Incorporation Of Tyrosine Research Articles

Incorporation of Tyrosine and Glutamine Residues into the Soluble Guanylate Cyclase Heme Distal Pocket Alters NO and O2 Binding

Nitric oxide (NO) is the physiologically relevant activator of the mammalian hemoprotein soluble guanylate cyclase (sGC). The heme cofactor of alpha1beta1 sGC has a high affinity for NO but has never been observed to form a complex with oxygen. Introduction of a key tyrosine residue in the sGC heme binding domain beta1(1-385) is sufficient to produce an oxygen-binding protein, but this mutation in the full-length enzyme did not alter oxygen affinity. To evaluate ligand binding specificity in full-length sGC we mutated several conserved distal heme pocket residues (beta1 Val-5, Phe-74, Ile-145, and Ile-149) to introduce a hydrogen bond donor in proximity to the heme ligand. We found that the NO coordination state, NO dissociation, and enzyme activation were significantly affected by the presence of a tyrosine in the distal heme pocket; however, the stability of the reduced porphyrin and the proteins affinity for oxygen were unaltered. Recently, an atypical sGC from Drosophila, Gyc-88E, was shown to form a stable complex with oxygen. Sequence analysis of this protein identified two residues in the predicted heme pocket (tyrosine and glutamine) that may function to stabilize oxygen binding in the atypical cyclase. The introduction of these residues into the rat beta1 distal heme pocket (Ile-145 --> Tyr and Ile-149 --> Gln) resulted in an sGC construct that oxidized via an intermediate with an absorbance maximum at 417 nm. This absorbance maximum is consistent with globin Fe(II)-O(2) complexes and is likely the first observation of a Fe(II)-O(2) complex in the full-length alpha1beta1 protein. Additionally, these data suggest that atypical sGCs stabilize O(2) binding by a hydrogen bonding network involving tyrosine and glutamine.

Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion.

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase–tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)–tRNATyr pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS–tRNATyr pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNATyr. The endogenous TyrRS and tRNATyr genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS–tRNATyr pair. In this engineered strain, 3-iodo-l-tyrosine and 3-azido-l-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-l-tyrosine and was also found to recognize 3-azido-l-tyrosine. The structural basis for the 3-azido-l-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.

Oxidation of tyrosine by permanganate in presence of cetyltrimethylammonium bromide

In this paper we report the effect of cetyltrimethylammonium bromide, CTAB on the oxidative degradation of tyrosine by permanganate. The reaction rate bears a first-order dependence on the [MnO 4 −] under pseudo-first-order conditions (large excess of [tyrosine] for at least 10 times over [MnO 4 −]) in presence of 10.0 × 10 −4 mol dm −3 CTAB. The effect of total [CTAB] on the reaction rate was determined. When [CTAB] was less than its critical micelle concentration (CMC) the rate constants ( k ψ) values decreased from 18.5 × 10 −4 to 7.2 × 10 −4 s −1. As the [CTAB] was greater than the CMC, the k ψ values increase from 7.2 × 10 −4 to 15.8 × 10 −4 s −1at room temperature. The premicellar environment of CTAB strongly inhibits the reaction rate where as increase in rate constant ascribed to the incorporation of tyrosine and MnO 4 − in to the Stern layer of CTAB micelles. The reaction has acid-dependent and acid-independent paths and, in the former case, the zero-order kinetics with respect to [H 2SO 4] shifted to fractional-order at higher [H 2SO 4]. Experiments have been done to confirm the nature of Mn(IV) formed during the reduction of permanganate by tyrosine. The mechanism with the observed kinetics has been proposed and discussed. The presence of –OH group is responsible for the higher reactivity of tyrosine which easily transfers the proton to MnO 4 −.

A Light‐Activated DNA Polymerase

When the time is right: The widely applied Thermus aquaticus DNA polymerase was rendered light-activatable by incorporation of the photocaged amino acid ortho-nitrobenzyl tyrosine in place of a key tyrosine residue in the active site (see picture). As the modified enzyme was completely inactive until irradiated with UV light, temporal regulation of polymerase activity was possible.

