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PubMed2003

Individual tyrosine side-chain contributions to circular dichroism of ribonuclease.

Experimental and theoretical studies using site-directed mutants of ribonuclease A (RNase A) offer more extensive information on the tyrosine side-chain contributions to the circular dichroism (CD) of the enzyme. Bovine pancreatic RNase A has three exposed tyrosine residues (Tyr73, Tyr76, and Tyr115) and three buried tyrosine residues (Tyr25, Tyr92 and Tyr97). The difference CD spectra between the wild type and the mutants at pH 7.0 (Deltaepsilon(277,wt) - Deltaepsilon(277,mut)) show bands with more negative DeltaDeltaepsilon(277) values for Y73F and Y115F than those for Y25F and Y92F and bands with positive DeltaDeltaepsilon(277) values for Y76F and Y97F. The theoretical calculations are in good semiquantitative agreement for all the mutants. The pH difference spectrum (pH 11.3-7.0) for the wild type shows a negative band at 295 nm and an enhanced positive band at 245 nm. The three mutants at buried tyrosine sites and one mutant at an exposed tyrosine site (Y76F) exhibit pH-difference spectra that are similar to that of the wild type. In contrast, two mutants at exposed tyrosine sites (Y73F and Y115F) exhibit diminished 295-nm negative bands and, instead of positive bands at 245 nm, negative bands are observed. Our results indicate that Tyr73 and Tyr115, two of the exposed tyrosine residues, are the largest contributors to the 277- and 245-nm CD bands of RNaseA, but the buried tyrosine residues and the one remaining exposed residue also contribute to these bands. Disulfide contributions to the 277- and 240-nm bands and the peptide contribution to the 240-nm band are confirmed theoretically.

Animals↗

PubMed1993

Localization of tyrosine hydroxylase in neuronal targets and efferents of the area postrema in the nucleus tractus solitarii of the rat.

Catecholamines in the nucleus tractus solitarii (NTS) have been implicated in autonomic responses to circulating hormones that act on neurons in the area postrema, the most caudal circumventricular organ in brain. We combined immunoperoxidase labeling of the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHAL) with immunogold-silver labeling of tyrosine hydroxylase to determine whether this enzymatic marker for catecholamines was present in efferents from the area postrema or their targets in the rat NTS. At survival periods of 10-12 days after PHAL injections into the area postrema, light microscopy revealed numerous varicose processes containing peroxidase reaction product for PHAL in the dorsomedial, medial, and commissural NTS. Some of these labeled processes were located near neuronal perikarya and processes containing immunogold-silver intensified reaction product for tyrosine hydroxylase. Electron microscopy of the commissural and dorsomedial NTS established that the majority of the labeling for PHAL was in axon terminals, whereas immunogold labeling for tyrosine hydroxylase was mainly in soma and dendrites. Only 3 out of 579 PHAL-labeled terminals also contained detectable tyrosine hydroxylase immunoreactivity. Fifty-eight percent (335/579) of the PHAL-labeled terminals formed synapses with recognized symmetric junctions, whereas the remainder lacked synaptic specializations within the examined series of serial sections. Of those PHAL terminals forming recognized symmetric junctions, 22% were on tyrosine hydroxylase-immunoreactive dendrites, 74% on unlabeled dendrites and 4% on unlabeled axon terminals. From a total of 1,250 observed contacts on tyrosine hydroxylase labeled dendrites, 88 (7%) contained PHAL, 9 (< 1%) contained TH, and 1,180 (93%) lacked detectable immunoreactivity and formed primarily symmetric synapses. We conclude that a few catecholamine, but mainly noncatecholamine efferents from the area postrema provide a monosynaptic, and most likely inhibitory input to target neurons both with and without tyrosine hydroxylase immunoreactivity in the dorsomedial and commissural NTS. Synapses between the efferent terminals from the area postrema and tyrosine hydroxylase labeled and unlabeled dendrites as well as unlabeled axons in these specific subnuclei of the NTS suggest multiple sites for modulation of gastric and cardiovascular reflexes in response to circulating peptides.

