Reduction of the suppressive effects of gurmarin on sweet taste responses by addition of beta-cyclodextrin

Cyclodextrins (CDs) have the remarkable ability to form inclusion complexes with a wide variety of guest molecules. In the present study, possible influences of CDs on gurmarin inhibition of the chorda tympani responses to sucrose were examined in C57BL mice. Responses to sucrose were suppressed to approximately 50% of control by treatment of the tongue with 30 micrograms/ml (approximately 7.1 microM) gurmarin. Rinsing the tongue with 15 mM beta-CD after gurmarin gave rapid recovery of the suppressed sucrose responses to approximately 85% of control, whereas 15 mM alpha- or gamma-CD did not. When gurmarin was mixed with beta-CD, the suppressive effects of gurmarin on sucrose responses were largely reduced. No such reduction was observed for mixtures with alpha- and gamma-CD. Gurmarin includes tyrosine and tryptophan residues whose aromatic rings are directed outward and can probably form inclusion complexes with beta-CD. Therefore, the observed reduction of the effects of gurmarin may be due to steric hindrances in inclusion complexes of gurmarin with beta-CD that may interfere with gurmarin binding to sweet taste receptors.      

Synthesis,characterization, and sweetness-suppressing activities of gurmarin analogues missing one disulfide bond

The sweetness-suppressing polypeptide gurmarin isolated from Gymnema sylvestre consists of 35 amino acid residues and includes three intramolecular disulfide bonds. The roles of the three disulfide bonds were investigated by replacing each with two alanine residues by solid-phase synthesis. Nine analogues of [Ala^3,18]gurmarin, [Ala^10,23]gurmarin, and [Ala^17,33]-gurmarin were obtained. Three analogues had native disulfide bonds, while the other six had non-native disulfide bonds. The three analogues with native disulfide bonds suppressed the response to sucrose, but not those to glucose, fructose, saccharin, or glycine in rats. In contrast, the six analogues with non-native disulfide bonds did not suppress the responses to any of these sweeteners. These results suggest that the native disulfide bonds of gurmarin are necessary for interaction with the receptor protein, and that the sucrose-specific receptor site is present in rats. © 1998 John Wiley & Sons, Inc. Biopoly 46: 65–73, 1998     

Role of hydrophobic amino acids in gurmarin,a sweetness-suppressing polypeptide

The sweetness-suppressing polypeptide gurmarin isolated from Gymnema sylvestre consists of 35 amino acid residues and contains three intramolecular disulfide bonds. Nuclear magnetic resonance analysis showed that the hydrophobic side chains of Tyr-13, Tyr-14, Trp-28, and Trp-29 in gurmarin are oriented outwardly. Together with the hydrophobic side chains of Leu-9, Ile-11, and Pro-12, they form a hydrophobic cluster, and therefore these hydrophobic groups are assumed to act as the site for interaction with the receptor protein. To examine the roles of these hydrophobic amino acids, they were replaced by Gly. The resulting [Gly^13,14,28,29]gurmarin and [Gly^{9,11,13,14,28,29}]gurmarin did not suppress the responses to sucrose, glucose, fructose, or Gly. This result strongly suggests that these hydrophobic amino acids are involved in the interaction with the receptor protein. © 1998 John Wiley & Sons, Inc. Biopoly 45: 231–238, 1998     

Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin

Gurmarin is a 35 amino acid peptide with three disulfide bridges in an inhibitor cystine knot. It is found in the plant Gymnema sylvestre, and has been identified as a sweet taste inhibitor in rodents. In this article we provide an efficient route for the synthesis of gurmarin by a controlled random oxidation strategy. We compared two oxidation procedures to form the three disulfide bridges. In the first, based on random oxidation, reduced gurmarin was synthesized using trityl for cysteine protection, and oxidized for 48h in a Tris-HCl buffer containing cystamine and reduced glutathione to facilitate disulfide scrambling. The second was based on step-wise deprotection followed by oxidation in which the cysteine pairs are orthogonally protected with tert-Butylthio, trityl and acetamidomethyl. To verify that the native gurmarin oxidation product was obtained, thermolysin cleavage was used. Cleavage of random oxidized gurmarin showed two possible disulfide combinations; the native and a non-native gurmarin disulfide isomer. The non-native isomer was therefore synthesized using the orthogonal deprotection-oxidation strategy and the native and the non-native gurmarin isomers were analyzed using UPLC. It was found that the random oxidation procedure leads to native gurmarin in high yield. Thus, the synthetic route was simple and significantly more efficient than previously reported syntheses of gurmarin and other cysteine rich peptides. Importantly, native gurmarin was obtained by random oxidation, which was confirmed by a synthetic approach for the first time.     

