Metabolism of chlorinated guaiacols by a guaiacol-degrading Acinetobacter junii strain.

The metabolism of chlorinated guaiacols by a pure bacterial strain identified by its ability to use guaiacol as the sole carbon and energy source was studied. This strain, identified as Acinetobacter junii 5ga, was unable to grow on several chlorinated guaiacols and catechols. However, strain 5ga grown on guaiacol degraded 4- and 5-chloroguaiacol and 4,5-dichloroguaiacol. Under the same conditions, these cells did not degrade 6-chloroguaiacol, 4,6-dichloroguaiacol, 4,5,6-trichloroguaiacol, or tetrachloroguaiacol, suggesting that the substitution at the 6 position in the ring prevents metabolism of the compound. Degradation of 4-chloroguaiacol was dependent on the initial ratio between the chlorinated compound and viable cells. Transient formation of chlorocatechols resulting from incubation of cells with 4-chloroguaiacol or 4,5-dichloroguaiacol was suggested by UV spectroscopy. Gas chromatography analyses of samples from cultures of strain 5ga grown on guaiacol and incubated with 4- and 4,5-dichloroguaiacol confirmed the presence of 4-chlorocatechol and 4,5-dichlorocatechol, respectively. The formation of the latter was corroborated by gas chromatography-mass spectrometry. Thus, this strain is able to initiate metabolism of specific chlorinated guaiacols by O-demethylation. The starting chlorinated guaiacols and their O-demethylated metabolites inhibited the growth of A. junii 5ga on guaiacol.     

Inhibitory effect of chlorinated guaiacols on methanogenic activity of anaerobic digester sludge

Of four chlorinated guaiacols, tetrachloroguaiacol at 62 M inhibited acetate methanogenesis, the strongest decreasing activity by 50%. 4,5,6-Trichloroguaiacol, 4,5-dichloroguaiacol, and 4-chloroguaiacol showed 50% inhibition at 0.13, 0.32, and 1.50 mM, respectively. Degradation test results of volatile fatty acids (acetic, propionic, and butyric acid) by anaerobic digester sludge (stored 5 weeks) indicated that syntrophic butyrate degraders of this sludge were more sensitive to tetrachloroguaiacol than acetoclastic methanogens and syntrophic propionate degraders.     

Anaerobic O-demethylation of chlorinated guaiacols byAcetobacterium woodii andEubacterium limosum

The acetogenic bacteriaEubacterium limosum andAcetobacterium woodii are strict anaerobes with the ability to metabolizeO-methyl substituents of aromatic compounds. In investigating the versatility of the O-demethylating activity of these acetogens, we examined the anaerobic O-demethylation of chlorinated guaiacols (2-methoxyphenols). Anaerobic cell suspensions of bothE. limosum andA. woodii were able to O-demethylate di-, tri- and tetrachloroguaiacols to the corresponding catechols. The chlorocatechols accumulated and were not further metabolized. A chlorine substituent in the positionortho to the methoxyl-group hindered, but did not completely inhibit O-demethylation. Similar O-demethylation of chloroguaiacols has been observed in anaerobic sediments. The O-demethylating enzyme(s) of both strains seem to be fairly non-specific.Abbreviations DCC Dichlorocatechol - DCG Dichloroguaiacol - TCC Trichlorocatechol - TCG Trichloroguaiacol - TeCC Tretrachlorocatechol - TeCG Tetrachloroguaiacol     

