Detection of an indole oxidizing system in maize leaves

An enzyme activity which brings about a rapid indole disappearance has been detected in cell free extracts of maize ($\text{Zea}\text{mays}$ L.) leaves. The indole utilization by this enzyme system is not dependent on L-serine and pyridoxal phosphate. It does not result in incorporation of (5-³H) indole or (1-¹⁴C) serine into tryptophan. There was no net tryptophan synthesis concomittant with indole disappearance. The enzyme activity is strongly inhibited by dithionite and diethyl-dithiocarbamate. The inhibition by the latter could be specifically removed by Cu²⁺. The activity of dialyzed enzyme could be restored by addition of Cu²⁺ and FAD. The products of indole oxidation were characterized as anthranilic acid and anthranil (2,1-benzisooxazole). The activity of the indole oxidizing system was 2 to 3 times higher in normal maize varieties (Ganga-2 and Ganga-5) than in Opaque-2.     

Indoleacetic Acid synthesis in soybean cotyledon callus tissue

Growth of an auxin-requiring soybean cotyledon callus tissue (Glycine max L., Merr. var. Acme) was promoted by tryptophan, tryptamine, indole, indoleacetamide and, to a very slight degree, anthranilic acid. When tryptophan-3-¹⁴C was supplied in the growth medium, labeled indoleacetic acid (IAA) was found in both the tissue and the medium. Medium, from which the cells had been removed, was also found to convert labeled tryptophan to IAA. Soybean callus contained 0.044 μmole/g free tryptophan, but this is apparently not available for conversion to IAA. These results suggest that while exogenously supplied trytophan could elevate a specific internal pool where IAA synthesis occurs some of the growth on a tryptophan medium can be accounted for by external conversion.     

Purification and properties of indole 2,3-dioxygenase from maize leaves

An indole 2,3-dioxygenase was purified ca 38-fold from maize leaves. The enzyme had an MW of about 98000, an optimum pH of 5.0 and the energy of activation was 9.1 kcal/mol. The K_max for indole was 1.4 × 10^?4 M. The enzyme was inhibited by diethyldithiocarbamate, salicylaldoxime and sodium dithionite. The inhibition by diethyldithiocarbamate was specifically reversed by Cu²⁺. The dialysed enzyme was stimulated by Cu²⁺. Four atoms of oxygen were utilized in the disappearance of 1 mole of indole. Inhibition of the enzyme by -SH compounds and -SH group inhibitors, and their partial removal by Cu²⁺ only, suggested the involvement of -SH groups in binding of Cu²⁺ at the catalytic site.     

The metabolism of [2-14C]indole in the rat

1. [2-¹⁴C]Indole has been synthesized from [¹⁴C]formate and o-toluidine via N[¹⁴C]-formyltoluidine. 2. When fed to rats, the ¹⁴C of [¹⁴C]indole (dose 70–80mg./kg. body wt.) is fairly rapidly excreted, and in 2 days an average of 81% appears in the urine, 11% in the faeces and 2·4% as carbon dioxide in the expired air. 3. Radioactivity is excreted in the urine as indoxyl sulphate (50% of the dose), indoxyl glucuronide (11%), oxindole (1·4%), isatin (5·8%), 5-hydroxyoxindole conjugates (3·1%), N-formylanthranilic acid (0·5%) and unchanged indole (0·07%). The faeces contain indoxyl sulphate (0·4% of the dose) and indole (0·2%), but the major metabolites have not been identified. 4. Fed to rats with biliary cannulae an average of 5·6% of a dose of [¹⁴C]indole (20–60mg./kg. body wt.) is excreted in the bile in 2 days. Radioactivity is present as indoxyl sulphate (0·8% dose) and 5-hydroxyoxindole conjugates (0·6%). 5. Rats further metabolize indoxyl into N-formylanthranilic acid and anthranilic acid, and oxindole into 5-hydroxyoxindole. 6. With rat-liver microsomes plus supernatant under aerobic conditions, indole gives indoxyl, oxindole, possibly isatin, N-formylanthranilic acid and anthranilic acid, but under anaerobic conditions gives only oxindole. Similarly, under aerobic conditions, oxindole gives 5-hydroxyoxindole, anthranilic acid and o-aminophenylacetic acid. 7. Indole is metabolized by two pathways, one via indoxyl to isatin, N-formylanthranilic acid and anthranilic acid, and the other via oxindole to 5-hydroxyoxindole and possibly to o-aminophenylacetic and anthranilic acid. 8. The following new compounds are described: 4-hydroxy-2-nitrophenylacetic acid, 3-, 4- and 5-benzyloxy-2-nitrophenylacetic acid, 5- and 7-hydroxyoxindole and 5-aminoacridine indoxyl sulphate.     

