Novel formation of alpha-amino acids from alpha-oxo acids and ammonia in an aqueous medium

In the course of a study of possible mechanisms for chemical evolution in the primeval sea, we found the novel formation of alpha-amino acids and N-acylamino acids from alpha-oxo acids and ammonia in an aqueous medium. Glyoxylic acid reacted with ammonia to form N-oxalylglycine, which gave glycine in a 5-39% yield after hydrolysis with 6N HC1. Pyruvic acid and ammonia reacted to give N-acetylalanine, which formed alanine in a 3-7% overall yield upon hydrolysis. The pH optima in these reactions were between pH 3 and 4. These reactions were further extended to the formation of other amino acids. Glutamic acid, phenylalanine and alanine were formed from alpha-ketoglutaric acid, phenylpyruvic acid and oxaloacetic acid, respectively, under similar conditions. N-Succinylglutamic acid was obtained as an intermediate in glutamic acid synthesis. Phenylacetylphenylalanineamide was also isolated as an intermediate in phenylalanine synthesis. Alanine, rather than aspartic acid, was produced from oxaloacetic acid. These reactions provide a novel route for the prebiotic synthesis of amino acids. A mechanism for the reactions will be proposed.     

Ascorbic acid induced degradation of beta-glucan: Hydroxyl radicals as intermediates studied by spin trapping and electron spin resonance spectroscopy

The formation of hydroxyl radicals in beta-glucan solutions treated with ascorbic acid and iron(II) was demonstrated by ESR spin trapping based methods. Two different spin traps were tested, namely DMPO which is commonly used to detect hydroxyl radicals, and POBN often used to detect carbon centered radicals. The experiments performed showed that the presence of iron(II) with DMPO led to low DMPO-OH adduct stability and further to DMPO dimerization. The level of hydroxyl radicals formed during the beta-glucan radical mediated degradation was evaluated using two ESR spin trapping methods based on the use POBN together with either 2% (v/v) EtOH or DMSO. The addition of ascorbic acid together with iron(II) in beta-glucan solution led to an immediate maximal production of hydroxyl radicals while the presence of ascorbic acid alone led to a progressive production of radical. Further hydroxyl radicals were found to be formed when iron(II) was added alone in beta-glucan solutions. The viscosity loss observed in the three last mentioned beta-glucan solutions were found to relate with the formation of hydroxyl radicals. These data confirm the involvement of hydroxyl radical in the beta-glucan degradation.     

Site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides and their thermal stability and nuclease-resistance properties.

We report here the site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides (ODNs). ODNs containing both 5-( N-aminohexyl)-carbamoyl-2'-deoxyuridine (H), which serves as a tether for the further conjugation of functional groups, and 5-(N,N-dimethylaminohexyl)carbamoyl-2'-deoxyuridine (D), which contributes to the thermal stability of the duplex and to the resistance to nucleolytic hydrolysis by nucleases, were synthesized. Functional groups such as folic acid and palmitic acid were site-specifically introduced into the terminus of the aminohexyl-linker of H. The thermal stability and resistance toward nuclease digestion of the modified ODNs were studied. We found that ODNs containing D and H formed stable duplexes with both the complementary DNA and RNA strands even when a bulky functional group such as folic acid, palmitic acid or cholesterol was attached to the terminus of the amino-linker. We also found that ODN analogues which contained D were more resistant to nucleolytic degradation by exo- and endonuclease than the unmodified ODN. Furthermore, duplexes formed by ODNs containing D and the complementary RNA could elicit RNase H activity.     

