Conversion of amino acids into aroma compounds by cell-free extracts of Lactobacillus helveticus

AIMS: Lactobacillus helveticus is an essential starter in Swiss-type cheeses such as Emmental. This study was to determine whether cell-free extracts of Lact. helveticus were able to convert free amino acids into neutral volatile aroma compounds at the pH and temperature occurring in cheese. METHODS AND RESULTS: A mix of branched-chain (Leu, Ile, Val), aromatic (Tyr, Phe) and sulphur (Met) amino acids was incubated for 7 days, at pH 5.7 and 24 degrees C, with cell-free extracts of six strains. The amino acids were all transaminated into the corresponding keto acids when an amino group acceptor (alpha-ketoglutaric acid) was provided. Phe and Tyr were transaminated the most efficiently, followed by Leu, Met, Ile and Val. Three major volatile compounds were detected by GC-MS: benzaldehyde, dimethyl disulphide and 2-methyl propanol. Whatever the strain, benzaldehyde was produced in the highest quantity (0.25-1 micromol l(-1) mg(-1) protein). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus helveticus intracellular enzymes could significantly contribute to the production of aroma compounds from amino acid catabolism.     

Gas chromatographic determination of D-/L-arabinitol ratio in healthy Polish children

The D-/L-arabinitol enantiomers ratio (a marker of disseminated candidiasis of Candida species) in urine was determined by gas chromatography (GC) in 198 healthy Polish children ranging in age from 0 to 18 years. The urine samples were dry and trifluoroacetic anhydride (TFAA)-treated. Enantiomers derivatives were separated on a chiral column (beta-Dex 120, 60 m x 0.25 mm I.D.). A glass "solid-phase" injector and electron capture detector (ECD) were used. The ECD response was linear with correlation coefficients 0.999. The limit of detection was 0.02 micromol/l. Good results in terms of reproducibility, accuracy (RSD<10%, bias<6%), and linearity were obtained from real urine samples containing up to 400 micromol/l D-arabinitol. TFA-arabinitol derivatives in biological samples were stable from 1 to 5 days (depending on the arabinitol contents), while TFA-arabinitol standard derivatives were stable for 2 weeks. The identity of D- and L-arabinitol were confirmed by GC-MS analysis. The mean D-/L-arabinitol ratios ranged from 2.48 to 1.65 in the examined groups. The D-/L-arabinitol ratio was found to be exponentially regressive with age. A few cases of diagnosis of disseminated candidiasis by the GC method and confirmed by blood culture are described. The described GC method was also used for monitoring antifungal treatment of patients with disseminated candidiasis.     

Cyclo(d‐Tyr‐d‐Phe): a new antibacterial,anticancer, and antioxidant cyclic dipeptide from Bacillus sp. N strain associated with a rhabditid entomopathogenic nematode

