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PubMed1996

Characterization of a melanosomal transport system in murine melanocytes mediating entry of the melanogenic substrate tyrosine.

In this study, we identify a transport system for tyrosine, the initial precursor of melanin synthesis, in the melanosomes of murine melanocytes. Melanosomes preloaded with tyrosine demonstrated countertransport of 10 microM [3H]tyrosine, indicating carrier-mediated transport. Melanosomal tyrosine transport was saturable, with an apparent Km for tyrosine transport of 54 microM and a maximal velocity of 15 pmol of tyrosine/unit of hexosaminidase/min. Transport was temperature-dependent (Ea = 7.5 kcal/mol) and showed stereospecificity for the l-isomer of tyrosine. Aromatic, neutral hydrophobic compounds (such as tryptophan and phenylalanine), as well as the small, bulky neutral amino acids (such as leucine, isoleucine, and methionine) competed for tyrosine transport. Tyrosine transport was inhibited by the classical system L analogue, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and by monoiodotyrosine, but not by cystine, lysine, glutamic acid, or 2-(methylamino)-isobutyric acid. Tyrosine transport showed no dependence on Na+ or K+, and did not require an acidic environment or the availability of free thiols. These results demonstrate the existence of a neutral amino acid carrier in murine melanocyte melanosomes which resembles the rat thyroid FRTL-5 lysosomal system h. This transport system is critical to the function of the melanosome since tyrosine is the essential substrate required for the synthesis of the pigment melanin.

Amino Acids↗
PubMed1997

Human phospholipase D1 can be tyrosine-phosphorylated in HL-60 granulocytes.

The human phospholipase D1 (hPLD1) has recently been cloned. Although recent data have implicated PLD in receptor-stimulated secretion, the regulation of the activity of PLD enzymes remains to be clarified. Purified hPLD1 is activated by several cytosolic cofactors among which are protein kinase Calpha, ARF, and RhoA. In human granulocytes, a strong correlation between tyrosine phosphorylation of proteins and PLD activity has been established. In this study, the presence of hPLD1 in HL-60 granulocytes and its phosphorylation on tyrosine residues have been studied. We generated antipeptide antibodies (Abs) specific for hPLD1 but not PLD2 as shown by Western blotting (WB) of recombinant PLD1 and PLD2. These Abs identified the presence of hPLD1 in HL-60 cells with the bulk of it being detected in the membranes and only a minor fraction in the cytosol. The hPLD1 Abs detected a major band at 120 kDa (PLD1a) and a minor band at 115 kDa (PLD1b). The specificity of the Abs was confirmed using PLD antisera neutralized with the immunizing peptides. The two forms of hPLD1 were consistently detected by immunoprecipitation under nondenaturing and denaturing conditions following a WB analysis with hPLD1 Abs. Following exposure of HL-60 cells to peroxides of vanadate (V4+-OOH), an inhibitor of tyrosine phosphatases, hPLD1 was immunoprecipitated under nondenaturing conditions from HL-60 cell lysates and assayed for tyrosine phosphorylation by WB. hPLD1 comigrated with a 120-kDa tyrosine phosphorylated protein by gel electrophoresis. Other tyrosine-phosphorylated peptides of 160, 140, 135, 90, and 75-80 kDa were also detected in hPLD1 immune complexes. hPLD1 and the associated tyrosine-phosphorylated proteins were not immunoprecipitated by neutralized hPLD1 Abs. Using denaturing conditions, the PLD immunoprecipitates were sequentially immunoblotted with anti-PLD1 and anti-phosphotyrosine Abs. PLD1a and PLD1b were detected, and the major PLD1a protein was superimposable with a major tyrosine-phosphorylated protein detected at 120 kDa. Conversely, PLD1a and PLD1b were recovered, at least in part, in the anti-phosphotyrosine immunoprecipitates. These results provide evidence that two PLD1 forms are expressed in human granulocytes. Furthermore, in response to stimulation by V4+-OOH, PLD1 was tyrosine-phosphorylated and associated with several, presently undefined, tyrosine-phosphorylated proteins.