Formation of kynurenic and xanthurenic acids from kynurenine and 3-hydroxykynurenine in the dinoflagellate Lingulodinium polyedrum: role of a novel,oxidative pathway

The dinoflagellate Lingulodinium polyedrum (syn. Gonyaulax polyedra) was used as a model organism for studying the effects of high and low physiological oxidative stress on the formation of kynurenic and xanthurenic acids from kynurenine and 3-hydroxykynurenine. Cell were incubated with the precursors and exposed to light (high physiological stress due to photosynthetically formed oxidants) or kept in darkness (low stress). In cultures of less than 0.5 ml cell volume/l of medium, cells took up approximately one half of 0.1 mM extracellular kynurenine within 18 h. The amino acid was partially converted to kynurenic acid, most of which was released to the medium; however, intracellular concentrations of the product were by approximately 10-fold higher than extracellular levels. Rates of kynurenic acid release exceeded by far those explained by kynurenine and tryptophan aminotransferase activities, the latter representing an additional source of kynurenic acid formation via indole-3-pyruvic acid. Light enhanced the release of kynurenic acid by approximately 4-fold; these rates were further increased by exposure to continuous light. Diurnal rhythmicity of kynurenic acid release was clearly exogenous and did not match with the circadian pattern of kynurenine or tryptophan aminotransferase activities; no rhythm was detected in constant darkness. Similar findings were obtained on turnover of 3-hydroxykynurenine to xanthurenic acid and release of the product to the medium. However, light/dark differences were relatively smaller, and additional products were formed, according to HPLC data obtained with electrochemical detection. Results are most easily explained on the basis of a recently discovered pathway of kynurenic acid formation from kynurenine, involving either non-enzymatic oxidation by H(2)O(2) or, at higher rates, enzymatic catalysis by hemoperoxidase. A corresponding mechanism may exist for the hydroxylated analogue.     

α1-Acute-phase globulins of rats. Microheterogeneity after isoelectric focusing

1. Improved resolution of mixtures of alpha(1)-globulins was obtained by the use of isoelectric focusing. 2. Because material recovered after isoelectric focusing in polyacrylamide gels behaved in a manner which suggested interaction with components derived from the gel, isoelectric focusing when used for preparative purposes was done in a matrix of Sephadex G-75. 3. By this means material from the individual bands formed by isoelectric focusing in 6m-urea could be isolated. The stability of these substances was examined by further isoelectric focusing. 4. Analysis of material that had been shown to be homogenous by isoelectric focusing in the absence of urea and of that from several individual bands derived from the same sample by isoelectric focusing in 6m-urea showed different proportions of sialic acid but no change in amino acid composition. 5. In the presence of 6m-urea the isoelectric points found were increased by 0.14-0.25 pH unit. After removal of most of the sialic acid with neuraminidase the increase was 0.36-0.72 pH unit. After treatment with 0.025m-H(2)SO(4) at 80 degrees C for 1h, which removed all the sialic acid, the increase was 0.40-0.87 pH unit. 6. Because removal of all the sialic acid did not decrease the number of bands formed by isoelectric focusing the observed heterogeneity could not be caused entirely by the presence of various proportions of sialic acid.     

Oxidation and chemiluminescence of catechol by hydrogen peroxide in the presence of Co(II) ions and CTAB micelles.

The oxidation of catechol in neutral and slightly alkaline aqueous solutions (pH 7-9.6) by excess hydrogen peroxide (0.002-0.09 mol/L) in the presence of Co(II) (2.10(-7)-2.10(-5) mol/L) is accompanied by abrupt formation of red purple colouration, which is subsequently decolourized within 1 h. The electron spectra of the reaction mixture are characterized by a broad band covering the whole visible range (400-700 nm), with maximum at 485 nm. The reaction is initiated by catechol oxidation to its semiquinone radical and further to 1,2-benzoquinone. By nucleophilic addition of hydrogen peroxide into the p-position of benzoquinone C=O groups, hydroperoxide intermediates are formed, which decompose to hydroxylated 1,4-benzoquinones. It was confirmed by MS spectroscopy that monohydroxy-, dihydroxy- and tetrahydroxy-1,4-benzoquinone are formed as intermediate products. As final products of catechol decomposition, muconic acid, its hydroxy- and dihydroxy-derivatives and crotonic acid were identified. In the micellar environment of hexadecyltrimethylammonium bromide the decomposition rate of catechol is three times faster, due to micellar catalysis, and is accompanied by chemiluminescence (CL) emission, with maxima at 500 and 640 nm and a quantum yield of 1 x 10(-4). The CL of catechol can be further sensitized by a factor of 8 (maximum) with the aid of intramicellar energy transfer to fluorescein.     

