Physiological role of tryptophanase in control of tryptophan biosynthesis in Bacillus alvei

Hoch, J. A. (University of Illinois, Urbana), and R. D. DeMoss. Physiological role of tryptophanase in control of tryptophan biosynthesis in Bacillus alvei. J. Bacteriol. 91:667-672. 1966.-Indole excretion occurred early in the exponential growth phase, and derived mainly from biosynthetic intermediates of tryptophan. Tryptophan cleavage by tryptophanase contributed about 1.5% of the indole excreted. In the presence of exogenous tryptophan (5 to 10 mug/ml), excretion of early indole was not observed. Experiments with isotopically labeled indole and tryptophan showed that a low rate of endogenous tryptophan biosynthesis occurred constantly during growth. Both exogenously and endogenously supplied tryptophan were degraded by tryptophanase. As a consequence, the intracellular tryptophan concentration appeared to be maintained at a constant low level. It was suggested that the action of tryptophanase is an example of an enzymatic mechanism which controls the level of a specific metabolite pool.     

Measurement of specific radioactivity by high-performance liquid chromatograph with a synchronized accumulating radioisotope detector: determination of turnover rate and pool size of tryptophan in rat

A high-performance liquid chromatograph with a synchronized accumulating radioisotope detector was used to determine the turnover rate and pool size of tryptophan in rat. The specific radioactivity could be followed for three half-lives on the final slope of the specific radioactivity curve following intravenous administration of 15 muCi of carrier free tryptophan labeled with carbon-14. Remarkable individual differences were noted in turnover rate and in pool size among rats.     

A fluorescence spectroscopy study on the interactions of the TAT-PTD peptide with model lipid membranes

Protein-transduction domains (PTDs) have been shown to translocate into and through the living cells in a rapid manner by an as yet unknown mechanism. Regardless of the mechanism of translocation, the first necessary step must be binding of the PTD peptide to the surface of the lipid membrane. We used fluorescence spectroscopy to study the interaction between PTD of the HIV-1 Tat protein (TAT-PTD; residues 47-60 of Tat, fluorescently labeled with tryptophan) and the lipid bilayer labeled with various fluorescence membrane probes. The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and an increase in anisotropy upon interaction with lipid bilayers. The fluorescence changes were linearly proportional to the density of negative charge in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. The value of one dissociation constant (Kd1 = 2.6 +/- 0.6 microM), which accounted for 24% of the interaction, was found to be independent of the negative charge density, suggesting its nonelectrostatic nature. The value of the second dissociation constant (Kd2), which accounted for 76% of the interaction, decreased linearly from 610 +/- 150 to 130 +/- 30 microM with an increase in negative charge density from 0 to 25 mol %, suggesting this interaction is electrostatic in nature. Even though the binding was predominantly electrostatic, it could not be reversed by high salt, indicating the presence of a second, irreversible, step in the interaction with lipid. When TAT-PTD was bound to lipid vesicles labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), fluorescence resonance energy transfer between the tryptophan and the probe occurred at a distance of 3.4 nm. No change in fluorescence anisotropy of either TMA-DPH or DPH was observed upon the interaction with TAT-PTD, indicating no significant disruption or perturbation of the lipid bilayer by the peptide. TAT-PTD did not cause dissipation of membrane potential (165 mV, negative inside). Inclusion of 3% pyrene-labeled phosphatidylglycerol (pyrene-PG) in the membrane revealed that TAT-PTD preferentially bound to the membrane in the liquid state. We conclude that membrane fluidity is an important physicochemical parameter, which may regulate binding of TAT-PTD to the membrane.     

Tryptophan Biosynthesis from Indole-3-Acetic Acid by Anaerobic Bacteria from the Rumen

Microbes in ruminal contents incorporated (14)C into cells when they were incubated in vitro in the presence of [(14)C]carboxyl-labeled indole-3-acetic acid (IAA). Most of the cellular (14)C was found to be in tryptophan from the protein fractions of the cells. Pure cultures of several important ruminal species did not incorporate labeled IAA, but all four strains of Ruminococcus albus tested utilized IAA for tryptophan synthesis. R. albus did not incorporate (14)C into tryptophan during growth in medium containing either labeled serine or labeled shikimic acid. The mechanism of tryptophan biosynthesis from IAA is not known but appears to be different from any described biosynthetic pathway. We propose that a reductive carboxylation, perhaps involving a low-potential electron donor such as ferredoxin, is involved.     

