Identification of pyridoxal phosphate-modified proteins using mass spectrometry.

RATIONALE: Pyridoxal 5'-phosphate (PLP) cooperates with a variety of enzymes in all organisms for many important biological processes. The development of mass spectrometry-based methodology for high-throughput modification analyses could provide an alternative way for PLP identification. The present study aims to identify PLP modification. METHODS: More PLP site-determining information was obtained by introducing multistage activation (MSA)-assisted collision-induced dissociation (CID). We then utilized immobilized metal ion affinity chromatography (IMAC) with Ti4+ to enrich the PLP peptides. In addition, alkaline phosphatase (ALP) was used to remove the phosphoryl group and further confirm the PLP modification site. RESULTS: MSA was able to greatly enhance the identification and localization of PLP modification. We applied this strategy to analyze PLP-modified proteins in Escherichia coli samples and accurately determine PLP site K270 in tryptophanase. CONCLUSIONS: MSA-assisted CID was used to provide better identification of PLP-modified peptides. Furthermore, tryptophanase with PLP modification at K270 in E. coli was identified with Ti4+ -IMAC enrichment followed by ALP treatment. This method provides a promising alternative for investigating biological functions of PLP-modified proteins.

Evaluation of the DIRAMIC system for detection of urinary tract infections and for Escherichia coli identification.

The use of the DIRAMIC system for the detection of urinary tract infections (UTI) and the possibility to identify Escherichia coli in the same culture media was evaluated. The results from DIRAMIC detection system were compared to counts of colony forming units per milliliter (CFU/ml) of urine inoculated in CLED Medium; 884 urine specimens were processed taking > or =10(4) CFU/ml as criteria of positive urine culture counts. For E. coli identification, substrates for the determination of beta-glucuronidase and tryptophanase were incorporated to the culture medium and named DETID-Ec. Outputs were compared to those from API RAPIDEC-ur strips. The DIRAMIC system can detect UTI, with a sensitivity and specificity of 82.25 and 94.49%, respectively. It was possible to identify E. coli during detection with 87.50% of sensitivity and 95.96% of specificity. The small volumes of culture medium used in the DIRAMIC system as well as the short times for the detection make the system a rapid and economical method for screening UTI. Furthermore, by using DETID-Ec culture medium the time and the number of biochemical tests necessary for the E. coli identification are lowered.

Effect of nondigestible oligosaccharides on large-bowel functions, blood lipid concentrations and glucose absorption in young healthy male subjects.

OBJECTIVE: To study the effect of the intake of 15 g nondigestible oligosaccharides per day on various parameters of large-bowel function, as well as on blood lipid concentrations and glucose absorption in man. DESIGN: Latin square, randomized, double-blind, diet-controlled. SETTING: Metabolic research unit. SUBJECTS: Twelve apparently healthy men (mean age 23 years), recruited from the Institute's pool of volunteers, no drop-outs. INTERVENTIONS: Four treatment periods of 3 weeks: inulin, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) and control; analyses of stool weight, intestinal transit, faecal pH, short-chain fatty acids, bile acids, faecal enzymes, blood lipids and glucose absorption. RESULTS: As compared to the control treatment: higher concentration of faecal acetate (inulin and GOS, P < 0.05) and valerate (inulin, P < 0.05), significantly lower concentration of faecal deoxycholic acid (inulin and FOS, P < 0.05 and P< 0.02, respectively) and beta-glucuronidase activity (inulin and GOS, P < 0.05 and P < 0.02 respectively). Other changes of faecal parameters and those of blood lipids and glucose absorption were statistically not significant. CONCLUSIONS: RESULTS indicate that nondigestible oligosaccharides are (partly) fermented in the human colon, but in healthy young men the effects are limited. Also the consumption of 15 g nondigestible oligosaccharides does not seem to alter blood lipid concentrations and glucose absorption in our young healthy adults.

[Rapid differentiation method for Shigella and Escherichia coli--application of Indole (tryptophanase), PGUA (beta-glucuronidase) and ONPG (beta-galactosidase) tests].