A new microbial cyclic dipeptide (diketopiperazine), cyclo(d ‐Tyr‐d ‐Phe) was isolated for the first time from the ethyl acetate extract of fermented modified nutrient broth of Bacillus sp. N strain associated with rhabditid Entomopathogenic nematode. Antibacterial activity of the compound was determined by minimum inhibitory concentration and agar disc diffusion method against medically important bacteria and the compound recorded significant antibacterial against test bacteria. Highest activity was recorded against Staphylococcus epidermis (1 µg/ml) followed by Proteus mirabilis (2 µg/ml). The activity of cyclo(d ‐Tyr‐d ‐Phe) against S. epidermis is better than chloramphenicol, the standard antibiotics. Cyclo(d ‐Tyr‐d ‐Phe) recorded significant antitumor activity against A549 cells (IC₅₀ value: 10 μM) and this compound recorded no cytotoxicity against factor signaling normal fibroblast cells up to 100 μM. Cyclo(d ‐Tyr‐d ‐Phe) induced significant morphological changes and DNA fragmentation associated with apoptosis in A549 cells. Acridine orange/ethidium bromide stained cells indicated apoptosis induction by cyclo(d ‐Tyr‐d ‐Phe). Flow cytometry analysis showed that the cyclo(d ‐Tyr‐d ‐Phe) did not induce cell cycle arrest. Effector molecule of apoptosis such as caspase‐3 was found activated in treated cells, suggesting apoptosis as the main mode of cell death. Antioxidant activity was evaluated by free radical scavenging and reducing power activity, and the compound recorded significant antioxidant activity. The free radical scavenging activity of cyclo(d ‐Tyr‐d ‐Phe) is almost equal to that of butylated hydroxyanisole, the standard antioxidant agent. We also compared the biological activity of natural cyclo(d ‐Tyr‐d ‐Phe) with synthetic cyclo(d ‐Tyr‐d ‐Phe) and cyclo(l ‐Tyr‐l ‐Phe). Natural and synthetic cyclo(d ‐Tyr‐d ‐Phe) recorded similar pattern of activity. Although synthetic cyclo(l ‐Tyr‐l ‐Phe) recorded lower activity. But in the case of reducing power activity, synthetic cyclo(l ‐Tyr‐l ‐Phe) recorded significant activity than natural and synthetic cyclo(d ‐Tyr‐d ‐Phe). The results of the present study reveals that cyclo(d ‐Tyr‐d ‐Phe) is more bioactive than cyclo(l ‐Tyr‐l ‐Phe). To the best of our knowledge, this is the first time that cyclo(d ‐Tyr‐d ‐Phe) has been isolated from microbial natural source and also the antibacterial, anticancer, and antioxidant activity of cyclo(d ‐Tyr‐d ‐Phe) is also reported for the first time. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and characterization of new radioligands for the mammalian melanin-concentrating hormone (MCH) receptor

Melanin-concentrating hormone (MCH) is a neuropeptide present in the brain of all vertebrates. For the characterization of MCH receptors, a monoiodinated [Phe13, Tyr19]-MCH radioligand analogue was developed. The high susceptibility of [125I]-[Phe13, Tyr19]-MCH to oxidative damage and its very lipophilic nature made it necessary to develop new MCH radioligands. To increase the stability, native methionines were replaced by non-sulphur containing amino acid residues. In one analogue, the L-enantiomer of the phenylalanine residue at position 13 was substituted by the D-enantiomer, which increased the relative affinity of the ensuing [125I]-[D-Phe13, Tyr19]-MCH about 7-fold. The different analogues were iodinated by an enzymatic reaction and used for binding studies with mouse melanoma cells. [125I]-[Met(O)4,8, Phe13, Tyr19]-MCH and [125I]-[Hse4,8, Phe13, Tyr19]-MCH showed only about 19% of total binding and [125I]-[Ser4,8, Phe13, Tyr19]-MCH displayed about 44% of total binding when compared with [125I]-[Phe13, Tyr19]-MCH. Non-specific binding for all tracers was below 11% of total binding of [125I]-[Phe13, Tyr19]-MCH binding. [125I]-[D-Phe13, Tyr19]-MCH was used for saturation binding studies and revealed a KD of 122.7 +/- 15.3 pmol/l. This radioligand was further characterized by association and dissociation binding studies.     

Inactivation of calpain I and calpain II by specificity-oriented tripeptidyl chloromethyl ketones

Three new tripeptidyl chloromethyl ketones, Leu-Leu-XCH2Cl, with X representing Phe, Tyr, or Lys, were synthesized and their potencies to inactivate calpains I and II were compared. They were designed to fulfil the specificity requirement of calpains established recently. When compared in terms of the dose for 50% inactivation, Leu-Leu-PheCH2Cl was the strongest inactivator, being 500-600 times more effective than tosyl-PheCH2Cl and 5-14 times more than N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]agmatine (E-64). The potency toward calpain, either I or II, decreased in the order Phe greater than Tyr greater than Lys derivatives greater than E-64, whereas that toward papain was E-64 greater than Lys greater than Phe greater than Tyr derivatives. From the determined kinetic parameters, the Phe derivative was 18.3 and 16.6 times more effective than E-64 on calpains I and II, respectively. Likewise, the rate of the alkylation reaction by these chloromethyl ketones with calpain I was 2-4 times greater than that with calpain II. Leu-Leu-PheCH2Cl and its N-dansylated product should be useful for highly selective affinity labeling of calpains I and II.     

