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R K Ibrahim

Publications and source records attributed to R K Ibrahim.

32 records · Page 2Linked to original sources
PubMed1988

Enzymatic synthesis of polymethylated flavonols in Chrysosplenium americanum. III. Purification and kinetic analysis of S-adenosyl-L-methionine:3-methylquercetin 7-O-methyltransferase.

An O-methyltransferase (OMT) which catalyzes the methylation of 3-methylquercetin to 3,7-dimethylquercetin, the second step of methyl transfers toward the biosynthesis of polymethylated flavonol glucosides, has been isolated from Chrysosplenium americanum shoot tips. The 7-OMT was purified by ammonium sulfate precipitation, gel filtration, chromatofocusing and ion-exchange chromatography using a fast protein liquid chromatography system. Compared with previously reported methods [1985) Arch. Biochem. Biophys. 238, 596-605), this protocol resulted in a highly purified enzyme preparation, free from other OMT activities, which allowed the study of its kinetic mechanism. Substrate interaction and product inhibition patterns obtained were consistent with an ordered bi bi mechanism, where S-adenosyl-L-methionine is the first substrate to bind to the enzyme and S-adenosyl-L-homocysteine is the last product released. However, the results obtained did not exclude the formation of one or more dead-end complex. The similarity in kinetic characteristics of this enzyme to those of the other Chrysosplenium OMTs suggests that methyltransferases of this tissue may have evolved from a common precursor.

Flavonoids↗
PubMed1988

Partial purification, characterization, and kinetic analysis of isoflavone 5-O-methyltransferase from yellow lupin roots.

An isoflavone 5-O-methyltransferase was partially purified from the roots of yellow lupin (Lupinus luteus) by fractional precipitation with ammonium sulfate, followed by gel filtration and ion-exchange chromatography using a fast-protein liquid chromatography system. This enzyme, which was purified 810-fold, catalyzed position-specific methylation of the 5-hydroxyl group of a number of substituted isoflavones. The methyltransferase had a pH optimum of 7 in phosphate buffer, an apparent pI of 5.2, a molecular weight of 55,000, no requirement for Mg2+, and was inhibited by various SH-group reagents. Substrate interaction kinetics of the isoflavonoid substrate and S-adenosyl-L-methionine gave converging lines which were consistent with a sequential bireactant binding mechanism. Furthermore, product inhibition studies showed competitive inhibition between S-adenosyl-L-methionine and S-adenosyl-L-homocysteine and noncompetitive inhibition between the isoflavone and either S-adenosyl-L-homocysteine or the 5-O-methylisoflavone. The kinetic patterns obtained were consistent with an ordered bi bi mechanism, where S-adenosyl-L-methionine is the first substrate to bind to the enzyme and S-adenosyl-L-homocysteine is the final product released. The physiological role of this enzyme is discussed in relation to the biosynthesis of 5-O-methylisoflavones of this tissue.

Cations, Divalent↗
PubMed1987

Enzymatic assay for flavonoid sulfotransferase.

A novel enzyme assay for flavonoid sulfotransferase is described. It makes use of tetrabutylammonium dihydrogen phosphate which forms a pair of ions with the flavonoid sulfate esters formed. This renders the sulfate ester soluble in organic solvents such as ethyl acetate, whereas the sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate, remains in the aqueous reaction mixture. The procedure is simple, rapid, and reproducible. It eliminates the need for chromatographic separation of the reaction products, except when their identification is required, and is suitable for use in the purification and kinetic studies of sulfotransferases.

Arylsulfotransferase↗
PubMed1985

Purification, properties and kinetic mechanism of flavonol 8-O-methyltransferase from Lotus corniculatus L.

A novel O-methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to the 8-hydroxyl group of flavonols was purified about 1200-fold from Lotus flower buds, by precipitation with ammonium sulfate and successive chromatography on columns of Sephadex G-100, S-adenosyl-L-homocysteine--Agarose, hydroxyapatite and Polybuffer ion exchanger. The enzyme exhibited strict specificity for position 8 of 8-hydroxyquercetin and 8-hydroxykaempferol, a pH optimum at 7.9, a pI value of 5.5, an Mr of 55 X 10(3) and required Mg2+ and SH groups for activity. The Km values for 8-hydroxykaempferol and S-adenosyl-L-methionine were 1.3 microM and 53 microM, respectively. The data obtained from substrate interaction and product inhibition studies are expected for a steady-state ordered bi-bi mechanism, with 8-hydroxyflavonol binding before S-adenosyl-L-methionine followed by the release of S-adenosyl-L-homocysteine and 8-methoxyflavonol. An alternative mechanism that may also fit the data is the mono-iso Theorell-Chance with the inverse binding sequence and an isomerization step of the free enzyme.