Covalent binding of folic acid to dimethylglycine dehydrogenase

Dimethylglycine dehydrogenase (EC 1.5.99.2) carries out the oxidative demethylation of dimethylglycine to sarcosine in liver mitochondria. In vivo, the enzyme uses tightly bound tetrahydropteroyl pentaglutamate (H₄PteGlu₅) as an acceptor of the one-carbon group generated during the reaction. The purified enzyme can use, but does not require, H₄PteGluB and under these conditions formaldehyde is the one-carbon unit produced. It is reported that folic acid may be covalently linked to dimethylglycine dehydrogenase in a specific and saturable manner so that only 1 mole of folic acid is bound per mole of enzyme. Covalently bound folic acid blocks the subsequent binding of H₄PteGlu, and does not inhibit the rate of dimethylglycine dehydrogenase activity in vitro.     

Indolic compounds in the leaves of Tecoma stans

Indole, tryptophan, tryptamine and skatole were isolated from the leaves of Tecoma stans. Anthranilic acid was also identified in its free form, in contrast to its glucoside, in Jasminum grandiflorum. The presence of both indole and anthranilic acid in the leaves of Tecoma stans indicates that they are the true substrate and product of indole oxygenase from the leaves of Tecoma stans.     

Enzymatic synthesis of cyclopeptine intermediates in Penicillium cyclopium

Cyclopeptine, a benzodiazepine alkaloid of Penicillium cyclopium, is formed from anthranilic acid, L-phenylalanine and the methyl group of L-methionine by cyclopeptine synthetase. The following partial activities of this enzyme system were determined in vitro: anthranilic acid and L-phenylalanine adenylyltransferase activity, binding of anthranilic acid and L-phenylalanine as thioesters to proteins, formation of thioester-bound N-methyl-L-phenylalanine and N-methyl-L-phenylalanylanthranilic acid. The obtained results indicate that cyclopeptine is formed via enzyme-bound intermediates by the thiotemplate mechanism of peptide biosynthesis.     

Purification and characterization of glyoxalase I from Hansenula mrakii

Glyoxalase I was purified from Hansenula mrakii IFO 0895 which was resistant to 25 mM methylglyoxal. The molecular weight of the purified enzyme was calculated to be 38,000 by both gel-filtration of Sephadex G-150 and SDS-PAGE. The enzyme was almost specific to methylglyoxal (K_m = 0.91 mM). The activity of the enzyme was not inhibited by metal ion chelators such as EDTA, which is a potent inhibitor for glyoxalase Is from other sources.     

Subunit structure of -acetohydroxy acid isomeroreductase from Salmonella typhimurium

α-Acetohydroxy acid isomeroreductase, purified from Salmonella typhimurium, has a molecular weight of 220,000. The native enzyme consists of a tetramer of four identical subunits on the basis of the following criteria: (1) SDS gel electrophoresis revealed a single component of molecular weight 55,000 (2) carboxypeptidase digestion of the enzyme revealed 4 moles of glycine released per mole of enzyme; (3) amino acid analysis of the native enzyme indicated 204 moles of lysine and arginine; (4) after tryptic digestion, a total of 51 peptides were detected by high voltage electrophoresis and descending chromatography. In the native enzyme, it was possible to tititrate 8 sulfhydryl groups per mole of enzyme. Neither the rate nor extent of sulfhydryl titration was affected by substrates or products. After denaturation with SDS or urea, 8 additional sulfhydryls per mole of enzyme were titrated.     

