A rapid procedure for detection of bacterial amino acid decarboxylases

The detection of decarboxylases of arginine, glutamic acid, histidine, lysine, ornithine, phenylalanine, tryptophan, and tyrosine in bacteria by thin-layer chromatography on polyamide sheets is described. The bacteria were grown on agar medium plates supplemented with eight amino acids at pH 5.5 for induction of amino acid decarboxylases, then transferred to amine-production media. The decarboxylation products in the spent media (amines and/or γ-amino-n-butyric acid) were dansylated and the dansyl derivatives were separated by thin-layer chromatography on polyamide sheets. This method requires only two separate incubations of the decarboxylase-induced bacteria in amine-production media for 1 h at 37°C for simultaneous detection of eight bacterial amino acid decarboxylases using 0.4 μl of the spent media.     

Polyamine content of the Plodia interpunctella granulosis virus and its larval host

The presence of polyamines in the granulosis virus and its larval host, Plodia interpunctella, was examined using thin-layer chromatography of the dansylated compounds. The uninfected and infected larvae contain the three polyamines, putrescine, spermidine, and spermine. Although purified granulosis virus infecting this host does not contain any typical polyamines, an acidsoluble dansylated compound with properties indicative of a polyamine was observed.     

Gas chromatographic–mass spectrometric method for quantitation of phenylalanine and tyrosine in serum

A validated gas chromatographic–mass spectrometric method for quantitation of phenylalanine and tyrosine in serum is described. Quantitation of phenylalanine and tyrosine with a non-labelled non-endogenous internal standard, -2-chlorophenylalanine, compared favourably with isotope dilution mass spectrometric quantitation. The 95% reference ranges for phenylalanine, tyrosine and the phenylalanine–tyrosine molar ratio in neonate cord blood serum were determined by isotope dilution mass spectrometry and were found to be 77.1–144.7, 33.3–109.3 μmol/l and 1.1–3.0, respectively.     

Detection and quantitation of 8-hydroxydeoxyguanosine 5'-monophosphate in X-irradiated calf-thymus DNA by fluorescence postlabeling

8-Hydroxydeoxyguanosine 5'-monophosphate (8-OH dGmp) was synthesized from deoxyguanosine 5'-monophosphate (dGmp) by ascorbic acid in the presence of hydrogen peroxide and labeled with dansyl chloride through a phosphoramidate linkage with ethylenediamine (EDA). A DNA model 8-OHd(TACG), isolated intact by high pressure liquid chromatography (HPLC) from x-irradiated d(TACG) and characterized by nmr, was digested enzymatically to 5'-mononucleotides. The modified nucleotide was enriched by HPLC and dansylated. Analysis of the dansylated product by HPLC, using a fluorescent detector, detected a peak with retention time corresponding to that of the dansyl labeled authentic marker. The same overall procedure was used to detect 8-OHdGmp from x-irradiated calf-thymus DNA. The content of 8-OHdGmp in the irradiated DNA increased linearly with increasing levels of x-irradiation in the dose range of 6-60 Gy.     

Photoaffinity labeling study of the interaction of calmodulin with the plasma membrane Ca2+ pump.

Bovine brain calmodulin was labeled with synthetic peptides corresponding to the calmodulin-binding domain of the erythrocyte plasma membrane Ca(2+)-ATPase. One 20-amino acid peptide and two 28-amino acid peptides were used, carrying L-4'-(1-azi-2,2,2-trifluoroethyl)phenylalanine residues in position 9 (peptides C20W* and C28W*) and position 25 (peptide C28WC*), respectively. The localization of the contact regions between calmodulin and the N- and C-terminal portions of the peptides was the aim of this study. The three peptides were N-terminally blocked with a 3H-labeled acetyl group to facilitate the identification of labeled fragments after isolation and digestion. The binding site for phenylalanine 25 was identified in the N-terminal domain of calmodulin while the phenylalanine derivative in position 9 labeled the C-terminal domain. Fluorescence studies using the dansylated N- and C-terminal halves of calmodulin and peptide C20W corresponding to the first 20 amino acids of the calmodulin-binding domain showed that only the C-terminal lobe of calmodulin had high affinity for the peptide (KD in the nanomolar range).     

