparation of particulate fraction, solubilization with l M NaCl, ammonium sulfate fractionation, diethylaminoethyl (DEAE)-cellulose chromatography, and calcium phosphate gel treatment. Hexulose-phosphate synthase has an absolute requirement for a bivalent metal ion—Mg 2+ and Mn 2+ being most effective and Co 2+ and Zn 2+ being much less effective. The enzyme has optimum activity, in the synthetic direction, at pH 7.0.

67] Pyruvate dehydrogenase complex from Escherichia coli

This chapter describes the assay method and purification procedures of pyruvate dehydrogenase complex isolated from Escherichia coli . The pyruvate dehydrogenase complex of E. coli consists of multiple copies of pyruvate decarboxylase and lipoamide dehydrogenase, which bind independently to dihydrolipoamide acetyltransferase. The purification involves fractionation with protamine sulfate, the addition of yeast ribonucleic acid, ultracentrifugation, and isoelectric precipitation. A further improvement is obtained by introducing several precipitation steps between pH 5.7 and pH 4.9 and by introducing calcium phosphate gel cellulose chromatography. Another method is more rapid and uses BioGel A-50m, followed by calcium phosphate gel cellulose chromatography. Two other principles for purifying the complex from E. coli using affinity chromatography are also described in the chapter. A continuous assay for the pyruvate decarboxylase is based on the decrease in absorbance at 420 nm because of the reduction of ferricyanide. The purification of pyruvate decarboxylase involves fractionation with protamine sulfate, the addition of yeast ribonucleic acid, ultracentrifugation, and isoelectric precipitation.

88] l-Glycol dehydrogenase from hen muscle

Publisher Summary This chapter describes the assay method, purification, and properties of L-glycol dehydrogenase isolated from the hen muscle. L-Glycol dehydrogenase is an enzyme purified from the hen muscle, which reversibly reduces uncharged vicinal dicarbonyls and α-hydroxycarbonyls to L(+)-glycols. L-glycol dehydrogenase activity is measured by determining the decrease in absorbance at 340 nm because of nicotinamide adenine dinucleotide phosphate dehydrogenase (NADPH) oxidation in a system contaning sodium-potassium phosphate buffer (300 μmol) at pH 7, NADPH (0.6 μ mol), and acetoin (12 μ mol); the total volume of the solution is 3 ml and the assay temperature is maintained at 25°C. Acetoin is washed three times with a large volume of peroxide-free anhydrous ether to remove the traces of diacetyl; after washing, it is stored at -18°C for at least 6 months. The steps involved in the purification of the enzyme are (1) aqueous extraction, (2) calcium phosphate gel adsorption, (3) sephadex G-100 gel filtration, (4) sephadex G-75 superfine gel filtration, (5) hydroxyapatite chromatography, and (6) electrofocusing.

Partial Purification of Enzymes of Bovine Kidney Mitochondria Activating Volatile Fatty Acids1,2

Mitochondria were prepared from kidney cortex tissue of lactating Holstein cows, and enzymes activating volatile fatty acids partially purified. Two fractions activating volatile fatty acids were obtained; one was similar to components isolated from bovine heart mitochondria activating acetate and propionate (acetyl CoA synthetase). The other was similar to components of bovine liver mitochondria and activated propionate, butyrate, and valerate but not acetate. On calcium phosphate gel chromatography this latter fraction could be separated into a distinct propionyl CoA synthetase (Michaelis-Menten constant for propionate 2.54×10−3 M) and a fraction activating butyrate and valerate. The role of a distinct propionyl CoA synthetase in kidney mitochondria remains to be elucidated because propionate is not a major substrate in peripheral blood of ruminants.We concluded that volatile fatty acid activation by bovine kidney mitochondria is due to an acetyl CoA synthetase, a propionyl CoA synthetase, and a butyrate and valerate-activating fraction probably composed of separate butyryl and valeryl CoA synthetases.

Effect of calcium phosphate and alumina C gamma gels on the mutagenicity and cytotoxicity of dimethylnitrosamine as studied in the CHO/HGPRT system.

When CaCl2 was added in increasing concentrations to a rat liver metabolic activation system (S9) buffered with sodium phosphate, the mutagenic activity and cytotoxicity of dimethylnitrosamine (DMN) in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system were greatly increased. This effect was not observed with an S9 mix buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). The calcium phosphate gel precipitate of the S9 mix possessed approximately 1/3 of the total activity of the mix, while the supernatant had only slight activity. However, when the calcium phosphate gel precipitate of a solution of S9 salts (without S9 protein) was added to the supernatant, the remaining 2/3 of the activity was recovered. Commercially obtained calcium phosphate, tricalcium phosphate, and alumina C gamma gels could substitute for CaCl2 in the S9 mix, but diethylaminoethyl cellulose (DEAE cellulose) could not. Alumina C gamma gel can exert its effect in the absence of both CaCl2 and phosphate in the S9 mix. Increasing the time of contact between the S9 protein and the S9 salts increased the efficacy with which the S9 mix activated DMN; this is indicative of an adsorptive process by calcium phosphate gel.

