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PubMed1990

Simultaneous determination of butyltin and phenyltin compounds in oysters by capillary gas chromatography.

A method is described for the simultaneous determination of butyl- and phenyltin compounds in oyster samples. The organotin compounds were extracted (as chlorides) from oyster homogenates with hydrochloric acid and benzene in the presence of 0.05% tropolone. These compounds were converted into pentyl derivatives with pentyl Grignard reagent and then analysed by capillary gas chromatography with a flame photometric detector equipped with a 393-nm filter. The recoveries of six organotin compounds added to oyster samples ranged from 71 to 74%. The detection limits of butyl- and phenyltin compounds were in the 5-9 pg range as tin. We detected significant amounts of three organotin compounds (di- and tributyltin and triphenyltin) in oyster samples.

Animals↗
PubMed1996

[Pharmacological therapeutic prospects of cerebral vasospasm].

New therapies of cerebral vasospasm aim to prevent the effects of subarachnoid haemorrhage. These effects result in red blood cell haemolysis and release of oxyhaemoglobin, free radicals formation and lipid peroxidations, imbalance in endothelial modulation of vasomotor tone and activation of the complement system. Low doses of fibrinolytic agents administered intrathecally accelerate the fibrinolysis of the clot and reduce the oxyhaemoglobin release. The tissue-type plasminogen activator has proven to be effective in preventing vasospasm, but the modalities of this therapy remain to be defined. Free radical reactions may be inhibited by free radical scavengers and inhibitors of lipid peroxidations. Tirilazad is a potent inhibitor of lipid peroxidations, which improves the patients' outcome and has gone to Phase III human trials. Superoxide dismutase and tropolone derivatives are currently evaluated in animal models. Vasomotor tone can be modified in experimental models either by blocking endothelin receptors (BQ-123), or by facilitating the release and enhancing the effect of nitric oxide using protein kinase C inhibitors, drugs that increase intracellular calcium (cyclopiazonic acid, LP-805) and free radicals scavengers (superoxide dismutase). These possibilities are being investigated. Finally, preliminary studies have demonstrated the efficacy of FUT-175, an inhibitor of the complement system, in the prevention of vasospasm. In the next years, these new therapies have to be validated by prospective and randomized clinical trials to propose guidelines for the management of patients at risk of cerebral vasospasm after aneurysmal rupture.

Antioxidants↗
PubMedDate not supplied

Esculetin antagonizes iron-chelating agents and increases the virulence of Listeria monocytogenes.

Iron is an essential compound for the growth and virulence of Listeria monocytogenes. In extracellular environments, iron often requires a siderophore to be acquired by microorganisms. Although it does not produce siderophores, L. monocytogenes can use some exogenous bacterial or fungal siderophores as well as a number of animal or plant o-diphenol compounds to overcome growth inhibition by the iron-chelating agents tropolone and 8-hydroxyquinoline. Esculin, a plant glycoside, can be hydrolysed by L. monocytogenes to the o-diphenol aglucon, esculetin. The latter neutralized in vitro growth inhibition induced by the iron-chelating agents. Furthermore, when injected into infected mice, esculetin enhanced mortality in a dose-dependent manner and increased bacterial counts in spleen induced by sublethal doses of L. monocytogenes. Esculetin apparently functioned as a siderophore for L. monocytogenes in murine tissues.

Animals↗
PubMed2001

Design, synthesis, and biological evaluation of a series of simple and novel potential antimalarial compounds.

A series of compounds bearing an endocyclic -N-O- moiety with potential antimalarial activity based on simple derivatives of the tropolone purpurogallin was prepared by means of a hetero Diels-Alder reaction using nitrosobenzene as a dienophile. The rationale behind the design of these compounds is presented, together with the synthetic route to derivatives bearing aromatic and aliphatic esters of the C4'-position hydroxyl group of the purpurogallin framework, as well as biological data obtained from in vitro assays against Plasmodium falciparum and Trypanosoma cruzi. Several of the new compounds have activities in the 3-9 microM range, and provide leads for the development of a novel class of antiparasitic drugs with improved biological and pharmacological properties.

Animals↗
PubMed2001

Synthesis of N-substituted N-nitrosohydroxylamines as inhibitors of mushroom tyrosinase.