Animals↗

PubMed1995

Dual mechanism of protein-tyrosine phosphorylation in concanavalin A-stimulated platelets.

Treatment of human platelets with the lectin Concanavalin A (Con A) resulted in the tyrosine phosphorylation of several proteins with molecular masses 65, 80, 85, 95, 120, 135, and 150 kDa. These proteins were divided in two groups: the first group included the 65-, 85-, 95-, and 120-kDa bands, which were tyrosine phosphorylated also in thrombin-stimulated platelets; the second group (80-, 135-, and 150-kDa bands) included proteins whose tyrosine phosphorylation was exclusively promoted by Con A, but not by thrombin. Members of the second group were rapidly dephosphorylated when the lectin was displaced from the cell surface by methyl alpha-D-mannopyranoside. Pretreatment of intact platelets with the prostacyclin analog iloprost, inhibited Con A-induced tyrosine phosphorylation of the first group of proteins, but had no effect on the tyrosine phosphorylation of the proteins of the second group. Succinyl-Con A, a dimeric derivative of the lectin, which binds to the platelet surface but does not promote clustering of the receptor, did not induce tyrosine phosphorylation of the second group of proteins, although phosphorylation of some members of the first group was observed. Our results demonstrate the presence of two different mechanisms leading to protein-tyrosine phosphorylation in Con A-stimulated platelets, and identify a new signal transduction pathway, promoted by the clustering of membrane glycoproteins, that produces tyrosine phosphorylation of specific substrates. This new pathway may be activated by platelet interaction with multivalent ligands, such as adhesive proteins, during adhesion, spreading, and aggregation.

Blood Platelets↗

PubMed1990

Phosphorylation of proteins of the postsynaptic density: effect of development on protein tyrosine kinase and phosphorylation of the postsynaptic density glycoprotein, PSD-GP180.

The effect of development on the tyrosine kinase activity of postsynaptic densities (PSDs) has been determined. PSDs were prepared from the forebrains of rats ranging in postnatal age from 13 to 90 days and the phosphorylation of both exogenous and endogenous substrates by tyrosine kinase measured. PSDs exhibited tyrosine kinase activity at all ages examined. Phosphorylation of the exogenous substrates polyglutamyltyrosine (4:1) and [val5] angiotensin II increased twofold between days 17 and 22 and then decreased between days 30 and 90 to levels slightly lower than those present at 13 days. The phosphorylation of endogenous PSD proteins on tyrosine residues, assessed by alkali digestion of polyacrylamide gels of 32P-labelled PSD proteins and by measuring the formation of [32P] phosphotyrosine by PSDs incubated in the presence of [gamma-32P] ATP, closely paralleled the changes in total tyrosine kinase activity. Tyrosine phosphorylation of the PSD-specific glycoprotein, PSD-GP180, also showed a transient increase between days 22 and 30, although its concentration within the PSD continued to increase slowly up to 90 days. The results indicate that the tyrosine kinase activity of PSDs is developmentally regulated and that tyrosine phosphorylation of PSD proteins is limited by enzyme rather than substrate availability.

Aging↗

PubMed2004

Effects of long-term oral administration of polymeric microcapsules containing tyrosinase on maintaining decreased systemic tyrosine levels in rats.

