scholarly journals Oxygenation and spontaneous deamination of 2-aminobenzenesulphonic acid in Alcaligenes sp. strain O-1 with subsequent meta ring cleavage and spontaneous desulphonation to 2-hydroxymuconic acid

1994 ◽  
Vol 300 (2) ◽  
pp. 429-436 ◽  
Author(s):  
F Junker ◽  
J A Field ◽  
F Bangerter ◽  
K Ramsteiner ◽  
H P Kohler ◽  
...  
Keyword(s):  
Phosphate Buffer ◽  
Enzyme Reaction ◽  
Anion Exchange Chromatography ◽  
Major Product ◽  
Exchange Chromatography ◽  
Ring Cleavage ◽  
Keto Form ◽  
Cell Extracts ◽  
Acid Semialdehyde ◽  
Spontaneous Reaction

2-Aminobenzenesulphonic acid (2AS) is degraded by Alcaligenes sp. strain O-1 via a previously detected but unidentified intermediate. A mutant of strain O-1 was found to excrete this intermediate, which was isolated and identified by m.s., 1H- and 13C-n.m.r. as 3-sulphocatechol (3SC). Proteins from cell extracts of strain O-1 were separated by anion-exchange chromatography. A multicomponent oxygenase was observed to convert 1 mol each of NADH, O2 and 2AS into 1 mol each of 3SC, NH3 and NAD+. The enzyme presumably catalysed formation of the ring of a 2-amino-2,3-diol moiety, and elimination in the amino group led to a rearomatization. 3SC was further degraded via meta ring cleavage, which could be prevented by inactivation of the 3-sulphocatechol-2,3-dioxygenase (3SC23O) with 3-chlorocatechol. In Tris buffer, the separated 3SC23O catalysed the reaction of 1 mol each of 3SC and O2 involving a transient yellow intermediate, and release of 1 mol of sulphite and two organic products. The major product was identified by n.m.r. and by g.c./m.s. as 5-carboxypenta-2,4-dien-5-olide (CPDO), an indicator of formation of 2-hydroxymuconic acid (2HM). The second product was identified as the Z,E isomer of 2HM by comparison with authentic material. When the CPDO in the product mixture was chemically hydrolysed to (Z,E)-2HM, 1 mol of (Z,E)-2HM/mol of 3SC was observed. If oxygenation of 3SC by 3SC23O was carried out in phosphate buffer, only a single product was detected, a keto form of 2HM. This dioate was also formed from authentic (Z,E)-2HM in phosphate buffer. Formation of the natural product (Z,E)-2HM from the xenobiotic, 3SC, seems to involve oxygenation to the unstable 2-hydroxy-6-sulphonomuconic acid semialdehyde, which hydrolyses spontaneously to 2HM. There would appear to be at least one spontaneous reaction per enzyme reaction in this pathway.

scholarly journals l-myo-inositol 1,4,5,6-tetrakisphosphate is present in both mammalian and avian cells

1988 ◽  
Vol 249 (1) ◽  
pp. 271-282 ◽  
Author(s):  
L Stephens ◽  
P T Hawkins ◽  
N Carter ◽  
S B Chahwala ◽  
A J Morris ◽  
...  
Keyword(s):  
High Performance ◽  
Periodic Acid ◽  
Platelet Activating Factor ◽  
Anion Exchange Chromatography ◽  
Total Radioactivity ◽  
Exchange Chromatography ◽  
Murine Bone Marrow ◽  
Cell Extracts ◽  
Inositol 1,4,5 Trisphosphate ◽  
Inositol Tetrakisphosphate