A series of N-substituted N-nitrosohydroxylamines including six new compounds were synthesized and examined for inhibition of mushroom tyrosinase. Corresponding hydroxylamines were reacted with n-butyl nitrite to give substituted nitrosohydroxylamines as their ammonium salt. The N-substituted hydroxylamines were prepared from the primary amines via the oxaziridine, or from the carbonyl compounds via the oxime. Most of the nitrosohydroxylamines tested inhibited mushroom tyrosinase. Among them, N-cyclopentyl-N-nitrosohydroxylamine exhibited the most potent activity (IC(50)=0.6 microM), as powerful as that of tropolone, one of the most powerful inhibitors. As removal of nitroso or hydroxyl moiety, the enzyme inhibitory activity was completely diminished. Both N-nitroso group and N-hydroxy group were suggested to be essential for the activity, probably by interacting with the copper ion at the active site of the enzyme. Lineweaver-Burk plotting showed that cupferron was a competitive inhibitor but that N-cyclopentyl-N-nitrosohydroxylamine was not.

Agaricales↗
PubMed2001

Hydroxy- or methoxy-substituted benzaldoximes and benzaldehyde-O-alkyloximes as tyrosinase inhibitors.

Several benzaldoximes, benzaldehyde-O-ethyloximes, and acetophenonoximes were synthesized and evaluated as tyrosinase inhibitors by an assay based on tyrosinase catalyzed L-DOPA oxidation. Whereas benzaldoxime itself is only a weak inhibitor, its derivatives with one or two hydroxy or methoxy moieties in para and meta positions depress tyrosinase activity. Acetophenonoximes and trisubstituted benzaldoximes show no inhibitory activity. The IC(50) of 3,4-dihydroxybenzaldehyde-O-ethyloxime (0.3 +/- 0.1 micromol L(-1)) is of the same magnitude as tropolone (0.13 +/- 0.08 micromol L(-1)), one of the best tyrosinase inhibitors known so far.

Enzyme Inhibitors↗
PubMed1992

Fluorescence stopped-flow study of the interaction of tubulin with the antimitotic drug MDL 27048.

The kinetics of the binding of MDL 27048 to tubulin have been studied by fluorescence stopped flow. The binding is accompanied by a fluorescence increase. The time course can be described by a sum of two exponentials, assumed to be due to the presence of two major tubulin isoforms. The observed rate constants depend in a nonlinear way on the concentration of MDL in pseudo-first-order conditions. This concentration dependence can be described by the presence of a fast equilibrium of low affinity, followed by an isomerization of the initial complex. The dissociation kinetics have been studied by displacement experiments, in which MTC was used as a competitive ligand. The reaction enthalpy change for the first binding equilibrium and the activation energies for the forward and reverse steps of the isomerization were determined from the temperature dependence. This was possible for the two tubulin isotype populations. The kinetics of the binding of MDL to tubulin are slowed down in the presence of 3',4',5'-trimethoxyacetophenone, a fast binding analog of the colchicine A-ring, but are not influenced by the binding of tropolone methyl ether, indicating that the binding site of MDL has the A-subsite in common with colchicine, but not the C-subsite.

Animals↗
PubMed1984

Interaction of tubulin with bifunctional colchicine analogues: an equilibrium study.

The interaction of tubulin with simple analogues of colchicine that contain both its tropolone and trimethoxyphenyl rings has been characterized, and the results were analyzed in terms of the simple bifunctional ligand model developed for the binding of colchicine [ Andreu , J. M., & Timasheff , S. N. (1982) Biochemistry 21, 534-543] on the basis of interactions of tubulin with single-ring analogues. The compound 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6- cycloheptatrien -1-one has been found to bind reversibly to 0.86 +/- 0.06 site of purified calf brain tubulin with an equilibrium constant of (4.9 +/- 0.3) X 10(5) M-1 (25 degrees C), delta H degrees app = -1.6 +/- 0.7 kcal mol-1, and delta S degrees app = 20.5 +/- 2.5 eu. The binding appears specific for the colchicine site. The closely related compound 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)-propionyl]amino] -2,4,6- cycloheptatrien -1-one interacts weakly with tubulin. Binding of the first analogue is accompanied by ligand fluorescence appearance, quenching of protein fluorescence, perturbation of the far-ultraviolet circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Substoichiometric concentrations of the analogue inhibit microtubule assembly in vitro. Excess analogue concentration under microtubule-promoting conditions induces an abnormal cooperative polymerization of tubulin, similar to that of the tubulin-colchicine complex.