Amino Acid Sequence↗
PubMed1999

Annexin VII and annexin XI are tyrosine phosphorylated in peroxovanadate-treated dogs and in platelet-derived growth factor-treated rat vascular smooth muscle cells.

The intraperitoneal administration of peroxovanadate results in the rapid accumulation of many tyrosine-phosphorylated proteins in the liver and kidney of treated animals. The availability of large pools of tyrosine-phosphorylated proteins derived from normal tissues facilitates the purification and identification of previously unknown targets for cellular tyrosine kinases. Using this procedure, we have thus far identified four proteins in the liver and kidney of peroxovanadate-treated dogs. Two of these, annexin VII and annexin XI, were novel and had not been previously reported to be substrates of tyrosine kinases while the remaining two, ezrin and clathrin, have been reported to be tyrosine phosphorylated in some cell culture systems. In the present study, isolated proteins were identified both by sequence analysis and immunological methods. Annexin VII and annexin XI are present in cultured rat vascular smooth muscle cells and both were tyrosine phosphorylated in response to a physiological ligand, platelet-derived growth factor-BB (PDGF-BB). Furthermore, the extent of tyrosine phosphorylation in response to PDGF-BB was augmented by the co-addition of peroxovanadate to cell cultures. In vitro phosphorylation assays showed that PDGF receptor, calcium-dependent tyrosine kinase (CADTK/Pyk-2), Src kinase, and epidermal growth factor receptor all were able to phosphorylate purified annexin VII and XI on tyrosine residues. These findings confirm the usefulness of phosphatase inhibition by peroxovanadate as a tool for identifying previously unknown physiological targets for cellular protein tyrosine kinases.

Amino Acid Sequence↗
PubMed2002

Protein kinase C-alpha and protein kinase C-epsilon are required for Grb2-associated binder-1 tyrosine phosphorylation in response to platelet-derived growth factor.

Grb2-associated binder-1 (Gab1) is an adapter protein related to the insulin receptor substrate family. It is a substrate for the insulin receptor as well as the epidermal growth factor (EGF) receptor and other receptor-tyrosine kinases. To investigate the role of Gab1 in signaling pathways downstream of growth factor receptors, we stimulated rat aortic vascular smooth muscle cells (VSMC) with EGF and platelet-derived growth factor (PDGF). Gab1 was tyrosine-phosphorylated by EGF and PDGF within 1 min. AG1478 (an EGF receptor kinase-specific inhibitor) failed to block PDGF-induced Gab1 tyrosine phosphorylation, suggesting that transactivated EGF receptor is not responsible for this signaling event. Because Gab1 associates with phospholipase Cgamma (PLCgamma), we studied the role of the PLCgamma pathway in Gab1 tyrosine phosphorylation. Gab1 tyrosine phosphorylation by PDGF was impaired in Chinese hamster ovary cells expressing mutant PDGFbeta receptor (Y977F/Y989F: lacking the binding site for PLCgamma). Pretreatment of VSMC with (a specific PLCgamma inhibitor) inhibited Gab1 tyrosine phosphorylation as well, indicating the importance of the PLCgamma pathway. Gab1 was tyrosine-phosphorylated by phorbol ester to the same extent as PDGF stimulation. Studies using antisense protein kinase C (PKC) oligonucleotides and specific inhibitors showed that PKCalpha and PKCepsilon are required for Gab1 tyrosine phosphorylation. Binding of Gab1 to the protein-tyrosine phosphatase SHP2 and phosphatidylinositol 3-kinase was significantly decreased by PLCgamma and/or PKC inhibition, suggesting the importance of the PLCgamma/PKC-dependent Gab1 tyrosine phosphorylation for the interaction with other signaling molecules. Because PDGF-mediated ERK activation is enhanced in Chinese hamster ovary cells that overexpress Gab1, Gab1 serves as an important link between PKC and ERK activation by PDGFbeta receptors in VSMC.