There is no effective treatment for melanoma, a fatal skin cancer occurring with increasing frequency. Dietary tyrosine restriction lowers systemic tyrosine and suppresses the growth of melanoma in mice, but this is not tolerated by human resulting in nausea, vomiting, and weight loss. We report here the successful use of oral polymeric microcapsules containing tyrosinase to lower the systemic tyrosine level in the rats. We found that microencapsulated tyrosinase incubated with intestinal content of rats selectively lowered the tyrosine level. We then studied the daily oral administration of microencapsulated tyrosinase in rats of one dose a day, two doses a day, and three doses a day over a period of up to 22 days. With three doses a day, the tyrosine levels in the test group decreased to 68.8% of the control group by day 4 and then decreased to 52.6% after this and remained at this level throughout the 22 days test period. This is the level shown earlier by other workers using dietary restriction of tyrosine to result in suppression of growth of melanoma. However, unlike dietary tyrosine restriction, oral tyrosinase microcapsules did not result in adverse effects nor significant differences in growth (weight gain) when compared to the control group. This approach can also be used for the lowering of systemic tyrosine in hypertyrosinemia, an inborn error of metabolism.

Administration, Oral↗

PubMed1999

Tyrosine modifications and inactivation of active site manganese superoxide dismutase mutant (Y34F) by peroxynitrite.

Recent studies from this laboratory have demonstrated that human manganese superoxide dismutase (MnSOD) is a target for tyrosine nitration in several chronic inflammatory diseases including chronic organ rejection, arthritis, and tumorigenesis. Furthermore, we demonstrated that peroxynitrite (ONOO-) is the only known biological oxidant competent to inactivate enzymatic activity, nitrate critical tyrosine residues, and induce dityrosine formation in MnSOD. To elucidate the differential contributions of tyrosine nitration and oxidation during enzymatic inactivation, we now compare ONOO- treatment of native recombinant human MnSOD (WT-MnSOD) and a mutant, Y34F-MnSOD, in which tyrosine 34 (the residue most susceptible to ONOO--mediated nitration) was mutated to phenylalanine. Both WT-MnSOD (IC50 = 65 microM, 15 microM MnSOD) and Y34F-MnSOD (IC50 = 55 microM, 15 microM Y34F) displayed similar dose-dependent sensitivity to ONOO--mediated inactivation. Compared to WT-MnSOD, the Y34F-MnSOD mutant demonstrated significantly less efficient tyrosine nitration and enhanced formation of dityrosine following treatment with ONOO-. Collectively, these results suggest that complete inactivation of MnSOD by ONOO- can occur independent of the active site tyrosine residue and includes not only nitration of critical tyrosine residues but also tyrosine oxidation and subsequent formation of dityrosine.

Catalytic Domain↗

PubMed1993

Effect of orthovanadate on tyrosine phosphorylation of P120 GTPase-activating protein in rat liver macrophages (Kupffer cells).

GTPase-activating protein (GAP), a protein capable of regulating the activity of p21ras protein, is phosphorylated on tyrosine residues following the activation of tyrosine kinase(s) associated with several growth factor receptors. The present study was designed to examine potential role of phosphotyrosine phosphatase in tyrosine phosphorylation of GAP. Addition of orthovanadate, a phosphate analogue known to inhibit phosphotyrosine phosphatase, to cultured liver macrophages induced tyrosine phosphorylation of numerous cellular proteins with a range of molecular weights between 30-130 kDa; one tyrosine-phosphorylated protein was identified as the 120 kDa GAP. The effect of orthovanadate on the tyrosine phosphorylation of GAP was time- and concentration-dependent. Quantitated data indicated that approximately 4% of the total content of cellular GAP was tyrosine-phosphorylated upon orthovanadate treatment. These observations suggest a potential regulatory role of phosphotyrosine phosphatase in the tyrosine phosphorylation of GTPase-activating protein in cellular signaling mechanisms in the hepatic macrophages.

Animals↗

PubMed1997

Evidence for an erbstatin-sensitive tyrosine kinase functioning in ascidian egg activation.