For the differentiation of Shigella from Escherichia coli, Indole (tryptophanase), PGUA (beta-glucuronidase) and ONPG (beta-galactosidase) tests were used. A total of 377 Shigella and 124 E. coli strains was examined for each sero- and biosero-type by using these tests. The results were as follows. 1) There were no Shigella strains showing positive reactions for both Indole and ONPG tests. 2) No E. coli strains with Shigella-like characteristics (negative for lysine-decarboxylase, motility and lactose-fermentation tests) showed negative results for both Indole and PGUA tests. 3) The abovementioned strains were classified into twelve types according to the results of these tests. Shigella strains, thus, were differentiated from antigenically Shigella-like E. coli strains. Additional use of these tests together with the conventional methods may valuable for the identification of Shigella strains.

Synthesis of serine-AMC-carbamate: a fluorogenic tryptophanase substrate.

A new fluorogenic substrate for the pyridoxal 5'-phosphate-dependent enzyme tryptophanase is described. L-Serine, which is linked to 7-amino-4-methylcoumarin through an O-carbamoyl tether, serves as a substrate for the enzyme. The released moiety, 7-amino-4-methylcoumarin (AMC), can be detected by either absorbance (355 nm) or fluorescence (excitation 365 nm/emission 440 nm). Kinetic constants were measured using each of these techniques: Km = 85 +/- 20 microM, Vmax = 2.9 +/- 0.4 mumol/min/mg (fluorescence) and Km = 129 +/- 21 microM, Vmax = 3.1 +/- 0.3 mumol/min/mg (absorbance). The Vmax for serine-AMC-carbamate is approximately 1.9 times faster than that of the natural substrate, tryptophan. Using fluorescence detection, solutions containing 10(-3) units of activity could be routinely assayed.

[Study of coenzyme reorientation in the active center of tryptophanase by linear dichroism method]. / Issledovanie reorientatsii kofermenta v aktivnom tsentre triptofanazy metodom lineinogo dikhroizma.

Tryptophanase from E.coli was oriented in a compressed slab of polyacrylamide gel and its linear dichroism (LD) and absorption spectra were measured. The free enzyme displays four LD bands at 305, 340, 425 and 490 nm. Two bands at 340 and 425 nm belong to the internal coenzyme-lysine aldimine. The 305 nm band apparently belongs to an aromatic amino acid residue; the sign and form of this band are changed upon the enzyme reaction with substrate analogs. The 490 nm band is present in the LD spectra of holo- and apoenzyme and disappears after treatment with NaBH4. It is suggested that the 490 nm band belongs to a quinoid enzyme subform. The reaction of tryptophanase with threo-beta-phenyl-DL-serine and L-threonine leads to formation of the external aldimine with a strong absorption band at 420-425 nm. The reduced LD (delta A/A) in this band is one order of magnitude greater than that in the 420 nm of the free enzyme. The complex with D-alanine is characterized by an intermediate LD value in the 425 nm band. In the presence of indole this complex displays the same LD as that observed with beta-phenylserine. The reaction of tryptophanase with L-alanine and oxindolyl-L-alanine leads to formation of the quinoid intermediate with a 500 nm absorption band. The LD value in this band differs from those in the absorption bands of the free enzyme. It is concluded that reorientations of the coenzyme occur in the course of the tryptophanase reaction.

[The rapid identification of E. coli by the detection of 3 enzymes]. / Zur schnellen Identifizierung von E. coli durch den Nachweis von 3 Enzymen.

232 isolates of gram-negative, oxidase-negative bacteria, isolated from various samples of different animal species, were tested with help of RAPIDEC coli for the production of beta-glucuronidase, beta-galactosidase and indole. The test correctly identified 164 of 175 E. coli strains (sensitivity 93.7%) and correctly indicated that 57 of 57 isolates of the family Enterobacteriaceae (7 Citrobacter sp., 18 Enterobacter sp., 16 Klebsiella sp., 10 Proteus sp., 2 Providencia sp. and 4 Salmonella sp.) were not E. coli (specificity 100%).

Characterization of the reactivity of sulphydryl groups in tryptophanase by a dual-monitoring high-performance liquid chromatographic system with a site-directed fluorescent reagent.