Pathway of indole metabolism by a denitrifying microbial community

The metabolism of indole in a mineral-salts medium inoculated with 9% anaerobically digested nitrate-reducing sewage sludge was studied. The sequential occurrence of four structurally-related compounds — oxindole, isatin, dioxindole, and anthranilic acid — was detected using high-performance liquid or thin-layer chromatography. Mass spectrometry and proton nuclear resonance were used to identify isatin and dioxindole isolated from the culture fluids. Prior exposure of the microorganisms to indole, oxindole, isatin, or anthranilic acid resulted in accelerated decomposition of these compounds in a pattern that was consistent with a proposed pathway for the metabolism of indole under denitrifying conditions.     

Production of Substituted l-Tryptophans by Fermentation

Claviceps purpurea has been shown to produce extracellular l-tryptophan from indole in stirred fermentors. The substrate specificity of this conversion was investigated by using substituted indoles, anthranilic acid, and 4-chloro-anthranilic acid. Addition of 2-, 4-, 5-, 6-, and 7-methyl indole or 6-chloroindole to C. purpurea C1M produced the corresponding substituted l-tryptophan. In contrast, addition of l-methyl, 6-trifluoromethyl, 6-nitro-, or 4-benzyloxy-substituted indoles, or anthranilic and 4-chloroanthranilic acids did not produce detectable amounts of the corresponding tryptophan.     

Neurospora crassa glutamine synthetase. Purification by affinity chromatography and characterization of subunit structure.

Neurospora crassa glutamine synthetase was purified to homogeneity by a procedure based on affinity chromatography. The enzyme is adsorbed to a matrix of anthranilic acid bound to Sepharose and eluted with AMP. Different experimental approaches indicate that the enzyme has an octameric structure formed by subunits of identical molecular weight.     

Oxidation of Indole and Indole Derivatives Catalyzed by Nonheme Chloroperoxidases

Indole, indolylacetic acid, and tryptophan were oxidized by chloroperoxidases isolated from strains of Streptomyces lividansand Pseudomonas pyrrocinia. Indigo (indoxyl), isatin, and anthranilic acid (intermediate products of oxidative degradation of indole and indole derivatives) were isolated from the reaction medium.     

Effect of amphotericin B on membrane-associated photosynthetic reactions in maize chloroplasts.

Superoxide dismutase of anaerobic purple sulfur bacterium, Chromatium vinosum, was purified to a homogeneous state. The enzyme contains two atoms of iron per mole and has a molecular weight of 41,000. It is composed of two identical subunits. Amino acid composition, absorption spectra, and the reaction rate constant with O₂^? are also similar to those of the Fe-superoxide dismutases from aerobes. The enzyme is sensitive to hydrogen peroxide and methylene blue-sensitized photooxidation. The functional and evolutional aspects of superoxide dismutase in anaerobes are discussed.     

Thermal denaturation of rat pulmonary and testicular angiotensin-converting enzyme isozymes. Effects of chelators and CoCl2

In an attempt to assess the biochemical consequences resulting from structural differences between rat pulmonary and testicular angiotensin-converting enzyme, the thermal stability of crude and purified preparations of each enzyme was compared. Structural heterology was verified by molecular weight determinations and by peptide mapping after limited proteolysis with Staphylococcus V8 proteinase. Thermal stability was monitored by changes in catalytic activity following incubations at 55°C in the presence of chelators and CoCl₂. Purified pulmonary angiotensin-converting enzyme was more sensitive to inhibition by the chelators EDTA and 1,10-phenanthroline and by the site-directed inhibitor captopril than was the testicular isozyme. Although the pulmonary holoenzyme was unaffected by cobalt, the testicular holoenzyme was inhibited by cobalt in a concentration-dependent manner. Crude pulmonary angiotensin-converting enzyme was significantly more resistant to thermal denaturation than its crude testicular counterpart. The differences in the thermal lability of each isozyme were still present in purified preparations, although the purified enzymes appeared to be more thermally stable than their crude counterparts. Both chelators and cobalt markedly potentiated the thermal denaturation of each isozyme. These data suggest that the structural heterology of the pulmonary and testicular isozymes may affect the interaction of zinc with the respective enzymes and that zinc may contribute to the structural integrity and thermal stability of angiotensin-converting enzyme in each tissue.     