Structural basis of binding of fluorescent, site-specific dansylated amino acids to human serum albumin

Human serum albumin (HSA) has two primary binding sites for drug molecules. These sites selectively bind different dansylated amino acid compounds, which-due to their intrinsic fluorescence-have long been used as specific markers for the drug pockets on HSA. We present here the co-crystal structures of HSA in complex with six dansylated amino acids that are specific for either drug site 1 (dansyl-l-asparagine, dansyl-l-arginine, dansyl-l-glutamate) or drug site 2 (dansyl-l-norvaline, dansyl-l-phenylalanine, dansyl-l-sarcosine). Our results explain the structural basis of the site-specificity of different dansylated amino acids. They also show that fatty acid binding has only a modest effect on binding of dansylated amino acids to drug site 1 and identify the location of secondary binding sites.     

Preparation of eight ovalbumin subfractions by combined lectin affinity chromatography

Ovalbumin was fractionated by successive lectin affinity chromatography using concanavalin A/Sepharose and wheat germ agglutinin/Sepharose. Eight glycoprotein fractions, all behaving as ovalbumin on polyacrylamide gel electrophoresis, were obtained. To characterize the carbohydrate chains, the asparaginyl-carbohydrates were prepared from the Pronase digests of the ovalbumin fractions and their dansyl derivatives were analyzed by high-performance liquid chromatography. The elution profile of the dansylated asparaginyl-carbohydrates from each subfraction was compared with that from the unfractionated ovalbumin. The results indicated that the above eight subfractions could be separated from each other according to their carbohydrate chains and that three of the subfractions were homogeneous with respect to their carbohydrate chains.     

Isolation and characterization of the bovine hypothalamic corticotropin-releasing factor

A 41 amino acid peptide with high intrinsic corticotropin-releasing activity was isolated from 1000 bovine hypothalami by means of immunoaffinity chromatography, gel filtration, and two steps of reverse phase HPLC. The primary structure of the amino terminal 39 amino acids was characterized by gas phase sequence analysis. The sequence of the amidated carboxyl terminal dipeptide was established by digestion of the intact natural product with Staphylococcus aureus V8 protease, dansylation of the digest and comparative reverse phase liquid chromatography studies with the synthetic dansylated dipeptides Ile-Ala-NH2, Ile-Ala-OH, Ala-Ile-NH2 and Ala-Ile-OH. The complete structure of the bovine corticotropin-releasing factor was established as: Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val- Leu- Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Asn-Asn-Arg-Lys-Leu- Leu- Asp-Ile-Ala-NH2 using approximately 650 pmol of material.     

Degradation of dansyl polyamines on high-performance thin-layer chromatographic plates

Using high-performance thin-layer chromatography with in situ quantitation to measure dansylated polyamines in the range of 1–20 pmol, we found that dansylated polyamines apparently react with the silica gel of the plates. The fluorescence of the dansyl polyamines diminished with increase in the time interval between application of a sample to the plate and start of the chromatographic separation. Conversely, the fluorescence at the site of application increased with the length of the time interval, indicating the formation of polar reaction products. If this reaction is not accounted for, considerable errors in quantitation of dansyl polyamines may occur.     

High-performance liquid chromatographic determination of urinary catecholamines by pre-column solid-phase dansylation on alumina

Sensitive and selective high-performance liquid chromatographic determination of catecholamines by pre-column solid-phase dansylation is described. After catecholamines are adsorbed on alumina, the amino groups not responsible for adsorption are dansylated by a solid-phase reaction. The excess reagent and fluorescent contaminants are washed out, and the dansylated catecholamines are eluted and separated by reversed-phase high-performance liquid chromatography. The four catecholamine derivatives can be separated within 10 min and no major interfering peak is observed on chromatograms. The response of each catecholamine is linear from 10 to 500 pmol per sample and the detection limit is 0.5 pmol. This method was applied to determination of catecholamines in human urine.     