The interaction of ethanol and exogenous arachidonic acid in the formation of leukotrienes and prostaglandin D2 in mastocytoma cells

The present studies were undertaken to examine the hypothesis that ethanol could affect cellular biosynthesis in the murine mastocytoma cell of prostaglandins and leukotrienes, oxidative metabolites of arachidonic acid, at concentrations that could be encountered in vivo as well as during in vitro experiments. The effects of ethanol which encompass these concentration ranges (200-1000 mg%) can be summarized as follows: first in the absence of exogenous arachidonic acid, ethanol caused a dose dependent decrease in the production of leukotrienes which was statistically significant at 200 mg%. At 1000 mg%, ethanol caused a 20-50% decrease in leukotrienes and a 21% decrease in the amount of prostaglandin D2 (PGD2) formed in these cells. Secondly, when cells were incubated with exogenous arachidonic acid (14 micrograms/ml), large increases in both PGD2 and leukotrienes occurred. Under these conditions, ethanol caused a further increase in the amount of leukotrienes and a small increase in the amount of PGD2 formed. This stimulatory effect was specific for ethanol since neither t-butanol nor n-butanol caused the enhanced production of leukotrienes with exogenous arachidonic acid. Thus, these experiments suggest that ethanol affects metabolism of arachidonic acid at reasonably low doses (200-400 mg%) of ethanol in a manner dependent on the free arachidonic acid in the tissue. Also, in vitro experiments in which ethanol is used as a solvent for arachidonic acid could be greatly affected by high levels of ethanol (500-1000 mg%) which are frequently utilized.     

Deamination of protein lysyl ε-amino groups by peroxidase in vitro

Peroxidase, catechol, and hydrogen peroxide were shown to react with proteins, causing a decrease in lysine detectable after acid hydrolysis. The loss of lysine did not occur in the presence of benzenesulfinic acid which suggested that the quinones formed by peroxidase had oxidized some lysyl residues to lysyl aldehyde that formed a cyclic ninhydrin negative Shiff's base. When peroxidase treated protein was oxidized with performic acid prior to hydrolysis a new ninhydrin positive compound was found, which was shown by cochromatography and mass spectroscopy to be α-aminoadipic acid. The α-aminoadipic acid recovered accounted for (20–40)% of the lysine lost.     

Increases in the number of adventitious roots caused by 2-thiouracil and 5-bromodeoxyuridine in Phaseolus mungo cuttings

A cutting of Phaseolus mungoproduced about 4 adventitious rootsat the basal 1 mm region when the basal part of the cuttingwas dipped in water. Rootlets became visible after a 36 hr lagperiod in untreated cuttings. Treatment with 2-thiouracil or5-bromodeoxyuridine increased the number of roots formed onthe cutting and prolonged the lag period. Effects of 2-thiouraciland 5-bromodeoxyuridine were reversed by simultaneously applieduracil and thymidine. The number of roots decreased and thelength of lag period was shortened. The increases in the numberof roots by 2-thiouracil or 5-bromodeoxyuridine was reducedby gibberellic acid, which did not cause a decrease in the numberof roots to be formed on control cuttings. Roots formed at thebasal region seem to suppress further root formation at theupper part of the hypocotyl. Inhibitors used here probably workby blocking the formation of these bottommost roots. (Received April 30, 1971; )     

Metabolism of arachidonic acid by peripheral and elicited rat polymorphonuclear leukocytes. Formation of 18- and 19-oxygenated dihydro metabolites of leukotriene B4