Use of directed mutagenesis to probe the role of tyrosine 198 in the catalytic mechanism of carboxypeptidase A

Derivatization of Tyr198 in carboxypeptidase A (CPA) results in lowered catalytic activity toward peptide substrates (Cueni, L., and Riordan, J.F. (1978) Biochemistry 17, 1834-1842). We have synthesized via directed mutagenesis a rat CPA variant [Phe198] CPA containing a Tyr198-to-Phe substitution in order to test whether the phenolic hydroxyl plays a critical role in catalysis. A double mutant [Phe193, Phe248]CPA in which both Tyr198 and Tyr248 have been replaced by phenylalanine has also been engineered. Enzymatic characterization of [Phe198]CPA indicates that the Tyr198 hydroxyl is not obligatory for the hydrolysis of peptide and ester substrates. Furthermore, parallel studies with [Phe198, Phe248]CPA show that simultaneous removal of both the Tyr198 and Tyr248 hydroxyls does not abolish catalytic activity. Analysis of the acetylated derivatives of [Phe198]CPA, [Phe248]CPA, and [Phe198, Phe248]CPA establishes that Tyr198 and Tyr248 are the active site tyrosines which are modified by N-acetylimidazole. In addition, the perturbations of enzymatic activity which accompany acetylation of native CPA can be largely assigned to derivatization of Tyr248. The changes in the kinetic constants of substrate hydrolysis due to the Tyr198-to-Phe substitution are manifested as small decreases in the kcat values, but the Km values are essentially unaffected. This exclusive effect on the kcat values suggests that the Tyr198 hydroxyl participates in catalysis by stabilizing the rate-determining transition-state complex.     

Effects of L-tyrosine and carbohydrate ingestion on endurance exercise performance.

To test the effects of tyrosine ingestion with or without carbohydrate supplementation on endurance performance, nine competitive cyclists cycled at 70% peak oxygen uptake for 90 min under four different feeding conditions followed immediately by a time trial. At 30-min intervals, beginning 60 min before exercise, each subject consumed either 5 ml/kg body wt of water sweetened with aspartame [placebo (Pla)], polydextrose (70 g/l) (CHO), L-tyrosine (25 mg/kg body wt) (Tyr), or polydextrose (70 g/l) and L-tyrosine (25 mg/kg body wt) (CHO+Tyr). The experimental trials were given in random order and were carried out by using a counterbalanced double-blind design. No differences were found between treatments for oxygen uptake, heart rate, or rating of perceived exertion at any time during the 90-min ride. Plasma tyrosine rose significantly from 60 min before exercise to test termination (TT) in Tyr (means +/- SE) (480 +/- 26 micromol) and CHO+Tyr (463 +/- 34 micromol) and was significantly higher in these groups from 30 min before exercise to TT vs. CHO (90 +/- 3 micromol) and Pla (111 +/- 7 micromol) (P < 0.05). Plasma free tryptophan was higher after 90 min of exercise, 15 min into the endurance time trial, and at TT in Tyr (10.1 +/- 0.9, 10.4 +/- 0.8, and 12.0 +/- 0.9 micromol, respectively) and Pla (9.7 +/- 0.5, 10.0 +/- 0.3, and 11.7 +/- 0.5 micromol, respectively) vs. CHO (7.8 +/- 0.5, 8.6 +/- 0.5, and 9.3 +/- 0.6 micromol, respectively) and CHO+Tyr (7.8 +/- 0.5, 8.5 +/- 0.5, 9.4 +/- 0.5 micromol, respectively) (P < 0.05). The plasma tyrosine-to-free tryptophan ratio was significantly higher in Tyr and CHO+Tyr vs. CHO and Pla from 30 min before exercise to TT (P < 0.05). CHO (27.1 +/- 0.9 min) and CHO+Tyr (26.1 +/- 1.1 min) treatments resulted in a reduced time to complete the endurance time trial compared with Pla (34.4 +/- 2.9 min) and Tyr (32.6 +/- 3.0 min) (P < 0.05). These findings demonstrate that tyrosine ingestion did not enhance performance during a cycling time trial after 90 min of steady-state exercise.     