It has been proposed that protein tyrosine phosphorylation plays an important role in sperm-induced egg activation. To obtain evidence for the involvement of tyrosine kinases at an early stage in the egg activation process, we analyzed the effects of tyrosine kinase inhibitors on surface contraction of fertilized eggs of the ascidian Ciona savignyi and analyzed their effects on cleavage. We found that only erbstatin analog inhibited surface contraction, which was observed 5 min after insemination. With respect to the cleavage that occurs around 50 min after insemination, tyrophostin A1 and genistein, together with erbstatin analog, showed inhibitory effects. In addition, transient tyrosine phosphorylation of at least five proteins was observed 2-5 min after insemination, followed by tyrosine phosphorylation of one protein 30-40 min after insemination. Among proteins tyrosine-phosphorylated at the former stage, tyrosine phosphorylation of a 75 kD protein was inhibited by erbstatin analog. Thus, an erbstatin-sensitive tyrosine kinase functions at an early stage in the ascidian egg activation process.

Animals↗

PubMed1999

Cholesterol efflux-mediated signal transduction in mammalian sperm: cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm capacitation.

We previously demonstrated that mouse sperm capacitation is accompanied by a time-dependent increase in protein tyrosine phosphorylation that is dependent on the presence of BSA, Ca2+, and NaHCO(3), all three of which are also required for this maturational event. We also demonstrated that activation of protein kinase A (PK-A) is upstream of this capacitation-associated increase in protein tyrosine phosphorylation. BSA is hypothesized to modulate capacitation through the removal of cholesterol from the sperm plasma membrane. In this report, we demonstrate that incubation of mouse sperm medium containing BSA results in a release of cholesterol from the sperm plasma membrane to the medium; release of this sterol does not occur in medium devoid of BSA. We next determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Blocking the action of BSA by adding exogenous cholesterol-SO-(4) to the BSA-containing medium inhibits the increase in protein tyrosine phosphorylation as well as capacitation. This inhibitory effect is overcome by (1) the addition of increasing concentrations of BSA at a given concentration of cholesterol-SO-(4) and (2) the addition of dibutyryl cAMP plus IBMX. High-density lipoprotein (HDL), another cholesterol binding protein, also supports the capacitation-associated increase in protein tyrosine phosphorylation through a cAMP-dependent pathway, whereas proteins that do not interact with cholesterol have no effect. HDL also supports sperm capacitation, as assessed by fertilization in vitro. Finally, we previously demonstrated that HCO-(3) is necessary for the capacitation-associated increase in protein tyrosine phosphorylation and demonstrate here, by examining the effectiveness of HCO-(3) or BSA addition to sperm on protein tyrosine phosphorylation, that the HCO-(3) effect is downstream of the site of BSA action. Taken together, these data demonstrate that cholesterol release is associated with the activation of a transmembrane signal transduction pathway involving PK-A and protein tyrosine phosphorylation, leading to functional maturation of the sperm.

Acrosome↗

PubMed2000

Role of tyrosine kinase in fibroblast compaction and cerebral vasospasm.

Hemolysate, a proposed causative agent for cerebral vasospasm following subarachnoid hemorrhage, produces contraction of cerebral arteries by activation of tyrosine kinases. In addition, hemolysate accelerates fibroblast collagen compaction that could play a role in cerebral vasospasm. We studied the effect of hemolysate on tyrosine phosphorylation and fibroblast collagen compaction in cultured dog cerebral and human dermal fibroblasts using tyrosine kinase inhibitors and tyrosine antibodies (Western blot). 1) Hemolysate was found to enhance tyrosine phosphorylation of two proteins approximately 64 and 120 kDa. The effect of hemolysate was time- and concentration-dependent. 2) Two main components in hemolysate, oxyhemoglobin and adenosine triphosphate (ATP), produced similar results to that of hemolysate. 3) Tyrosine kinase inhibitor genistein and tyrphostin A51 (30 microM) markedly reduced the effect of hemolysate on tyrosine phosphorylation. 4) In another study, hemolysate increased fibroblast collagen compaction and the effect of hemolysate was reduced by genistein and tyrphostin A51. We conclude that hemolysate activates tyrosine kinase that may lead to acceleration of fibroblast compaction. This effect of hemolysate may contribute to cerebral vasospasm.