Metabolism of chloroguaiacols by Rhodococcus chlorophenolicus

Summary A polychlorophenol degrader, Rhodococcus chlorophenolicus, was shown to metabolize five different chlorinated guaiacols, namely tetrachloroguaiacol, 3,4,6-trichloroguaiacol, 3,5,6-trichloroguaiacol, 3,5-dichloroguaiacol and 3,6-dichloroguaiacol. Seven different intermediate metabolites, each with three hydroxyl or methoxyl groups, were identified. Four of these metabolites were also dehalogenation products, three carrying one chlorine atom less than the parent compound, and one metabolite from tetrachloroguaiacol where two chlorine atoms had been removed. Tetrachloroguaiacol was shown to undergo reductive dehalogenation. Demethylation of guaiacol to catechol was observed with the dichloroguaiacols, but not with polychloroguaiacols.Abbreviations DCG dichloroguaiacol - TCG trichloroguaiacol - TeCG tetrachloroguaiacol - DCC dichlorocatechol - TCC trichlorocatechol - TeCC tetrachlorocatechol - TCP trichlorophenol - TeCP tetrachlorophenol - PCP pentachlorophenol. An example of numeration - 346-TCG 3,4,6-trichloroguaiacol - GLC gas liquid chromatography     

Metabolism of mono-and dichlorinated guaiacols by Rhodococcus ruber CA16

The metabolism of chloroguaiacols by a soil bacterium was studied. The strain was isolated by enrichment with guaiacol as the sole carbon and energy source, and identified as a Rhodococcus ruber CA16. None of seven chlorinated, guaiacols supported bacterial growth. However, ultraviolet spectroscopy chloride release, and oxygen consumption showed that resting cells grown on guaiacol degraded completely 4-chloroguaiacol 5-chloroguaiacol and 6-chloroguaiacol and, to a lesser extent, 4,5-dichloroguaiacol Gas chromatographic analysis suggested microbial formation of 4-chlorocatechol and 4,5-dichlorocatechol from 4-chloroguaiacol and 4,5-dichloroguaiacol, respectively. Although mono-and dichloroguaiacols did not affect the strain's ability to grow on guaiacol, chlorocatechols completely arrested growth. The role of chlorocatechols in chloroguaiacol metabolism by this guaiacol-degrading bacterial strain is discussed.     

Biodegradation of chlorophenols by mixed and pure cultures from a fluidized-bed reactor

An aerobic, continuous-flow fluidized-bed reactor was established with inoculum from activated sludge, and fed a mixture of 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetrachlorophenol (TeCP) and pentachlorophenol (PCP) as the sole sources of carbon and energy for 2 years. Experiments with the enrichment were performed with material from the reactor. Later, degradation experiments were completed using pure cultures of bacteria that were isolated from suspended samples of the carrier biofilm. In batch-bottle bioassays, the reactor enrichment degraded PCP, TeCP and TCP both in mineral salts (MS) and tryptone-yeast extract-glucose (TGY) media. ortho-Methoxylated chlorophenols including 4,5-dichloroguaiacol (4,5-DCG), tetrachloroguaiacol (TeCG) and trichlorosyringol (TCS) resisted biodegradation by the enrichment both in MS and TGY media, whereas 5,6-dichlorovanillin (5,6-DCV) was readily transformed to an unidentified metabolite. Experiments with ¹⁴C labeled chlorophenols showed mineralization of 2,4-dichlorophenol (DCP) and 2,3,5-TCP to ¹⁴CO₂ by the enrichment. Material from the suspended biofilm after continuous chlorophenol feeding for 2 years was inoculated onto TGY-agar plates, and showed predominantly two colony, types accounting for over 99% of the total colony counts. The two colony types, were equal in abundance. Six Gram-negative, oxidase- and catalase-positive, non-fermentative small rods were isolated in TGY agar media supplemented with 10 mg/l of TeCP or PCP. All isolates formed colonies in TGY plus 150 mg/l of PCP. The isolates degraded TCP and TeCP but not PCP. In mixtures of isolated bacteria the rates of chlorophenol degradation were similar to those observed with individual isolates. Three isolates were identified as Pseudomonas saccharophila and three were an unidentified species of Pseudomonas.     

Hydroxylation and dechlorination of chlorinated guaiacols and syringols by Rhodococcus chlorophenolicus.

