Short-term effect of stress on tyrosine hydroxylase activity

The short-term influences of stress on the activities of tyrosine hydroxylase in vivo and in vitro were examined in mice. The in vivo tyrosine hydroxylase activity was estimated by the rate of dopa accumulation which was measured at 30 min after the injection of NSD-1015 (100 mg kg), an aromatic l-amino acid decarboxylase inhibitor, intraperitoneally and was compared with tyrosine hydroxylase activity measured in vitro. For the in vivo assay, both the accumulation of dopa (tyrosine hydroxylase activity) and that of 5-hydroxytryptophan (tryptophan hydroxylase activity) and the levels of monoamines and the metabolites (noradrenalin, adrenalin, dopamine, normetanephrine, 3-methoxytyramine and serotonin) and those of precursor amino acids, tyrosine and tryptophan, were investigated in ten different brain regions and in adrenals. The amount of dopa accumulation in the brain as a consequence of decarboxylase inhibition, in vivo tyrosine hydroxylase activity, was significantly increased by stress, in nerve terminals (striatum, limbic brain, hypothalamus, cerebral cortex and cerebellum) and also in adrenals. The effect of stress on tyrosine hydroxylase activity in vitro at a subsaturating concentration of 6-methyltetrahydropterin cofactor was also observed in nerve terminals (striatum, limbic brain, hypothalamus, and cerebral cortex). The amount of 5-hydroxytryptophan accumulation, the in vivo tryptophan hydroxylase activity, was also significantly increased in bulbus olfactorius, limbic brain, cerebral cortex, septum and lower brain stem. The influence of stress was also observed on the levels of precursor amino acids, tyrosine and tryptophan and monoamines in specific brain parts. These results suggest that the stress influences both catecholaminergic neurons and serotonergic neurons in nerve terminals in the brain. This effect was also observed on tyrosine hydroxylase activity in vitro in nerve terminals. However, in adrenals, the influence by stress was not observed on the in vitro activity, although dopa accumulation was increased.     

Studies on the mechanism of the stimulation of tyrosine aminotransferase activity in vivo by pyrimidine analogs: the role of enzyme synthesis and degradation

The pyrimidine analogs, 5-fluoroorotate and 5-azacytidine, have been shown to stimulate the basal level as well as the cortisone, tryptophan, and casein hydrolysate-induced levels of the rat liver enzyme, tyrosine aminotransferase. This stimulation was most marked in the case of dietary and hormonal induction when the analog was given 4–6 hr prior to the administration of the inducer. When tryptophan induced tyrosine aminotransferase, maximal stimulation by the analog occurred if it were given 2 hr prior to the administration of the amino acid. The optimal stimulatory dose of 5-azacytidine was 5 mg/kg body weight whereas 5-fluoroorotate gave its highest stimulation at a dose of 60 mg/kg. Of several orotic acid analogs tested, only the chloro-analog had an effect similar to the fluoro-congener.Utilizing quantitative immunochemical precipitation and pulse labeling in vivo, it was demonstrated that the administration of 5-fluoroorotate or 5-azacytidine at doses of 60 and 5 mg per kg, respectively, while causing a stimulation in the basal level of tyrosine aminotransferase, did not result in any change in the rate of enzyme synthesis. Furthermore, after cortisone induction of the enzyme, the delayed administration of these analogs caused either a further stimulation in the level of the enzyme or the maintenance of a high level while the enzyme activity decayed in animals not given the analogs. The rates of synthesis either showed no change or a decrease while the amount of enzyme was increasing. Prelabeling of the enzyme in vivo after induction with cortisone and followed by the administration of 5-fluoroorotate resulted in a marked decrease in the t$\text{1}\text{2}$ of the decay rate of the enzyme measured either by loss of radioactivity or by loss of enzyme activity. These studies suggest that these analogs act in some manner to prevent enzyme turnover by an inhibition of enzyme degradation.     