Animals↗

PubMed1987

Saccharomyces cerevisiae strains sensitive to inorganic mercury. III. Tyrosine uptake.

In Saccharomyces cerevisiae, the HGS2-1 allele confers sensitivities to inorganis mercury (Ono and Sakamoto 1985) and to excess fermentable sugars such as glucose (Sakamoto et al. 1985); exogenous tyrosine antagonizes both inorganic mercury and excess glucose. In this study, the inorganic mercury sensitive strain has been shown to have about twice more glucose-1,6-bisphosphate and slightly less pyruvate than the normal strains, suggesting that the inorganic mercury sensitive strain has the reduced aldolase activity. It has been also shown that the growth retarded cells accumulate trehalose, by which the lower level of glucose-6-phosphate in the inorganic mercury sensitive strain is accounted for, and that inorganic mercury, presumably excess glucose also, causes growth inhibition via depletion of cellular tyrosine. The mechanism how cellular tyrosine is depleted by inorganic mercury or excess glucose is accounted for by the facts that (1) the tyrosine uptake activity is decreased with increase of glucose concentration in growth medium, (2) HGS2-1 enhances the effect of glucose on the tyrosine uptake activity, and (3) inorganic mercury inhibits the tyrosine uptake system by binding to its SH-group(s). Thus, it is concluded that the role of tyrosine is not to detoxify inorganic mercury nor excess fermentable sugars but simply to counteract depletion of cellular tyrosine induced by them.

Biological Transport↗

PubMed1986

Tyrosine loading in patients with hepatic cirrhosis: lack of effect on plasma catecholamines.

Plasma norepinephrine concentrations are often elevated in patients with hepatic cirrhosis in relation to the stage of disease and possibly in response to a decrease in "effective" arterial blood volume. Since tyrosine, the precursor for catecholamines, is said to influence the rate of catecholamine biosynthesis within the central nervous system and peripheral sympathetic structures, we tested whether basal hypertyrosinemia and increased plasma tyrosine levels after oral loading with l-tyrosine are associated with elevated plasma catecholamine concentrations. Baseline norepinephrine (NE) and epinephrine (E) were significantly higher in 17 patients with decompensated cirrhosis, as compared with 11 healthy controls (NE: 809 +/- 108 pg/ml vs 295 +/- 16 pg/ml; E: 69 +/- 9 pg/ml vs 36 +/- 8 pg/ml). No significant correlation between the basal plasma tyrosine and norepinephrine level could be demonstrated in patients with cirrhosis (r = 0.04). Oral tyrosine loading (100 mg/kg b.w.) administered in six equal doses did not change the level of catecholamines, whereas plasma tyrosine increased two- to three-fold. Even a large single dose (14 g l-tyrosine) failed to alter plasma catecholamines in six cirrhotic patients with marked ascites. We therefore conclude that the enhanced availability of tyrosine in cirrhotics does not influence catecholamine biosynthesis in peripheral sympathetic neurons.

Blood Pressure↗

PubMed1983

Development of hypertension in spontaneously hypertensive rats fed L-tyrosine-supplemented diets.

The blood pressure of spontaneously hypertensive rats (SHR) was measured by tail-plethysmography. Feeding SHR a diet supplemented with 0.6 g% L-tyrosine, for 15 weeks after weaning, resulted in a slower increase of blood pressure than in rats fed the control diet (no tyrosine added). The blood pressure stabilized, after about 8 weeks, at values lower by about 10 mm Hg than in the control SHR group. Diets with a higher content of free L-tyrosine (1.2 or 2.4 g%) produced no greater hypotensive effects, despite the fact that the plasma level of the amino acid, at the time of blood pressure measurements, was related to the tyrosine content of the diet. In addition, providing 2.4 g% free L-tyrosine to the diet of SHR with established hypertension, produced within a few days a decrease of blood pressure similar to the one recorded in rats fed the tyrosine-supplemented diet during the whole period of development of hypertension. A maximal effect of L-tyrosine, in decreasing the blood pressure of SHR, is thus obtained at relatively low concentrations of the amino acid in the diet, and after a short period of consumption. However, this effect is rather small, and rapidly reversed upon removing free L-tyrosine from the diet.