Interplay of anionic charge, poly(ethylene glycol), and iodinated tyrosine incorporation within tyrosine‐derived polycarbonates: Effects on vascular smooth muscle cell adhesion, proliferation, and motility

Regulation of smooth muscle cell adhesion, proliferation, and motility on biomaterials is critical to the performance of blood-contacting implants and vascular tissue engineering scaffolds. The goal of this study was to examine the underlying substrate-smooth muscle cell response relations, using a selection of polymers representative of an expansive library of multifunctional, tyrosine-derived polycarbonates. Three chemical components within the polymer structure were selectively varied through copolymerization: (1) the content of iodinated tyrosine to achieve X-ray visibility; (2) the content of poly(ethylene glycol) (PEG) to decrease protein adsorption and cell adhesivity; and (3) the content of desaminotyrosyl-tyrosine (DT), which regulates the rate of polymer degradation. Using quartz crystal microbalance with dissipation, we quantified differential serum protein adsorption behavior because of the chemical components DT, iodinated tyrosine, and PEG: increased PEG content within the polymer structure progressively decreased protein adsorption but the simultaneous presence of both DT and iodinated tyrosine reversed the effects of PEG. The complex interplay of these components was next tested on the adhesion, proliferation, and motility behavior cultured human aortic smooth muscle cells. The incorporation of PEG into the polymer reduced cell attachment, which was reversed in the presence of iodinated tyrosine. Further, we found that as little as 10% DT content was sufficient to negate the PEG effect in polymers containing iodinated tyrosine, whereas in non-iodinated polymers, the PEG effect on cell attachment was reversed. Cross-functional analysis of motility and proliferation revealed divergent substrate chemistry related cell response regimes. For instance, within the series of polymers containing both iodinated tyrosine and 10% of DT, increasing PEG levels lowered smooth muscle cell motility without a change in the rate of cell proliferation. In contrast, for non-iodinated tyrosine and 10% of DT, increasing PEG levels increased cell proliferation significantly while reducing cell motility. Clearly, the polycarbonate polymer library offers a sensitive platform to modulate cell adhesion, proliferation, and motility responses, which, in turn, may have implications for controlling vascular remodeling in vivo. Additionally, our data suggests unique biorelevant properties following the incorporation of iodinated subunits in a polymeric biomaterial as a potential platform for X-ray visible devices.

Noninvasive Imaging of Protein Metabolic Labeling in Single Human Cells Using Stable Isotopes and Raman Microscopy

We have combined nonresonant Raman microspectroscopy and spectral imaging with stable isotope labeling by amino acids in cell culture (SILAC) to selectively detect the incorporation of deuterium-labeled phenylalanine, tyrosine, and methionine into proteins in intact, single HeLa cells. The C-D stretching vibrational bands in these amino acids are observed in the 2100-2300 cm(-1) spectral region that is devoid of vibrational contributions from other, nondeuterated intracellular constituents. We found that incubation with deuterated amino acids for 8 h in cell culture already led to clearly detectable isotope-related signals in Raman spectra of HeLa cells. As expected, the level of isotope incorporation into proteins increased with incubation time, reaching 55% for deuterated phenylalanine after 28 h. Raman spectral imaging of HeLa cells incubated with deuterium-labeled amino acids showed similar spatial distributions for both isotope-labeled and unlabeled proteins, as evidenced by Raman ratio imaging. The SILAC-Raman methodology presented here combines the strengths of stable isotopic labeling of cells with the nondestructive and quantitative nature of Raman chemical imaging and is likely to become a powerful tool in both cell biology applications and research on tissues or whole organisms.

Sigma Receptor Antagonists Inhibit Human Lens Cell Growth and Induce Pigmentation

The expression of the Sigma 1 receptor and the ability of receptor antagonists to inhibit growth and induce pigment formation were investigated in human lens epithelial cells. Capsular bags were formed for experimental purposes by performing sham cataract operations on donor lenses. The resultant bags were cultured in Eagle's minimum essential medium (EMEM) alone or supplemented with the Sigma receptor antagonists rimcazole (3 microM) and BD1047 (10 microM). Cell growth was monitored by phase microscopy. Tyrosine incorporation was quantified by culturing in the presence of 14-C tyrosine for 24 hours. At the end of the culture period, some bags were fixed in 4% paraformaldehyde for electron microscopy, and others were plunged into liquid nitrogen for later immunoblot and PCR analyses. Protein levels of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TYRP2) were quantified by Western blot analysis. The presence of pigment granules within epithelial cells were monitored by phase and electron microscopy techniques. The Sigma-1 receptor was expressed in native human lens cells and in cultured capsular bag cells. The Sigma receptor antagonists BD1047 and rimcazole inhibited lens cell growth and, surprisingly, lens cells accumulated pigment granules in the presence of the antagonists. The antagonists raised preexisting levels of TYR and TYRP1, whereas there was no change in TYRP2. The human lens normally expresses components of the melanin synthesis pathway, and this suggests a possible origin for the pigment granules that have been observed under certain conditions in the human lens. Exposure of lens cells to Sigma receptor antagonists leads to growth inhibition and pigment granule production.