Inhibitory effect of gurmarin on palatal taste responses to amino acids in the rat

Gurmarin (10 microg/ml), a protein extracted from Gymnema sylvestre, depressed significantly (40-50%) the phasic taste responses to sugars (sucrose, fructose, lactose, and maltose) and saccharin sodium recorded from the greater superficial petrosal nerve (GSP) innervating palatal taste buds in the rat. However, no significant effect of gurmarin was observed for taste responses to NaCl, HCl, and quinine hydrochloride. Phasic responses to D-amino acids that taste sweet to humans (His, Asn, Phe, Gln) were also depressed, but gurmarin treatment was without significant effect on taste responses to D-Trp and D-Ala, six L-amino acids (His, Asn, Phe, Gln, Trp, and Ala), and two basic amino acid HCl salts (Arg and Lys). With the exception of D-Trp, these inhibitory effects of gurmarin on GSP taste responses were related to the rat's preference for these substances.     

Efficient racemization of N‐phenylacetyl‐D‐glufosinate for L‐glufosinate production

Most amino acids contain chiral centres and exist as both D‐enantiomer and L‐enantiomer. The optically pure enantiomer is often more valuable than the racemate. Enzymatic resolution provides an effective strategy to obtain optically pure amino acids but often results in large amounts of unwanted isomer. In this study, optically pure L‐glufosinate (L‐PPT) was obtained by coupling amidase‐mediated hydrolysis of N‐phenylacetyl‐D,L‐glufosinate with racemization of N‐phenylacetyl‐D‐glufosinate (NPDG), which exclusively exhibits effective herbicidal properties compared with its D‐enantiomer. To improve the yield of L‐PPT, the racemization reaction conditions were optimized, and through single‐factor experiments, the optimal reaction temperature, reaction time, and mole ratio of phenylacetic acid to NPDG were determined to be 150°C, 30 minutes, and 1.5, respectively. The response surface methodology was applied to further optimize the racemization conditions, and the final yield of L‐PPT reached 96.13% with optimum reaction temperature of 154°C, reaction time of 23 minutes, and phenylacetic acid/NPDG mole ratio of 1.7, respectively. Moreover, adding a small amount of acetic anhydride further raised the yield of L‐PPT to 97.02%.     

Beta-cyclodextrin inhibits the sweet taste suppressing activity of gurmarin by the formation of an inclusion complex with aromatic residues in gurmarin.

Our recent study in mice revealed that the inhibitory activity of gurmarin on the sweet taste responses was reduced significantly by the presence of beta-cyclodextrin (beta-CD). To investigate the mechanism involved in the action of beta-CD, physicochemical experiments were performed on the interaction of CDs with gurmarin examining the effect of CDs on the UV absorption spectrum of gurmarin and on the elution behavior in gel filtration (or exclusion) chromatography. Among the three kinds of cyclodextrins tested, beta-CD induced significant changes in the UV absorption spectrum of gurmarin that were characteristic of those found in the inclusion complex formation of tyrosine and tryptophan with beta-CD. The abnormal retention behavior of gurmarin in gel filtration resulting from hydrophobic interaction with the gel matrix reverted to normal in the presence of beta-CD in the elution buffer. These results suggest that the unique domain of gurmarin, in which five aromatic amino acid residues are all directed outwardly and form a hydrophobic cluster, is a possible site of interaction with the gurmarin-sensitive sweet taste receptor molecules in rodents.     

Induction of salivary gurmarin-binding proteins in rats fed gymnema-containing diets.

Gymnema sylvestre, a tropical plant, contains gurmarin that selectively suppresses sucrose responses of the chorda tympani nerve in rats and mice. We investigated preference for taste solutions and saliva composition in rats fed a diet containing this plant (gymnema diet). Preference for 0.01 M sucrose and a mixture of 0.03 M sucrose and 0.03 mM quinine-HCl significantly decreased at 1-2 days after the start of the gymnema diet and subsequently returned closely to the control levels within about a week. There was no significant change in preference for NaCl, monosodium glutamate and quinine-HCl during feeding trials. Submandibular saliva of rats fed the gymnema diet for 4 and 14 days showed an inhibitory effect on immunoreaction between gurmarin and antigurmarin serum. Analyses using electrophoresis and affinity chromatography indicated that the saliva contains gurmarin binding proteins with molecular weights of 15, 16, 45, 60 and 66 kDa. These results suggest that reduction of preference for sucrose was probably caused by gurmarin contained in the gymnema diet and subsequent restoration of the preference may be due to suppression of the effect of gurmarin by salivary gurmarin-binding proteins induced by the gymnema diet.     