Animals↗

PubMed1996

The characterization of tyrosine phosphorylation of 78 and 92 kDa proteins in rat basophilic RBL-2H3 cells.

Previously, we have demonstrated that tyrosine phosphorylation of 78 and 92 kDa proteins in rat basophilic leukemia cells (RBL-2H3) is involved in a signal transduction system for high-affinity IgE receptor (Fc epsilon RI)-mediated histamine secretion. However, it is not clarified whether the tyrosine phosphorylation of 78 and 92 kDa proteins in RBL-2H3 cells is regulated by activation of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3-kinase). In this study, therefore, the effect of depletion of PKC in RBL-2H3 cells, or the influence of PKC, PI3-kinase and tyrosine kinase inhibitors on histamine release from RBL-2H3 cells was examined. The elimination of PKC in RBL-2H3 cells induced significant suppression of histamine release, although the tyrosine phosphorylation of 78 and 92 kDa proteins was not inhibited. The inhibition of histamine release was also observed by the treatment with a PKC inhibitor such as H-7, calphostin C, a PI3-kinase inhibitor such as wortmannin or a tyrosine kinase inhibitor such as ST638, genistein, hervimycin A, although the tyrosine phosphorylation of both proteins was inhibited by only ST638. These results suggest that the 78 kDa protein in RBL-2H3 cells is not identical to the protein-tyrosine kinase PTK72 and the tyrosine phosphorylation of 78 and 92 kDa proteins in RBL-2H3 cells occurs upstream of PKC and PI3-kinase activation or is regulated independently of the PKC- and PI3-kinase-dependent signaling pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2000

Tyrosine phosphorylation of the human guanylyl cyclase C receptor.

Tyrosine phosphorylation events are key components of several cellular signal transduction pathways. This study describes a novel method for identification of substrates for tyrosine kinases. Co-expression of the tyrosine kinase EphB1 with the intracellular domain of guanylyl cyclase C (GCC) in Escherichia coli cells resulted in tyrosine phosphorylation of GCC, indicating that GCC is a potential substrate for tyrosine kinases. Indeed, GCC expressed in mammalian cells is tyrosine phosphorylated, suggesting that tyrosine phosphorylation may play a role in regulation of GCC signalling. This is the first demonstration of tyrosine phosphorylation of any member of the family of membrane-associated guanylyl cyclases.

Animals↗

PubMed2004

Role of zinc in regulation of protein tyrosine phosphatase activity in osteoblastic MC3T3-E1 cells: zinc modulation of insulin-like growth factor-I's effect.

Zinc, an essential trace element, has been demonstrated to stimulate bone growth in animal and human. The cellular mechanism by which zinc stimulates bone growth has not been fully clarified. The effect of hormone and zinc on protein tyrosine phosphatase activity in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers, and then exchanged to culture medium containing either vehicle, zinc sulfate or various hormones in the absence of 10% FBS. After medium change, cells were cultured for 48 h. Protein tyrosine phosphatase activity in the lysate of cells was significantly increased by culture with zinc (10(-6) - 10(-4) M). The effect of zinc in increasing the enzyme activity was completely blocked by culture with cycloheximide (10(-7 )M), an inhibitor of protein synthesis, or 5, 6-dichloro-l-beta-D- riboifuranosylbenzimidarzole (DRB) (10(-6) M), an inhibitor of translational activity. Addition of calcium chloride (10 microM) into the reaction mixture caused a significant increase in protein tyrosine phosphatase activity; this increase was completely blocked in the presence of trifluoperazine (50 microM), an antagonist of calmodulin. Culture with zinc caused a significant increase in Ca2+/calmodulin-dependent protein tyrosine phosphatase activity in osteoblastic cells. Protein tyrosine phosphatase activity was significantly raised by culture with parathyroid hormone (human PTH [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33]; 10(-7) M), 17beta-estradiol (10(-7) M), insulin-like growth factor-I (IGF-I; 10(-8) M) or insulin (10(-8) M). The enzyme activity was not significantly enhanced by the addition of calcium (10 microM) into the reaction mixture. The effect of PTH or IGF-I in increasing protein tyrosine phosphatase activity was completely blocked by culture with DRB. The IGF-I-induced increase in enzyme activity was significantly enhanced by culture with zinc. Such an effect was not seen in the case of PTH. Moreover, the effect of IGF-I in increasing proliferation of osteoblastic cells was significantly enhanced by culture with zinc. The effect of PTH was not enhanced by zinc. This study demonstrates that protein tyrosine phosphatase activity in osteoblastic cells is enhanced by various bone anabolic factors, and that zinc modulates the effect of IGF-I on protein tyrosine phosphatase activity and cell proliferation.