Animals↗
PubMed1984

Carbon-13 nuclear magnetic resonance study of microtubule protein: evidence for a second colchicine site involved in the inhibition of microtubule assembly.

A 13C nuclear magnetic resonance study of bovine microtubule protein was carried out at 43 kG in the presence and absence of colchicine 13C labeled at the tropolone methoxy. Analysis indicated that tubulin has at least two colchicine binding sites: a quasi-irreversibly bound, high-affinity site (i.e., the KD less than 5 microM site generally accepted as the site of colchicine action) as well as a low-affinity site(s) (KD approximately 650 microM) with which free colchicine rapidly exchanges (greater than 100 s-1). The methoxy resonance is broadened to different apparent extents as a result of binding at these two sites (50- vs. 150-Hz broadening for the high- and low-affinity sites, respectively) but undergoes no change in chemical shift upon binding. The low-affinity sites are interpreted to be analogous to the sites deduced by Schmitt and Atlas [Schmitt, H., & Atlas, D. (1976) J. Mol. Biol. 102, 743-758] from labeling studies using bromocolchicine. These sites are likely to be the sites responsible for the abrupt halt in microtubule assembly ("capping") observed at high colchicine concentrations (greater than 20 microM)--a qualitatively different behavior from that observed at low colchicine concentrations [Sternlicht, H., Ringel, I., & Szasz, J. (1983) Biophys. J. 42, 255-267]. Carbon-13 spectra from the aliphatic carbons of microtubule protein consists of narrow resonances--many with line widths less than 30 Hz--superimposed on a broad background. The narrow resonances were assigned to flexible regions in nontubulin proteins [microtubule-associated proteins (MAPs)], in accord with an earlier 1H nuclear magnetic resonance study of microtubule protein [Woody, R. W., Clark, D. C., Roberts, G. C. K., Martin, S. R., & Bayley, P. M. (1983) Biochemistry 22, 2186-2192]. This assignment was supported by 13C NMR analysis of phosphocellulose-purified (MAP-depleted) tubulin as well as heat-stable MAPs. Aliphatic carbons in the MAP preparations were characterized by narrow resonances indicative of carbons with considerable motional freedom whereas the aliphatic regions of phosphocellulose-purified tubulin were, for the most part, characterized by broad resonances indicative of carbons with restricted mobility. However, a moderately narrow resonance (approximately less than 50-Hz line width) coincident with the C gamma resonance of glutamate was detected in 13C NMR spectra of tubulin which indicated that a fraction of the glutamic acid residues is relatively mobile.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1996

Stoichiometric and substoichiometric inhibition of tubulin self-assembly by colchicine analogues.

The mechanism of the stoichiometric and substoichiometric inhibitions of tubulin self-assembly by several structural analogues of colchicine (COL) was investigated. The inhibition data were analyzed in terms of a simple model that takes into consideration Kg, the normal microtubule growth constant, equal to Cr-1 (Cr is the critical concentration for microtubule formation), and Kb, the binding constant of the drug to tubulin. In this manner, the value of the microtubule inhibition constant (Ki), which is the binding constant of the tubulin-drug complex to the end of a growing microtubule (which stops the microtubule growth), was determined. The results of the analysis of microtubule inhibition by the various colchicine analogues show that all the inhibitions can be expressed reasonably by this model. The strongest inhibitors found were colchicine (COL), allocolchicine (ALLO), and the biphenyl keto analogue 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl (TKB), which had essentially identical values of Ki = (2.1 +/- 0.3) x 10(6) M(-1). MTC, the two-ring analogue of colchicine, was weaker (Ki = 5.6 x 10(5) M(-1). A most striking result was that tropolone methyl ether (TME), which is ring C of COL, and which binds very weakly to tubulin (Kb = 3.5 x 10(2) M(-1)), is a substoichiometric inhibitor. Its Ki value of 8.7 x 10(5) M(-1) makes it identical in strength to MTC, suggesting that ring A makes little or no contribution to the induction of assembly inhibition. The three biphenyls, which bind to tubulin with similar affinity, spanned the spectrum from strong substoichiometric inhibition (TKB) to stoichiometric inhibition for 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB) and an intermediate mode for the methoxy derivative 2,3,4,4'-tetramethoxy-1,1'-biphenyl (TMB). The extent of tubulin bound to drugs at 50% inhibition (r) was ca. 2% for TKB, ALLO, and COL, i.e. one liganded tubulin for every 40-50 molecules of free protein (substoichiometric). This ratio was 1:1.5 for TCB (stoichiometric) and 1:6 for TMB (intermediate). For TME, which is a single ring compound, it was 1:25. The progression of the stoichiometries varied directly with Ki and was totally unrelated to the values of Kb, which indicated the control of the stoichiometry by Ki and the close thermodynamic linkage between r and Ki. Comparison of the inhibitory capabilities of the various drugs identified the need for strong substoichiometric inhibition of a carbonyl group on ring C or C'. Furthermore, this group must be properly oriented by interaction with the protein or by the structural rigidity imparted by ring B, as in ALLO. The simple linked equilibrium model developed in this paper permits the alignment of drugs along a continuum that ranges from stoichiometric to strong substoichiometric modes of microtubule inhibition. Furthermore, it shows that the previously identified two classes are the two ends of a monotonously progressing spectrum described by a single mechanism of action.