L-tryptophan inhibition of tyrosine aminotransferase degradation in rat liver in vivo

The administration of l-tryptophan to both intact and adrenalectomized animals results in a marked increase in the activity of tyrosine aminotransferase. Maximal increases in enzyme activity are stimulated by doses of l-tryptophan much lower than those required for maximal stimulation of tryptophan oxygenase activity in vivo. When l-tryptophan was administered to animals that had been given cortisone 5 hr earlier, a further sustained increase in enzyme activity was demonstrated. 5-Hydroxy-dl-tryptophan and indole administration in amounts equimolar to l-tryptophan also result in similar increases in activity whereas α-methyl-dl-tryptophan produces little or no increase.Utilizing pulse-labeling in vivo with quantitative immunochemical precipitation of tyrosine aminotransferase by specific antisera, it was demonstrated that the administration of tryptophan caused an increase in enzyme amount with no concomitant increase in the rate of enzyme synthesis. In animals given cortisone, subsequent injections of tryptophan caused the amount of enzyme to continue to increase while both the amount of enzyme in control animals, as well as the rates of synthesis in both tryptophan-treated and control animals, decreased in a parallel fashion. Prelabeling of tyrosine aminotransferase in vivo after the enzyme had been induced with cortisone demonstrated that the subsequent administration of tryptophan caused a marked inhibition in the decay of the radioactive enzyme, as well as in enzyme activity. These data support the proposal that the amino acid, tryptophan, has a special role both in the maintenance of hepatic protein synthesis and in the regulation of specific enzyme degradation in rat liver.     

The action in vivo of a structural analogue of GABA: hydrazinopropionic acid

Abstract— The action of hydrazinopropionic acid in vivo on the metabolism of amino acids in the CNS of mice was studied over a period of 24 hr. At 82 μmoles/kg, a transient fourfold rise in the levels of tyrosine occurred followed by a more moderate and prolonged increase in the levels of GABA. When the dose of hydrazinopropionate was raised to 123 μmoles/kg, the changes in the levels of tyrosine were identical with those at the lower dose, while levels of GABA rose further before reaching a steady state. Levels of glutamate decreased concomitantly. In addition, the concentrations of alanine, β-aminoisobutyric acid and an unknown compound tended to rise. A doubling of the dose to 246 μmoles/kg enhanced the effects obtained with the lower doses but did not produce any new changes in the patterns of amino acids of the CNS. The elevation in the concentrations of alanine paralleled changes in the levels of tyrosine. The changes in the concentrations of the unknown compound, tentatively identified as α-aminoadipate, and of β-aminoisobutyric acid resembled those of GABA. The results are interpreted to indicate an inhibition by hydrazinopropionic acid of tyrosine aminotransferase and aminobutyrate aminotransferase.     

γ-aminobutyric acid in the central nervous system of metamorphosing and mature Manduca sexta

We have begun to examine the factors controlling the accumulation of the neurotransmitter γ-aminobutyric acid (GABA) in the central nervous system (CNS) of the sphinx moth Manduca sexta. Analysis of soluble amino acids in CNS structures from mature moths outlines the regional distribution of GABA. Analysis of amino acids in the antennal lobes (the primary olfactory centres) of Manduca during metamorphosis reveals that GABA accumulates gradually and continuously through most of adult development until eclosion; within 18 hr after eclosion, levels of GABA abruptly increase 27–50%. The activity of the biosynthetic enzyme glutamic acid decarboxylase (EC 4.1.1.15), assayed in extracts of antennal lobes from developing moths, does not change after eclosion. Extracts of hemolymph from mature moths contain low levels of glutamate ( <0.2 mM) and higher levels of certain other amino acids such as serine, glutamine and proline. The concentration of proline in hemolymph increases up to 2-fold after eclosion. Glutamate, glutamine and proline are interconvertible in the CNS, and each can serve as precursor for GABA synthesis both in vivo and in vitro. The efficiency of the precursor role in vitro is similar for each amino acid, as estimated from the ratio of the specific radioactivities of GABA and glutamic acid in the ganglion derived from each precursor. Exogenous proline and glutamine can equilibrate rapidly with the ganglionic pools of the same amino acids while glutamic acid is relatively excluded. Taken together, the findings of this study show that proline and glutamine may contribute substantially to synthesis of GABA in the CNS of M. sexta.     