3H]tryptophan-[14C]proline dual label method for the simultaneous determination of collagen and noncollagen protein production

A new method for the simultaneous determination of newly synthesized collagen and noncollagen proteins has been developed. Because tryptophan is not found in collagen noncollagen proteins were specifically labeled with [3H]tryptophan. [14C]Proline was used to label both groups of proteins. To calculate the 14C-labeled noncollagen protein the 3H radioactivity of the protein mixture was divided by the ratio of 3H:14C in noncollagen protein of a representative sample. This value was obtained by collagenase digestion. The remaining 14C radioactivity in the protein mixture was attributed to [14C]collagen. There was a very good correlation between the dual label method and the widely used collagenase digestion method for the measurement of collagen and noncollagen protein production and for the calculation of the relative rate of collagen synthesis. This new method provides a simple and accurate analysis of collagen production, and it is suitable for rapid processing of a large number of biological samples.     

Nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol-binding protein II produced in Escherichia coli. Analysis of the apoprotein and the holoprotein containing bound all-trans-retinol and all-trans-retinal

Rat cellular retinol-binding protein II (CRBP II) is a 15.6-kDa intestinal protein which binds all-trans-retinol and all-trans-retinal but not all-trans-retinoic acid. We have previously analyzed the interaction of Escherichia coli-derived rat apoCRBP II with several retinoids using fluorescence spectroscopic techniques. Interpretation of these experiments is complicated, because the protein has 4 tryptophan residues. To further investigate ligand-protein interactions, we have utilized 19F nuclear magnetic resonance (NMR) spectroscopy of CRBP II labeled at its 4 tryptophan residues with 6-fluorotryptophan. Efficient incorporation of 6-fluorotryptophan (93%) was achieved by growing a tryptophan auxotroph of E. coli harboring a prokaryotic expression vector with a full-length rat CRBP II cDNA on defined medium supplemented with the analog. Comparison of the 19F NMR spectra of 6-fluorotryptophan-substituted CRBP II with and without bound all-trans-retinol revealed that resonances corresponding to 2 tryptophan residues (designated WA and WB) undergo large downfield changes in chemical shifts (2.0 and 0.5 ppm, respectively) associated with ligand binding. In contrast, 19F resonances corresponding to two other tryptophan residues (WC and WD) undergo only minor perturbations in chemical shifts. The 19F NMR spectra of 6-fluorotryptophan-substituted CRBP II complexed with all-trans-retinal and all-trans-retinol were very similar, suggesting that the interactions of these two ligands with the protein are similar. Molecular model building, based on the crystalline structures of two homologous proteins was used to predict the positions of the 4 tryptophan residues of CRBP II and to make tentative resonance assignments. The fact that ligand binding produced residue-specific changes in the chemical shifts of resonances in CRBP II suggests that NMR analysis of isotopically labeled retinoid-binding proteins expressed in E. coli will provide an alternate, albeit it complementary, approach to fluorescence spectroscopy for examining the structural consequences of their association with ligand.     

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.     

Determination of deuterium-labeled tryptophan in proteins by means of high-performance liquid chromatography and thermospray mass spectrometry

In order to develop direct methods for determining the extent of metabolic incorporation of isotopically labeled amino acids into a protein, the determination of deuterated tryptophan in [2H5]tryptophan-bacteriorhodopsin was investigated. The isotopically modified protein was subjected to alkaline hydrolysis. After phenyl isothiocyanate derivatization of the hydrolysate, the mixture was separated by reversed-phase liquid chromatography. Field desorption mass spectrometry and thermospray mass spectrometry were investigated for their ability to determine the ratio between [2H5]tryptophan and total tryptophan in the collected fractions. In order to check the procedure a set of known tryptophan/[2H5]tryptophan mixtures were passed through the same derivatization, HPLC separation, and lyophilization procedure as used for the biological samples.     