When myo-[3H]inositol-prelabelled primary-cultured murine bone-marrow-derived macrophages were challenged with platelet-activating factor (PAF; 200 ng/ml), there was a rapid (2.5-fold at 10 s) rise in the intracellular concentration of D-myo-[3H]inositol 1,4,5-trisphosphate, followed by a rise in myo-[3H]inositol tetrakisphosphate. myo-[3H]Inositol tetrakisphosphate fractions were isolated by high-performance anion-exchange chromatography from myo-[3H]inositol-prelabelled chick erythrocytes and primary-cultured macrophages. In both cases [3H]iditol and [3H]inositol were the only significant products (greater than 90% of recovered radioactivity) after oxidation to completion with periodic acid, reduction with NaBH4 and dephosphorylation with alkaline phosphatase. The presence of [3H]inositol after this procedure is consistent with the occurrence of [3H]inositol 1,3,4,5-tetrakisphosphate in the cell extracts, whereas [3H]iditol could only be derived from D- or L-inositol 1,4,5,6-tetrakisphosphate. When [3H]inositol tetrakisphosphate fractions obtained from (A) unstimulated macrophages, (B) macrophages that had been stimulated with PAF for 40s or (C) chick erythrocytes were subjected to the above procedure, radioactivity was recovered in these polyols in the following proportions: A, 60-90% in iditol, with 10-40% in inositol; B, total radioactivity increased by a factor of 9.8, 94% being recovered in inositol and 8% in iditol; C, 70-80% in iditol and 20-30% in inositol. [3H]Iditol derived from myo-[3H]inositol tetrakisphosphate fractions from macrophages and chick erythrocytes was oxidized to sorbose by L-iditol dehydrogenase (L-iditol:NAD+2-oxidoreductase, 1.1.1.14) at the same rate as authentic L-iditol. D-[14C]Iditol, derived from D-myo-inositol 1,4,5-trisphosphate, was not oxidized by L-iditol dehydrogenase. This result indicates that the [3H]iditol was derived from L-myo-inositol inositol 1,4,5,6-tetrakisphosphate. The data are consistent with rapid PAF-sensitive synthesis of D-myo-[3H]inositol 1,3,4,5-tetrakisphosphate in macrophages, and demonstrate that L-myo-inositol 1,4,5,6-tetrakisphosphate is synthesized in both mammalian and avian cells. The levels of L-myo-[3H]inositol 1,4,5,6-tetrakisphosphate in primary-cultured macrophages are not acutely sensitive to PAF.

scholarly journals Protein phosphatase 2A1 is the major enzyme in vertebrate cell extracts that dephosphorylates several physiological substrates for cyclin-dependent protein kinases.

1993 ◽  
Vol 4 (7) ◽  
pp. 669-677 ◽  
Author(s):  
P Ferrigno ◽  
T A Langan ◽  
P Cohen
Keyword(s):  
Rat Liver ◽  
Protein Phosphatase ◽  
Human Fibroblasts ◽  
High Mobility ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
High Mobility Group Protein ◽  
Cell Extracts ◽  
Dependent Protein

Okadaic acid (2 nM) inhibited by 80-90% the protein phosphatase activities in diluted extracts of rat liver, human fibroblasts, and Xenopus eggs acting on three substrates (high mobility group protein-I(Y), caldesmon and histone H1) phosphorylated by a cyclin-dependent protein kinase (CDK) suggesting that a type-2A phosphatase was responsible for dephosphorylating each protein. This result was confirmed by anion exchange chromatography of rat liver and Xenopus extracts, which demonstrated that the phosphatases acting on these substrates coeluted with the two major species of protein phosphatase 2A, termed PP2A1 and PP2A2. When matched for activity toward glycogen phosphorylase, PP2A1 was five- to sevenfold more active than PP2A2 and 35-fold to 70-fold more active than the free catalytic subunit (PP2Ac) toward the three CDK-labeled substrates. Protein phosphatases 1, 2B, and 2C accounted for a negligible proportion of the activity toward each substrate under the assay conditions examined. The results suggest that PP2A1 is the phosphatase that dephosphorylates a number of CDK substrates in vivo and indicate that the A and B subunits that are associated with PP2Ac in PP2A1 accelerate the dephosphorylation of CDK substrates, while suppressing the dephosphorylation of most other proteins. The possibility that PP2A1 activity is regulated during the cell cycle is discussed.

Influence of Posture on Free Calcium and Related Variables

1980 ◽  
Vol 26 (6) ◽  
pp. 781-781
Author(s):  
Brian W Renoe ◽  
Jay M McDonald ◽  
Jack H Ladenson
Keyword(s):  
Phosphate Buffer ◽  
Water Solution ◽  
Internal Standard ◽  
Anion Exchange Chromatography ◽  
Sample Volume ◽  
Exchange Chromatography ◽  
Free Calcium ◽  
Reference List ◽  
Extraction And Purification