Animals↗
PubMed2006

Detection and purification of tyrosine-sulfated proteins using a novel anti-sulfotyrosine monoclonal antibody.

Protein tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST1 and TPST2) that catalyze the transfer of sulfate to tyrosine residues in secreted and transmembrane proteins. Tyrosine sulfation plays a role in protein-protein interactions in several well defined systems. Although dozens of tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of tyrosine sulfation in biological systems requires the development of new tools for the detection and study of tyrosine-sulfated proteins. We have developed a novel anti-sulfotyrosine monoclonal antibody (called PSG2) that binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independently of sequence context. We show that it can detect tyrosine-sulfated proteins in complex biological samples and can be used as a probe to assess the role of tyrosine sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 showed that certain sperm/epididymal proteins are undersulfated in Tpst2(-/-) mice. This indicates that TPST1 and TPST2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2(-/-) males. PSG2 should be widely applicable for identification of tyrosine-sulfated proteins in other systems and organisms.

Animals↗
PubMed2000

Tyrosine nitration by peroxynitrite formed from nitric oxide and superoxide generated by xanthine oxidase.

Peroxynitrite (ONOO(-)) is a potent nitrating and oxidizing agent that is formed by a rapid reaction of nitric oxide (NO) with superoxide anion (O(2)). It appears to be involved in the pathophysiology of many inflammatory and neurodegenerative diseases. It has recently been reported (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) that ONOO(-) generated at neutral pH from NO and O(2) (NO/O(2)) was substantially less efficient than preformed ONOO(-) at nitrating tyrosine. Here we re-evaluated tyrosine nitration by NO/O(2) with a shorter incubation period and a more sensitive electrochemical detection system. Appreciable amounts of nitrotyrosine were produced by ONOO(-) formed in situ (2.9 micrometer for 5 min; 10 nm/s) by NO/O(2) flux obtained from propylamine NONOate (CH(3)N[N(O)NO](-) (CH(2))(3)NH(2)(+)CH(3)) and xanthine oxidase using pterin as a substrate in phosphate buffer (pH 7.0) containing 0.1 mm l-tyrosine. The yield of nitrotyrosine by this NO/O(2) flux was approximately 70% of that produced by the same flux of preformed ONOO(-) (2.9 micrometer/5 min). When hypoxanthine was used as a substrate, tyrosine nitration by NO/O(2) was largely eliminated because of the inhibitory effect of uric acid produced during the oxidation of hypoxanthine. Tyrosine nitration caused by NO/O(2) was inhibited by the ONOO(-) scavenger ebselen and was enhanced 2-fold by NaHCO(3), as would be expected, because CO(2) promotes tyrosine nitration. The profile of nitrotyrosine and dityrosine formation produced by NO/O(2) flux (2.9 micrometer/5 min) was consistent with that produced by preformed ONOO(-). Tyrosine nitration predominated compared with dityrosine formation caused by a low nanomolar flux of ONOO(-) at physiological concentrations of free tyrosine (<0.5 mm). In conclusion, our results show that NO generated with O(2) nitrates tyrosine with a reactivity and efficacy similar to those of chemically synthesized ONOO(-), indicating that ONOO(-) can be a significant source of tyrosine nitration in physiological and pathological events in vivo.

Humans↗
PubMed1998

Tyrosine phosphorylation and src family kinases control keratinocyte cell-cell adhesion.