Sulphydryl groups of E. coli tryptophanase (L-tryptophan indole lyase, E.C. 4.1.99.1) were made to react with a fluorescent maleimide derivative, N-(4-anilino-1-naphthyl)maleimide(ANM). By carefully controlling the reaction conditions it was possible to limit the extent of sulphydryl group modification. The modified enzyme was digested with (L-1-tosylamide-2-phenylethyl chloromethyl ketone)-trypsin. The fluorescent peptides obtained were analysed by reversed-phase high-performance liquid chromatography on a C18 column with a dual-monitoring system consisting of a UV and a fluorescence monitor connected in tandem. This was followed by the determination of the amino acid composition of the fluorescent peptides. Comparison of these results with the known, complete primary structure of tryptophanase from the K-12 strain of E. coli allowed the assignment of position 298 to the cysteine residue, which is more selectively modified by ANM under the conditions chosen and is involved in the maintenance of the catalytic activity.

The microbial metabolism of tryptophan in rats fed a diet containing 7.5% saccharin in a two-generation protocol.

Sodium saccharin was fed at 7.5% in the diet to rats in a two-generation protocol. Saccharin-treated animals in both generations showed increased urinary excretion of indican. During lactation, the pups of saccharin-fed dams were exposed to elevated levels of indican via the milk. Establishment of the gut flora in pups at weaning in the presence of saccharin was associated with increased caecal size and caecal protein, decreased caecal tryptophanase activity, and increased urine volume and urinary indican excretion. Pups from dams fed saccharin from birth only, showed more-variable responses during the first weeks of life than pups from dams fed saccharin from before conception, due to variations in tryptophanase activity. The various biochemical and physiological changes were detected soon after the pups were weaned, and were found equally in both males and females. After adjustment for body weight, the changes detected were greatest during the first month after weaning.

Evaluation of the rapid NFT system for identification of gram-negative, nonfermenting rods.

This study evaluated the ability of the Rapid NFT system (API System SA, Montalieu-Vercieu, France) to accurately identify 262 clinically isolated, gram-negative, nonfermentative rods without additional tests. Identifications were classified as correct; low discrimination, with a spectrum of two or more possibilities (additional tests necessary for accurate identification); and incorrect. Correct identification rates were analyzed in two categories: (i) correct to species or biotype for all organism groups except Alcaligenes faecalis-odorans, Moraxella, Pseudomonas testosteroni-alcaligenes-pseudoalcaligenes, and Acinetobacter calcoaceticus biotype haemolyticus-alcaligenes (in this category, the latter four genus-biotype group identifications were taken as correct) and (ii) correct to species or biotype in all cases, including the above four groups. In category i, 87.4% of the strains were correctly identified, with 4.2% low discrimination and 8.4% incorrect. When the criteria of category ii were used, 71.8% of the strains were correctly identified, with 19.9% low discrimination. The Rapid NFT system provided excellent species identification of Pseudomonas and Flavobacterium spp., Bordetella bronchiseptica, and Achromobacter xylosoxidans strains. Within Acinetobacter calcoaceticus, differentiation between biotypes anitratus and lwoffi was satisfactory, but the system did not differentiate between biotypes haemolyticus and alcaligenes. Species resolution within the genera Moraxella and Alcaligenes was incomplete. All Alcaligenes faecalis strains were misidentified and accounted for 50% of misidentifications with the Rapid NFT system; however, these results may reflect taxonomic differences rather than true misidentifications. The Rapid NFT system is easy to inoculate and interpret and represents a worthwhile advance in the identification of gram-negative, nonfermentative rods.

The effects of saccharin on the metabolism of dietary tryptophan to indole, a known cocarcinogen for the urinary bladder of the rat.