Purification and characterisation of 6 G-fructosyltransferase from the roots of asparagus ( Asparagus officinalis L.)

A fructosyltransferase that catalyses the transfer of the terminal (2 → 1)-β-linked d-fructosyl group of fructo-oligosaccharides [1 F(1-β- d-fructofuranosyl) m sucrose, m > 0] to HO-6 of the glucosyl group of similar saccharides [1 F(1-β- d-fructofuranosyl) n sucrose, n > 0] has been purified (760-fold) from an extract of the roots of asparagus ( Asparagus officinalis L.) by successive fractionation with ammonium sulfate, treatment with calcium phosphate gel, and then chromatography on octyl-Sepharose, DEAE-cellulose, Sephadex G-200, and raffinose-coupled Sepharose 6B. The enzyme, tentatively termed 6 G-fructosyltransferase, was homogeneous in disc electrophoresis, had a mol. wt. of ~69,000 and an optimum pH of ~5.5, was stable at pH 5.0–6.0 on heating for 20 mins at 45° and for 10 min at 20–37°, and was inhibited by Hg 2+, p-chloromercuribenzoate, and Ag +.

Simple and rapid purification of tryptophan 5-monooxygenase from rabbit brain by affinity chromatography.

A simple and rapid method for isolating tryptophan 5-monooxygenase [L-tryptophan, tetrahydropteridine:oxygen oxidoreductase (5-hydroxylating), EC 1.14.16.4] was reported. The method involves adsorption on calcium phosphate gel and affinity chromatography on agarose coupled with dimethyltetrahydropteridine. Tryptophan 5-monooxygenase was purified 1,100-fold from a rabbit brain extract to a specific activity of 15.9 nmol/min.mg, which is far higher than that of the highly purified preparation reported by Tong and Kaufman ((1975) J. Biol. Chem. 250, 4152-4158). It was also demonstrated that this method was applicable to the purification of rat brain tryptophan 5-monooxygenase.

Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases.

An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively.

Peroxisomal β-ketothiolase

β-Ketothiolase was found in rat liver peroxisomes that were isolated by sucrose density gradient centrifugation. The presence of dithiothreitol was essential for measuring the peroxisomal thiolase, but dithiothreitol had little effect upon the thiolase activity in the mitochondria or cytosol. Dithiothreitol was not used during the isolation procedures. Acetoacetyl CoA and β-ketolauryl CoA, which was synthesized chemically, were used as substrates. The peroxisomal thiolase was active with β-ketolauryl CoA but had almost no activity with acetoacetyl CoA as the substrate. Thiolase activities in the mitochondrial and cytosolic fractions utilized both long- and short-chain substrates. The thiolases in the various fractions bound to and were purified by chromatography on calcium phosphate gel cellulose columns. The peroxisomal thiolase did not bind to phosphocellulose, whereas the mitochondrial and cytosolic activities could be chromatographed on phosphocellulose. Peroxisomal β-ketolauryl CoA thiolase activity was inhibited about 20% by 25 m m Mg 2+, and more activity was measured in a phosphate than in a Tris buffer. In control rat livers, the total activity of β-ketolauryl CoA thiolase was 3.4 μmol/min per gram of liver in the peroxisomes and 4.9 μmol/min per gram of liver in the mitochondria, which indicates that peroxisomes contain the capacity for 40% of the total thiolase activity associated with β-oxidation systems. In addition, 6.3 μmol/min per gram of liver of β-ketolauryl CoA thiolase was found in the soluble fraction and chromatographed differently from the peroxisomal enzyme on phosphocellulose. Clofibrate in the rat diet for 6 or 21 days resulted in about a 15-fold increase in peroxisomal thiolase when assayed with β-ketolauryl CoA and somewhat less of an increase in the other fractions.

Purification of 20α-hydroxysteroid oxidoreductase from bovine fetal erythrocytes

NADPH-dependent 20α-hydroxysteroid oxidoreductase (20α-HSD; EC 1.1.1.149) from bovine fetal erythrocytes was obtained for the first time free of hemoglobin by a new 2,500-fold purification scheme. This was achieved by a sequence of calcium phosphate gel adsorption, ammonium sulfate fractionation, and affinity chromatography. The present results lead us to believe that the NADPH-dependent 3β-hydroxysteroid oxidoreductase activity, which was co-purified with 20α-activity, may originate at the active site of 20α-HSD (2).