Improved glutathione production by gene expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To utilize Pichia pastoris to produce glutathione, an intracellular expression vector harboring two genes (gsh1 and gsh2) from Saccharomyces cerevisiae encoding enzymes involved in glutathione synthesis and regulated by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was transformed into P. pastoris GS115. Through Zeocin resistance and expression screening, a transformant that had higher glutathione yield (217 mg/L) in flask culture than the host strain was obtained. In fed-batch culture process, this recombinant strain displayed high activity for converting precursor amino acids into glutathione. The glutathione yield and biomass achieved 4.15 g/L and 98.15 g (dry cell weight, DCW)/L, respectively, after 50 h fermentation combined with addition of three amino acids (15 mmol/L glutamic acid, 15 mmol/L cysteine, and 15 mmol/L glycine).     

Synthesis and characterization of the sweet protein brazzein

The sweet protein brazzein isolated from the fruit of the African plant, Pentadiplandra brazzeana Baillon is 2000-500 times sweeter than sucrose, and consists of 54 amino acid residues with four intramolecular disulfide bonds. Brazzein was prepared by the fluoren-9-yl-methoxycarbonyl solid-phase method, and was identical to natural brazzein by high performance liquid chromatography, mass spectroscopy, peptide mapping, and taste evaluation. The D enantiomer of brazzein was also synthesized, and was shown to be the mirror image of brazzein. The D enantiomer (ent-brazzein) was devoid of any sweetness and was essentially tasteless. © 1996 John Wiley & Sons, Inc.     

Location of the Disulfide Bonds of the Sweetness-suppressing Polypeptide Gurmarin

The sweetness-suppressing polypeptide gurmarin has been isolated from the leaves of Gymnema sylvestre and consists of 35 amino acid residues including three intramolecular disulfide bonds. The primary structure has already been determined. The positions of the disulfide bonds were located, by a combination of mass spectrometric analysis and sequencing of cystine-containing pep tides obtained by thermolysin-catalyzed hydrolysis of gurmarin, to be at Cys3–Cys18, Cys10–Cys23, and Cys17–Cys33.     

Synergistic responses of the chorda tympani to mixtures of umami and sweet substances in rats

It has been known that umami substances such as monosodium L-glutamate (MSG) and 5'-inosine monophosphate (IMP) elicit a unique taste called 'umami' in humans. One of the characteristics of the umami taste is synergism: the synergistic enhancement of the magnitude of response produced by the addition of 5'-ribonucleotides to MSG. In addition to this well-documented synergism, we report here for the first time on another type of synergism between a glutamate receptor agonist, L-AP4, and sweet substances, by analyzing the chorda tympani responses in rats. The results are as follows: (i) when L-AP4 was mixed with one of the sweet substances, such as sucrose, glucose, fructose and maltose, large synergistic responses were observed. (ii) These synergistic responses, except to L-AP4 + sucrose, were not suppressed by sweet taste suppressants, gurmarin and pronase E. (iii) These synergistic responses were not suppressed by either metabotropic or ionotropic glutamate receptor antagonists. (iv) Fibers that responded well to the binary mixtures of L-AP4 and sweet substances also responded well to NaCl and HCl, but very weakly to sucrose. These findings are different from the characteristics of synergism between glutamate and IMP. The multiple transduction mechanisms for the umami taste in rat taste cells are discussed.     

Induction of salivary kallikreins by the diet containing a sweet-suppressive peptide, gurmarin, in the rat

Gymnema sylvestre (gymnema) contains gurmarin that selectively inhibits responses to sweet substances in rodents. The present study investigated possible interaction between gurmarin and the submandibular saliva in rats fed diet containing gymnema. Electrophoretic analyses demonstrated that relative amounts of two proteins in the saliva clearly increased in rats fed the gymnema diet. However, rats previously given section of the bilateral glossopharyngeal nerve showed no such salivary protein induction. Analyses of amino acid sequence indicate that two proteins are rat kallikrein 2 (rK2) and rat kallikrein 9 (rK9). rK2 and rK9, a family of serine proteases, have a striking resemblance of cleavage site in the protein substrates. Interestingly, gurmarin possesses comparable residues with those rK2 and rK9 prefer. The kallikreins significantly inhibited immunoreaction between gurmarin and antigurmarin antiserum. These results suggest that rK2 and rK9 increased by chemosensory information for the gymnema diet via the glossopharyngeal nerve might cleave gurmarin or at least cause specific binding with it.     