Reevaluation of amino acid stimulation of protein synthesis in murine- and human-derived skeletal muscle cells assessed by independent techniques

Murine L6 and human rhabdomyosarcoma cells were cultured standardized in low (0.28 mM) and normal (9 mM) amino acid (AA) concentrations to reevaluate by independent methods to what extent AA activate initiation of protein synthesis. Methods used were incorporation of radioactive AA into proteins, distribution analysis of RNA in density gradient, and Western blots on initiation factors of translation of proteins in cultured cells as well as in vivo (gastrocnemius, C57Bl mice) during starvation/refeeding. Incorporation rate of AA gave incorrect results in a variety of conditions, where phenylalanine stimulated the incorporation rate of phenylalanine into proteins, but not of tyrosine, and tyrosine stimulated incorporation of tyrosine but not of phenylalanine. Similar problems were observed when [35S]methionine was used for labeling of fractionated cellular proteins. However, the methods entirely independent of labeled AA incorporation indicated that essential AA activate initiation of translation, whereas nonessential AA did not. Branched-chain AA and glutamine, in combination with some other AA, also stimulated initiation of translation. Starvation/refeeding in vitro agreed qualitatively with results in vivo evaluated by initiation factors. Insulin at physiological concentrations (100 microM/ml) did not stimulate global protein synthesis at low or normal AA concentrations but did so at supraphysiological levels (3 mU/ml), confirmed by independent methods. Our results reemphasize that labeled AA should be used with caution for quantification of protein synthesis, since the precursor pool(s) for protein synthesis is not in complete equilibrium with surrounding AA. "Flooding" tracee experiments did not overcome this problem.

TRNA-like structure regulates translation of Brome mosaic virus RNA.

For various groups of plant viruses, the genomic RNAs end with a tRNA-like structure (TLS) instead of the 3' poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLS(TYMV) of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3' TLS(BMV) (about 200 nucleotides) with determinants for tyrosylation. We discovered TLS(BMV)-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLS(BMV) tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLS(BMV) in trans. Intriguingly, a subdomain of the TLS(BMV) could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLS(BMV) during the BMV infection cycle.

Incorporation of 3-nitrotyrosine into the C-terminus of alpha-tubulin is reversible and not detrimental to dividing cells.

The C-terminus of the alpha-chain of tubulin is subject to reversible incorporation of tyrosine by tubulin tyrosine ligase and removal by tubulin carboxypeptidase. Thus, microtubules rich in either tyrosinated or detyrosinated tubulin can coexist in the cell. Substitution of the terminal tyrosine by 3-nitrotyrosine has been claimed to cause microtubule dysfunction and consequent injury of epithelial lung carcinoma A549 cells. Nitrotyrosine is formed in cells by nitration of tyrosine by nitric oxide-derived species. We studied properties of tubulin modified by in vitro nitrotyrosination at the C-terminus of the alpha-subunit, and the consequences for cell functioning. Nitrotyrosinated tubulin was a good substrate of tubulin carboxypeptidase, and showed a similar capability to assemble into microtubules in vitro to that of tyrosinated tubulin. Tubulin of C6 cells cultured in F12K medium in the presence of 500 micro m nitrotyrosine became fully nitrotyrosinated. This nitrotyrosination was shown to be reversible. No changes in morphology, proliferation, or viability were observed during cycles of nitrotyrosination, denitrotyrosination, and re-nitrotyrosination. Similar results were obtained with CHO, COS-7, HeLa, NIH-3T3, NIH-3T3(TTL-), and A549 cells. C6 and A549 cells were subjected to several passages during 45 days or more in the continuous presence of 500 micro m nitrotyrosine without noticeable alteration of morphology, viability, or proliferation. The microtubular networks visualized by immunofluorescence with antibodies to nitrotyrosinated and total tubulin were identical. Furthermore, nitrotyrosination of tubulin in COS cells did not alter the association of tubulin carboxypeptidase with microtubules. Our results demonstrate that substitution of C-terminal tyrosine by 3-nitrotyrosine has no detrimental effect on dividing cells.