Study of the brain serotonergic system with labeled alpha-methyl-L-tryptophan

α-Methyl- l -tryptophan (α-MTrp) is an artificial amino acid and an analog of tryptophan (Trp), the precursor of the neurotransmitter serotonin (5-HT). In this article we have summarized available data, which suggest that the measurement of the unidirectional uptake of α-MTrp and its conversion to 5-HT synthesis rates is a valid approach for the determination of brain 5-HT synthesis rates. The main feature on which the model is based is the trapping of labeled α-MTrp in brain tissue. An overview of opposing opinions, which suggest that there is a need for a metabolic conversion of tracer, is also presented and discussed critically. As with all biological modeling there is likely to be room for improvements of the proposed biological model. In addition, there are a limited number of clearly defined circumstances in which the method is confounded by the metabolism of labeled α-MTrp via the kynurenine pathway. Nonetheless, a significant body of evidence suggests that labeled α-MTrp is a useful tracer to study brain 5-HT synthesis in most circumstances. Calculation of 5-HT synthesis rates depends on the plasma-free tryptophan concentration, which, according to the balance of arguments in the literature, is a more appropriate parameter than the total-plasma tryptophan. The method, as proposed by us, can be used in conjunction with autoradiographic measurements in laboratory animals, and with positron emission tomography in large animals and humans. We review studies in animals looking at the normal control of 5-HT synthesis and the way in which it is altered by drugs, as well as initial studies investigating healthy humans and patients with neuropsychiatric disorders.     

The effect of tryptrophan on nucleocytoplasmic translocation of RNA in rat liver.

The effect of the administration of tryptophan on the transport of nuclear poly (A)-containing mRNA to the cytoplasm in rat liver was investigated. Administration of tryptophan to fasted rats pretreated with cordycepin and actinomycin D led to decreased levels of nuclear poly (A)-mRNA and a concomitant increase in the levels of polyribosomal poly (A)-mRNA in the cytoplasm as determined by measuring in vivo incorporation of labeled precursors into hepatic RNA. Using isolated hepatic nuclei of rats prelabeled in vivo with [14C]orotic acid, there was greater release of labeled poly(A)-mRNA into the incubation medium from nuclei of tryptophan-treated rats than from nuclei of control animals. The increased release of RNA from hepatic nuclei of tryptophan-treated animals was not related to the cell sap present in the media since cell saps from livers of control and experimental rats gave similar results. These results support earlier findings which suggest that in the rat tryptophan increases the rate of translocation of hepatic poly(A)-mRNA from nucleus to cytoplasm.     

Dietary tryptophan does not alter the function of brain serotonin neurons

The hypothesis that alterations in dietary tryptophan modify the functional activity of brain serotonin-containing neurons was tested by recording the electrophysiological activity of single serotonergic cells in awake, behaving cats after meal ingestion of diets containing varying proportions of tryptophan and the neutral amino acids that compete with tryptophan for uptake into the brain. The data revealed that while the various diets produced significant changes in brain serotonin and its major metabolite, 5-hydroxyindoleacetic acid, there was no change in the activity of serotonin-containing dorsal raphe cells following meal ingestion. Furthermore, a pulse injection of tritiated labeled tryptophan following the various diets produced no significant change in the release of tritiated serotonin into the lateral ventricles, while tritiated 5-hydroxyindoleacetic acid was significantly increased. These data suggest that dietary tryptophan does not alter the functional activity of central serotonergic neurons, in contrast with current popular beliefs that such dietary manipulations alter brain function.     

Fluorimetric studies of actin labeled with dansyl aziridine.

F-actin has been specifically labeled with a fluorescent probe, dansyl aziridine, at cysteine-373 of the protein. The fluorescence property of the conjugated probe serves as a spectroscopic indicator of several processes in which actin participates. The sulfhydryl modification does not impair the G-F transformation of actin, nor does it affect the complex formation of actin and myosin or the dissociation of the complex by ATP as judged by viscosity measurements. However, both labeled actin and actin modified by N-ethylmaleimide, which also reacts at cysteine-373, stimulate the Mg²⁺-ATPase of myosin only about 75% as well as unmodified actin. The probe attached to actin exhibits a 65-nm blue shift of its emission maximum from 560 to 495 nm and a sixfold fluorescence enhancement indicating that it is located in a hydrophobic environment. The excitation spectrum of labeled actin indicates that a tryptophan and a tyrosine residue are close to the probe and transfer excitation energy to the dansyl fluorophore. Upon depolymerization of F-actin, the fluorescence intensity of labeled actin increases about 20%. The fluorescence of labeled actin is also enhanced by the addition of EDTA, ATP, and pyrophosphate, but Mg²⁺ antagonizes this effect reversibly. However, in the presence of 10 mm orthophosphate buffer (pH 7.4) these effects disappear. When labeled F-actin binds with myosin subfragment-1 (SF-1) or heavy meromyosin (HMM), the fluorescence of the actin adduct is enhanced. The fluorescence properties of labeled acto-SF-1 and acto-HMM become insensitive to EDTA and polyphosphates even in the absence of orthophosphate. These results suggest that the two-stranded helical structure of the F-actin filament is stabilized by the presence of phosphate and/or the binding of the myosin “head”.     