3T3 Cells↗

PubMed1986

Evaluations of tyrosine apodecarboxylase assays for pyridoxal phosphate.

The alternate procedures used in the tyrosine apodecarboxylase assays for pyridoxal 5'-phosphate were evaluated to determine optimal conditions. Two preparations of tyrosine apodecarboxylase from Streptococcus faecalis were used: a cell suspension and a partially purified cell-free form. The activity of the decarboxylase was measured in two different assays using [14C]tyrosine or [3H]tyrosine as substrate. The presence of serum proteins caused greater inhibition of the assay for serum pyridoxal phosphate using [14C]tyrosine as substrate than the assay with [3H]tyrosine. In contrast, addition of deproteinized serum extract did not appear to inhibit either assay. The rate of reconstitution of the apodecarboxylase in the cell suspension was at least four times slower than that of the cell-free enzyme. The rate of reconstitution of the cell-free enzyme was faster in acetate than in citrate buffer. Inorganic sulfate or phosphate, at normal plasma concentrations, did not alter either the reconstitution rate of tyrosine decarboxylase or the final activity obtained in the assays using either substrate. The tyrosine apodecarboxylase assay for pyridoxal phosphate can be optimized by using deproteinized sera or plasma and incubating the cell-free apoenzyme with the coenzyme in acetate buffer for a time sufficient to obtain maximum reconstitution.

Acetates↗

PubMed1992

Ethanol modulates epidermal growth factor-stimulated tyrosine kinase and phosphorylation of PLC-gamma 1.

A431 cells have an abundance of Epidermal Growth Factor (EGF) receptors which possess intrinsic tyrosine kinase activity. Treatment of membranes isolated from A431 cells with EGF caused a 2-3 fold increase in phosphorylation of a synthetic peptide (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly) which is a substrate for tyrosine kinase. Treatment of these membranes with 0.1 to 100 mM ethanol altered basal tyrosine kinase activity in a biphasic manner; increase at 10 mM and decrease at 100 mM ethanol. The treatment of the membranes with the same concentrations of ethanol also altered EGF's ability to stimulate tyrosine kinase activity: increase at 0.1 mM ethanol and decrease at 10 mM. Strikingly, EGF-stimulated tyrosine kinase was more sensitive to ethanol than the basal activity. Experiments with other alcohols showed a relationship between chain length and the inhibitory ability of the alcohol. These data demonstrate a biochemical effect of low concentrations of ethanol on tyrosine kinase. Interestingly, ethanol treatment of A431 cells inhibited EGF-stimulated phosphorylation of PLC-gamma 1 which is a substrate for EGF receptor tyrosine kinase. It is concluded that ethanol at low concentrations has significant modulatory effect on basal and EGF-stimulated tyrosine kinase, as well as PLC-gamma 1 phosphorylation.

Amino Acid Sequence↗