Production of tyrosine and other aromatic compounds from phenylalanine by rumen microorganisms

Summary Rumen contents from three fistulated Japanese native goats fed Lucerne hay cubes (Medicago sativa) and concentrate mixture were collected to prepare the suspensions of mixed rumen bacteria (B), mixed protozoa (P) and a combination of the two (BP). Microbial suspensions were anaerobically incubated at 39°C for 12h with or without 1 MM ofl-phenylalanine (Phe). Phe, tyrosine (Tyr) and other related compounds in both supernatant and microbial hydrolysates of the incubations were analyzed by HPLC. Tyr can be produced from Phe not only by rumen bacteria but also by rumen protozoa. The production of Tyr during 12h incubation in B (183.6 mol/g MN) was 4.3 times higher than that in P. One of the intermediate products between Phe and Tyr seems to bep-hydroxyphenylacetic acid. The rate of the net degradation of Phe incubation in B (76.O mol/g MN/h) was 2.4 times higher than in P. In the case of all rumen microorganisms, degraded Phe was mainly (>53%) converted into phenylacetic acid. The production of benzoic acid was higher in P than in B suspensions. Small amount of phenylpyruvic acid was produced from Phe by both rumen bacteria and protozoa, but phenylpropionic acid and phenyllactic acid were produced only by rumen bacteria.     

Phenylalanine and tyrosine kinetics in compensated liver cirrhosis: effects of meal ingestion

We explored the mechanism(s) of increased aromatic amino acids concentrations in liver cirrhosis using phenylalanine (Phe) and tyrosine (Tyr) isotope infusions in male patients with compensated cirrhosis (five in Child Class A, three in B) and in eight matched healthy controls, in both postabsorptive and fed states. After a baseline period, a standard liquid mixed meal was fed continuously over 4 h. Both a "plasma" and an intracellular model were employed. In the patients, steady-state Phe and Tyr concentrations were approximately 30-50% greater, and rates of Phe appearance (Ra) (plasma model), Tyr Ra, and Phe hydroxylation (Hy; both models) were approximately 25 to >100% greater than in controls in both states. Meal ingestion increased (P<0.05 or less vs. basal) Phe and Tyr concentrations, Phe and Tyr Ra, Phe Hy, and % Tyr Ra not deriving from Hy in both groups. Hy and Tyr Ra remained>50% greater (P<0.04 to P<0.01) in patients, whereas Phe Ra was more modestly increased. Phe utilization for protein synthesis increased similarly in both groups. Tyr clearance was normal, whereas Phe clearance tended to be lower (P=0.09, intracellular model) in the patients. In summary, in compensated liver cirrhosis studied under fasted and fed states, 1) Tyr Ra is increased; 2) Phe Hy and Phe Ra (plasma model) are increased; 3) Tyr clearance is normal; and 4) Phe clearance is slightly decreased. In conclusion, in cirrhosis increased total tyrosine Ra and hydroxylation contribute to fasting and postmeal hypertyrosinemia, whereas the mechanism(s) responsible for the hyperphenylalaninemia may include both increased production and decreased disposal.     