The influence of phenylalanine on accumulation of rosmarinic and caffeic acids by Lavandula vera MM cell culture

Lavandula vera MM cell suspension culture was grown in Linsmayer-Skoog medium with different concentrations of phenylalanine. Adding phenylalanine to the medium (0.1–0.5 g/l) enhanced accumulation of caffeic acid in parallel with rosmarinic acid. When 0.3 g phenylalanine/l was added, the yield of rosmarinic and caffeic acids reached 87 mg/l and 60 mg/l respectively, compared with 68 mg/l and 4 mg/l in controls (without phenylalanine).     

Hydrophilic interaction and reversed-phase ultraperformance liquid chromatography TOF-MS for serum metabonomic analysis of myocardial infarction in rats and its applications

An ultra performance liquid chromatography coupled to mass spectrometry-based metabonomic approach, which utilizes both reversed-performance (RP) chromatography and hydrophilic interaction chromatography (HILIC) separations, has been developed to characterize the global serum metabolic profile associated with myocardial infarction (MI). The HILIC was found necessary for a comprehensive serum metabonomic profiling, providing complementary information to RP chromatography. By combining with partial least squares discriminant analysis, 21 potential biomarkers in rat serum were identified. To further elucidate the pathophysiology of MI, related metabolic pathways have been studied. It was found that MI was closely related to disturbed sphingolipid metabolism, phospholipid catabolism, fatty acid transportation and metabolism, tryptophan metabolism, branched-chain amino acids metabolism, phenylalanine metabolism, and arginine and proline metabolism. With the presented metabonomic method, we systematically analyzed the therapeutic effects of Traditional Chinese Medicine Sini decoction (SND). The results demonstrated that SND administration could provide satisfactory effects on MI through partially regulating the perturbed metabolic pathways.     

Incorporation of non-acetylated hexosamines into plasma membrane glycoproteins of liver cells after galactosamine injection

The following procedure for the detection of non-acetylated amino sugars in the plasma membrane was established: i) derivatization of free amino groups with dansyl-chloride, ii) hydrolysis with 3 M HCl (for 4 h at 105 degrees C) to liberate the dansylated carbohydrate moieties from the plasma membrane, iii) purification of the dansylated amino sugars by paper chromatography and subsequent analysis by thin-layer chromatography. Using this procedure, plasma membranes from rat liver were analysed after injection of D-[14C]galactosamine. For this purpose, rats were divided into three groups: the first received D-galactosamine.HCl at a dose of 2 mg/kg b.w., the second at a dose of 75 mg/kg b.w. and the third at a hepatitis-inducing dose of 260 mg/kg b.w.. In all three groups the majority of the protein-bound radioactivity in the plasma membrane was not dansylated, thus representing N-acetylated amino sugars. At a dose of 2 mg/kg, only 0.34% of the protein-bound radioactivity in the plasma membrane reacted with dansyl-chloride. At a dose of 70 mg/kg this value increased to 1.9%. At 260 mg/kg the value was 3.6%. These results indicate that the incorporation of non-acetylated amino sugar into the plasma membrane was dose-dependent and reached 90 pmol per mg plasma membrane protein during galactosamine injury. However, this incorporation of non-acetylated amino sugars into the plasma membrane did not represent a pathological mechanism responsible for the onset of the galactosamine-induced liver injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation and isolation of dansyladenylates as fluorescent substrates in reactions of the energy metabolism

A method was developed for preparation of dansylated derivatives of adenine nucleotides characterized by fluorescence when being irradiated with UV-light. The involvement of dansylated ATP, ADP and AMP as substrate analogues in energy metabolism is demonstrated in the ATPase, hexokinase, pyruvate kinase and adenylate kinase reactions. The kinetics of the reactions is discussed.     

Estrogen determination using liquid chromatography with precolumn fluorescence labeling

A liquid chromatography procedure is described for the determination of some estrogens using fluorescence detection. The estrogens are labeled by precolumn derivatization with 5-dimethylaminonaphthalene-1-sulfonylchloride (dansyl chloride) and chromatographed on a reversed-phase, C-18 column with a mobile phase consisting of methanol, water, and acetic acid. The eluted analytes are measured with a fluorescence detector using excitation and emission wavelengths of 350 and 540 nm, respectively. The chief advantage of this new procedure is its sensitivity, requiring smaller amounts of sample to detect and quantitate estrogens in biological materials. We could detect less than 400 pg of estriol. With our procedure, this corresponded to about 25 ng in the final reaction mixture, before derivatization. The use of smaller sample volumes could improve this limit. Linearity for dansylated estriol, estrone, and estradiol was excellent over the estrogen range below 100 μg in the sample. This corresponds to approximately 1.7 μg on the column. Within-run precision was better than 5% for the full extraction and derivatization procedure for estriol from pregnancy urine samples. Chromatography is complete within 10 min for dansylated estriol, estrone, and estradiol.     