Adult male rats were fed diets containing 0 to 10% saccharin, 2% tryptophan, or 2% tryptophan plus 5% saccharin ad libitum for 1 to 2 months. Saccharin produced a dose-related increase in the urinary excretion of indican which is the main metabolite of indole. The renal clearances of both indican and saccharin were reduced at high plasma concentrations (200 to 300 micrograms/ml) of saccharin, suggesting saturation of renal tubular secretion. The increased amounts of indican in the urine, and of indole in the cecum arose from accumulation of protein and tryptophan in the cecum rather than an increase in the enzyme tryptophanase which metabolizes tryptophan to indole. The high levels of protein in the ceca of rats fed saccharin-containing diets were associated with a dose-related increase in the weight of the contents and wall of the cecum. Administration of 2% tryptophan in the diet increased significantly the amounts of tryptophan in the cecum and in plasma, but produced only small increases in the size of the cecum, the amount of indole present, and the excretion of indican. The effects of saccharin and tryptophan were additive. These effects are consistent with saccharin having a major effect on protein digestion in the intestine such that increased amounts of tryptophan are available in the cecum for microbial metabolism to indole. The greater formation of indole and excretion of indican in the urine suggest that saccharin increases the catabolism of dietary tryptophan to metabolites with known cocarcinogenic activity toward the rat bladder.

Nucleotide sequence of the structural gene for tryptophanase of Escherichia coli K-12.

The tryptophanase structural gene, tnaA, of Escherichia coli K-12 was cloned and sequenced. The size, amino acid composition, and sequence of the protein predicted from the nucleotide sequence agree with protein structure data previously acquired by others for the tryptophanase of E. coli B. Physiological data indicated that the region controlling expression of tnaA was present in the cloned segment. Sequence data suggested that a second structural gene of unknown function was located distal to tnaA and may be in the same operon. The pattern of codon usage in tnaA was intermediate between codon usage in four of the ribosomal protein structural genes and the structural genes for three of the tryptophan biosynthetic proteins.

[A medium for a simultaneous assay of l-triptophane desaminase, beta-galactosidase, motility and indole production (author's transl)]. / Un nuovo terreno per la determinazione simultanea della triptofano desaminasi, della beta-galattosidasi, della motilità e dell'indolo.

The author describes a medium for a simultaneous assay, in a single test-tube, of 1-triptophane desaminase, beta-galactosidase, motility and indole production.

[Physical and chemical properties of tryptophenases of five species of Enterobacteriaceae]. / Propriétés physiques et chimiques des tryptophanases de cinq espèces d'Entérobactériaceae

The molecular weight, sedimentation coefficient, and amino acids composition were determined on five tryptophanases (TPases) from Escherichia coli B and E. aurescens, Shigella alkalescens, and Proteus vulgaris and P. morganii. These TPases have identical sedimentation profile and coefficient (9.6 S), and the same molecular weight (220 000). Each enzyme is constituted of four identical subunits having a molecular weight of 55 000. The amino acids composition of these TPases is very similar, with the exception of P. morganii and P. vulgaris TPases which present significative variations in basic amino acids and tryptophan content. The species differentiation of the coli group cannot be made on their TPase characteristics only, contrary to P. morganii and P. vulgaris which can be differentiated between them and from the coli group.

[Differentiation of tryptophanases of five species of Enterobacteriaceae by sulfhydryl groups]. / Distinction des tryptophanases de cinq espèces d'Entérobactériaceae par les groupements sulfhydryles

We have studied the sulfhydryl groups (-SH) on the tryptophanases (TPases) from Escherichia coli B. and E. aurescens, Shigella alkalescens, and Proteus vulgaris and P. morganii. The coli group and the P. morganii apo TPases have 20 -SH groups per mole of enzyme, where as P. vulgaris apoTPase has 16. In coli group TPases, there are 16 -SH groups on the mole surface and they are all implicated in the activity and the enzyme-substrate bond. Proteus morganii TPase has 8 surface -SH groups, 4 of which are implicated in the activity; the remaining 12 -SH groups are located inside the mole and take part in the activity and the enzyme-substrate bond. Proteus vulgaris TPase has 4 surface -SH groups which are constructive of the enzyme structure, whereas the 12 remaining -SH groups are located inside the mole and take part in the activity and the enzyme-substrate bond. It is concluded that Proteus TPases are molecules which have inverted quaternary structure in comparison to those of the coli group. The studied TPases have four subunits, each of them is constituted of one polypeptidic chain having a molecular weight of 55,000.