We show that Rhodococcus chlorophenolicus PCP-I, a polychlorophenol degrader, also degrades various chlorine-substituted guaiacols (2-methoxyphenols) and syringols (2,6-dimethoxyphenols). The substrates investigated were tetrachloroguaiacol, 3,4,6- and 3,5,6-trichloroguaiacol, 3,5- and 3,6-dichloroguaiacol, trichlorosyringol, and 3,5-dichlorosyringol. The first step was a hydroxylation, probably in a position para to the preexisting hydroxyl. Tetrachloroguaiacol and trichlorosyringol, with a chlorine substituent in the para position, were both hydroxylated and dechlorinated. The optimum temperature for degradation of polychlorinated guaiacols and syringols was 37 to 41 degrees C. Degradation of polychlorinated phenols, guaiacols, and syringols by R. chlorophenolicus was inducible, and induction was controlled coordinately.     

Methylation of halogenated phenols and thiophenols by cell extracts of gram-positive and gram-negative bacteria

O-methylation of 2,6-dibromophenol was studied in cell extracts prepared from Rhodococcus sp. strain 1395. O-methylation activity was enhanced by the addition of S-adenosyl-l-methionine but was not affected by the addition of 5-methyltetrahydrofolate nor by up to 10 mM MgCl(2) or EDTA. By using 2,6-dibromophenol, 4,5,6-trichloroguaiacol, and pentachlorothiophenol as the substrates, O-methylation activity was also demonstrated in extracts from two other Rhodococcus sp. strains, an Acinetobacter sp. strain, and a Pseudomonas sp. strain. A diverse range of chloro- and bromophenols, chlorothiophenols, chloro- and bromoguaiacols, and chloro- and bromocatechols were assayed as the substrates by using extracts prepared from strain 1395; all of the compounds were methylated to the corresponding anisoles, veratroles, or guaiacols, which have been identified previously from experiments using whole cells. The specific activity of the enzyme towards the thiophenols was significantly higher than it was towards all the other substrates-high activity was found with pentafluorothiophenol, although the activity with pentafluorophenol was undetectable with the incubation times used. For the chlorophenols, the position of the substituents was of cardinal importance. The enzyme had higher activity towards the halogenated catechols than towards the corresponding guaiacols, and selective O-methylation of the 3,4,5-trihalogenocatechols yielded predominantly the 3,4,5-trihalogenoguaiacols. As in experiments with whole cells, neither 2,4-dinitrophenol, hexachlorophene, nor 5-chloro- or 5-bromovanillin was O-methylated. The results showed conclusively that the methylation reactions were enzymatic and confirmed the conclusion from extensive studies using whole cells that methylation of halogenated phenols may be a significant alternative to biodegradation.     

Hydroxylation and dechlorination of chlorinated guaiacols and syringols by Rhodococcus chlorophenolicus

We show that Rhodococcus chlorophenolicus PCP-I, a polychlorophenol degrader, also degrades various chlorine-substituted guaiacols (2-methoxyphenols) and syringols (2,6-dimethoxyphenols). The substrates investigated were tetrachloroguaiacol, 3,4,6- and 3,5,6-trichloroguaiacol, 3,5- and 3,6-dichloroguaiacol, trichlorosyringol, and 3,5-dichlorosyringol. The first step was a hydroxylation, probably in a position para to the preexisting hydroxyl. Tetrachloroguaiacol and trichlorosyringol, with a chlorine substituent in the para position, were both hydroxylated and dechlorinated. The optimum temperature for degradation of polychlorinated guaiacols and syringols was 37 to 41 degrees C. Degradation of polychlorinated phenols, guaiacols, and syringols by R. chlorophenolicus was inducible, and induction was controlled coordinately.     