Buffers↗
PubMed1985

Enzymatic synthesis of polymethylated flavonols in Chrysosplenium americanum. I. Partial purification and some properties of S-adenosyl-L-methionine:flavonol 3-, 6-, 7-, and 4'-O-methyltransferases.

Four novel flavonol O-methyltransferases (OMTs) were partially purified from Chrysosplenium americanum by precipitation with ammonium sulfate, successive chromatography on Sephacryl S-200 and hydroxylapatite, and chromatofocusing on Polybuffer ion exchanger. They exhibited strict position specificity for positions 3 of quercetin, 7 of 3-methylquercetin, 4' of 3,7-dimethylquercetin, and 6 of 3,7,3'-trimethylquercetagetin. None of these enzymes reacted with phenylpropanoids, flavones, dihydroflavonols, or any of their glucosides. Except for the 7-OMT whose activity was lost during chromatofocusing, the other enzymes had apparent pI values of 4.8, 5.4, and 5.7 for the 3-, 4'-, and 6-OMTs, respectively. They had similar molecular weights (Mr 57,000) and their pH optima varied between 7.8 and 9.0, with a shift in optimal activity from lower to higher pH with increasing level of substrate methylation. Unlike the 3 and 4' enzymes, the 6-OMT showed an absolute requirement for Mg2+ whose activation was saturable and was inhibited by EDTA. The in vitro stepwise O-methylation of quercetin----3-methylquercetin----3,7-dimethylquercetin----3 ,7, 4'-trimethylquercetin tends to suggest a coordinated sequence of methyl transfers in this tissue.

Cations, Divalent↗
PubMed1985

Enzymatic synthesis of polymethylated flavonols in Chrysosplenium americanum. II. Substrate interaction and product inhibition studies of flavonol 3-, 6-, and 4'-O-methyltransferases.

The steady-state kinetic behavior of three position-specific O-methyltransferases (3-, 4'-, and 6-OMTs) was compared with reference to substrate inhibition patterns in Chrysosplenium americanum. The 6-OMT was severely inhibited by the flavonoid substrate at concentrations close to Km, whereas the other two enzymes were less affected by their respective flavonoid substrates. Substrate interaction kinetics for the 6-OMT gave converging lines consistent with a sequential binding mechanism, whereas the data generated for the 3- and 4'-OMTs could be fitted to the equation for a ping-pong mechanism or to that of a sequential binding mechanism where Kia was much smaller Ka. More information on the mechanism of reaction was obtained from product inhibition studies. The three enzymes exhibited competitive inhibition patterns between S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH), whereas other patterns were either noncompetitive or uncompetitive. The steady-state kinetic properties of the 3-, 4'-, and 6-OMTs were consistent with a sequential ordered reaction mechanism, in which SAM and SAH were leading reaction partners and included an abortive EQB complex. Product inhibition constants were sufficiently low to suggest that these may be important in regulating the pathway of polymethylated flavonoid synthesis. It was suggested that due to their greater sensitivity to inhibition by SAH, the OMTs involved in earlier steps of the methylation sequence may regulate the rate of synthesis of final products in Chrysosplenium.

Flavonoids↗
PubMed1984

Kinetic mechanism of a flavonol-ring-B O-glucosyltransferase from Chrysosplenium americanum.

The kinetic mechanism of a flavonol-ring-B O-glucosyltransferase from Chrysosplenium americanum was investigated. Substrate interaction kinetics for the flavonol and UDPG gave converging lines, which were consistent with a sequential bireactant binding mechanism. They also showed substrate inhibition with respect to the flavonol. Intercept and slope replots were linear and gave a KA of 250 microM and a KB of 10 microM. Product-inhibition studies showed competitive inhibition between UDPG and UDP (KiQ 20 microM) and non-competitive inhibition between the flavonol substrate and its glucoside (KiP 1 mM). Kinetic patterns were consistent with an ordered bi-bi mechanism, where UDPG is the first substrate to bind to the enzyme and UDP is the final product released. The high KiP value, as compared with that of KB, indicates that the reaction is not inhibited by the glucosylated products formed and conforms with the accumulation of flavonol glucosides in C. americanum.

Glucosyltransferases↗
PubMed1983

Purification and Properties of Flavonol-Ring B Glucosyltransferase from Chrysosplenium americanum.

A novel glucosyltransferase which catalyzed the transfer of glucose from UDP-glucose to positions 2' and 5' of partially methylated flavonols was isolated from the shoots of Chrysosplenium americanum Schwein ex Hooker. It was purified 225-fold by ammonium sulfate precipitation and successive chromatography on Sephadex G-100, hydroxyapatite, and polybuffer ion exchanger. This glucosyltransferase appeared to be a single polypeptide with an apparent molecular weight of 42,000 daltons, pH optimum of 7.5 to 8.0, and an isoelectric point of 5.1. It had low but similar K(m) values for the 2' and 5' positions of flavonol substrates and the cosubstrate UDP-glucose and was inhibited by both reaction products, the glucosides formed, and UDP.Glucosyltransferase activity was independent of divalent cations, was not inhibited by EDTA, but showed requirement for SH groups. The differential effect on enzyme activity of metal ions, especially cupric ion, and various SH group reagents seemed to indicate the involvement of two active sites in the glucosylation reaction; the site specific for 2' activity being more susceptible than that of the 5' activity. The substrate specificity expressed by this glucosyltransferase and the requirement of at least two para-oriented B-ring substituents (at 2' and 5') for activity support this view.