Performance of piglets in response to the standardized ileal digestible phenylalanine and tyrosine supply in low-protein diets

Reducing the CP level of the diet allows for a reduction in N excretion without limiting performance as long as the amino acid (AA) requirements are covered. The availability of crystalline AA has permitted for a considerable reduction in the CP level of diets, practically used in pig nutrition. The adoption of low CP diets and the extent to which the CP content can be reduced further depends on the knowledge of the minimum levels of indispensable AA that maximize growth. The standardized ileal digestible (SID) Phe : Lys and Tyr : Lys requirements and the possibility to substitute Tyr by Phe have never been studied in piglets. The objectives of this study were to estimate these requirements in 10 to 20 kg pig as well as to determine the extent to which Phe can be used to cover the Tyr requirement. In three dose–response studies, six pigs within each of 14 blocks were assigned to six low CP diets (14.5% CP) sub-limiting in Lys at 1.00% SID. In experiment 1, the SID Phe : Lys requirement estimate was assessed by supplementing a Phe-deficient diet with different levels of l-Phe to attain 33%, 39%, 46%, 52%, 58%, and 65% SID Phe : Lys. Because Phe can be used for Tyr synthesis, the diets provided a sufficient Tyr supply. A similar approach was used in experiment 2 with six levels of l-Tyr supplementation to attain 21%, 27%, 33%, 39%, 45% and 52% SID Tyr : Lys. Phenylalanine was supplied at a level sufficient to sustain maximum growth (estimated in experiment 1). The SID Phe : Lys and SID Tyr : Lys requirements for maximizing daily gain were 54% and 40% using a curvilinear-plateau model, respectively. A 10% deficiency in Phe and Tyr reduced daily gain by 3.0% and 0.7%, respectively. In experiment 3, the effect of the equimolar substitution of dietary SID Tyr by Phe to obtain 50%, 57%, and 64% SID Phe : (Phe+Tyr) was studied at two limiting levels of Phe+Tyr. From 57% to 64% SID Phe : (Phe+Tyr), performance was slightly reduced. In conclusion, it is recommended not to use a Phe+Tyr requirement in the ideal AA profile but rather use a SID Phe : Lys of 54% and a SID Tyr : Lys of 40% to support maximal growth.     

Endomorphin 2 analogues containing Dmp residue as an aromatic amino acid surrogate with high mu-opioid receptor affinity and selectivity

To investigate the effectiveness of a 2',6'-dimethylphenylalanine (Dmp) residue as an aromatic amino acid surrogate, endomorphin 2 (EM(2): Tyr-Pro-Phe-Phe-NH(2)) analogues were prepared, in which the constitutive aromatic amino acids (Tyr(1), Phe(3), or Phe(4)) were replaced by Dmp or its isomer, D-Dmp. Replacement of Phe(3) by Dmp increased the affinity over 10-fold for both mu- and delta-opioid receptors, without affecting receptor selectivity. In contrast, replacement of Phe(4) considerably reduced the mu-receptor affinity and selectivity. These data indicated that the Dmp-substitution of Phe(3), but not Phe(4), in EM(2) is favorable for improving mu-receptor specificity. Inversion of the chirality of the substituted Dmp residue resulted in marked decrease in the mu-receptor affinity. Replacement of Tyr(1) by Dmp yielded an analogue that exhibited only a limited decrease in mu-receptor affinity and GPI potency, despite the lack of a phenolic hydroxyl group at the N-terminal residue. In contrast, D-Dmp(1)- or Phe(1)-substitution of Tyr(1) resulted in a significant decrease in mu-receptor affinity and GPI potency. These results suggested that the Dmp residue can mimic Tyr(1), which is one of the critical structural elements of opioid peptides.     