Animals↗
PubMed1996

Kinetics of association and dissociation of two enantiomers, NSC 613863 (R)-(+) and NSC 613862 (S)-(-) (CI 980), to tubulin.

The kinetics of binding of R- and S-enantiomers were studied by the fluorescence stopped-flow technique. For the R-enantiomer, the time course of the increase in fluorescence is best fitted by a sum of two exponentials. In pseudo-first-order conditions, the first observed rate constant showed a linear concentration dependence whereas the second showed a hyperbolic one. The dissociation rate constants were determined independently by displacement experiments with 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC). The two exponential phases were assumed to be due to a two-step binding mechanism: an initial binding followed by a conformational change. This is different from colchicine and MTC binding, where the two phases show a hyperbolic concentration dependence and are attributed to the parallel binding to different isoforms of tubulin [Banerjee, A., & Luduena, R. F. (1992) J. Biol. Chem. 267, 13335-13339]. R-isomer binding did not discriminate between the tubulin isoforms. The temperature dependence of all the rate constants were measured, and the entire thermodynamic reaction path was constructed. For the S-isomer, the direct fluorescence stopped-flow study showed that the signals were largely imputable to the fluorescence of the binding at low-affinity sites [Leynadier, D., Peyrot, V., Sarrazin, M., Briand, C., Andreu, J. M., Rener, G. A., & Temple, C., Jr. (1993) Biochemistry 32, 10674-10682]. Therefore, we exploited the competition between R- and S-isomers to determine the binding kinetics of the S-isomer to the R-site. The observed rate constants for competitive binding showed a linear concentration dependence, thus allowing us to calculate the association rate constant of the S-isomer to the R-site. The kinetics of displacement of the S-isomer by MTC allowed the dissociation rate constant for the S-isomer to be determined. The binding of both enantiomers to tubulin in presence of tropolone methyl ether (analog of the colchicine C ring) was decreased, indicating the involvement of the C subsite.

Animals↗
PubMed1998

Linkages in tubulin-colchicine functions: the role of the ring C (C') oxygens and ring B in the controls.