In their progression from the basal to upper differentiated layers of the epidermis, keratinocytes undergo significant structural changes, including establishment of close intercellular contacts. An important but so far unexplored question is how these early structural events are related to the biochemical pathways that trigger differentiation. We show here that beta-catenin, gamma-catenin/plakoglobin, and p120-Cas are all significantly tyrosine phosphorylated in primary mouse keratinocytes induced to differentiate by calcium, with a time course similar to that of cell junction formation. Together with these changes, there is an increased association of alpha-catenin and p120-Cas with E-cadherin, which is prevented by tyrosine kinase inhibition. Treatment of E-cadherin complexes with tyrosine-specific phosphatase reveals that the strength of alpha-catenin association is directly dependent on tyrosine phosphorylation. In parallel with the biochemical effects, tyrosine kinase inhibition suppresses formation of cell adhesive structures, and causes a significant reduction in adhesive strength of differentiating keratinocytes. The Fyn tyrosine kinase colocalizes with E-cadherin at the cell membrane in calcium-treated keratinocytes. Consistent with an involvement of this kinase, fyn-deficient keratinocytes have strongly decreased tyrosine phosphorylation levels of beta- and gamma-catenins and p120-Cas, and structural and functional abnormalities in cell adhesion similar to those caused by tyrosine kinase inhibitors. Whereas skin of fyn-/- mice appears normal, skin of mice with a disruption in both the fyn and src genes shows intrinsically reduced tyrosine phosphorylation of beta-catenin, strongly decreased p120-Cas levels, and important structural changes consistent with impaired keratinocyte cell adhesion. Thus, unlike what has been proposed for oncogene-transformed or mitogenically stimulated cells, in differentiating keratinocytes tyrosine phosphorylation plays a positive role in control of cell adhesion, and this regulatory function appears to be important both in vitro and in vivo.

Animals↗
PubMed1994

Effects of tyrosine-->phenylalanine mutations on auto- and trans-phosphorylation reactions catalyzed by the insulin receptor beta-subunit cytoplasmic domain.

Activation of the insulin receptor kinase is closely associated with autophosphorylation of several tyrosine residues in the cytoplasmic domain of the receptor's two beta-subunits. To determine the contribution of these tyrosine phosphorylations to autoactivation of the receptor kinase, we have blocked phosphorylation at specific tyrosine by replacing these tyrosine residues, individually and in combination, with phenylalanine in a soluble 45-kD analog of the cytoplasmic insulin receptor kinase domain (CIRK). Kinetic studies of auto- and transphosphorylation with this panel of mutated CIRKs indicate that: (i) None of the tyrosines (953, 960, 1,146, 1,150, 1,151, 1,316, or 1,322) are necessary for catalysis: all single Y-->F mutants retain the ability to autoactivate comparable to the parent CIRK. (ii) Two of the tyrosine autophosphorylation sites, either tyrosine 1,150 or 1,151, contribute most (70-80%) of the autoactivation, because replacement of these two tyrosines by phenylalanine was the minimal change that abolishes autoactivation. (iii) A mutant CIRK having all seven reported tyrosine phosphorylation sites replaced by phenylalanine retained basal kinase activity but was incapable of autoactivation. These findings imply that autoactivation can occur without phosphorylation having occurred at any single site (953, 960, 1,146, 1,150, 1,151, 1,316, or 1,322), and autophosphorylation need not follow an ordered, sequential pathway beginning, for example, at tyrosine 1,146 as proposed for the intact insulin receptor.

Animals↗
PubMed1991

Temperature-dependent tyrosine phosphorylation of microtubule-associated protein kinase in epidermal growth factor-stimulated human fibroblasts.