Tyrosine requirements in children with classical PKU determined by indicator amino acid oxidation

Tyrosine (Tyr) is an essential amino acid in phenylketonuria (PKU) because of the limited hydroxylation of phenylalanine (Phe) to Tyr. The recommended intakes for Tyr in PKU are at least five times the recommended phenylalanine intakes. This suggests that Phe and Tyr contribute approximately 20 and 80%, respectively, of the aromatic amino acid (AAA) requirement (REQ). In animals and normal humans, dietary Tyr was shown to spare 40-50% of the Phe requirement, proportions that reflect dietary and tissue protein composition. We tested the hypothesis that the Tyr REQ in PKU would account for 45% of the total AAA REQ by indicator amino acid oxidation (IAAO). Tyr REQ was determined in five children with PKU by examining the effect of varying dietary Tyr intake on lysine oxidation and the appearance of (13)CO(2) in breath (F(13)CO(2)) under dietary conditions of adequate energy, protein (1.5 g x kg(-1) x day(-1)), and phenylalanine (25 mg x kg(-1) x day(-1)). Lysine oxidation and F(13)CO(2) were determined using a primed 4-h oral equal-dose infusion of L-[1-(13)C]lysine. Lysine oxidation and F(13)CO(2) decreased linearly as Tyr intake increased, to a break point that was interpreted as the mean dietary Tyr requirement (16.3 and 19.2 mg x kg(-1) x day(-1), respectively). At Tyr intakes of >16.3 and 19.2 mg x kg(-1) x day(-1), lysine oxidation and F(13)CO(2), respectively, were low and constant. This represents 40.4 and 44.4%, respectively, of the total AAA intake. The current recommendations for Tyr intake in PKU patients appear to be overestimated by a factor of approximately 5. This study is the first application of the IAAO technique in a pediatric population and in humans with an inborn error of metabolism.     

Effect of feeding precursors on phenylalanine ammonia-lyase activity and coumarin accumulation in leaves of <Emphasis Type="Italic">Matricaria chamomilla</Emphasis> L.

Four-week-old chamomile (Matricaria chamomilla) plants were exposed for 72 h to 0.01, 0.1 and 1 mM phenylalanine (Phe) or tyrosine (Tyr). Phe at all concentrations significantly increased phenylalanine ammonia-lyase (PAL) activity (by 30, 76 and 90%, respectively) as well as accumulation of coumarin-related compounds (herniarin and its precursors (Z)- and (E)-2-β-D-glucopyranosyloxy-4-methoxycinnamic acids). Free Phe content increased significantly at the highest dose tested. Lower Tyr concentrations (0.01 and 0.1 mM) significantly increased PAL activity and increased free Tyr content, however free Phe content decreased. This indicated that Tyr-mediated stimulation of PAL is coupled to Phe consumption. Notwithstanding, Tyr had no effect on coumarin accumulation. Therefore we speculate that in chamomile a regulation/signalling mechanism could be operating in the pathway leading to coumarin synthesis. The malondialdehyde accumulation, an usual marker of stress in plants, was not significantly changed by amino acid supplements, suggesting that membrane damage is not the signal causing coumarin accumulation. In parallel experiment we observed that neither lower (0.25 × full strength), nor higher (3 × full strength) nitrogen concentration of nutrient solution compared to normal (1 × full strength, 205 mg N l^-1) solution used for Phe/Tyr supply affected herniarin and GMCAs accumulation. This indicates that Phe had stimulatory effect on PAL activity and coumarin metabolism.     

Comparison of plasma and urinary phytoestrogens in Japanese and Finnish women by time-resolved fluoroimmunoassay