Microbial removal of chlorinated phenols during aerobic treatment of effluents from radiata pine kraft pulps bleached with chlorine-based chemicals,with or without hemicellulases

  The removal of chlorophenolic compounds from kraft mill effluents bleached with chlorine (cBKME) or chlorine plus hemicellulases (bBKME) was studied in reactors of aerobic treatment lagoons. In these laboratory models, a stable microbial population removed biochemical oxygen demand at similar rates of the mill lagoon. Complete removal of nine chlorophenols and chloroguaiacols during microbial treatment of these effluents was detected by gas chromatography. Abiotic removal was only observed with 2,4-dichlorophenol and 2,4,5-trichlorophenol. There were no significant differences in degradative ability between microorganisms acclimated to grow in reactors fed with cBKME or bBKME. The latter had a lower content of adsorbable organic halogen and chlorophenols than cBKME. Microorganisms acclimated to cBKME or bBKME were only able to grow on phenol or guaiacol as sole carbon source. However, these microorganisms removed (0.1–0.5 mM) 4-chlorophenol, 2,4-dichlorophenol and 2,4-dichlorophenoxyacetate with BKME as primary carbon source. Under these conditions, 2,4,6- and 2,4,5-trichlorophenol, 4,5-dichloroguaiacol, 4,5,6-trichloroguaiacol and tetrachloroguaiacol were not removed. These results suggest that the microbial removal of bleaching chlorophenols and chloroguaiacols during aerobic treatment, probably takes place only because of their very low concentration (1–200 ppb) in BKME. Received: 12 February 1996 / Received revision: 10 June 1996 / Accepted: 22 June 1996     

Hexahydrobenzofuranoid neolignans from an Aniba species

The trunk wood of an Amazonian Aniba species contains three novel neolignans: (2R, 3R, 3aS, 5R)-3a-allyl-5-methoxy-2-(3,4,5-trimethoxyphenyl)-3-methyl-2,3,3a,4,5,6-hexahydro-6-oxobenzofuran (canellin-D), (2R,3R,3aS,5R)-3a-allyl-5,7-dimethoxy-2-(3-methoxy-4,5-methylenedioxyphenyl)-3-methyl-2,3,3a,4,5,6-hexahydro-6-oxobenzofuran (canellin-E) and (2S,3S,3aS,5R)-3a-allyl-5-methoxy-2-(3-methoxy-4,5-methylenedioxyphenyl)-3-methyl-2,3,3a,4,5,6-hexahydro-6-oxobenzofuran (armenin-C). The absolute stereochemistries of these and of all other known hexahydro-6-oxobenzofurans were determined by CD comparisons with model compounds.     

Study of Biochemical Pathways and Enzymes Involved in Pyrene Degradation by Mycobacterium sp. Strain KMS

Pyrene degradation is known in bacteria. In this study, Mycobacterium sp. strain KMS was used to study the metabolites produced during, and enzymes involved in, pyrene degradation. Several key metabolites, including pyrene-4,5-dione, cis-4,5-pyrene-dihydrodiol, phenanthrene-4,5-dicarboxylic acid, and 4-phenanthroic acid, were identified during pyrene degradation. Pyrene-4,5-dione, which accumulates as an end product in some gram-negative bacterial cultures, was further utilized and degraded by Mycobacterium sp. strain KMS. Enzymes involved in pyrene degradation by Mycobacterium sp. strain KMS were studied, using 2-D gel electrophoresis. The first protein in the catabolic pathway, aromatic-ring-hydroxylating dioxygenase, which oxidizes pyrene to cis-4,5-pyrene-dihydrodiol, was induced with the addition of pyrene and pyrene-4,5-dione to the cultures. The subcomponents of dioxygenase, including the alpha and beta subunits, 4Fe-4S ferredoxin, and the Rieske (2Fe-2S) region, were all induced. Other proteins responsible for further pyrene degradation, such as dihydrodiol dehydrogenase, oxidoreductase, and epoxide hydrolase, were also found to be significantly induced by the presence of pyrene and pyrene-4,5-dione. Several nonpathway-related proteins, including sterol-binding protein and cytochrome P450, were induced. A pyrene degradation pathway for Mycobacterium sp. strain KMS was proposed and confirmed by proteomic study by identifying almost all the enzymes required during the initial steps of pyrene degradation.     