Treatment of normal human fibroblasts with epidermal growth factor (EGF) results in the rapid (0.5 min) and simultaneous tyrosine phosphorylation of the EGF receptor (EGFr) and several other proteins. An exception to this tyrosine phosphorylation wave was a protein (42 kDa) that became phosphorylated on tyrosine only after a short lag time (5 min). We identified this p42 kDa substrate as the microtubule-associated protein (MAP) kinase using a monoclonal antibody to a peptide corresponding to the C-terminus of the predicted protein (Science 249, 64-67, 1990). EGF treatment of human fibroblasts at 37 degrees C for 5 min resulted in the tyrosine phosphorylation of 60-70% of MAP kinase as determined by the percent that was immunoprecipitated with antiphosphotyrosine antibodies. Like other tyrosine kinase growth factor receptors, the EGFr is activated and phosphorylated at 4 degrees C but is not internalized. Whereas most other substrates were readily tyrosine phosphorylated at 4 degrees C, MAP kinase was not. When cells were first stimulated with EGF at 4 degrees C and then warmed to 37 degrees C without EGF, tyrosine phosphorylation of MAP kinase was again observed. Treatment of cells with the protein kinase C activator phorbol myristate acetate (PMA) also resulted in the tyrosine phosphorylation of MAP kinase, and again only at 37 degrees C. Tryptic phosphopeptide maps demonstrated that EGF and PMA both induced the phosphorylation of the same peptide on tyrosine and threonine. This temperature and PMA sensitivity distinguishes MAP kinase from most other tyrosine kinase substrates in activated human fibroblasts.

Amino Acid Sequence↗
PubMed1997

Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3'5'-monophosphate-dependent pathway.

Mammalian sperm capacitation, defined as an obligatory maturational process leading to the development of the fertilization-competent state, results from a poorly understood series of morphological and molecular events. We report here that ejaculated bovine sperm, incubated under conditions that support capacitation in vitro, display a reproducible pattern of protein tyrosine phosphorylations that are regulated by a cAMP-dependent pathway. The appearance of these tyrosine phosphorylated proteins correlated temporally with the time course of capacitation induced by heparin, and these phosphorylations displayed a similar heparin concentration dependence. Glucose, which inhibits capacitation, inhibited these protein tyrosine phosphorylations in media containing heparin. The biologically active cAMP analogues (dibutyryl cAMP [db-cAMP], 8-bromo cAMP, sp-cAMPS) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) induced the same protein tyrosine phosphorylation patterns as seen with heparin. Moreover, these cAMP agonists could overcome the inhibition of the heparin-induced tyrosine phosphorylations by glucose. In contrast, Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS), a protein kinase A (PK-A) antagonist, blocked the capacitation-associated increases in protein tyrosine phosphorylation. This cAMP regulation of the protein tyrosine phosphorylation pattern is mediated by PK-A since N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide-dihydrochloride (H89), another inhibitor of PK-A, inhibited the heparin-induced protein tyrosine phosphorylation pattern in a concentration-dependent manner in either the absence or presence of db-cAMP, IBMX, and glucose. These data support a model for sperm capacitation that includes protein tyrosine phosphorylation as an important regulatory pathway, and a role for cAMP/PK-A in the regulation of this pathway leading to capacitation. These studies are the first to report a unique interrelationship between tyrosine kinase/phosphatase and cAMP signaling pathways at the level of PK-A in bovine sperm capacitation.

1-Methyl-3-isobutylxanthine↗
PubMed2003

Role of constitutively activated protein tyrosine kinases in malignant myeloproliferative disorders: an update.

Modern molecular technology helped identify more than 10 protein tyrosine kinases related to myeloid malignancies, which allowed the development of small molecule inhibitors targeting deregulated protein tyrosine kinase activity. Protein tyrosine kinase deregulation can occur as a consequence of fusion gene formation because of chromosomal translocations, or as distinct gain-of-function point mutations. Although the tyrosine kinase inhibitor imatinib mesylate (Gleevec) targeting the ABL protein tyrosine kinase has revolutionized current chronic myeloid leukemia therapy, it became rapidly evident that overcoming the multiple cellular resistance mechanisms will be very challenging. To develop efficient therapeutic alternatives, one must understand the complex signal transduction mechanisms involved in transformation by deregulated protein tyrosine kinases. This article reviews the most recently identified molecular mechanisms involved in cell transformation by the BCR/ABL protein tyrosine kinase fusion and presents new members of the increasing family of deregulated protein tyrosine kinases involved in myeloproliferative disorders. In addition, the article discusses new, promising small molecule protein tyrosine kinase inhibitors and the molecular mechanism that may lead to resistance to these drugs. Finally, the article highlights putative alternative strategies that could be used to block signal transduction pathways of deregulated protein tyrosine kinase activity.

