Spontaneous mutation in Big Blue transgenic mice: analysis of age, gender, and tissue type.

A lacI-containing transgenic mouse mutation detection system (Big Blue) was used to determine the frequency and spectrum of spontaneous mutation in two rapidly dividing tissues (male germ cells and thymus) and one slowly dividing tissue (brain) at 3 and 10 months of age. By screening 9.4 million lambda plaques, a total of 343 circular mutant plaques were recovered from the three tissues.

Tryptophan fluorescence of human phenylalanine hydroxylase produced in Escherichia coli.

Human phenylalanine hydroxylase (hPAH) contains three tryptophan residues (W120, W187, and W326). All three tryptophan residues were mutated to phenylalanine either as single mutants or in combination, and one tryptophan was also mutated to isoleucine. The mutant enzymes were expressed in Escherichia coli and purified as fusion proteins with maltose-binding protein and a linker region containing a recognition site for the serine protease factor Xa.

p53 wild-type and p53 nullizygous Big Blue transgenic mice have similar frequencies and patterns of observed mutation in liver, spleen and brain.

Transgenic mouse mutation detection systems offer a powerful tool for analysis of spontaneous and induced mutations in vivo. Mice doubly transgenic for a null mutation of the p53 tumor suppressor gene and a lambda shuttle vector harboring the lacI gene were utilized to examine the rate and pattern of spontaneous somatic mutation of the lacI transgene in vivo. Three somatic tissues were examined: liver, spleen and brain.

Resonance Raman studies of catecholate and phenolate complexes of recombinant human tyrosine hydroxylase.

Human tyrosine hydroxylase isoform 1 (hTH1) was expressed in Escherichia coli, purified as the apoenzyme, and reconstituted with iron. The resonance Raman spectra of hTH1 complexed with dopamine, noradrenaline, tyramine, and catechol have been studied and compared to those obtained for TH isolated from bovine adrenal glands or rat phaeochromocytoma tissue. A TH-phenolate complex is reported for the first time.

Phosphorylation and activation of human tyrosine hydroxylase in vitro by mitogen-activated protein (MAP) kinase and MAP-kinase-activated kinases 1 and 2.

Mitogen-activated protein-kinase (MAP) kinase-activated protein kinases 1 and 2 (MAPKAP kinase-1, MAPKAP kinase-2), were found to phosphorylate bacterially expressed human tyrosine hydroxylase in vitro at comparable rates to other proteins thought to be physiological substrates of these protein kinases. The phosphorylation of all four alternatively spliced forms of human tyrosine hydroxylase by MAPKAP kinases-1 and -2 reached plateau values at 1 mol/mol subunit and 2 mol/mol subunit, respectively; the sites of phosphorylation were identified as Ser40 (MAPKAP kinase-1) and Ser19 and Ser40 (MAPKAP kinase-2). In contrast to calmodulin-dependent protein kinase-II, which phosphorylates Ser19 faster than Ser40, MAPKAP kinase-2 phosphorylated Ser40 about twice as fast as Ser19.

Conformation and interaction of phenylalanine with the divalent cation at the active site of human recombinant tyrosine hydroxylase as determined by proton NMR.

Recombinant human tyrosine hydroxylase has been purified as a metal-free apoenzyme (apo-hTH1) which tightly binds one Fe2+, Co2+, or Zn2+ per subunit with activation only by Fe2+ and competitive inhibition by the other cations. L-tyrosine and L-phenylalanine are alternative substrates for this enzyme, giving similar Vmax values, although the KM value for phenylalanine is about 8-fold greater than for tyrosine. Apo-hTH1 enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 values of the protons of enzyme-bound phenylalanine both in the presence and in the absence of the oxidized form of the cofactor L-erythro-7,8-dihydrobiopterin (BH2), which was used as an inactive analog of the natural cofactor (6R)-1-erythro-tetrahydrobiopterin (BH4).

The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and 1H-NMR spectroscopy.

Three isoforms of human tyrosine hydroxylase were expressed in Escherichia coli and purified to homogeneity as the apoenzymes (metal-free). The apoenzymes exhibit typical tryptophan fluorescence emission spectra when excited at 250-300 nm. The emission maximum (342 nm) was not shifted by the addition of metal ions, but reconstitution of the apoenzymes with Fe(II) at pH 7-9 reduced the fluorescence intensity by about 35%, with an end point at 1.

Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation. Structure/activity studies and mechanistic implications.

Three isozymes of human tyrosine hydroxylase (hTH1, hTH2 and hTH4) were expressed in Escherichia coli and purified to homogeneity. Natural catecholamines and related synthetic compounds were found to be potent inhibitors, competitive to the tetrahydrobiopterin cofactor, of all the isozymes. Combining visible spectroscopy and equilibrium-binding studies, it was found that catecholamines bind to hTH1 and hTH2 with a stoichiometry of about 1.

7-substituted pterins in humans with suspected pterin-4a-carbinolamine dehydratase deficiency. Mechanism of formation via non-enzymatic transformation from 6-substituted pterins.

A recently described new form of hyperphenylalaninemia is characterized by the excretion of 7-substituted isomers of biopterin and neopterin and 7-oxo-biopterin in the urine of patients. It has been shown that the 7-substituted isomers of biopterin and neopterin derive from L-tetrahydrobiopterin and D-tetrahydroneopterin and are formed during hydroxylation of phenylalanine to tyrosine with rat liver dehydratase-free phenylalanine hydroxylase. We have now obtained identical results using human phenylalanine hydroxylase.