Nuclear magnetic resonance and molecular genetic studies of the membrane-bound D-lactate dehydrogenase of Escherichia coli

In this study we demonstrate the potential of combining fluorine-19 nuclear magnetic resonance (NMR) spectroscopy with molecular genetics. We are using the membrane-bound enzyme D-lactate dehydrogenase of Escherichia coli as a model system to characterize interactions between proteins and lipids. We have labeled D-lactate dehydrogenase with 4-, 5-, and 6-fluorotryptophans and obtained high-resolution fluorine-19 NMR spectra showing five resonances, in agreement with the five tryptophan residues expected from the DNA sequence. The five 19F resonances in the spectra have been assigned to the specific tryptophan residues in the primary sequence of D-lactate dehydrogenase by site-directed oligonucleotide mutagenesis of the cloned gene. We observe large differences in the relative fluorine-19 chemical shifts of each tryptophan residue when labeled by different isomers of fluorotryptophan. We have determined by NMR methods that two tryptophans are exposed to the solvent and that none of the tryptophan residues are within 10 A of the lipid phase. On the basis of 19F NMR spectroscopy of the labeled tryptophan residues, the conformation of D-lactate dehydrogenase is similar in aqueous solution and in the presence of a variety of lipids and detergents. This result indicates that the presence of lipids or detergents is not required to maintain the tertiary structure of this membrane-bound enzyme. In contrast, Triton X-100 induces a change to an abnormal conformation of the enzyme as judged from both NMR spectroscopy and the effect of temperature on the maximal velocity of the enzyme in the presence of this detergent.     

Chiral separation of tryptophan enantiomers by liquid chromatography with BSA-silica stationary phase

The separation of tryptophan enantiomers was carried out with medium-pressure liquid chromatography using BSA (bovine serum albumin)-bonded silica as a chiral stationary phase. The influence of various experimental factors such as pH and ionic strength of mobile phase, separation temperature, and the presence of organic additives on the resolution was studied. In order to expand this system to preparative scale, the loadability of sample and the stability of stationary phase for repeated use were also examined. The separation of tryptophan enantiomers was successful with this system. The data indicated that a higher separation factor (α) was obtained at a higher pH and lower temperature and ionic strength in mobile phase. Addition of organic additives (acetonitrile and 2-propanol) in mobile phase contributed to reduce the retention time of L-tryptophan. About 30% of the separation factor was reduced after 80 days of repeated use.     

19F NMR study of the leucine-specific binding protein of Escherichia coli: mutagenesis and assignment of the 5-fluorotryptophan-labeled residues

The Escherichia coli L-leucine receptor is an aqueous protein and the first component in the distinct transport pathway for hydrophobic amino acids. L-leucine binding induces a conformational change, which enables the receptor to dock to the membrane components. To investigate the ligand-induced conformational change and binding properties of this protein, we used (19)F NMR to probe the four tryptophan residues located in the two lobes of the protein. The four tryptophan residues were labeled with 5-fluorotryptophan and assigned by site-directed mutagenesis. The (19)F NMR spectra of the partially ligand free proteins show broadened peaks which sharpen when L-leucine is bound, showing that the labeled wild-type protein and mutants are functional. The titration of L-phenylalanine into the 5-fluorotryptophan labeled wild-type protein shows the presence of closed and open conformers. Urea-induced denaturation studies support the NMR results that the wild-type protein binds L-phenylalanine in a different manner to L-leucine. Our studies showed that the tryptophan to phenylalanine mutations on structural units linked to the binding pocket produce subtle changes in the environment of Trp18 located directly in the binding cleft.     