Mass-spectrometric determination of the structure of glycopeptides from the bacterial cell wall (illustrated by Lactobacillus bulgaricus)

Mass spectrometry has been applied to the structural analysis of one of the glycopeptides from blastolysin, antitumor bacterial preparation isolated from the Lactobacillus bulgaricus cell wall. The glycopeptide (MW 10,000) was subjected to partial acid hydrolysis (6 N HCl, 100 degrees C) and the resulting products were dansylated or trifluoroacetylated and methylated or deuteromethylated. The mixture of these derivatives was examined by high-performance liquid chromatography or gas chromatography followed by mass spectrometry using electron impact and ammonia chemical ionization techniques.     

Submergence(Fermentation)Culture of Anisodus acutanguhts Cells and Identification of Hyoscyamine and Scopolamine in the Cultured Ceils

The growth rate of Anisodus acutangulus cells in the submergence culture was 1.5 g dry wt/l/day, a rate 3 times as that in the suspension culture and more than 10 times over that of the solid static culture: However, the contents of hyoscyamine (0.203 mg/g dry wt) and scopolamine (0.178 mg/g dry. wt) in submergence culture were only slightly higher than those in the two other cultures. But when the 12-day-old submergence culture was supplemented with phenylalanine (5m mol/l) and kinetin (0.1mg/l), it was observed that not only the cell growth rate was increased but also the cellular content of hyoscyamine was raised to a level of 0.217 mg/g dry wt., and that scopolamine to 0.412 mg/g dry wt. The contents of these two alkaloids represented 1.1 and 2.3 times respectively the value of the culture without phenylalanine and kinetin supplements. The optimum date for harvesting the A. acutangulus culture Was on the 14th day of the culture. The monomers of hyoscyamine and scopolamine isolated from the cultured cells were purified and recrystalized, and then identifited as the two compounds in question by the thin-layer chromatography, melting point determination, and ultraviolet, infra-red and nuclear magnetic resonance spectra. In this paper, we also summarize and discuss the results of A. acutangulus culture experiments performed in the past 8 years. Our finding seems to inclieate that following a pilot production trial, the tissue culture method could well be employed to produce hyoscyamine and scopolamine from A. acutangulus cells on an industrial scale.     

Simultaneous high-performance liquid chromatographic analysis of pregabalin, gabapentin and vigabatrin in human serum by precolumn derivatization with o-phtaldialdehyde and fluorescence detection

A rapid, simple and robust method is presented for the simultaneous determination of the gamma-amino-n-butyric acid (GABA) derivatives pregabalin (PGB), gabapentin (GBP) and vigabatrin (VGB) in human serum by high-performance liquid chromatography (HPLC). Serum is deproteinized with trichloroacetic acid and aliquots of the supernatant are precolumn derivatized with o-phtaldialdehyde (OPA) and 3-mercaptopropionic acid. Separation is achieved on a Alltima 3C18 column using isocratic elution; the drugs are monitored using fluorescence detection. Norvaline is used as an internal standard. Within-day precision (COV; n = 10) is 1.2% for PGB (serum concentration 10.0 mg/l), 1.1% for GBP (serum concentration 15.8 mg/l) and 0.3% for VGB (serum concentration 15.5 mg/l). The method is linear up to at least 63 mg/l for PGB, 40 mg/l for GBP and 62 mg/l for VGB. Lower limits of quantitation (LOQ) are 0.13 mg/l for PGB, 0.53 mg/l for GBP and 0.06 mg/l for VGB. No interferences were found from commonly coadministered antiepileptic drugs (AEDs) and from endogenous amino acids. Experimental design in combination with statistical evaluation (ANOVA) was used to study the robustness of chromatography and sample preparation. The method is very suitable for routine therapeutic drug monitoring and for pharmacokinetic studies.     