Regulation of tryptophan and tyrosine hydroxylase

The synthesis of the neurotransmitters serotonin, norepinephrine, and the dopamine is regulated by the initial amino acid hydroxylases. Little is known about the factors that regulate the level of tryptophan hydroxylase in tissue. However, the level of tyrosine hydroxylase is regulated by transsynaptic induction. Acute regulation of in vivo hydroxylase activity appears to be by substrate availability in the case of tryptophan hydroxylase and possibly by feedback inhibition with tyrosine hydroxylase. A newly described phenomenon which has been termed “receptor mediated feedback inhibition” involving neuronal feedback regulation of the activity of both tyrosine and tryptophan hydroxylase may also have an important role.     

Effects of polyamines on tyrosine hydroxylase activity in adrenals

The ability of polyamines (putrescine, spermidine, and spermine) to modify tyrosine hydroxylase (TH) activity was examined in crude or purified enzyme preparation and in adrenal tissue slices. Polyamines showed biphasic effects on TH activity in vitro at physiological pH 7.0, with an inhibitory effect at low concentrations (<1 mM) and a stimulatory effect at high concentrations. The degree of both inhibition and stimulation produced by polyamines at low and high concentrations, respectively, were proportional to the number of the amino group in the polyamines (putrescine < spermidine < spermine). The degree of inhibition by polyamines was much greater with purified enzyme than with crude enzyme preparations. Tyrosine hydroxylation in situ in adrenal tissue slices was stimulated by polyamines without inhibition at any concentrations tested. This evidence suggests that TH molecules in vivo could interact with polyamines or polyamine-like substances which inhibit the TH activity at physiological concentrations less than 1 mM.     

Expression pattern and substrate specificity of Clonorchis sinensis tyrosinases

Tyrosinase (TYR) is a copper-containing glycoenzyme that mediates hydroxylation of tyrosine into dihydroxyphenylalanine and oxidation of dihydroxyphenylalanine into dihydroxyphenylalanine quinone. TYRs play pivotal roles in eggshell sclerotisation of trematode parasites, while their comprehensive biochemical properties remain elusive. We characterised genes encoding four TYRs (CsTYR1–4) of Clonorchis sinensis, a causative agent of human hepatobiliary disease. These genes shared tightly conserved amino acid residues, two copper binding catalytic motifs and a cysteine-rich epidermal growth factor-like domain. The native and recombinant CsTYRs showed high reactivity against diphenol compounds, especially those with hydroxyl groups in ortho-positions (catechol and l-dihydroxyphenylalanine), but showed minimal activity toward monophenol compounds. Diphenolase activity was enhanced by increased pH of the reaction buffer from 5.0 to 7.0. The temporal induction of CsTYR expression coordinated with the sexual maturation of the worm; enzyme activity was mainly in the vitelline glands and intrauterine immature eggs proximal to the ovary. The primary structures and functional domains of CsTYRs showed significant similarities to those of the vertebrate orthologs, whereas the amino acids shared with the nematode and insect proteins were largely restricted in the bicopper active center. Unlike highly diverged TYR homologs in vertebrates, multiple paralogs have not yet evolved into the separate lineages in trematode genomes, suggesting that duplication of TYR genes might relate to increased genic dosage/redundancy in trematodes. In vitro treatment of copper chelator, diethyldithiocarbamic acid, inhibited generation of phenotypically normal egg. TYR proteins are essential for C. sinensis reproduction, thus might be targeted for therapeutic and vaccine strategies against clonorchiasis, which is prevalent in several Asian countries and is one of the most important predisposing factors for human cholangiocarcinoma. The close phylogenetic relationships between trematode and vertebrate homologs also provide a molecular clue to understand the multifaceted evolutionary pathway of TYR homologs across animal taxa.     

Acute effects of aspartame on large neutral amino acids and monoamines in rat brain

The dipeptide aspartame (APM; aspartylphenylalanine methylester), an artificial sweetener, was studied $\text{in vivo}$ for its ability to influence brain levels of the large neutral amino acids and the rates of hydroxylation of the aromatic amino acids. The administration by gavage of APM (200 mg/kg) caused large increments in blood and brain levels of phenylalanine and tyrosine by 60 minutes. Brain tryptophan level was occasionally reduced significantly, but the brain levels of the branched-chain amino acids were always unaffected. Smaller doses (50, 100 mg/kg) also raised blood and brain tyrosine and phenylalanine, but did not reduce brain tryptophan levels. At the highest dose (200 mg/kg), APM gavage caused an insignificant increase in dopa accumulation (after NSD-1015), and a modest reduction in 5-hydroxytryptophan accumulation. No changes in the brain levels of serotonin, 5-hydroxyindoleacetic acid, dopamine, dihydroxyphenylacetic acid, homovanillic acid, or norepinephrine were produced by APM administration (200 mg/kg). These results thus indicate that APM, even when administered in amounts that cause large increments in brain tyrosine and phenylalanine, produce minimal effects on the rates of formation of monoamine transmitters.     