Previous studies have shown that leukotriene B4 is metabolized by polymorphonuclear leukocytes (PMNL) by a 20-hydroxylase, a 19-hydroxylase, and a reductase. We have now identified for the first time LTB4 metabolites formed by a combination of the reductase and omega-oxidation pathways. We have also discovered that rat PMNL metabolize LTB4 by a novel pathway to 18-hydroxy products. Dihydro metabolites of LTB4 have formerly been reported only after incubation of exogenous LTB4 with PMNL, but we have now shown that they are formed to the same extent from endogenous arachidonic acid after stimulation of PMNL with the ionophore, A23187. The following metabolites have been identified after incubation of either LTB4 or arachidonic acid with rat PMNL: 10,11-dihydro-LTB4, 10,11-dihydro-12-epi-LTB4, 10,11-dihydro-12-oxo-LTB4, 19-hydroxy-LTB4, 19-hydroxy-10,11-dihydro-LTB4, 19-oxo-10,11-dihydro-LTB4, 18-hydroxy-LTB4, 18-hydroxy-10,11-dihydro-LTB4, and 18-hydroxy-10,11-dihydro-12-oxo-LTB4. Negligible amounts of 20-hydroxylated products were formed. Incubation of PMNL with 10,11-dihydro-LTB4 resulted in the formation of all of the above dihydro metabolites. However, none of the omega-oxidized metabolites of LTB4 was further metabolized to a significant extent when incubated with PMNL, possibly at least partially because they were not substrates for a specific LTB4 uptake mechanism. We found that the biosynthesis and metabolism of LTB4 is considerably enhanced in PMNL from an inflammatory site (carrageenan-induced pleurisy) compared with peripheral PMNL. When arachidonic acid was the substrate, the greatest increase was observed for products formed by the reductase pathway, which were about eight times higher in pleural PMNL. The rates of formation of both LTA hydrolase and omega-hydroxylase products were about three times higher, whereas the total amounts of 5-lipoxygenase products were about twice as high in pleural PMNL. The amounts of products formed by the above enzymatic pathways reached maximal levels about 4-6 h after injection of carrageenan and then declined.     

Formation of isodialuric acid lesion within DNA oligomers via one-electron oxidation of 5-hydroxyuracil: characterization, stability and excision repair

5-Hydroxyuracil is a major oxidized nucleobase that can be generated by the action of ^•OH radical and one-electron oxidants. The latter modified base that exhibits a low ionization potential is highly susceptible to further degradation upon exposure to various oxidants. Emphasis was placed in thiswork on the formation and characterization of one-electron oxidation products of 5-hydroxyuracil within DNA fragments of defined sequence. For this purpose, 5-hydroxyuracil containing single- and double-stranded oligonucleotides of various lengths were synthesized and then exposed to the oxidizing action of iridium salts. Isodialuric acid was found to be formed almost quantitatively by a one-electron oxidation mechanism for which relevant information was inferred from a freeze-quenched ESR study. Information on the stability of isodialuric acid thus formed and its conversion products in aqueous solutions was also gained from experiments performed at acidic, neutral and alkali pH’s. Moreover, biochemical features dealing with the substrate specificity of several bacterial and yeast base excision repair enzymes to remove isodialuric acid from site-specifically modified DNA fragments were determined.     

大鼠胃泌酸腺细胞膜和肝匀浆对五肽胃泌素及其类似物的降解

 本文用HPLC分离五肽胃泌素(Pentagastrin,Boc-β-Ala-Trp-Met-Asp-Phe·NH_2,PG_(1-5))及其N-端无保护基的TFA·五肽(β-Ala-Trp-Met-Asp-Pne·NH_2·TFA,TFA·PA_(1-5))被胃泌酸细胞膜和肝匀浆降解之产物,并对它们的氨基酸组成作了分析,又用薄层层析作了进一步验证,从而提出了酶促降解时可能的酶切位点。实验结果表明,N-端的Boc-基团能大幅度增加对酶降解的抵抗力,这从一个方面解释了为什么商品五肽胃泌素的泌酸活性大于其它胃泌素类似物的原因。     