Abstract Vol. 25 p 1766: Second paragraph, line four, the reference numbers cited should be (5-11); numbers of all subsequent citations above 10 in text should be increased by one, to correspond with the list of references. p 1828: In Figure 2, the label on the ordinate is not clear. It reads “counts/10 s × 10−3.” p 1930: Under “Determination of Concentrations in Human Plasma,” the injection of dihydroergotamine should be 0.5 mL of a 1 g/L solution. p 1931: Nine to 12 determinations of binding in the absence of nonradioactive standard were also used to construct the standard curves. p 1932: In the paragraph preceding Results and in the caption to Figure 3, the amount of compound 2 is 0.5 mg, not 1 mg. p 2020: In the preparation of phosphate buffer, the second sentence should read: “Dissolve 2.84 g of sodium diphosphate in 100 mL of de-ionized water (solution 2).” p 2021: In the section on extraction of serum under Procedures, the 10 µL of internal standard and 200 µL of sample should be added to 200 µL of phosphate buffer. Vol. 26 p99: In the caption for Figure 3A, read “r = 0.99,” not “r = 0.09.” p. 176: The sample volume referred to in the fourth paragraph of column three should be 0.15 µL, not “150 mL.” p 193: Add to reference list: 8. Goutte-Coussieu, C., Habrioux, G., Eichenberger, D., and Jayle, M. F., Rapid fluorometry of estrogens in nonpregnancy urine, with use of chloroform extraction and purification by anion-exchange chromatography. Clin. Chem. 25, 230 (1979). 9. Jarrige, P., Purification et propriétés des sulfatases du suc d’Helix pomatia. Bull. Soc. Chim. Biol. (Paris) 7-8,761 (1963). 10. Ittrich, G., Untersuchungen über Extraktion des roten Kober-Farbstoffs durch organische Lösungsmittel zur Ostrogenbestimmung im Harn. Acta Endocrinol. (Copenhagen) 35, 34 (1960). 11. Adessi, G., Lallier, J. C., Goutte-Coussieu, C., and Jayle, M. F., Méthode de dosage automatique par spectrofluorimétrie des estrogénes urinaires au cours et en dehors de la grossesse. Ann. Biol. Clin. 37, 89 (1979). 12. Schöller, R., Contrôle de Qualité en Hormonologie. I.-Stéroides Urinaires. Editions Sepe, Paris-Fresnes, 1976, p 51. p. 354: In Table 1, last line, columns 2 and 4, substitute “0.12” for “0.16.”

scholarly journals Guanosine 5′-[γ-thio]triphosphate-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate in HL-60 granulocytes. Evidence that the guanine nucleotide acts by relieving phospholipase C from an inhibitory constraint

1990 ◽  
Vol 271 (3) ◽  
pp. 743-748 ◽  
Author(s):  
M Camps ◽  
C F Hou ◽  
K H Jakobs ◽  
P Gierschik
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Anion Exchange Chromatography ◽  
Major Product ◽  
Exchange Chromatography ◽  
High Protein ◽  
High Protein Concentration ◽  
Inositol Phosphate Accumulation ◽  
Stimulation Of

Myeloid differentiated human leukaemia (HL-60) cells contain a soluble phospholipase C that hydrolysed phosphatidylinositol 4.5-bisphosphate and was markedly stimulated by the metabolically stable GTP analogue guanosine 5′-[gamma-thio]triphosphate (GTP[S]). Half-maximal and maximal (up to 5-fold) stimulation of inositol phosphate formation by GTP[S] occurred at 1.5 microM and 30 microM respectively. Other nucleotides (GTP, GDP, GMP, guanosine 5′-[beta-thio]diphosphate. ATP, adenosine 5′-[gamma-thio]triphosphate, UTP) did not affect phospholipase C activity, GTP[S] stimulation of inositol phosphate accumulation was inhibited by excess GDP, but not by ADP. The effect of GTP[S] on inositol phosphate formation was absolutely dependent on and markedly stimulated by free Ca2+ (median effective concn. approximately 100 nM). Analysis of inositol phosphates by anion-exchange chromatography revealed InsP3 as the major product of GTP[S]-stimulated phospholipase C activity. In the absence of GTP[S], specific phospholipase C activity was markedly decreased when tested at high protein concentrations, whereas GTP[S] stimulation of the enzyme was markedly enhanced under these conditions. As both basal and GTP[S]-stimulated inositol phosphate formation were linear with time whether studied at low or high protein concentration, these results suggest that (a) phospholipase C is under an inhibitory constraint and (b) GTP[S] relieves this inhibition, most likely by activating a soluble GTP-binding protein.

Influence of Posture on Free Calcium and Related Variables

1980 ◽  
Vol 26 (6) ◽  
pp. 781-781
Author(s):  
Brian W Renoe ◽  
Jay M McDonald ◽  
Jack H Ladenson
Keyword(s):  
Phosphate Buffer ◽  
Water Solution ◽  
Internal Standard ◽  
Anion Exchange Chromatography ◽  
Sample Volume ◽  
Exchange Chromatography ◽  
Free Calcium ◽  
Reference List ◽  
Extraction And Purification