Purification and characterization of the blue-green rat phaeochromocytoma (PC12) tyrosine hydroxylase with a dopamine-Fe(III) complex. Reversal of the endogenous feedback inhibition by phosphorylation of serine-40.

Tyrosine hydroxylase (TH) was purified from tumours of rat phaeochromocytoma (PC12) cells by a three-step purification procedure giving 30 mg of pure enzyme in 3 days. The enzyme sedimented with an S(eo),w value of 9.2 S and revealed an apparent subunit molecular mass of 62 kDa with a minor 60 kDa component.

Inactivation of purified phenylalanine hydroxylase by dithiothreitol.

Purified rat liver phenylalanine hydroxylase is inactivated in vitro by ascorbate and thiol compounds, dithiothreitol being the most effective inhibitor, with a second order rate constant for the inactivation of 0.066 +/- 0.002 mM-1.

Phosphorylation of human recombinant tyrosine hydroxylase isoforms 1 and 2: an additional phosphorylated residue in isoform 2, generated through alternative splicing.

The single human tyrosine hydroxylase (TH) gene generates four different mRNA species through alternative splicing events. TH-1 and TH-2 mRNAs are expressed mostly in the brain. We have produced large amounts of the corresponding proteins in Escherichia coli to analyze their respective molecular characteristics.

Recombinant human tyrosine hydroxylase isozymes. Reconstitution with iron and inhibitory effect of other metal ions.

Human tyrosine 3-monooxygenase (tyrosine hydroxylase) exists as four different isozymes (TH1-TH4), generated by alternative splicing of pre-mRNA. Recombinant TH1, TH2 and TH4 were expressed in high yield in Escherichia coli. The purified isozymes revealed high catalytic activity [when reconstituted with Fe(II)] and stability at neutral pH.

EPR and 1H-NMR spectroscopic studies on the paramagnetic iron at the active site of phenylalanine hydroxylase and its interaction with substrates and inhibitors.

The paramagnetic iron at the active site of highly purified, catalytically active phenylalanine hydroxylase was studied by EPR at 3.6 K and one-dimensional 1H-NMR spectroscopy at 293 K. The EPR-detectable iron of the bovine enzyme was found to be present as a high-spin form (S = 5/2) in different ligand field symmetries depending on medium conditions (buffer ions) and the presence of ligands known to bind at the active site.

Cooperative homotropic interaction of L-noradrenaline with the catalytic site of phenylalanine 4-monooxygenase.

Catecholamines (adrenaline, noradrenaline and dopamine) are potent inhibitors of phenylalanine 4-monooxygenase (phenylalanine hydroxylase, EC 1.14.16.

Phenylalanine as substrate for tyrosine hydroxylase in bovine adrenal chromaffin cells.

Incubation of bovine chromaffin cells with L-[14C]phenylalanine resulted in label accumulation in catecholamines at about 30% of the rate seen with L-tyrosine as precursor. Studies with purified tyrosine hydroxylase (EC 1.14.

pH-dependent release of catecholamines from tyrosine hydroxylase and the effect of phosphorylation of Ser-40.

Bovine adrenal tyrosine hydroxylase (TH) is isolated in a partially inhibited state with the feed-back inhibitors adrenaline and noradrenaline tightly coordinated to high-spin (S = 5/2) Fe(III) at the active site. In addition to the charge-transfer interaction with iron, an additional charged group in the polypeptide chain, with an apparent pKa of about 5.3 at 4 degrees C, is involved in the binding of catecholamines.

Identification of protein phosphatase 2A as the major tyrosine hydroxylase phosphatase in adrenal medulla and corpus striatum: evidence from the effects of okadaic acid.

(i) The major sites on bovine adrenal tyrosine hydroxylase (TH) phosphorylated by calmodulin-dependent multiprotein kinase (CaM-MPK) and cyclic AMP-dependent protein kinase were shown to be Ser-19 and Ser-40, respectively, while Ser-40 was also phosphorylated slowly by CaM-MPK. (ii) Type 2A and type 2C phosphatases accounted for approximately 90% and approximately 10% of TH phosphatase activity, respectively, in extracts of adrenal medulla and corpus striatum assayed at near physiological free Mg2+ (1 mM), while type 1 and type 2B phosphatases had negligible activity towards TH. (iii) Incubation of adrenal chromaffin cells with okadaic acid increased TH phosphorylation by 206% and activity by 77%, establishing that type 2A phosphatases play a major role in regulating TH in vivo.

[From butterflies to neurobiology and the diagnosis of AIDS. The 100th anniversary of the discovery of pteridines].

The first report on the isolation of pteridines from biological materials was published in 1889. During the last 100 years a large number of pteridines have been isolated from many different organisms and have been shown to be involved in several biochemical processes. The best characterized biologically occurring unconjugated pteridine, i.

Resonance Raman studies on the blue-green-colored bovine adrenal tyrosine 3-monooxygenase (tyrosine hydroxylase). Evidence that the feedback inhibitors adrenaline and noradrenaline are coordinated to iron.

Tyrosine 3-monooxygenase (tyrosine hydroxylase) is a non-heme iron, tetrahydropterin-dependent enzyme which catalyzes the rate-limiting step in the biosynthesis of catecholamines. The highly purified bovine adrenal enzyme contains an unusual blue-green chromophore with lambda max at around 700 nm (epsilon = 1.3 (mM subunit enzyme)-1 cm-1).