Linkages between structural components of colchicine (COL) and its biphenyl analogues (allocolchicine, ALLO, and its analogues) in the binding to tubulin and its functional consequences were scrutinized. Three ring ALLO analogues with the carbomethoxyl in position 4' of ring C' replaced by a carbomethyl (KAC) and methoxy (MAC) groups were synthesized. The binding properties and consequences of binding (microtubule inhibition, abnormal polymerization, and induction of GTPase activity) were compared within the series of three ring and two ring compounds, as well as between pairs consisting of a two ring and a three ring compound with identical groups in position 4'. Binding measurements showed that the binding of KAC to the COL binding site proceeded with similar chemical characteristics as that of its two ring analogue (TKB), but with the kinetic characteristics of ALLO. The binding constant of KAC was found to be 1.9 x 10(6) M-1 and that of MAC was 4.6 x 10(5) M-1. The binding strength of the three ring analogues in descending order was KAC > ALLO > MAC, with increments similar to the biphenyl compounds, TKB > TCB > TMB. The difference in binding affinities between the pairs of three ring and two ring molecules was invariant (delta delta G degree = -1.3 +/- 0.2 kcal/mol-1), showing that in all cases ring B makes only an entropic contribution by suppressing free rotation about the biaryl bond. In the case of microtubule inhibition, all three ring compounds inhibited strongly with similar potencies, even though the spread in inhibition strength between the corresponding two ring molecules was > 3.3 kcal mol-1 of free energy. This difference was interpreted in terms of the ability of the various molecules to maintain tubulin in the proper conformation for binding in abnormal geometry to the growth end of a microtubule. This ability attains a maximal plateau value for three ring compounds, independently of the oxygen-containing group in ring C' (or C) and is maintained for the methyl ketone whether in a two or three ring compound. The induction of the GTPase activity was found to follow in general the binding affinity, with the exception that molecules that contained a methyl ketone were stronger GTPase inducers than expected from their alignment according to binding affinity. The finding that the binding of tropolone methyl ether (ring C of COL) induced a GTPase activity shows that ring C contains the ability to induce both substoichiometric microtubule inhibition and GTPase activity. Rings A and B act only as anchors in the binding, with ring A making an energetic contribution, while the effect of ring B is only entropic. It was concluded that both microtubule assembly inhibition and induction of GTPase activity were modulated by the same postbinding conformational change in tubulin. The difference between the strengths of these activities induced by ligands reflects the difference between a narrow allosteric effect between two well-defined sites in the case of GTPase activity and a broad effect aimed at the multiple sites involved in the incorporation of a tubulin protomer into the microtubule structure. Thus, there seems to be a loose thermodynamic linkage between binding and GTPase activity, while there is none between binding and microtubule inhibition, the two phenomena being linked only kinetically.

Acetamides↗
PubMedDate not supplied

Improved fermentation processes for NS0 cell lines expressing human antibodies and glutamine synthetase.

To meet the increasing requirement for therapeutic antibodies to conduct clinical trials, an enhanced culture medium and fed-batch process was developed for GS-NS0 cell lines. This process was shown to produce high concentrations of monoclonal antibodies for several cell lines expressing different antibodies. Cells were adapted to growth in a glutamine- and serum-free medium containing bovine serum albumin (BSA), cholesterol, and transferrin. A number of amino acids were found to be depleted during cell culture. The concentrations of these amino acids were increased, and further cell culture analyses were performed. This process of cell growth and analysis was repeated over multiple cycles until no depletion was detected. This resulted in an amino acid supplement that was shown to be generic and enhanced antibody productivity up to 5-fold for the three cell lines tested. Transferrin was replaced using tropolone, a lipophilic iron chelator and ferric ammonium citrate. Cell growth was equivalent to that in transferrin-containing medium over the wide ranges tested. A concentrated feed solution, based on the amino acid supplement and the components of the serum- and protein-free supplements, was formulated. Addition of this feed in response to metabolic requirements resulted in a harvest titer a further 2-fold higher than the enhanced culture medium. Harvest antibody titers of up to 600 mg/L were achieved for three cell lines expressing different antibodies, representing an increase of 10-fold over the starting concentrations.

Amino Acids↗
PubMed2001

Crossover phase behavior (discotic to calamitic) in liquid-crystalline copper complexes. Dependence on the length and position of alkoxy chains in new polycatenar bis[5-(dialkoxybenzylidine)aminotropololonato]copper(II) complexes.

New polycatenar metallomesogens have been synthesized containing a tropolone core joined to benzylidine terminal rings. Variation of the position, number, and nature of alkoxy substituents on the terminal rings allows for the control of the phase behavior of the compounds. In particular, for copper complexes with 3,4-dialkoxy substitution, a change from calamitic to discotic (columnar) behavior is seen as the chain lengths are increased.

Journal Article↗
PubMed2005

Six-coordinate titanium complexes of a tripodal aminetris(phenoxide) ligand: synthesis, structure, and dynamics.