Cell Transformation, Neoplastic↗
PubMed1996

Phosphorylation of each of the distal three tyrosines of the CD28 cytoplasmic tail is required for CD28-induced T cell IL-2 secretion.

Signaling by the CD28 T cell costimulatory receptor is known to involve recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase) which is dependent upon phosphorylation of tyrosine 173 of the CD28 cytoplasmic tail, present in a YMNM motif. However, whether this phosphorylation is required for CD28 costimulation and whether or not phosphorylation of any of the other three tyrosines of the CD28 cytoplasmic tail (tyrosines 188, 191 and 200) is also important for CD28 induced responses is unclear. To address this we examined the ability of chimeric receptors, consisting of the extracellular plus transmembrane membrane domain of human CD8 alpha linked to different mutated human CD28 cytoplasmic tails, to induce IL-2 secretion in Jurkat T leukemia cells in the presence of PMA and ionomycin. A receptor in which tyrosine 173 of the CD28 tail was mutated to phenylalanine was able to induce IL-2. By contrast, receptors which contained single tyrosine 188, 191 or 200 to phenylalanine substitutions were unable to induce IL-2. These results imply that in this system phosphorylation of tyrosine 173 and hence activation of PI3-kinase is not required for CD28 induced IL-2 secretion. Further, they imply that phosphorylation of each of tyrosines 188, 191 and 200 is necessary for this response. Despite an apparent requirement for phosphorylation of all three of these tyrosines, however, receptors which contain tyrosine only at positions 191 or 200 and a truncated receptor which does not contain tyrosine 200 induce normal IL-2. These last findings, therefore, illustrate the complexity of CD28 mediated activation signals.

CD28 Antigens↗
PubMed1989

Sulfation of two tyrosine-residues in human complement S-protein (vitronectin).

Human S-protein (vitronectin) and hemopexin, two structurally related plasma proteins of similar molecular mass and abundance, were analyzed for tyrosine sulfation. Both proteins were synthesized and secreted by the human hepatoma-derived cell line Hep G2, as shown by immunoprecipitation from the culture medium of [35S]methionine-labelled cells. When Hep G2 cells were labelled with [35S]sulfate, S-protein, but not hemopexin, was found to be sulfated. Half of the [35S]sulfate incorporated into S-protein was recovered as tyrosine sulfate. The stoichiometry of tyrosine sulfation was approximately two mol tyrosine sulfate/mol S-protein. Examination of the S-protein sequence for the presence of the known consensus features for tyrosine sulfation revealed three potential sulfation sites at positions 56, 59 and 401. Tyrosine 56 is the most probable site for stoichiometric sulfation, followed by tyrosine 59 which appears more likely to become sulfated than tyrosine 401. Tyrosines 56 and 59 are located in the anionic region of S-protein which has no homologous counterpart in hemopexin. We discuss the possibility that tyrosine sulfation of the anionic region of S-protein may stabilize the conformation of S-protein in the absence of thrombin-antithrombin III complexes and may play a role in its binding to thrombin-antithrombin III complexes during coagulation.

Amino Acid Sequence↗
PubMed1996

Enzymatic and non-enzymatic oxygenation of tyrosine.