Two forms of glutamine synthetase in leaves of Cucurbita pepo

It has been shown that the leaves of pumpkin ( Cucurbita pepo) contain two molecular forms of glutamine synthetase (GS), one occurring in the cytosol (GS 1)and the other in the chloroplasts (GS 2). The activities of both forms were greater when ammonium ion was infiltrated into the leaves and this was shown to be due to de novo synthesis. The two synthetases were purified by ammonium sulphate fractionation, ion exchange chromatography on DEAE-cellulose, selective adsorption on calcium phosphate gel, and preparative polyacrylamide gel electrophoresis. The MWs of GS 1 and GS 2, estimated by gel filtration on Sephacryl S-200, were 480 000 and 370 000 respectively. During polyacrylamide gel electrophoresis in the presence of SDS both GS 1 and GS 2 were dissociated into polypeptide chains with MWs of 58 000 and 50 000 respectively, suggesting that GS, 1 and GS 2 are octamers consisting of identical monomers. The synthetases showed noticeable differences in their amino acid composition. In GS 1 and GS 2 the proportions of α- helical segments were 34 and 17 % respectively. In the presence of Mg 2+, the pH optima for GS 1 and GS 2 were 7.25 and 7.75 respectively, and K m values toward l-glutamate were 13 and 46 mM respectively. From the experimental data it is inferred that GS 1 and GS 2 are isoenzymes.

Isoenzymes of wheat o-diphenolase revealed by column isoelectric focusing

Common wheat o-diphenolase at different purification steps has been submitted to column isoelectric focusing electrophoresis. The fraction purified with calcium phosphate gel shows multiple forms focused at pI 3.60, 4.95, 6.80, and 9.60. The enzyme activity and the purification degree of the four enzymatic components obtained have been calculated. The major fraction recovered after purification by chromatography on DEAE-cellulose does not display other multiple forms; however, it has some enzymatically inactive proteins well separated in the pH-gradient. The enzymatic protein, focused at pI 9.60, thus achieves a 1753-fold purification over the crude extract. The application of the technique allows a characterization of the wheat o-diphenolase and its recovery in highly purified form.

Purification and characterization of phenylalanine 4-monooxygenase from rat liver

Phenylalanine 4-monooxygenase ( l-phenylalanine, tetrahydropteridine:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1) was purified approx. 600-fold to apparent homogeneity with a 48% yield from rat liver. Two distinct active forms were separable by calcium phosphate gel chromatography and numbered based on their order of elution from the gel column. The predominant form, Form I, had ane stimated molecular weight of about 240 000. The enzyme gave a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular weight of which was estimated to be approx. 51 000, indicating that the enzyme might be composed of four identical subunits. The molecular properties of Form I were: sedimentation coefficient, 10.1 S; Stokes radius, 55 Å; diffusion coefficient, 3.90 · 10 −7 cm 2/s; frictional ratio, 1.33 and isoelectric point, pH 5.6. The enzyme contained approx. 0.6 mol of iron and 0.3 mol of phosphate/mol of subunit of the enzyme. No. significant differences in kinetic properties of the two forms, Form I and Form II, were observed. Amino acid analysis studies revealed that the amino acid composition of Form I was essentially identical with that of Form II, indicating that both forms might be the products of the same gene. There were, however, minor differences in the phosphate content and the isoelectric point between the two forms

Resolution and reconstitution of succinate-cytochrome c reductase. Preparations and properties of high purity succinate dehydrogenase and ubiquinol—cytochrome c reductase

An improved method was developed to sequentially fractionate succinate-cytochrome c reductase into three reconstitutive active enzyme systems with good yield: pure succinate dehydrogenase, ubiquinone-binding protein fraction and a highly purified ubiquinol-cytochrome c reductase (cytochrome b- c 1 III complex). An extensively dialyzed succinate-cytochrome c reductase was first separated into a succinate dehydrogenase fraction and the cytochrome b- c 1 complex by alkali treatment. The resulting succinate dehydrogenase fraction was further purified to homogeneity by the treatment of butanol, calcium phosphate gel adsorption and ammonium sulfate fractionation under anaerobic condition in the presence of succinate and dithiothreitol. The cytochrome b- c 1 complex was separated into cytochrome b- c 1 III complex and ubiquinone-binding protein fractions by careful ammonium acetate fractionation in the presence of deoxycholate. The purified succinate dehydrogenase contained only two polypeptides with molecular weights of 70 000 and 27 000 as revealed by the sodium dodecyl sulfate polyacrylamide gel electrophoretic pattern. The enzyme has the reconstitutive activity and a low K m ferricyanide reductase activity of 85 μmol succinate oxidized per min per mg protein at 38°C. Chemical composition analysis of cytochrome b- c 1 III complex showed that the preparation was completely free of contamination of succinate dehydrogenase and ubiquinone-binding protein and was 30% more pure than the available preparation. When these three components were mixed in a proper ratio, a thenoyl-trifluoroacetone- and antimycin A-sensitive succinate-cytochrome c reductase was reconstituted.

Partial purification and some properties of wheat (Triticum aestivum) o‐diphenolase

Abstracto‐Diphenolase extracted from Triticum aestivum (cv. Nettuno) and purified in various steps gave a 250‐fold purification over the crude extract. This purified enzyme showed maximum relative activity towards 4‐methylcatechol, generally high or moderate activity towards di‐ and polyphenols, and very low activity towards monophenols. Total activity, determined at each step of purification, showed that treatment with ammonium sulphate and calcium phosphate gel gave rise to an activation of o‐diphenolase, thus revealing a latency of enzymatic activity. In the pH profile activity towards 4‐methylcatechol two optima at pH 5.3 and 6.9 were observed.