O-Methylation of Chlorinated para-Hydroquinones by Rhodococcus chlorophenolicus

Rhodococcus chlorophenolicus PCP-I, a degrader of polychlorinated phenols, guaiacols (2-methoxyphenols), and syringols (2,6-dimethoxyphenols), was shown to O-methylate the degradation intermediate, a chlorinated para-hydroquinone, into 4-methoxyphenol. O-methylation was constitutively expressed, whereas the degradation of chlorophenols and chlorohydroquinones was inducible in R. chlorophenolicus. The O-methylating reaction required two hydroxyl groups in positions para to each other. R. chlorophenolicus selectively methylated the hydroxyl group flanked by two chlorine substituents. Tetrachlorohydroquinone, trichlorohydroquinone, and 2,6-dichlorohydroquinone were methylated into tetrachloro-4-methoxyphenol, 2,3,5-trichloro-4-methoxyphenol, and 3,5-dichloro-4-methoxyphenol, respectively. Chlorohydroquinones with only one chlorine adjacent to a hydroxyl group were methylated only in trace amounts, and no metabolite was formed from hydroquinone. The degradation intermediates formed in hydroxylation of tetrachloroguaiacol and trichlorosyringol by R. chlorophenolicus were O-methylated into two isomeric trichlorodimethoxyphenols and two isomeric dichlorotrimethoxyphenols, respectively. R. chlorophenolicus also degraded the polychlorinated methylation products (tetrachlorinated and trichlorinated 4-methoxyphenols), but not mono- and dichlorinated 4-methoxyphenols.     

Three oxygenated cyclohexenone derivatives produced by an endophytic fungus

Three cyclohexenone derivatives, (4S,5S,6S)-5,6-epoxy-4-hydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (1), (4R,5R)-4,5-dihydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (2), and (4R,5S,6R)-4,5,6-trihydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (3), were isolated from unpolished rice fermented with an xylariaceous endophytic fungus (strain YUA-026). The structures of three compounds were established on the basis of spectroscopic analyses and chemical conversion. The minimum inhibitory concentrations of 1 and 3 were 100 microg/ml and 400 microg/ml against Staphylococcus aureus, 100 microg/ml and 200 microg/ml against Pseudomonas aeruginosa, and 200 microg/ml and >400 microg/ml against Candida albicans, respectively. In addition, 1 and 3 exhibited phytotoxic activity against lettuce.     

Study of biochemical pathways and enzymes involved in pyrene degradation by Mycobacterium sp. strain KMS

Pyrene degradation is known in bacteria. In this study, Mycobacterium sp. strain KMS was used to study the metabolites produced during, and enzymes involved in, pyrene degradation. Several key metabolites, including pyrene-4,5-dione, cis-4,5-pyrene-dihydrodiol, phenanthrene-4,5-dicarboxylic acid, and 4-phenanthroic acid, were identified during pyrene degradation. Pyrene-4,5-dione, which accumulates as an end product in some gram-negative bacterial cultures, was further utilized and degraded by Mycobacterium sp. strain KMS. Enzymes involved in pyrene degradation by Mycobacterium sp. strain KMS were studied, using 2-D gel electrophoresis. The first protein in the catabolic pathway, aromatic-ring-hydroxylating dioxygenase, which oxidizes pyrene to cis-4,5-pyrene-dihydrodiol, was induced with the addition of pyrene and pyrene-4,5-dione to the cultures. The subcomponents of dioxygenase, including the alpha and beta subunits, 4Fe-4S ferredoxin, and the Rieske (2Fe-2S) region, were all induced. Other proteins responsible for further pyrene degradation, such as dihydrodiol dehydrogenase, oxidoreductase, and epoxide hydrolase, were also found to be significantly induced by the presence of pyrene and pyrene-4,5-dione. Several nonpathway-related proteins, including sterol-binding protein and cytochrome P450, were induced. A pyrene degradation pathway for Mycobacterium sp. strain KMS was proposed and confirmed by proteomic study by identifying almost all the enzymes required during the initial steps of pyrene degradation.     