Development of an autophagic system in differentiating cells of the cellular slime moldDictyostelium discoideum

Summary Changes in an autophagic system during differentiation of cells ofDictyostelium discoideum, NC-4 were studied under light and electron microscopes, and it was demonstrated cytochemically that acid phosphatase was almost exclusively localized in food and autophagic vacuoles. Autophagic vacuoles first appeared during formation of loose aggregates, coupled with the defecation of food vacuoles. Autophagic vacuoles seem to originate from flat sacs which segregate parts of the cytoplasm. No acid phosphatase was detected in the vacuoles when first formed, but activity appeared later probably due to fusion with Golgi-like vesicles. When starved cells were not allowed to aggregate due to a low cell density, they formed no autophagic vacuoles but retained many food vacuoles. This indicates that the formation of autophagic vacuoles is not simply due to starvation, but to cell interaction mediated by cell contact. Autophagic vacuoles containing acid phosphatase rapidly increased in number in all cells in the early stage of aggregation. After papillae formed, however, they selectively decreased in the prespore cells, but developed further and grew larger in the prestalk cells.     

Initial reactions in the biodegradation of 1-chloro-4-nitrobenzene by a newly isolated bacterium, strain LW1

Bacterial strain LW1, which belongs to the family Comamonadaceae, utilizes 1-chloro-4-nitrobenzene (1C4NB) as a sole source of carbon, nitrogen, and energy. Suspensions of 1C4NB-grown cells removed 1C4NB from culture fluids, and there was a concomitant release of ammonia and chloride. Under anaerobic conditions LW1 transformed 1C4NB into a product which was identified as 2-amino-5-chlorophenol by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. This transformation indicated that there was partial reduction of the nitro group to the hydroxylamino substituent, followed by Bamberger rearrangement. In the presence of oxygen but in the absence of NAD, fast transformation of 2-amino-5-chlorophenol into a transiently stable yellow product was observed with resting cells and cell extracts. This compound exhibited an absorption maximum at 395 nm and was further converted to a dead-end product with maxima at 226 and 272 nm. The compound formed was subsequently identified by 1H and 13C NMR spectroscopy and mass spectrometry as 5-chloropicolinic acid. In contrast, when NAD was added in the presence of oxygen, only minor amounts of 5-chloropicolinic acid were formed, and a new product, which exhibited an absorption maximum at 306 nm, accumulated.     

Arachidonic acid metabolism in rabbit renal cortex. Formation of two novel dihydroxyeicosatrienoic acids

[1-14C]Eicosatetraenoic (arachidonic) acid was incubated with a low speed (17,000 X g) rabbit renal cortical supernatant or with a cortical microsomal suspension fortified with NADPH for 15 min at 37 degrees C. The products which were less polar than prostaglandins on reversed phase high performance liquid chromatography were identified by gas chromatography-mass spectrometry. Both the fortified microsomes and the low speed supernatant formed significant amounts of two novel metabolites, 11,12-dihydroxy-5,8,14-eicosatrienoic acid and 14,15-dihydroxy-5,8,11-eicosatrienoic acid. Other identified products were 19- and 20-hydroxyeicosatetraenoic acid, 19-oxoeicosatetraenoic acid, and in the low speed supernatant, eicosatetraen-1,20-dioic acid. The metabolites were not formed in significant amounts by high speed cortical supernatant or by nonfortified cortical microsomes. Carbon monoxide inhibited formation of these compounds, indicating that they may be formed by the cytochrome P-450-linked renal monooxygenase systems.     