Abstract Vol. 25 p 1766: Second paragraph, line four, the reference numbers cited should be (5-11); numbers of all subsequent citations above 10 in text should be increased by one, to correspond with the list of references. p 1828: In Figure 2, the label on the ordinate is not clear. It reads “counts/10 s × 10−3.” p 1930: Under “Determination of Concentrations in Human Plasma,” the injection of dihydroergotamine should be 0.5 mL of a 1 g/L solution. p 1931: Nine to 12 determinations of binding in the absence of nonradioactive standard were also used to construct the standard curves. p 1932: In the paragraph preceding Results and in the caption to Figure 3, the amount of compound 2 is 0.5 mg, not 1 mg. p 2020: In the preparation of phosphate buffer, the second sentence should read: “Dissolve 2.84 g of sodium diphosphate in 100 mL of de-ionized water (solution 2).” p 2021: In the section on extraction of serum under Procedures, the 10 µL of internal standard and 200 µL of sample should be added to 200 µL of phosphate buffer. Vol. 26 p99: In the caption for Figure 3A, read “r = 0.99,” not “r = 0.09.” p. 176: The sample volume referred to in the fourth paragraph of column three should be 0.15 µL, not “150 mL.” p 193: Add to reference list: 8. Goutte-Coussieu, C., Habrioux, G., Eichenberger, D., and Jayle, M. F., Rapid fluorometry of estrogens in nonpregnancy urine, with use of chloroform extraction and purification by anion-exchange chromatography. Clin. Chem. 25, 230 (1979). 9. Jarrige, P., Purification et propriétés des sulfatases du suc d’Helix pomatia. Bull. Soc. Chim. Biol. (Paris) 7-8,761 (1963). 10. Ittrich, G., Untersuchungen über Extraktion des roten Kober-Farbstoffs durch organische Lösungsmittel zur Ostrogenbestimmung im Harn. Acta Endocrinol. (Copenhagen) 35, 34 (1960). 11. Adessi, G., Lallier, J. C., Goutte-Coussieu, C., and Jayle, M. F., Méthode de dosage automatique par spectrofluorimétrie des estrogénes urinaires au cours et en dehors de la grossesse. Ann. Biol. Clin. 37, 89 (1979). 12. Schöller, R., Contrôle de Qualité en Hormonologie. I.-Stéroides Urinaires. Editions Sepe, Paris-Fresnes, 1976, p 51. p. 354: In Table 1, last line, columns 2 and 4, substitute “0.12” for “0.16.”

scholarly journals Separation of Sb (V) and Sb (III) antimony compounds using anion exchange chromatography technique

2020 ◽  
Vol 12 (3) ◽  
pp. 164-169
Author(s):  
Herlinawati Herlinawati ◽  
◽  
Buchari Buchari ◽  
M. Bachri Amran ◽  
◽  
...  
Keyword(s):  
Anion Exchange ◽  
Flow Rate ◽  
Phosphate Buffer ◽  
Mobile Phase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Capacity Factor ◽  
Optimum Conditions ◽  
Chromatography Method ◽  
Theoretical Plates

Separation of Sb (V) and Sb (III) antimony compounds using anion exchange chromatography technique have been done. To obtain the separation of Sb (V) and Sb (III) antimony compounds which is good in this study have been studied several parameters of separation in anion exchange chromatography technique. Parameters that influence the process of separation of Sb (V) and Sb (III) antimony compounds is the concentration and pH of the mobile phase (eluent) have been evaluated. The separation of Sb (V) and Sb (III) antimony compounds is good and optimum obtained using an eluent 200 mM phosphate buffer at pH 7 with a flow rate of 1 mL/min. Based on the optimum conditions for the separation of Sb (V) and Sb (III) antimony compounds with anion exchange chromatography method has generated value the capacity factor (k ') Sb (V) and Sb (III) obtained are respectively 1.77 and 3.01. While the value of selectivity (α), Number of theoretical plates (N) and Resolution (Rs) obtained are respectively 1.70; 369.48; and 1.48.

Extracellular polysaccharides from suspension cultures of Nicotiana plumbaginifolia

2020 ◽  
Author(s):  
Ian Sims ◽  
A Bacic
Keyword(s):  
Uronic Acid ◽  
Cell Suspension Cultures ◽  
Anion Exchange Chromatography ◽  
Nicotiana Plumbaginifolia ◽  
Suspension Cultures ◽  
Exchange Chromatography ◽  
Arabinogalactan Protein ◽  
Extracellular Polysaccharides ◽  
Selective Precipitation ◽  
The Individual

The soluble polymers secreted by cell-suspension cultures of Nicotiana plumbaginifolia contained 78% carbohydrate, 6% protein and 4% inorganic material. The extracellular polysaccharides were separated into three fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7 and the individual polysaccharides in each fraction were then isolated by selective precipitation and enzymic treatment. Monosaccharide and linkage compositions were determined for each polysaccharide after reduction of uronic acid residues and the degree of esterification of the various uronic acid residues in each polysaccharide was determined concurrently with the linkage types. Six components were identified: an arabinoxyloglucan (comprising 34% of the total polysaccharide) and a galactoglucomannan (15%) in the unbound neutral fraction, a type II arabinogalactan (an arabinogalactan-protein, 11%) and an acidic xylan (3%) in the first bound fraction, and an arabinoglucuronomannan (11%) and a galacturonan (26%) in the second bound fraction. © 1995.

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