Comparative mechanistic and substrate specificity study of inositol polyphosphate 5-phosphatase Schizosaccharomyces pombe Synaptojanin and SHIP2

Inositol-5-phosphatases are important enzymes involved in the regulation of diverse cellular processes from synaptic vesicle recycling to insulin signaling. We describe a comparative study of two representative inositol-5-phosphatases, Schizosaccharomyces pombe synaptojanin (SPsynaptojanin) and human SH2 domain-containing inositol-5-phosphatase SHIP2. We show that in addition to Mg2+, transition metals such as Mn2+, Co2+, and Ni2+ are also effective activators of SPsynaptojanin. In contrast, Ca2+ and Cu2+ are inhibitory. We provide evidence that Mg2+ binds the same site occupied by Ca2+ observed in the crystal structure of SPsynaptojanin complexed with inositol 1,4-bisphosphate (Ins(1,4)P2). Ionizations important for substrate binding and catalysis are defined for the SPsynaptojanin-catalyzed Ins(1,4,5)P3 reaction. Kinetic analysis with four phosphatidylinositol lipids bearing a 5-phosphate and 54 water-soluble inositol phosphates reveals that SP-synaptojanin and SHIP2 possess much broader substrate specificity than previously appreciated. The rank order for SPsynaptojanin is Ins(2,4,5)P3 > phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) approximately Ins(4,5)P2 approximately Ins(1,4,5)P3 approximately Ins(4,5,6)P3 > PtdIns(3,5)P2 approximately PtdIns(3,4,5)P3 approximately Ins(1,2,4,5)P4 approximately Ins(1,3,4,5)P4 approximately Ins-(2,4,5,6)P4 approximately Ins(1,2,4,5,6)P5. The rank order for SHIP2 is Ins(1,2,3,4,5)P5 > Ins(1,3,4,5)P4 > PtdIns(3,4,5)P4 approximately PtdIns(3,5)P2 approximately Ins(1,4,5,6)P4 approximately Ins(2,4,5,6)P4. Because inositol phosphate isomers elicit different biological activities, the extended substrate specificity for SPsynaptojanin and SHIP2 suggest that these enzymes likely have multiple roles in cell signaling and may regulate distinct pathways. The unique substrate specificity profiles and the importance of 2-position phosphate in binding also have important implications for the design of potent and selective SPsynaptojanin and SHIP2 inhibitors for pharmacological investigation.     

Rubrifacine fromErwinia rubrifaciens

For the main component of the pigments ofErwinia rubrifaciens, we suggest the hitherto unknown structure of a 3-(3-carboxymethyl-4,5-dihydro-4,5-dioxo-2-pyrrolyl)-4,5,6-trihydroxy-pyridine-2-acetic acid. Its isolation and characterization are described. Inasmuch as arylpyrrolindiones are known to be red, the proposed structure can account for the color of the pigment. The 2,3,4-trihydroxypyridine moiety probably inhibits the electron transport in mitochondria. This could explain the toxic action ofErwinia rubrifaciens onJuglans regia.     