The role of reductants in the tyrosine hydroxylase reaction.

The role of several reducing systems in the tyrosine hydroxylase reaction has been studied. A significant dependence upon the reducing systems beyond that required to regenerate the oxidized cofactor has been observed. 2-Mercaptoethanol, NADPH, and ascorbate are each effective at reducing the cofactor, but their abilities to stimulate tyrosine hydroxylase vary over a threefold range. NADPH is a suitable reductant for the tyrosine hydroxylase reaction, even in the absence of pteridine reductase. A reducing system containing ascorbate, ferrous ion, and catalase gives unusually high enzyme activity and low blanks. This ascorbate system, in addition to being useful for in vitro enzyme assays, may serve as a model for the in vivo reaction. Ascorbate may play an important role in the hydroxylation of tyrosine in catecholaminergic tissues. This study demonstrates that an efficient reductant for the tyrosine hydroxylase reaction must, in addition to reducing the pterin cofactor, also interact effectively with the enzyme itself.     

Megalodiscus temperatus: absorption and incorporation of tritiated tyrosine, thymidine, and adenosine

Autoradiographic and liquid scintillation counting studies indicate adults of Megalodiscus temperatus can absorb large amounts of thymidine and adenosine, but use little of the thymidine in in vitro exposures of up to 72 hr. Labeling of worms exposed to ³H-adenosine in periods as short as 3 hr was achieved especially over young eggs, testes, and the ovary. Large amounts of tyrosine are found in the free amino acid pool of this worm but little is incorporated into tissues for exposure periods of up to 24 hr. Ligation experiments and autoradiograms of frozen-dried worms show thymidine and adenosine can enter via the tegument. Adenosine added to the exposure medium did not inhibit absorption of tyrosine but did markedly reduce the uptake of ³H-thymidine in 8-hr in vitro exposures. Bacteria adhering to the glycocalyx of M. temperatus were identified as Escherichia coli, but their role in uptake phenomena is unknown. The association of thymidine with the lymph vessels indicates a circulatory or excretory role for this system in M. temperatus. Adenosine was considered the best compound of the three to use for labeling cells for studies on the reproductive system.     

The effects of tetrahydroisoquinolinecarboxylic acids on tyrosine 3-monooxygenase

The effects of tetrahydroisoquinolinecarboxylic acids, derived from dopamine and various phenylpyruvates, on the enzyme tyrosine 3-monooxygenase have been investigated. Using a partially purified tyrosine 3-monooxygenase from bovine adrenal medulla, 3′,4′-deoxynorlaudanosolinecarboxylic acid was found to be a mixed inhibitor against the cofactor (K_i = 122 μM), equipotent with norepinephrine. Norlaudanosolinecarboxylic acid inhibited tyrosine 3-monooxygenase competitively with respect to the cofactor (K_i = 126 μM). When tyrosine 3-monooxygenase activity in catecholamine-free striatal homogenates was studied, again 3′,4′-deoxynorlaudanosolinecarboxylic acid (K_i = 40 μM) behaved as a mixed inhibitor whereas norlaudanosolinecarboxylic acid (K_i = 136 μM) was competitive. When the rat striatal tyrosine 3-monooxygenase was subjected to phosphorylating conditions in vitro, decreases in the K_i of norlaudanosolinecarboxylic acid and in that of 3′,4′-deoxynorlaudanosolinecarboxylic acid were observed, whereas the K_i of dopamine was increased. Tyrosine 3-monooxygenase activity in rat striatal synaptosomes was also inhibited by 3′,4′-deoxynorlaudanosolinecarboxylic acid (IC₅₀ = 100 μm) and phosphorylating conditions affected only that inhibition produced by dopamine, but not that by the tetrahydroisoquinolinecarboxylic acids. The results are discussed in relation to the structure of the tetrahydroisoquinolinecarboxylic acids and their possible role in vivo.     