Anaerobic transformation of alkanes to fatty acids by a sulfate-reducing bacterium,strain Hxd3

Strain Hxd3, an alkane-degrading sulfate reducer previously isolated and described by Aeckersberg et al. (F. Aeckersberg, F. Bak, and F. Widdel, Arch. Microbiol. 156:5-14, 1991), was studied for its alkane degradation mechanism by using deuterium and (13)C-labeled compounds. Deuterated fatty acids with even numbers of C atoms (C-even) and (13)C-labeled fatty acids with odd numbers of C atoms (C-odd) were recovered from cultures of Hxd3 grown on perdeuterated pentadecane and [1,2-(13)C(2)]hexadecane, respectively, underscoring evidence that C-odd alkanes are transformed to C-even fatty acids and vice versa. When Hxd3 was grown on unlabeled hexadecane in the presence of [(13)C]bicarbonate, the resulting 15:0 fatty acid, which was one carbon shorter than the alkane, incorporated a (13)C label to form its carboxyl group. The same results were observed when tetradecane, pentadecane, and perdeuterated pentadecane were used as the substrates. These observations indicate that the initial attack of alkanes includes both carboxylation with inorganic bicarbonate and the removal of two carbon atoms from the alkane chain terminus, resulting in a fatty acid one carbon shorter than the original alkane. The removal of two terminal carbon atoms is further evidenced by the observation that the [1,2-(13)C(2)]hexadecane-derived fatty acids contained either two (13)C labels located exclusively at their acyl chain termini or none at all. Furthermore, when perdeuterated pentadecane was used as the substrate, the 14:0 and 16:0 fatty acids formed both carried the same numbers of deuterium labels, while the latter was not deuterated at its carboxyl end. These observations provide further evidence that the 14:0 fatty acid was initially formed from perdeuterated pentadecane, while the 16:0 fatty acid was produced after chain elongation of the former fatty acid with nondeuterated carbon atoms. We propose that strain Hxd3 anaerobically transforms an alkane to a fatty acid through a mechanism which includes subterminal carboxylation at the C-3 position of the alkane and elimination of the two adjacent terminal carbon atoms.     

Arachidonic acid metabolism in rat basophilic leukemia (RBL-1) cells

Rat basophilic leukemia (RBL-1) cells metabolized arachidonic acid through more than one enzymatic pathway. The major cyclooxygenase product was prostaglandin (PG) D₂ as established by chromatographic and chemical behavior and the effect on platelet aggregation. PGD₂ formation from exogenous arachidonic acid was inhibited by indomethacin, 1 μg/ml. RBL-1 incubated with exogenous arachidonic acid also formed SRS-A the synthesis of which was not inhibited by indomethacin. However, the SRS-A activity was blocked by the specific receptor antagonist FPL 55712. [¹⁴C]arachidonic acid was effectively incorporated into the phospholipids of RBL-1 cells. Challenge of such prelabelled cells or unlabelled cells with A 23187 caused release of PGD₂, SRS-A and another presently unidentified product. However, with A 23187 as a stimulus, the RBL-1 cyclo-oxygenase could not be blocked by low concentrations of indomethacin. This work further substantiates our earlier findings that SRS-A formed from arachidontic acid is not a cyclooxegenase product.     

High resolution proton NMR studies of gangliosides. Structure of two types of GD3 lactones and their reactivity with monoclonal antibody R24

Ganglioside GD3 was converted at room temperature to two stable lactones, denoted as GD3 lactones I and II. The reaction sequence was presumed to be GD3----GD3 lactone I----GD3 lactone II based on the time course of their production. Lactone I behaved as a monosialoganglioside and lactone II as a neutral species. The two lactones were isolated by DEAE-Sephadex column chromatography. The positions of the inner ester linkages were investigated by two-dimensional J-correlated proton NMR spectroscopy. An ester linkage was most likely formed between the carboxyl group of the external sialic acid residue and C9-OH of the internal sialic acid residue in lactone I. In addition to this ester linkage, a second ester linkage between the carboxyl group of the internal sialic acid and C2-OH of the galactose residue was likely formed in lactone II. The structural changes induced by lactonization were further examined by their reactivity with the monoclonal antibody R24 (Puckel, C. S., Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., and Old, L. J. (1982) J. Exp. Med. 155, 1133-1147), which reacted with GD3. R24 was found to bind weakly to GD3 lactone I, but not to GD3 lactone II. The results suggest that the monoclonal antibody requires both sialic acid residues for high affinity binding, and the complete lactonization results in a loss of negative charges and/or a change in the overall conformation of the oligosaccharide moiety which may account for the loss of binding.     