Annexin VI is attached to transverse-tubule membranes in isolated skeletal muscle triads

Annexin VI is a 68-kDa protein of the Annexin family, a group of Ca2+-dependent phospholipid-binding proteins widely distributed in mammalian tissues including skeletal muscle. We investigated a) which membrane system contributes Annexin VI to skeletal muscle triads, and b) whether Annexin VI removal affects triad integrity or function. Annexin VI was present in isolated triads and transverse tubules but not in heavy sarcoplasmic reticulum vesicles, indicating that Annexin VI binds to either free or triad-attached transverse tubules. Extraction with EGTA of Annexin VI from triads did not alter their migration as a single band in sucrose density gradients or their ouabain binding-site density, indicating that triad integrity does not require Annexin VI. Caffeine-induced Ca2+ release kinetics and Ca2+ uptake rates were likewise not affected by Annexin VI removal from triads, suggesting that Annexin VI is not involved in these functions. Annexin VI purified from rabbit skeletal muscle displayed Ca2+-dependent binding to liposomes containing phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine. Binding saturated at 1/20 molar ratio phosphatidylinositol 4,5-bisphosphate/phosphatidylcholine and was optimal at free [Ca2+] > or = 20 mM. Extraction of Annexin VI from triads did not affect the generation of phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, or phosphatidic acid by endogenous lipid kinases, suggesting that despite its capacity to bind to negatively charged phospholipids, Annexin VI does not affect the kinase activities responsible for their generation.     

Characterization of catechol derivative removal by lignin peroxidase in aqueous mixture

The use of lignin peroxidase (LIP) as an alternative method for the removal of four catechols (1,2-dihydroxybenzene): catechol (CAT), 4-chlorocatechol (4-CC), 4,5-dichlorocatechol (4,5-DCC) and 4-methylcatechol (4-MC) typical pollutants in wastewater derived from oil and paper industries, was evaluated. The removal of 2 mM catecholic substrates by 1 μM LIP after 1 h was in the following order: 4,5-DCC (95%) > 4-CC(90%) > CAT(55%) > 4-MC(43%). Except for 4-MC, all reactions were accompanied by the formation of insoluble products, leading to LIP precipitation. LIP was exposed to soluble or insoluble product-dependent inactivation, depending on the substrates tested, immediately at the start of the reactions. Despite immediate enzyme inactivation, removal of catecholic substrates continued, resulting in oligomeric product formation. Major oxidation products analyzed were compatible with dimeric, trimeric and tetrameric structures. Ether linkages and a benzoquinone structure were detected in two purified oligochlorocatechols.Catechol derivatives removal initiated by LIP, seems to be different for each catecholic substrate in terms of substrate consumption and transformation, and of enzyme activity.     

Phosphoinositide interconversion in thrombin-stimulated human platelets

Stimulation of platelets and other secretory cells by agonists results in the degradation of phosphoinositides by phospholipase C. Kinetic studies suggest that hydrolysis of phosphatidylinositol 4,5-diphosphate (PI-4,5-P2) is an initial event in this process. Platelets contain much larger amounts of phosphatidylinositol (PI) than PI-4,5-P2, and approximately 50% of total phosphoinositides are degraded upon stimulation. We have investigated whether degradation of PI occurs by direct phospholipase C hydrolysis or by phosphorylation to PI-4,5-P2 followed by phospholipase C action on the latter compound. When platelets are incubated for 3 min with 32Pi prior to stimulation, the phosphoinositides are labeled to different specific activities. Under these nonequilibrium conditions, the time course of change in specific activity reflects turnover. The rise in specific activity of phosphatidylinositol 4-phosphate (PI-4-P) is similar in stimulated and unstimulated cells, indicating that there is little increase in the conversion of PI to PI-4-P during thrombin stimulation. In addition, the specific activity of the 4-phosphate in PI-4-P during thrombin stimulation is less than both the 5-phosphate of PI-4,5-P2 and the phosphate group of phosphatidic acid, indicating that the 4-phosphate moiety is not labeled to equilibrium with ATP. This finding is inconsistent with a rapid flux of PI via PI-4-P to PI-4,5-P2 during thrombin stimulation, in which case the 4-phosphate would be at maximum specific activity. We, therefore, conclude that the bulk of PI breakdown that occurs in thrombin-stimulated platelets occurs via direct phospholipase C hydrolysis of PI.