Incorporation of caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor.

The nonsense codon suppression technique was used to incorporate o-nitrobenzyl cysteine or o-nitrobenzyl tyrosine (caged Cys or Tyr) into the 9' position of the M2 transmembrane segment of the gamma-subunit of the muscle nicotinic ACh receptor expressed in Xenopus oocytes. The caged amino acids replaced an endogenous Leu residue that has been implicated in channel gating. ACh-induced current increased substantially after ultraviolet (UV) irradiation to remove the caging group. This represents the first successful incorporation of caged Cys into a protein in vivo and the first incorporation of caged amino acids within a transmembrane segment of a membrane protein. The bulky nitrobenzyl group does not prevent the synthesis, assembly, or trafficking of the ACh receptor. When side chains were decaged using 1-ms UV light flashes, the channels with caged Cys or caged Tyr responded with strikingly different kinetics. The increase in current upon photolysis of caged Cys was too rapid for resolution by the voltage-clamp circuit [time constant (tau) <10 ms], whereas the increase in current upon photolysis of caged Tyr was dominated by a phase with tau approximately 500 ms. Apparently, the presence of a bulky o-nitrobenzyl Tyr residue distorts the receptor into an abnormal conformation. Upon release of the caging group, the receptor relaxes, with tau approximately 500 ms, into a conformation that allows the channel to open. Tyr at the 9' position of the gamma-subunit greatly increases the ability of ACh to block the channel by binding within the channel pore. This is manifested in two ways. 1) A "rebound," or increase in current, occurs upon removal of ACh from the bathing medium; and 2) at ACh concentrations >400 microM, inward currents are decreased through the mutated channel. The ability to incorporate caged amino acids into proteins should have widespread utility.

Incorporation of nitrotyrosine into α-tubulin by recombinant mammalian tubulin-tyrosine ligase

Tubulin-tyrosine ligase (TTL, EC 6.3.2.25) from porcine brain, which catalyses the readdition of tyrosine to the C-terminus of detyrosinated α-tubulin, was cloned and expressed in Escherichia coli as a glutathione S-transferase-fusion protein. Upon cleavage of the immobilised fusion protein, an electrophoretically homogeneous enzyme was obtained. Recombinant TTL, which exhibited similar catalytic properties as the mammalian enzyme purified from brain tissue, was capable of using nitrotyrosine as an alternative substrate in vitro. Incorporation of tyrosine into tubulin was competitively inhibited by nitrotyrosine with an apparent K i of 0.24 mM. The TTL-catalysed incorporation of nitrotyrosine as sole substrate into α-tubulin was clearly detectable at concentrations of 10 μM by immunological methods using nitrotyrosine specific antibodies. However, in competition with tyrosine 20-fold higher concentrations of nitrotyrosine were necessary before its incorporation became evident. Analysis of the C-terminal peptides of in vitro modified α-tubulin by MALDI-MS confirmed the covalent incorporation of nitrotyrosine into tubulin by TTL. In contrast to the C-terminal tyrosine, pancreatic carboxypeptidase A was incapable of cleaving nitrotyrosine from the modified α-tubulin.

EFFECTS OF CILIARY NEUROTROPHIC FACTOR (CNTF) ON PROTEIN TURNOVER IN CULTURED MUSCLE CELLS

The goal of the study was to evaluate the mechanism by which ciliary neurotrophic factor (CNTF) regulated protein metabolism in skeletal muscle. L8 myotubes were cultured and effects of various times and doses of CNTF on protein synthesis and degradation were evaluated. Effects of CNTF on turnover of specific pools of proteins (myofibrillar and non-myofibrillar) were also evaluated. Protein synthesis was assayed by incorporation of radioactive tyrosine into muscle proteins. Degradation was assessed by release of labelled tyrosine from pre-labelled myotubes. Effects of CNTF on protein turnover were found to be time- and dose-dependent. CNTF (1 and 10ng/ml) increased myofibrillar protein synthesis after 12h of exposure but had no effect on non-myofibrillar protein synthesis. Longer exposures of CNTF (24h) reduced non-myofibrillar protein synthesis and had no effect on myofibrillar protein synthesis. High concentrations of CNTF (10 and 20ng/ml) reduced myofibrillar protein degradation but had no effect on degradation of non-myofibrillar proteins. To evaluate the mechanism by which CNTF exerts control of protein turnover, we completed a Northern blot for CNTF receptor α-subunit (CNTFRα). This was non-detectable via conventional northern analysis. Use of RT-PCR, however, confirmed expression of CNTFRα, albeit at a low level compared to rat skeletal muscle. This low expression of the receptor in L8 myotubes may explain the limited effect of CNTF in vitro compared to the larger effects typically detected in vivo. CNTF regulated protein turnover through control of protein synthesis and degradation. Effects were dose and timedependent. These observations may explain ability of CNTF to exert both anabolic and catabolic actions in vivo.