In vivo regulation of phenylalanine hydroxylation to tyrosine, studied using enrichment in apoB-100

Phenylalanine hydroxylation is necessary for the conversion of phenylalanine to tyrosine and disposal of excess phenylalanine. Studies of in vivo regulation of phenylalanine hydroxylation suffer from the lack of a method to determine intrahepatocyte enrichment of phenylalanine and tyrosine. apoB-100, a hepatic export protein, is synthesized from intrahepatocyte amino acids. We designed an in vivo multi-isotope study, [(15)N]phenylalanine and [2H2]tyrosine to determine rates of phenylalanine hydroxylation from plasma enrichments in free amino acids and apoB-100. For independent verification of apoB-100 as a reflection of enrichment in the intrahepatocyte pool, [1-(13)C]lysine was used as an indicator amino acid (IAA) to measure in vivo changes in protein synthesis in response to tyrosine supplementation. Adult men (n = 6) were fed an amino acid-based diet with low phenylalanine (9 mg.kg(-1).day(-1), 4.54 mumol.kg(-1).,h(-1)) and seven graded intakes of tyrosine from 2.5 (deficient) to 12.5 (excess) mg.kg(-1).day(-1). Gas chromatography-quadrupole mass spectrometry did not detect any tracer in apoB-100 tyrosine. A new and more sensitive method to measure label enrichment in proteins using isotope ratio mass spectrometry demonstrated that phenylalanine hydroxylation measured in apoB-100 decreased linearly in response to increasing tyrosine intake and reached a break point at 6.8 mg.kg(-1).day(-1). IAA oxidation decreased with increased tyrosine intake and reached a break point at 6.0 mg.kg(-1).day(-1). We conclude: apoB-100 is an accurate and useful measure of changes in phenylalanine hydroxylation; the synthesis of tyrosine via phenylalanine hydroxylation is regulated to meet the needs for protein synthesis; and that plasma phenylalanine does not reflect changes in protein synthesis.     

Role of tyrosine, DOPA and decarboxylase enzymes in the synthesis of monoamines in the brain of the locust

The metabolic transformation of tyrosine (TYR) by the decarboxylase and hydroxylase enzymes was investigated in the central nervous system of the locust, Locusta migratoria. It has been demonstrated that the key amino acids, 3,4-dihydroxyphenylalanine (DOPA), 5-hydroxytryptophan (5HTP) and tyrosine are decarboxylated in all part of central nervous system. DOPA and 5HTP decarboxylase activities show parallel changes in the different ganglia, but the rank order of the activity of TYR decarboxylase is different. Enzyme purification has revealed that the molecular weights of TYR decarboxylase and DOPA/5HTP decarboxylase are 370,000 and 112,000, respectively. The decarboxylation of DOPA by DOPA/5HTP decarboxylase is stimulated, whereas the decarboxylation of DOPA by TYR decarboxylase is inhibited in the presence of the cofactor pyridoxal-5'-phosphate. TYR hydroxylase could not be detected and 3H-TYR is found to be metabolised to tyramine (TA), but not to DOPA. The haemolymph contains a significant concentration of DOPA (120 pmol/100 microl haemolymph), and the ganglia incorporates DOPA from the haemolymph by a high affinity uptake process (K(M)=12 microM and V(max)=24 pmol per ganglion/10 min). Our results suggest that no tyrosine hydroxylase is present in the locust CNS and the DOPA uptake into the ganglia by a high affinity uptake process as well as the DOPA decarboxylase enzyme may be responsible for the regulation of the ganglionic dopamine (DA) level. Two types of decarboxylases exist, one of them decarboxylating DOPA and 5HTP (DOPA/5HTP decarboxylase), other decarboxylating TYR (TYR decarboxylase). The DOPA/5HTP decarboxylase enzyme present in the insect brain may correspond to the 5HTP/DOPA decarboxylase in vertebrate brain, whereas TYR decarboxylase is characteristic only for the insect brain.     