Effect of serum on phenylalanine hydroxylase levels in cultured hepatoma cells.

Continued high levels of phenylalanine hydroxylase in cultured H4-II-E-C3 rat hepatoma cells require either serum or glucocorticoids in the culture medium. Upon withdrawal of serum, cellular phenylalanine hydroxylase levels decay exponentially with a half-life of 22 hours for about 60 hours, after which time a low, constant enzyme content persists for at least 96 hours. This decline of phenylalanine hydroxylase is fully reversible; normal enzyme levels are restored in a time- and dosage-dependent fashion upon addition of serum to basal cultures. The serum factor is nondialyzable and moderately heat-stable. The stimulation by serum of the phenylalanine hydroxylas content of basal cultures is blocked by 3-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide and requires ongoing cellular protein synthesis. When added to the enzyme-assay mixture in vitro, serum does not alter the phenylalanine hydroxylase activity of extracts from basal cultures. Three lines of evidence suggest that serum contains a nonsteroidal phenylalanine hydroxylase stimulatory components(s): (a) glucocorticoid antagonists inhibit less than one-half of the biological activity of serum; (b) exhaustive extraction of endogenous serum glucocorticoids with charcoal reduces the activity of serum to about one-half of control values; and (c) the stimulatory effects of charcoal reduces the values; and (c) the stimulatory effects of charcoal-extracted serum and hydrocortisone are additive. The phenylalanine hydroxylase stimulatory activities of the charcoal-extracted sera from four mammalian species and from three stages in development in one mammalian species are comparable. A survey of partially purified preparations of a number of known hormones failed to reveal any one capable of elevating the phenylalanine hydroxylas levels of basal cultures in a manner comparable to that of charcoal-extracted serum.     

L-Phenylalanine ammonia-lyase from Phaseolus vulgaris. Characterisation and differential induction of multiple forms from elicitor-treated cell suspension cultures

L-Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified over 200-fold from cell cultures of bean (phaseolus vulgaris L.) exposed to elicitor heat-released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. Four forms of the enzyme, with identical Mr but differing apparent pI values of 5.4, 5.2, 5.05 and 4.85, were observed following the final chromatofocussing stage of the purification. A preparation (purified 43-fold by ammonium sulphate precipitation, gel-filtration and ion-exchange chromatography) containing all four forms exhibited apparent negative rate cooperativity with respect to substrates. However, the individual forms displayed normal Michaelis-Menten kinetics, with Km values of 0.077 mM, 0.122 mM, 0.256 mM and 0.302 mM in order of decreasing apparent pI value. A preparation purified 200-fold and containing all four forms was used to immunise rabbits for the production of anti-(phenylalanine ammonia-lyase) serum. The antiserum was characterised by: immunotitration experiments; solid phase enzyme-linked immunosorbent assays; comparison of immunoprecipitates of 35S-labelled phenylalanine ammonia-lyase subunits (synthesized both in vivo and in vitro) on both one-dimensional and two-dimensional polyacrylamide gels after immunoprecipitation with the bean antiserum or antisera raised against pea and parsley phenylalanine ammonia-lyase preparations and immune blotting. SDS/polyacrylamide gels and SDS/polyacrylamide gel electrophoresis followed by immune blotting, indicated that the Mr of newly synthesized (in vivo and in vitro) bean phenylalanine ammonia-lyase subunits is 77000; a 70000-Mr form is readily generated as a partial degradation product during purification. Immunoprecipitates of bean phenylalanine ammonia-lyase synthesized both in vivo and in vitro showed the presence of multiple subunit types of identical Mr but differing in pI. Furthermore, treatment of bean cultures with Colletotrichum elicitor resulted in a 10-fold increase in phenylalanine ammonia-lyase extractable activity within 8 h, and chromatofocussing analysis indicated that this was associated with differential increased appearance of the high-pI, low-Km forms as compared to the two higher Km forms. This differential induction was further confirmed by immune blotting of crude extracts subjected to isoelectric focussing.