Effects of serum deprivation, insulin and dexamethasone on polysome percentages in C2C12 myoblasts and differentiating myoblasts

An increase in the rate of protein synthesis in living cells can be achieved by regulating the quantity of mRNA, ribosomes, and enzymes available for translation or by regulating the efficiency at which existing components are used. Efficiency can be measured by comparing the number of ribosomes actively engaged in the synthesis of protein (polysomes) to the pool of free ribosomes. The objective of this study was to determine the percentage of ribosomes found as polysomes in C2C12 cells deprived of serum or exposed to insulin or dexamethasone 24 h before and after being stimulated to differentiate. Individual 60 mm culture dishes were exposed to serum-free control medium, medium containing serum, insulin, or dexamethasone for a period of 1 h or 2 h and then quickly frozen. The ribosomes and polysomes from these cells were separated by ultracentrifugation on 15 to 60% sucrose gradients and the absorbance across the gradient at 254 nm was recorded. Polysome percentages were determined as the area under the polysome peak divided by the total area under the curve. Serum deprivation caused a 12% decline in the percentage of ribosomes found as polysomes (P< 0.01). Dexamethasone caused a quadratic decline (P< 0.05) in polysome percentage, while insulin yielded a quadratic increase (P< 0.05). Protein synthesis assays measuring3H-tyrosine uptake showed similar responses. These changes occurred in the absence of any differences in total RNA concentration. It was concluded that differentiation and the absence of serum in the media reduced the rate of recruitment of ribosomes for protein synthesis. Insulin increased ribosome recruitment which was also observed by a similar increase in incorporation of radio-labeled tyrosine.

Schistosoma mansoni: females enhance [14C]-tyrosine incorporation in males maintained in vitro.

Incorporation of radiolabelled tyrosine was examined in males of Schistosoma mansoni maintained in vitro. Adult worms were recovered from mice and only those that were paired were used. Males and females were separated and placed in 4 degrees C RPMI 1640 containing 10% fetal calf serum (FCS). Individual males were incubated for 1 h either alone or with a female. Other males were incubated in a medium containing 40 ng ml-1 recombinant tumour necrosis factor alpha (rTNF alpha). Subsequently, males were incubated with or without females for 1 h in a medium containing 20 muCi ml-1 [14C]-tyrosine. All incubations were carried out in RPMI 1640 containing 10% FCS at 37 degrees C under 5% CO2. Untreated single males incorporated small amounts of labelled tyrosine. Pretreatment of single males with rTNF alpha did not affect their tyrosine incorporation (P = 0.693). However, males maintained in vitro with females incorporated 221% more tyrosine than the controls (P = 0.00008), suggesting that a chemical stimulus from the female affects tyrosine incorporation in males.

Adsorption dynamics of l-glutamic acid copolymers at a heptane/water interface

Random copolymers of glutamic acid (glu–ala, glu–leu, glu–phe, glu–tyr) were employed to investigate the relationship between side chain structure and peptide charge on adsorption behavior at an oil/water boundary. Adsorption of a series of glutamate copolymers at a heptane/water interface was examined by the dynamic pendant-drop method to determine interfacial tension. Incorporation of leucine or phenylalanine into a glutamate copolymer results in greater tension reduction than incorporation of alanine or tyrosine. These effects are amplified at pH values near the isoelectric point of glutamate, where macroscopic adsorbed films of glu–leu and glu–phe exhibit gel-like properties in response to interfacial area compression. Differences in interfacial tension behavior of glu–tyr and glu–phe indicate the importance of the tyrosine p-hydroxyl group on adsorption and aggregation at the oil/water interface.