Factors affecting 'Hass' avocado fruit size: Carbohydrate, abscisic acid and isoprenoid metabolism in normal and phenotypically small fruit

Carbohydrate and abscisic acid (ABA) metabolism were investigated in normal and phenotypically small 'Hass' avocado ( Persea americana Mill.) fruit in an attempt to link alterations in sugar and ABA content with changes in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) activity and fruit size. The small-fruit phenotype was characterized by reduced seed HMGR activity, increased seed insoluble acid invertase ( β - d -fructofuranosidase, EC 3.2.1.26), decreased sucrose synthase (SS; UDP- d -glucose: d -fructose-2- α -glucosyl-transferase, EC 2.4.1.13) activity, decreased sucrose content, and increased glucose as a proportion of the total soluble sugar. Sucrose phosphate synthase (SPS; UDP- d -glucose: d -fructose 6-phosphate 2- α - d -glucosyltransferase, EC 2.4.1.14) activity was unaffected in seed but reduced in mesocarp of the small fruit. In addition, the small-fruit variant displayed enhanced respiration and both seed and mesocarp tissue showed increased ABA metabolism. Applied ABA caused an increase in insoluble acid invertase activity in seed tissue of normal fruit while mevastatin reduced HMGR activity in this tissue, caused sucrose depletion and increased the proportion of glucose from 5 to 57% of total soluble sugars. Exogenous glucose suppressed HMGR activity in seed tissue whereas in mesocarp tissue, HMGR activity was reduced to 38% of the control after 6 h but enhanced by 46% by 48 h. Glucose increased ABA biosynthesis and turnover in competent tissues. These results suggest that ABA turnover is mediated, in part, by carbohydrate content and composition which also affects HMGR activity. It is proposed that sugar and ABA signals act in concert to modulate expression and/or activity of HMGR in the control of 'Hass' avocado fruit growth and final fruit size.     

γ-Glutamylcysteinylserine — A New Homologue of Glutathione in Plants of the Family Poaceae*

In addition to glutathione (γ-GluCysGly), many species of the family Poaceae have another tripeptide which has the amino acid sequence γ-GluCysSer. This thiol was isolated from etiolated leaves of wheat (Triticum aestivum L. cv. Star). Its structure was elucidated by quantitative amino acid analysis after total hydrolysis and by partial hydrolysis with carboxypeptidase A and γ-glutamyltranspeptidase. The content of γ-GluCysSer in the leaves of T. aestivum is increased by incubation with sulfate and is severely diminished by incubation with buthionine sulfoximine, a specific inhibitor of γ-glutamylcysteine synthetase. Oxidized γ-GluCysSer is reduced by yeast glutathione reductase with a rate somewhat lower than for glutathione, but the new tripeptide is not a substrate of glutathione-S-transferase from equine liver. Besides homoglutathione (γ-GluCysßAla), a tripeptide found in plants of the order Fabales, the tripeptide γ-GluCysSer is the second homologue of glutathione detected in plants.     

Effect of Sugars and Amino Acids on Androgenesis of Cucumis sativus

The effects of sugars (sucrose, maltose, glucose and fructose) and amino acids (glutamine, glycine, arginine, asparagine and cysteine) on embryogenesis and plantlet regeneration from cultured anthers of Cucumis sativus L. cv. Calypso and Green Long were studied. Type and concentration of sugar and amino acid influenced embryogenesis. Among the different sugars tested, sucrose was the best for embryo induction with an optimal concentration of 0.25 M. Maximum of 72 and 80 embryos per 60 anthers of Calypso and Green Long, respectively, were induced on embryo induction medium [B5 (Gamborg, Miller and Ojima (1968) Exp. Cell Res. 50: 151–158) supplemented with 2.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 μM 6-benzyladenine (BA)] containing 0.25 M sucrose. The addition of amino acids to the embryo induction medium improved embryo yield with a combination of amino acids (glutamine, glycine, arginine, asparagine and cysteine of 1.0 mM each) giving the best response. Embryo differentiation was achieved on B5 medium supplemented with 0.25 μM of α-naphthaleneacetic acid (NAA), 0.25 μM kinetin (KN) and 0.09 M sucrose. Embryos were converted on B5 medium supplemented with abscisic acid (ABA) (10 μM) and 0.09 M sucrose. Embryos that developed on B5 medium supplemented with a combination of amino acids (glutamine, glycine, arginine, asparagine and cysteine of 1.0 mM each) exhibited the highest plantlet regeneration frequency.     