The five-coordinate titanium(IV) alkoxide LTi(O(t)Bu) (LH(3) = tris(2-hydroxy-3,5-di-tert-butylbenzyl)amine) is protonolyzed readily by the conjugate acids of monoanionic bidentate ligands, both symmetrical (tropolone, acetylacetone, di-p-toluoylmethane) and unsymmetrical (8-hydroxyquinoline, salicylaldehyde, 2,6-diformyl-p-cresol, anthrarufin). The geometry of these complexes, which is pseudo-octahedral with the tripodal ligand adopting a chiral, propeller-like conformation, has been confirmed in four cases by X-ray crystallography. Variable-temperature NMR spectroscopy indicates that the six-coordinate complexes undergo two dynamic processes. First, the ligands undergo a twisting motion that results in racemization, a process which is over 10(4) times faster than in five-coordinate complexes. The rate acceleration upon binding of an equatorial ligand is ascribed to steric repulsions with one of the cis phenoxides; the dynamics of a binuclear dibenzyl phosphate-bridged compound, which has a unique conformation of the tripodal ligand, indicates that flexing the cis phenoxide is the rate-limiting step in racemization. Second, the complexes undergo a process that interchanges the inequivalent arms of the tripodal ligand. This process involves a trigonal twist that shifts the bidentate ligand between clefts in the tripod. The intermediate geometry in the reaction appears to be a transition state and not a long-lived intermediate, as judged from the relative rates of interconversion of tripod arms and chelate ends in the ditoluoylmethane complex. Tripod arm interchange takes place without partial dissociation of the bidentate chelate, a reaction that has been observed on a slower time scale in one case.

Journal Article↗
PubMed2000

Purification and characterization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab).

The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparent homogeneity by a combination of ammonium sulfate precipitation, DEAE-Sephacel chromatography, phenyl agarose chromatography, and Sephadex G-200 gel filtration. The purified enzyme has a molecular weight of 120 +/- 3 kDa and is a tetramer of 30 +/- 1.5 kDa. Native polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of a single isoform with an observed pH optimum of 4.0. 4-Methyl catechol is the best substrate, followed by catechol, and L-3,4-dihydroxyphenylalanine, all of which exhibited a phenomenon of inhibition by excess substrate. No activity was detected toward chlorogenic acid, catechin, caffeic acid, gallic acid, and monophenols. Tropolone, both a substrate analogue and metal chelator, proved to be the most effective competitive inhibitor with an apparent K(i) of 5.8 x 10(-)(7) M. Ascorbic acid, metabisulfite, and cysteine were also competitive inhibitors.

Amino Acid Sequence↗
PubMed2000

Fast apple (Malus x domestica) and tobacco (Nicotiana tobacum) leaf polyphenol oxidase activity assay for screening transgenic plants.

A spectrophotometric assay method for the analysis of polyphenol oxidase (PPO), in apple and tobacco leaves, has been optimized to increase efficiency in the screening of large numbers of transgenic plants. Crude protein extracts from leaf punches were prepared in a FastPrep homogenizer. The addition of Triton X-100 during extraction resulted in 44 and 74% increases in the PPO activity recovered, from apple and tobacco, respectively. The enzyme kinetics differed markedly between apple and tobacco. Apple leaf PPO was isolated in a latent state and was activated by the addition of SDS. In contrast, tobacco PPO activity was inhibited by SDS, particularly at acidic pH. Apple PPO showed a pronounced pH optimum around pH 6, whereas the pH profile for tobacco PPO was much flatter, with a broad optimum around pH 4. The calculated Km' value for apple PPO, using 4-methylcatechol as substrate, was 8.1, and for tobacco the Km was 4.3. The PPO reaction was strongly inhibited by tropolone, a Cu competitor, and restored by the addition of Cu2+. Several factors affecting variability in leaf PPO activity levels in plants are discussed.

Biotechnology↗
PubMed2003

Partial purification of latent persimmon fruit polyphenol oxidase.

Persimmon fruit polyphenol oxidase (PPO) was partially purified using a combination of phase partitioning with Triton X-114 and ammonium sulfate fractionation between 50 and 75%. The enzyme, which showed both monophenolase and diphenolase activities, was partially purified in a latent form and could be optimally activated by the presence of 1 mM sodium dodecyl sulfate (SDS) with an optimum pH of 5.5. In the absence of SDS, the enzyme showed maximum activity at acid pH. SDS-PAGE showed the presence of a single band when L-DOPA was used as substrate. The apparent kinetic parameters of the latent enzyme were determined at pH 5.5, the V(m) value being 15 times higher in the presence of SDS than in its absence, whereas the K(M) was the same in both cases, with a value of 0.68 mM. The effect of several inhibitors was studied, tropolone being the most active with a K(i) value of 0.45 microM. In addition, the effect of cyclodextrins (CDs) was studied, and the complexation constant (K(c)) between 4-tert-butylcatechol (TBC) and CDs was calculated using an enzymatic method. The value obtained for K(c) was 15580 M(-1).

Ammonium Sulfate↗