Tyrosinase isolated from cultured human melanoma cells was studied for tyrosine oxygenation activity. L-Tyrosine and D-tyrosine were used as substrates and dopa was measured with HPLC and electrochemical detection as the product of oxygenation. Incubations were performed in the presence or absence of dopamine as co-substrate. Oxygenation of L-tyrosine occurred only in the presence of dopamine as co-substrate. No oxygenation of D-tyrosine was found, and we conclude that human tyrosinase is characterised by exclusive specificity for the L-isomer of tyrosine in its oxygenase function. It has recently been suggested that superoxide anion is a preferential oxygen substrate for human tyrosinase. Incubations were therefore performed with L- and D-tyrosine, human tyrosine, and xanthine/xanthine oxidase in the system, generating superoxide anion and hydrogen peroxide. Considerable formation of dopa was observed, but the quantity was the same irrespective of whether D-tyrosine or L-tyrosine was used as the substrate. Furthermore, formation of dopa occurred in a xanthine/xanthine oxidase system when bovine serum albumin (BSA) was substituted for tyrosinase. Our results provide no evidence that superoxide anion is an oxygen substrate for human tyrosinase. In the incubate containing xanthine/xanthine oxidase, catalase completely inhibited dopa formation, and superoxide dismutase and mannitol each strongly inhibited dopa formation. The results are compatible with hydroxyl radicals being responsible for the formation of dopa, since such radicals may be secondarily formed in the presence of superoxide anion and hydrogen peroxide.

Animals↗
PubMed1990

Human placental L-tyrosine transport: a comparison of brush-border and basal membrane vesicles.

1. The mechanisms responsible for L-tyrosine transport at both the maternal-facing and fetal-facing surfaces of the human full-term placenta have been studied using isolated brush-border and basal membrane vesicles under conditions where a direct comparison of the transport properties of the two membranes can be made. 2. Brush-border vesicle uptake of L-tyrosine was substantially into an osmotically active space. Transport was Na(+)-independent, N-ethylmaleimide-sensitive (half-maximal inhibition, Ki = 1.1 mM), and insensitive to pH over the range 5.5-8.5. The initial rate of brush-border L-tyrosine uptake as a function of concentration showed saturation and obeyed Michaelis-Menten kinetics with Michaelis constant (Km) and maximum velocity (Vmax) values of 54.2 microM and 1.28 pmol (mg protein)-1 s-1, respectively. Influx of L-tyrosine was stereospecific and was virtually completely abolished by L-phenylalanine, L-tryptophan, L-leucine or by 2-aminobicycloheptane-2-carboxylic acid. These properties suggest that system L is responsible for brush-border L-tyrosine transport. 3. Basal membrane transport of L-tyrosine was more complex and uptake was slower than that found in the brush border. Although, as in the brush-border membranes, uptake was completely Na(+)-independent, N-ethylmaleimide was a less effective inhibitor, there was stimulation of transport at more alkaline pH and uptake did not show marked stereospecificity. An apparent Km of 168.9 microM and a Vmax of 0.31 pmol (mg protein)-1 s-1 were calculated for basal L-tyrosine transport. There was clear inhibition by L- and D-tyrosine, L-phenylalanine and L-tryptophan. 2-Aminobicycloheptane-2-carboxylic acid was not as effective. 4. These findings suggest the existence of non-identical carrier-mediated transport systems for L-tyrosine in brush-border and basal membranes. Brush-border transport resembles that by system L; L-tyrosine transport at the basal membrane may be via system t.

Amino Acids↗
PubMed1980

Isoniazid interaction with tyrosine as a possible mode of action of the drug in mycobacteria.

The antitubercular drug isoniazid (INH) was hown by radio-chromatographic studies to react with tyrosine in growth medium. Exogenous tyrosine added to the growth medium interfered with the inhibitory action of INH on Mycobacterium phlei. These observations were confirmed by difference spectra studies which showed that tyrosine would react with INH as long as the tyrosine phenolic hydroxyl group was not blocked. These results led to the hypothesis that INH could exert its influence by interfering with tyrosine residues in mycobacterial proteins. N-acetylimidazole, a tyrosine-acetylating agent, mimicked the action of INH on the reduced nicotinamide adenine dinucleotide oxidase and dehydrogenase activity in electron transport particles from wild-type and INH-resistant M. phlei. Pyrazinamide, a drug structurally related to INH, also mimicked its effect on electron transport particles. To confirm that INH could react with tyrosine in proteins, purified enzymes with known tyrosine positions were tested. Bovine carboxypeptidase A with tyrosine at the active site was inhibited by INH and N-acetylimidazole, whereas the controls, yeast alcohol dehydrogenase and ribonuclease A, were not. It is therefore proposed that tyrosine residues in proteins may serve as the target for INH action in mycobacteria.