Tubulin-tyrosine ligase catalyzes covalent binding of 3-fluoro-tyrosine to tubulin: kinetic and [ 19F]NMR studies

The use of 3-fluoro-tyrosine as an alternative substrate for the enzyme tubulin:tyrosine ligase which catalyzes the incorporation of tyrosine into the α-tubulin subunit was investigated. The incorporation of tyrosine into tubulin was inhibited competitively by 3-fluoro-tyrosine with an apparent K i of ∼ 25 μM. The affinity for this analog was similar to that of tyrosine, confirming that the hydrogen at position 3 of the aromatic ring is not essential for the reaction catalyzed by TTLase. The incorporation of 3-fluoro-tyrosine into the C-terminus of the α-turbulin subunit was demonstrated through [ 19F]NMR spectroscopy. The 3-fluoro-tyrosine signal at −58.6 ppm (trifluoroacetic acid as external standard), with a bandwidth of 24.7 Hz presented a chemical shift of 0.75 ppm upfield and an enlargement in the bandwidth (30.5 Hz) when incorporated into tubulin. These results strongly suggest that this amino acid is exposed to the solvent in tubulin. Tubulin covalently labeled with 3-fluoro-tyrosine was competent to polymerize into microtubules. The use of fluorinated tubulin in [ 19F]NMR spectroscopy for studying questions concerning protein conformation and interactions will be discussed.

Insulin-like growth factor-1 and insulin resistance in skeletal muscles of adult and old rats.

A study was designed to compare the effects, in vitro, of insulin-like growth factor-1 (IGF-1) and insulin on rat epitrochlearis muscle metabolism during aging (1, 6-8, or 18-20 months). Our results showed that in young epitrochlearis, IGF-1 was equipotent to insulin in stimulating 2-deoxy-glucose and aminoisobutyric acid transport but more potent in increasing tyrosine incorporation into protein. Both insulin and IGF-1 action on glucose transport was decreased in adult compared with young muscle. Whereas an insulin resistance of amino acid transport and protein synthesis was also recorded in adult rat muscle, the stimulatory effect of IGF-1 on these processes was abolished. Thus the degree of resistance observed varied both with the agonist and with the subsequent metabolic process observed. Whereas modifications of IGF-1 action in mature animals may be correlated in part to the dramatic decrease of IGF-1 receptors (80%), no similar observations were recorded for the insulin receptor. Since muscle IGF-1 receptor gene expression did not decrease in parallel with receptor number, an alteration in IGF-1 receptor messenger RNA (mRNA) translation or receptor degradation may be hypothetized. We concluded that: 1) In contrast to glucose transport, intracellular IGF-1 and insulin postreceptor pathways leading to amino acid uptake and protein metabolism differ. 2) Modification in postbinding events might be involved in decreased insulin- and IGF-1-stimulated muscle metabolism during aging.

Biosynthesis of the pseudobactin chromophore from tyrosine

Biosynthetic studies have shown the incorporation of DL-[2,3,3- 2H 3]-tyrosine into the chromophore of pseudobactin, a siderophore produced by Pseudomonas flourescens B10. A subsequent feeding using DL-[2′,5′,6′- 2H 3]-3,4-dihydroxyphenylalanine showed no incorporation of the label, suggesting that oxidation of the aryl ring occurs after the incorporation of tyrosine. The 13C NMR assignments of pseudobactin are also reported herein.

Direct NMR evidence for the equivalent participation of L-phenylalanine and L-tyrosine in the biosynthesis of the intermolecular Diels–Alder type adducts of prenylchalcone and prenylated 2-arylbenzofuran in Morus alba cell cultures

L-[3-13C]Phenylalanine and L-[3-13C]tyrosine were administered to Morus alba cell cultures, to produce intermolecular Diels–Alder type adducts of a prenylchalcone and a 2-arylbenzofuran such as chalcomoracin (1) as well as the adducts of two molecules of prenylchalcones such as kuwanon J (2) in high yields. The 13C NMR spectra of 1 and 2 isolated from the cultures revealed that both amino acids were incorporated intact into chalcomoracin (1) and kuwanon J (2). This is the first example of direct NMR evidence for the almost equivalent incorporation of phenylalanine and tyrosine into the shikimate metabolites. This finding suggests the participation of a biosynthetic route from phenylalanine via trans-cinnamate to p-coumarate and from tyrosine to p-coumarate in this plant.