Time-resolved fluoroimmunoassays (TR-FIA), with europium labeled phytoestrogens as tracers, were developed for the quantitative measurement of genistein, daidzein and enterolactone in plasma and urine for the purpose of screening large populations and studies on possible correlation between the values in biological fluids and the risk of western diseases. The mean values of the three phytoestrogens in plasma as determined by TR-FIA were similar to those obtained by gas chromatography-mass spectrometry (GC-MS). The urinary excretion levels of total individual phytoestrogens were higher than those obtained by GC-MS, with the exception of the daidzein values. However, comparing the assay results obtained by the present method and those obtained by GC-MS, a strong correlation was evident (r = 0.87 - 0.99, p < 0.001). We measured plasma levels of genistein, daidzein and enterolactone in 111 healthy Japanese women The mean and median levels of genistein were 406.8 and 306.3 nmol/l, respectively, and those of daidzein were 118.4 and 76.8 nmol/l, respectively. These levels are higher than those reported for Americans and Western Europeans. Isoflavone intake as calculated from dietary records (genistein: mean, 86.5 mircomol/day and daidzein: mean, 57.4 micromol/day) was correlated with the plasma concentrations observed (genistein: r = 0.287, p < 0.01 and daidzein: r = 0.313, p < 0.01). Plasma enterolactone levels were low in Japanese women (mean, about 10 nmol/l). The levels of urinary excretions of genistein, daidzein were also measured and it was found that, in the majority, the levels ranged between 5-25 and 5-50 micromol/24 h, respectively. In contrast, healthy Finnish women showed very low values of isoflavones (below 10 nmol/l in plasma (n = 87) and below 0.6 micromol/24 h in urine (n = 126) for both compounds) and high levels of enterolactone in both plasma and urine (plasma: mean, 25 nmol/l and urine: majority range, 1-7 micromol/24 h).     

Engineering of substrate specificity of D-amino acid oxidase from the yeast Trigonopsis variabilis: Directed mutagenesis of Phe258 residue

Natural D-amino acid oxidases (DAAO) are not suitable for selective determination of D-amino acids due to their broad substrate specificity profiles. Analysis of the 3D-structure of the DAAO enzyme from the yeast Trigonopsis variabilis (TvDAAO) revealed the Phe258 residue located at the surface of the protein globule to be in the entrance to the active site. The Phe258 residue was mutated to Ala, Ser, and Tyr residues. The mutant TvDAAOs with amino acid substitutions Phe258Ala, Phe258Ser, and Phe258Tyr were purified to homogeneity and their thermal stability and substrate specificity were studied. These substitutions resulted in either slight stabilization (Phe258Tyr) or destabilization (Phe258Ser) of the enzyme. The change in half-inactivation periods was less than twofold. However, these substitutions caused dramatic changes in substrate specificity. Increasing the side chain size with the Phe258Tyr substitution decreased the kinetic parameters with all the D-amino acids studied. For the two other substitutions, the substrate specificity profiles narrowed. The catalytic efficiency increased only for D-Tyr, D-Phe, and D-Leu, and for all other D-amino acids this parameter dramatically decreased. The improvement of catalytic efficiency with D-Tyr, D-Phe, and D-Leu for TvDAAO Phe258Ala was 3.66-, 11.7-, and 1.5-fold, and for TvDAAO Phe258Ser it was 1.7-, 4.75-, and 6.61-fold, respectively.     

The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.

The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1' subsite. Thermolysin and TLP-ste variants with identical S1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P1' position.     

Inter- and intra-individual variation in plasma and red blood cell vitamin E after supplementation

To establish the range of individual blood responses to supplemental vitamin E, 30 healthy subjects ingested 75 mg of deuterium-labelled alpha-tocopherol with a standard breakfast. Blood was collected at 6, 9, 12, 27 and 51 h post ingestion and deuterated (d6) and non-deuterated (do) alpha-tocopherol concentrations were determined in plasma and red blood cells (RBC) by GC-MS. To examine intra-individual responses, 6 of these subjects were re-examined at 6-month intervals over a 30-month period. Post ingestion, the amount of d6-alpha-tocopherol in blood increased rapidly with time with maximal concentrations seen at 12 h (plasma) and 27 h (RBC) in most subjects. At these times, d6-alpha-tocopherol concentration ranged from 0.3-12.4 micromol/l in plasma and 0.6-4.09 micromol/l packed cell in RBC. Area under the curve calculations indicated inter-individual differences of alpha-tocopherol uptake to be 40-fold for plasma (12.9-493.3 micromol h/l) and 6-fold for RBC (24.4-146.1 micromol h/l packed RBC). Intra-individual variation in alpha-tocopherol uptake was small in comparison and remained relatively constant over the 30-month period. We conclude that vitamin E uptake varies widely in the normal population, although it is comparatively stable for an individual over time. These differences likely arise from variations in the regulation of vitamin E uptake and metabolism between subjects. Factors regulating this process must be better understood before the optimal intake of vitamin E can be ascertained.     