Maximizing production of glutathione by amino acid modulation and high-cell-density fed-batch culture of Saccharomyces cerevisiae

In this paper the high-cell-density fed-batch culture and optimal amino acid modulation were combined together to enhance glutathione production in Saccharomyces cerevisiae T65. Ethanol concentration in the broth was an important parameter for feedback control in fed-batch culture. Low ethanol concentration was propitious to both the cell growth and glutathione synthesis. The feedback control of a low ethanol concentration was an efficient way to realize high-cell-density culture and the biomass reached 140 g/L after 57 h fermentation. With optimal amino acid addition to elevate the glutathione content continually, the maximum glutathione yield achieved 2190 mg/L.     

Determination of the X-ray structure of the snake venom protein omwaprin by total chemical synthesis and racemic protein crystallography

The 50‐residue snake venom protein L ‐omwaprin and its enantiomer D ‐omwaprin were prepared by total chemical synthesis. Radial diffusion assays were performed against Bacillus megaterium and Bacillus anthracis; both L ‐ and D ‐omwaprin showed antibacterial activity against B. megaterium. The native protein enantiomer, made of L ‐amino acids, failed to crystallize readily. However, when a racemic mixture containing equal amounts of L ‐ and D ‐omwaprin was used, diffraction quality crystals were obtained. The racemic protein sample crystallized in the centrosymmetric space group P2₁/c and its structure was determined at atomic resolution (1.33 Å) by a combination of Patterson and direct methods based on the strong scattering from the sulfur atoms in the eight cysteine residues per protein. Racemic crystallography once again proved to be a valuable method for obtaining crystals of recalcitrant proteins and for determining high‐resolution X‐ray structures by direct methods.     

Characterization of Leuconostoc mesenteroides NRRL B-512F dextransucrase (DSRS) and identification of amino-acid residues playing a key role in enzyme activity

Dextransucrase (DSRS) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes the synthesis of soluble dextran from sucrose or oligosaccharides when acceptor molecules, like maltose, are present. The L. mesenteroides NRRL B-512F dextransucrase-encoding gene (dsrS) was amplified by the polymerase chain reaction and cloned in an overexpression plasmid. The characteristics of DSRS were found to be similar to the characteristics of the extracellular dextransucrase produced by L. mesenteroides NRRL B-512F. The enzyme also exhibited a high homology with other glucosyltransferases. In order to identify critical amino acid residues, the DSRS sequence was aligned with glucosyltransferase sequences and four amino acid residues were selected for site- directed mutagenesis experiments: aspartic acid 511, aspartic acid 513, aspartic acid 551 and histidine 661. Asp-511, Asp-513 and Asp-551 were independently replaced with asparagine and His-661 with arginine. Mutation at Asp-511 and Asp-551 completely suppressed dextran and oligosaccharide synthesis activities, showing that at least two carboxyl groups (Asp-511 and Asp-551) are essential for the catalysis process. However, glucan-binding properties were retained, showing that DSRS has a two-domain structure like other glucosyltransferases. Mutations at Asp-513 and His-661 resulted in greatly reduced dextransucrase activity. According to amino acid sequence alignments of glucosyltransferases, α-amylases or cyclodextrin glucanotransferases, His-661 may have a hydrogen-bonding function. Received: 16 April 1997 / Received revision: 17 June 1997 / Accepted: 23 June 1997     

Feeding responses by female Pieris brassicae butterflies to carbohydrates and amino acids

Most Lepidoptera feed during the adult stage on carbohydrate‐rich food sources, primarily floral nectar. However, little is known about the factors leading to the acceptance of a possible food source. It is reported that butterflies select for nectar rich in sucrose and amino acids. This suggests that the insects have developed a sensitivity to these nectar compounds. We tested females of the large cabbage white, Pieris brassicae L. (Lepidoptera: Pieridae) for their responses to 10 different nectar‐ or honeydew‐sugars after either tarsal or proboscis stimulation. In no‐choice experiments, food‐deprived P. brassicae showed the strongest response to sucrose, followed by fructose. Other sugars, including galactose, glucose, maltose, mannose, melezitose, melibiose, raffinose and trehalose, did not elicit a feeding response. Mixtures of essential or common non‐essential amino acids did not stimulate feeding. In a choice situation, P. brassicae preferred sucrose over fructose, whereas they accepted a sucrose and amino acid solution equally to a plain sucrose solution. The results indicate that for P. brassicae, feeding is mainly elicited by sucrose and fructose.