Culture Media↗
PubMed1973

Specificity of the tyrosine-phenylalanine transport system in Bacillus subtilis.

l-Tyrosine and l-phenylalanine enter cells of Bacillus subtilis via a system of active transport that exhibits complex kinetic behavior. The specificity of the transport system was characterized both at low concentrations of transport substrate (where affinity for l-tyrosine or l-phenylalanine is high but capacity is low) and at high concentrations (where affinity is low but capacity is high). Specificity was not found to differ significantly as a function of either l-tyrosine or l-phenylalanine concentration. Kinetic analysis showed that the relationship between the uptake of l-phenylalanine and l-tyrosine is strictly competitive. Neither l-tyrosine nor l-phenylalanine uptake was competitively inhibited by other naturally occurring l-amino acids, indicating the importance of the phenyl side chain to uptake specificity. Hence, it is concluded that l-tyrosine and l-phenylalanine are transported by a common system that is specific for these two amino acids. The abilities of analogue derivatives of l-tyrosine and l-phenylalanine to inhibit the uptake of l-[(14)C]tyrosine and l-[(14)C]phenylalanine competitively were determined throughout a wide range of substrate and inhibitor concentrations. In this manner, the contributions of the side chain, the alpha-amino group and the carboxyl group to uptake specificity were established. It is concluded that the positively charged alpha-amino group contributes more significantly to uptake specificity than does the negatively charged carboxyl group. The recognition of a phenyl ring is an essential feature of specificity; other amino acids with aromatic side chains, such as the indole and imidazole rings of l-tryptophan and l-histidine, do not compete with l-tyrosine and l-phenylalanine for uptake. The presence of the p-hydroxy substitutent in the side chain (as in l-tyrosine) enhances the uptake of the aryl amino acid analogues investigated.

Amino Acids↗
PubMed1997

Tyrosine phosphorylation events during coxsackievirus B3 replication.

In order to study cellular and viral determinants of pathogenicity, interactions between coxsackievirus B3 (CVB3) replication and cellular protein tyrosine phosphorylation were investigated. During CVB3 infection of HeLa cells, distinct proteins become phosphorylated on tyrosine residues, as detected by the use of antiphosphotyrosine Western blotting. Two proteins of 48 and 200 kDa showed enhanced tyrosine phosphorylation 4 to 5 h postinfection (p.i.), although virus-induced inhibition of cellular protein synthesis had already occurred 3 to 4 h p.i. Subcellular fractionation experiments revealed distinct localization of tyrosine-phosphorylated proteins of 48 and 200 kDa in the cytosol and membrane fractions of infected cells, respectively. In addition, in Vero cells infected with CVB3, echovirus (EV)11, or EV12, increased tyrosine phosphorylation of a 200-kDa protein was detected 6 h p.i. Herbimycin A, a specific inhibitor of Src-like protein tyrosine kinases, was shown to inhibit virus-induced tyrosine phosphorylations and to reduce the production of progeny virions. In contrast, in cells treated with the inhibitors staurosporine and calphostin C, the synthesis of progeny virions was not affected. Immunoprecipitation experiments suggested that the tyrosine-phosphorylated 200-kDa protein in CVB3-infected cells is of cellular origin. In summary, these investigations have begun to unravel the effect of CVB3 as well as EV11 and EV12 replication on cellular tyrosine phosphorylation and support the importance of tyrosine phosphorylation events for effective virus replication. Such cellular phosphorylation events triggered in the course of enterovirus infection may enhance virus replication.

Animals↗