Probing aromatic, hydrophobic, and steric effects on the self-assembly of an amyloid-β fragment peptide

Aromatic amino acids have been shown to promote self-assembly of amyloid peptides, although the basis for this amyloid-inducing behavior is not understood. We adopted the amyloid-β 16-22 peptide (Aβ(16-22), Ac-KLVFFAE-NH(2)) as a model to study the role of aromatic amino acids in peptide self-assembly. Aβ(16-22) contains two consecutive Phe residues (19 and 20) in which Phe 19 side chains form interstrand contacts in fibrils while Phe 20 side chains interact with the side chain of Va l18. The kinetic and thermodynamic effect of varying the hydrophobicity and aromaticity at positions 19 and 20 by mutation with Ala, Tyr, cyclohexylalanine (Cha), and pentafluorophenylalanine (F(5)-Phe) (order of hydrophobicity is Ala < Tyr < Phe < F(5)-Phe < Cha) was characterized. Ala and Tyr position 19 variants failed to undergo fibril formation at the peptide concentrations studied, but Cha and F(5)-Phe variants self-assembled at dramatically enhanced rates relative to wild-type. Cha mutation was thermodynamically stabilizing at position 20 (ΔΔG = -0.2 kcal mol(-1) relative to wild-type) and destabilizing at position 19 (ΔΔG = +0.2 kcal mol(-1)). Conversely, F(5)-Phe mutations were strongly stabilizing at both positions (ΔΔG = -1.3 kcal mol(-1) at 19, ΔΔG = -0.9 kcal mol(-1) at 20). The double Cha and F(5)-Phe mutants showed that the thermodynamic effects were additive (ΔΔG = 0 kcal mol(-1) for Cha 19,20 and -2.1 kcal mol(-1) for F(5)-Phe 19,20). These results indicate that sequence hydrophobicity alone does not dictate amyloid potential, but that aromatic, hydrophobic, and steric considerations collectively influence fibril formation.     

Fluorescence quenching method for the determination of carbazochrome sodium sulfonate with aromatic amino acids

In Britton‐Robinson (BR) buffer medium (pH 3.3), carbazochrome sodium sulfonate (CSS) can react with some aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) to form a 1:1 complex by electrostatic attraction, aromatic stacking interaction and Van der Waals' force, resulting in fluorescence quenching of these amino acids. Maximum quenching wavelengths were located at 352 nm (CSS‐Trp system), 303 nm (CSS‐Tyr system) and 284 nm (CSS‐Phe system), respectively. The fluorescence quenching value (ΔF) was proportional to the concentration of CSS in a certain range. The fluorescence quenching method for the determination of CSS showed high sensitivity, with detection limits of 31.3 ng/mL (CSS‐Trp system), 44.6 ng/mL (CSS‐Tyr system) and 315.0 ng/mL (CSS‐Phe system), respectively. The optimum conditions of the reaction conditions and the effect of coexisting substances were investigated and results showed that the method had good selectivity. The method was successfully applied for the rapid determination of CSS in blood and urine samples. Based on the bimolecular quenching constant K_q, the effect of temperature and Stern‐Volmer plots, this study showed that quenching of fluorescence of amino acids by CSS was a static quenching process. Copyright © 2012 John Wiley & Sons, Ltd.