Regulation of synaptosomal tyrosine hydroxylation in different brain regions with noradrenergic innervation

Tyrosine hydroxylation rate was measured by a modified tritium release assay at the physiological pH of 7.4 in synaptosomes prepared from cerebellum, hippocampus and hypothalamus. Incubation in the presence of 2 mM 8 bromo cAMP increased tyrosine hydroxylation in all three regions. An almost identical activation was seen after membrane depolarization by 50 mM K⁺. Removal of Ca²⁺ from the incubation medium had no significant effect on the activation produced by either agent, however it did significantly increase the control tyrosine hydroxylation rate in the hypothalamus. The combined effect of 8 Br cAMP and high K⁺ was found to be additive in the cerebellum and hippocampus but not in the hypothalamus. A reduction in tyrosine hydroxylation was observed if incubation was carried out in the presence of 1 μM noradrenaline; the degree of inhibition was similar in the three regions. 2 mM 8 Br. cAMP added to the noradrenaline restored tyrosine hydroxylation to control levels in synaptosomes from the hypothalamus, but not the hippocampus and cerebellum. Tyrosine hydroxylase in the hypothalamus is associated with dopaminergic nerve terminals as well as noradrenergic nerve terminals derived from more than one cell group, the hippocampus and cerebellum however both receive their noradrenergic input entirely from the locus coeruleus. Differences between synaptosomes from the three brain regions may therefore reflect differences in the nature of the enzyme as well as local regulatory mechanisms.     

Comprehensive ADP‐ribosylome analysis identifies tyrosine as an ADP‐ribose acceptor site

Despite recent mass spectrometry (MS)‐based breakthroughs, comprehensive ADP‐ribose (ADPr)‐acceptor amino acid identification and ADPr‐site localization remain challenging. Here, we report the establishment of an unbiased, multistep ADP‐ribosylome data analysis workflow that led to the identification of tyrosine as a novel ARTD1/PARP1‐dependent in vivo ADPr‐acceptor amino acid. MS analyses of in vitro ADP‐ribosylated proteins confirmed tyrosine as an ADPr‐acceptor amino acid in RPS3A (Y155) and HPF1 (Y238) and demonstrated that trans‐modification of RPS3A is dependent on HPF1. We provide an ADPr‐site Localization Spectra Database (ADPr‐LSD), which contains 288 high‐quality ADPr‐modified peptide spectra, to serve as ADPr spectral references for correct ADPr‐site localizations.     

Pancreatic acinar cells utilize tyrosine to synthesize L-dihydroxyphenylalanine

The pancreatic β cells can synthesize dopamine by taking L-dihydroxyphenylalanine, but whether pancreatic acinar cells synthesize dopamine has not been confirmed. By means of immunofluorescence, the tyrosine hydroxylase -immunoreactivity and aromatic amino acid decarboxylase (AADC)- immunoreactivity were respectively observed in pancreatic acinar cells and islet β cells. Treatment with L-dihydroxyphenylalanine, not tyrosine, caused the production of dopamine in the incubation of INS-1 cells (rat islet β cell line) and primary isolated islets, which was blocked by AADC inhibitor NSD-1015. However, only L-dihydroxyphenylalanine, but not dopamine, was detected when AR42J cells (rat pancreatic acinar cell line) were treated with tyrosine, which was blocked by tyrosine hydroxylase inhibitor AMPT. Dopamine was detected in the coculture of INS-1 cells with AR42J cells after treatment with tyrosine. In an in vivo study, pancreatic juice contained high levels of L-dihydroxyphenylalanine and dopamine. Both L-dihydroxyphenylalanine and dopamine accompanied with pancreatic enzymes and insulin in the pancreatic juice were all significantly increased after intraperitoneal injection of bethanechol chloride and their increases were all blocked by atropine. Inhibiting TH with AMPT blocked bethanechol chloride-induced increases in L-dihydroxyphenylalanine and dopamine, while inhibiting AADC with NSD-1015 only blocked the dopamine increase. Bilateral subdiaphragmatic vagotomy of rats leads to significant decreases of L-dihydroxyphenylalanine and dopamine in pancreatic juice. These results suggested that pancreatic acinar cells could utilize tyrosine to synthesize L-dihydroxyphenylalanine, not dopamine. Islet β cells only used L-dihydroxyphenylalanine, not tyrosine, to synthesize dopamine. Both L-dihydroxyphenylalanine and dopamine were respectively released into the pancreatic duct, which was regulated by the vagal cholinergic pathway. The present study provides important evidences for the source of L-dihydroxyphenylalanine and dopamine in the pancreas.     