Zinc content, metal chelator inactivation and catalytic inhibition of D-lactate dehydrogenase from the barnacle, Balanus nubilus Darwin.

Purified NAD-linked d-lactate dehydrogenase from the depressor muscle of the giant barnacle, Balanus nubilus Darwin, is inactivated when incubated with the metal chelators o-phenanthroline and EDTA. M-Phenanthroline and p-phenanthroline, which lack metal chelating ability, are ineffective in inactivating the enzyme. Inactivated enzyme can be reactivated by the addition of zinc ions to the assay mixture. Atomic absorption spectrophotometric analysis of purified B. nubilusd-lactate dehydrogenase revealed that this enzyme contains stoichiometric amounts of zinc (2 g-atoms per mol of subunit), unlike other lactate dehydrogenases, which lack zinc. Zinc appears to be required for maximal catalytic activity. Aromatic, nitrogen-containing metal chelators and their nonchelating analogs are effective instantaneous inhibitors of B. nubilusd-lactate dehydrogenase. These compounds bind at the coenzyme binding site, as the mode of inhibition is distinctly competitive with respect to NADH. The different effects of metal chelators and their nonchelator analogs suggest that time-dependent inactivation (chelation of the enzyme zinc ions) and instantaneous inhibition (competition with NADH binding) have independent mechanisms.     

Tryptophan Biosynthesis in Cell Cultures of Nicotiana tabacum

Some of the general features of the pathway for l-tryptophan biosynthesis in cell cultures of Nicotiana tabccum var. Wisc. 38 have been investigated. The results of both isotope competition and direct-labeling experiments show that shikimic acid, anthranilic acid, indoleglycerol phosphate, and indole can serve as precursors to l-tryptophan in these cells, indicating that, in terms of its biochemical intermediates, the pathway is similar to that described for the bacteria and fungi.     

Determination of dissociation constants of crystalline-alpha-chymotrypsin complexes

As a first step in investigations of the properties of crystalline enzymes, the binding of indole, N-formyl-l-phenylalanine, and N-formyl-l-p-iodophenylalanine to α-chymotrypsin crystals, and the binding of indole to tosyl-α-chymotrypsin crystals, has been studied. The methods used were spectrophotometric measurements of the concentration of indole in the supernatant, or measurements of the concentration of radioactively labeled indole in both the supernatant and the crystal. The dissociation constants of the specific binding site of the crystalline enzyme have been determined for indole and N-formyl-l-phenylalanine. It was found that indole does not bind to tosyl-α-chymotrypsin crystals and that N-formyl-p-iodophenylalanine does not bind to the substrate binding site of the crystalline enzyme.The information obtained from these simple equilibrium measurements is in agreement with X-ray diffraction studies. The approach is, therefore, capable of determining whether or not compounds bind to the active site of a crystalline enzyme, and whether the occupancy of this site is sufficient for structure determinations using X-ray diffraction methods.     

Biosynthesis of 2-aminobenzaldehyde in flowers of Robinia pseudoacacia and Philadelphus coronarius

Feeding experiments with 13C- and fluorine-labelled precursors were performed to reveal the biosynthesis of 2-aminobenzaldehyde in flowers of Robinia pseudoacacia and Philadelphus coronarius. The results are in agreement with the transformation of anthranilic acid to indole followed by oxidative ring opening and hydrolysis of the resulting N-formyl-2-aminobenzaldehyde. This route differs from that observed in Hebeloma sacchariolens (Basidiomycetes) in which anthranilic acid is directly reduced to 2-aminobenzaldehyde.     

Oxidation of indole and its derivatives by heme-independent chloroperoxidases]

Indole, indolylacetic acid, and tryptophan were oxidized by cloroperoxidases isolated from strains of Streptomyces lividans and Pseudomonas pyrrocinia. Indigo (indoxyl), isatin, and anthranilic acid (intermediate products of oxidative degradation of indole and indole derivatives) were extracted from the reaction medium.