Efficient isotopic tryptophan labeling of membrane proteins by an indole controlled process conduct

A protocol for the efficient isotopic labeling of large G protein‐coupled receptors with tryptophan in Escherichia coli as expression host was developed that sufficiently suppressed the naturally occurring L‐tryptophan indole lyase, which cleaves tryptophan into indole, pyruvate, and ammonia resulting in scrambling of the isotopic label in the protein. Indole produced by the tryptophanase is naturally used as messenger for cell–cell communication. Detailed analysis of different process conducts led to the optimal expression strategy, which mimicked cell–cell communication by the addition of indole during expression. Discrete concentrations of indole and ¹⁵N₂‐L‐tryptophan at dedicated time points in the fermentation drastically increased the isotopic labeling efficiency. Isotope scrambling was only observed in glutamine, asparagine, and arginine side chains but not in the backbone. This strategy allows producing specifically tryptophan labeled membrane proteins at high concentrations avoiding the disadvantages of the often low yields of auxotrophic E. coli strains. In the fermentation process carried out according to this protocol, we produced ～15 mg of tryptophan labeled neuropeptide Y receptor type 2 per liter medium. Biotechnol. Bioeng. 2013; 110: 1681–1690. © 2013 Wiley Periodicals, Inc.     

Labeling of subunit b of the ATP synthase from Escherichia coli with a photoreactive phospholipid analogue

Purified ATP synthase (F1F0) from Escherichia coli K12 was labeled with the hydrophobic photoreactive label 1-palmitoyl 2-(2-azido-4-nitro)benzoyl sn-glycero-3-[3H]phosphocholine in reconstituted proteoliposomes. The F0-subunit b was predominantly labeled. A very low amount of label was detected on the other F0-subunits a and c. The label in subunit b could be traced back by proteolytic digestion to the NH2-terminal fragment 1 to 53 which contains the stretch of hydrophobic amino acid residues 1 to 32. By sequencing the intact protein, the distribution of label among the amino acids in this segment was determined. Cysteine 21 was predominantly labeled. Other labeled amino acids occurred at the NH2-terminal (Asn-2) and at position 26 (tryptophan). Due to the restricted mobility of the label in the lipid bilayer, these residues are suggested to be located in or close to the polar head of the lipid bilayer. These results will be compared with predictions for the arrangement of the polypeptide b derived from the hydrophobicity profile.     

Linear mapping of tryptophan residues in Vesiculovirus M and N proteins by partial chemical cleavage.

Nonlimit chemical cleavage at tryptophan residues of protein labeled at the amino terminus afforded a simple procedure for generating specific fragments and for mapping tryptophan positions. A comparison of the matrix (M) and nucleocapsid (N) proteins of four members of the Vesiculovirus group by this procedure suggests considerable conservation of tryptophan number and location in the four serotypes examined.     

Resonance energy transfer between tryptophan 57 in the epsilon subunit and pyrene maleimide labeled gamma subunit of the chloroplast ATP synthase

The intrinsic fluorescence of the catalytic portion of the chloroplast ATP synthase (CF1) is quenched when cysteine 322, the penultimate amino acid of the gamma subunit, is specifically labeled with pyrene maleimide (PM). The epsilon subunit of CF1 contains the only two residues of tryptophan, which dominate the intrinsic fluorescence of unlabeled CF1. CF1 deficient in the epsilon subunit (CF1-epsilon) was reconstituted with mutant epsilon subunits in which phenylalanine replaced tryptophan at position 15 (epsilonW15F) and position 57 (epsilonW15/57F). CF1(epsilonW15F) containing a single tryptophan, epsilonW57, was labeled with PM at gammaC322. Resonance energy transfer (RET) from epsilonW57 to PM on gammaC322 occurred with an efficiency of energy transfer of 20%. RET was also observed from epsilonW57 to PM attached to the disulfide thiols of the gamma subunit (gammaC199,205) with an efficiency of approximately 45%. The R(o) (the distance at which the efficiency of energy transfer is 50%) for the epsilonW57 and PM donor/acceptor pair is 30 A, indicating that both gammaC322 and gammaC199,205 must be within 40 A of epsilonW57. These RET measurements show that both gammaC322 and gammaC199,205 are located near the base of the alpha/beta hexamer. This places the C-terminus of CF1 gamma much closer to epsilon than hypothesized based on homology to crystal structures of mitochondrial F1. These new RET measurements also allow the alignment of the predicted epsilon subunit structure. The orientation is similar to that predicted from cross-linking and mutational studies for the epsilon subunit of Escherichia coli F1.