In vivo incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin

In vitro incorporation of [¹⁴C]tyrosine into the C-terminal position of the α subunit of tubulin was not affected by 4 mm cycloheximide. This inhibitor of protein synthesis was used for in vivo experiments. The in vivo incorporation of [¹⁴C]tyrosine into soluble brain protein of cycloheximide-treated rats was 10% of that of untreated rats. Treatment with vinblastine sulfate of the soluble brain protein showed that the incorporation of [¹⁴C]tyrosine into tubulin was higher in cycloheximide-treated than in untreated rats with respect to the incorporation into the total soluble protein. In the case of cycloheximide-treated rats, about 60% of the radioactivity incorporated into protein was released by the action of carboxypeptidase A, whereas 10% was liberated from the protein of untreated rats. The radioactive compound released by the action of carboxypeptidase A was identified as [¹⁴C]tyrosine. The α and β subunits of tubulin from animals that received [¹⁴C]tyrosine were separated by polyacrylamide gel electrophoresis. The radiosactivity ratio of $\text{α}\text{β}$ subunits of tubulin from cycloheximide-treated rats was threefold higher than that of untreated rats. When a mixture of [¹⁴C]amino acids was injected, the radioactivity ratio of $\text{α}\text{β}$ subunits of tubulin was similar for cycloheximide-treated and untreated rats. The results reported are consistent with the assumption that the α subunit of tubulin can be tyrosinated in vivo.     

Mutation of serine 395 of tyrosine hydroxylase decouples oxygen-oxygen bond cleavage and tyrosine hydroxylation

Ser395 and Ser396 in the active site of rat tyrosine hydroxylase are conserved in all three members of the family of pterin-dependent hydroxylases, phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase. Ser395 is appropriately positioned to form a hydrogen bond to the imidazole nitrogen of His331, an axial ligand to the active site iron, while Ser396 is located on the wall of the active site cleft. Site-directed mutagenesis has been used to analyze the roles of these two residues in catalysis. The specific activities for formation of dihydroxyphenylalanine by the S395A, S395T, and S396A enzymes are 1.3, 26, and 69% of the wild-type values, respectively. Both the S395A and S396A enzymes bind a stoichiometric amount of iron and exhibit wild-type spectra when complexed with dopamine. The K(M) values for tyrosine, 6-methyltetrahydropterin, and tetrahydrobiopterin are unaffected by replacement of either residue with alanine. Although the V(max) value for tyrosine hydroxylation by the S395A enzyme is decreased by 2 orders of magnitude, the V(max) value for tetrahydropterin oxidation by either the S395A or the S396A enzyme is unchanged from the wild-type value. With both mutant enzymes, there is quantitative formation of 4a-hydroxypterin from 6-methyltetrahydropterin. These results establish that Ser395 is required for amino acid hydroxylation but not for cleavage of the oxygen-oxygen bond, while Ser396 is not essential. These results also establish that cleavage of the oxygen-oxygen bond occurs in a separate step from amino acid hydroxylation.     

Selective inhibition of proline hydroxylation by 3,4-dehydroproline

The effect of proline analogs on peptidyl proline hydroxylation has been studied in vivo using aerated root slices of Daucus carota. One analog, 3,4-dehydroproline, acted at micromolar concentrations to rapidly and selectively inhibit peptidyl proline hydroxylation. A structurally altered hydroxyproline-rich cell wall glycoprotein was synthesized and secreted by dehydroproline-treated tissue. The capacity to hydroxylate proline recovered slowly following a short pulse treatment with the analog, with a halftime for recovery of about 24 hours. Recovery was not altered by supplying exogenous proline. Dehydroproline had little effect on the induction of nitrate reductase by nitrate, nor on wound-induced increases in amino acid uptake and protein synthesis. In contrast, other proline analogs inhibit proline hydroxylation only at millimolar concentrations. It is hypothesized that dehydroproline acts as an enzyme-activated suicide inhibitor of prolyl hydroxylase. This analog should become a useful tool for elucidating the functional significance of hydroxyproline-rich glycoproteins.