Journal Article↗
PubMedDate not supplied

Effect of aflatoxicosis on the kinetic behaviour of ceftiofur in chickens.

The kinetic behaviour of ceftiofur sodium was studied in aflatoxin treated chickens for 30 days and in non-treated chickens, following oral, intramuscular and intravenous administrations of 10 mg kg(-1) bodyweight of ceftiofur. Aflatoxicosis resulted in a more significant decrease in ceftiofur serum concentration in the treated than in non-treated chickens following oral and intravenous administrations. The kinetic behaviour showed that following intravenous injection the elimination half life time t0.5 (el) was significantly shorter in the treated chickens (1.75+/-0.03 hours) than in non-treated chickens (4.23+/-0.05 hours). Following oral administration, the kinetic behaviour revealed a longer absorption half-life [t0.5 (ab), 62.74+/-1.59 minutes] in the treated chickens than in non-treated (50.46+/-5.07 minutes), with lower Cmax 23.25+/-0.42 microg ml(-1) at long tmax (3.05+/-0.07 hour) in treated chickens than in non-treated (Cmax 27.83+/-1.28 at tmax 2.39+/-0.07 hours).

Animals↗
PubMedDate not supplied

Characterization of a monoclonal antibody specific to a flavonol 2'-O-glucosyltransferase.

A monoclonal antibody to a partially purified preparation of 2'-O-glucosyltransferase was produced by in vitro immunization of spleen cells from BALB/c mice, followed by fusion with mouse myeloma cells. Hybridoma culture supernatants were screened by enzyme-linked immunosorbent assay for (i) their ability to produce immunoglobulins and (ii) their immunoreactivity with a partially purified enzyme preparation. The majority of the immunoglobulin-producing hybridomas were IgM secretors. Two highly immunoreactive IgM-secreting clones were chosen for further characterization. The supernatant fraction from a culture of one of these clones displayed 50% inhibition of the 2'-O-glucosyltransferase activity. The native form of the 2'-O-glucosyltransferase was essential for recognition, suggesting that the epitope recognized by the antibody is a conformational discontiguous one.

Animals↗
PubMedDate not supplied

Evidence for the existence of meta and para directing O-methyltransferases in tobacco cell cultures.

The O-methyltransferase of tobacco cell culture was resolved to its meta and para directing forms by chromatography on DEAE-cellulose. Despite similarities in molecular weights and pI values of the two forms, however, evidence from pH optima, SH-group inhibitors, methylation ratios, SDS-acrylamide gels and mixed substrate experiments indicates the existence of two discrete enzymes acting at the meta and para positions of caffeic acid and quercetin, respectively; though the latter enzyme was less substrate specific than its meta counterpart.

Cells, Cultured↗
PubMedDate not supplied

Activation of isoflavone biosynthesis in excised cotyledons of Lupinus seedlings by jasmonoids and excess light.

Exogenous jasmonic acid (JA) and methyl jasmonate (MJ) induced accumulation of isoflavone constituents in cotyledons prepared from imbibed seeds of white lupin (Lupinus albus L.). Exogenous 0.2 mM MJ enhanced the levels of 7-O-(6"-O-malonyl)glucosylgenistein and 7-O-glucosylgenistein in the cotyledons of etiolated seedlings that had been incubated in the dark for 48 h. Regarding isoflavone induced by excision and slicing in the cotyledons as background level, the effect of light was 2- to 3-fold higher than that of 0.2 mM MJ. Cotyledons exposed to MJ along with a 24-h light period displayed a higher level of isoflavone accumulation than that of light alone. Total molar amounts of isoflavone accumulated in the cotyledons treated with MJ under continuous light were approximately the sum of those induced by MJ alone and light alone, respectively. The additive-like effect of MJ and light on isoflavone accumulation in lupin tissues suggested the presence of two different signaling systems independently responsible for those two stimuli. Excised cotyledons from etiolated yellow lupin (L. luteus L. cv. Topaz) seedlings also supported this hypothesis. The cotyledons could accumulate both an isoflavone and a flavone, and MJ selectively increased some of the isoflavone constituents, whereas light enhanced the levels of both. The selective accumulation mechanism of isoflavonoids in cotyledons, in which jasmonoids are involved, clearly differed from that activated by light.

Chromatography, High Pressure Liquid↗