Formation of 14,15-hepoxilins of the A(3) and B(3) series through a 15-lipoxygenase and hydroperoxide isomerase present in garlic roots.

We report herein for the first time the formation by freshly grown garlic roots and the structural characterization of 14,15-epoxide positional analogs of the hepoxilins formed via the 15-lipoxygenase-induced oxygenation of arachidonic acid. These compounds are formed through the combined actions of a 15(S)-lipoxygenase and a hydroperoxyeicosatetraenoic acid (HPETE) isomerase. The compounds were formed when either arachidonic acid or 15-HPETE were used as substrates. Both the "A"-type and the "B"-type products are formed although the B-type compounds are formed in greater relative quantities. Chiral phase high performance liquid chromatography analysis confirmed the formation of hepoxilins from 15(S)- but not 15(R)-HPETE, indicating high stereoselectivity of the isomerase. Additionally, the lipoxygenase was of the 15(S)-type as only 15(S)-hydroxyeicosatetraenoic acid was formed when arachidonic acid was used as substrate. The structures of the products were confirmed by gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives as well as after characteristic epoxide ring opening catalytically with hydrogen leading to dihydroxy products. That 15(S)-lipoxygenase activity is of functional importance in garlic was shown by the inhibition of root growth by BW 755C, a dual cyclooxygenase/lipoxygenase inhibitor and nordihydroguaiaretic acid, a lipoxygenase inhibitor. Additional biological studies were carried out with the purified intact 14(S), 15(S)-hepoxilins, which were investigated for hepoxilin-like actions in causing the release of intracellular calcium in human neutrophils. The 14,15-hepoxilins dose-dependently caused a rise in cytosolic calcium, but their actions were 5-10-fold less active than 11(S), 12(S)-hepoxilins derived from 12(S)-HPETE. These studies provide evidence that 15(S)-lipoxygenase is functionally important to normal root growth and that HPETE isomerization into the hepoxilin-like structure may be ubiquitous; the hepoxilin-evoked release of calcium in human neutrophils, which is receptor-mediated, is sensitive to the location within the molecule of the hydroxyepoxide functionality.     

Kinetic and mechanistic analysis of Trypanosoma cruzi trans-sialidase reveals a classical ping-pong mechanism with acid/base catalysis

The trans-sialidase from Trypanosoma cruzi catalyzes the transfer of a sialic acid moiety from sialylated donor substrates to the terminal galactose moiety of lactose and lactoside acceptors to yield alpha-(2,3)-sialyllactose or its derivatives with net retention of anomeric configuration. Through kinetic analyses in which the concentrations of two different donor aryl alpha-sialoside substrates and the acceptor substrate lactose were independently varied, we have demonstrated that this enzyme follows a ping-pong bi-bi kinetic mechanism. This is supported for both the native enzyme and a mutant (D59A) in which the putative acid/base catalyst has been replaced by the demonstration of the half-reaction in which a sialyl-enzyme intermediate is formed. Mass spectrometric analysis of the protein directly demonstrates the formation of a covalent intermediate, while the observation of release of a full equivalent of p-nitrophenol by the mutant in a pre-steady state burst provides further support. The active site nucleophile is confirmed to be Tyr342 by trapping of the sialyl-enzyme intermediate using the D59A mutant and sequencing of the purified peptic peptide. The role of D59 as the acid/base catalyst is confirmed by chemical rescue studies in which activity is restored to the D59A mutant by azide and a sialyl azide product is formed.