Torgeir Flatmark

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 ≈90% and ≈ 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.

Crystal structure of the ternary complex of the catalytic domain of human phenylalanine hydroxylase with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine, and its implications for the mechanism of catalysis and substrate activation.

Phenylalanine hydroxylase catalyzes the stereospecific hydroxylation of L-phenylalanine, the committed step in the degradation of this amino acid. We have solved the crystal structure of the ternary complex (hPheOH-Fe(II).BH(4).THA) of the catalytically active Fe(II) form of a truncated form (DeltaN1-102/DeltaC428-452) of human phenylalanine hydroxylase (hPheOH), using the catalytically active reduced cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and 3-(2-thienyl)-L-alanine (THA) as a substrate analogue. The analogue is bound in the second coordination sphere of the catalytic iron atom with the thiophene ring stacking against the imidazole group of His285 (average interplanar distance 3.8A) and with a network of hydrogen bonds and hydrophobic contacts. Binding of the analogue to the binary complex hPheOH-Fe(II).BH(4) triggers structural changes throughout the entire molecule, which adopts a slightly more compact structure. The largest change occurs in the loop region comprising residues 131-155, where the maximum r.m.s. displacement (9.6A) is at Tyr138. This loop is refolded, bringing the hydroxyl oxygen atom of Tyr138 18.5A closer to the iron atom and into the active site. The iron geometry is highly distorted square pyramidal, and Glu330 adopts a conformation different from that observed in the hPheOH-Fe(II).BH(4) structure, with bidentate iron coordination. BH(4) binds in the second coordination sphere of the catalytic iron atom, and is displaced 2.6A in the direction of Glu286 and the iron atom, relative to the hPheOH-Fe(II).BH(4) structure, thus changing its hydrogen bonding network. The active-site structure of the ternary complex gives new insight into the substrate specificity of the enzyme, notably the low affinity for L-tyrosine. Furthermore, the structure has implications both for the catalytic mechanism and the molecular basis for the activation of the full-length tetrameric enzyme by its substrate. The large conformational change, moving Tyr138 from a surface position into the active site, may reflect a possible functional role for this residue.

Electron carriers of the bovine adrenal chromaffin granules.

Abstract Chromaffin granules of bovine adrenal medulla essentially free of contamination from mitochondria, lysosomes and fragments of endoplasmic reticulum, have been lysed by hypo-osmotic shock. The membrane fraction has been shown to contain a b -type cytochrome (cytochrome b 561 ), flavoprotein(s) and NADH: (acceptor) oxidoreductase (EC 1.6.99.3) with ferricyanide, 2,6-dichlorophenolindophenol (DCIP), bovine heart ferricytochrome c and the endogenous ferricytochrome b 561 as acceptors. Furthermore, the membrane fraction catalyzes the rapid oxidation of bovine heart ferrocytochrome c . This enzymic activity, which is completely inhibited by CN(3 · 10 −4 M) and CO, is not attributed to cytochrome a + a 3 (ferrocytochrome c : O 2 oxidoreductase, EC 1.9.3.1).

On the mechanism of induction of the enzyme systems for peroxisomal β-oxidation of fatty acids in rat liver by diets rich in partially hydrogenated fish oil

In this paper, we describe the early biochemical changes in liver cells that occur in rats fed a semisynthetic diet containing 20% (w/w) partially hydrogenated fish oil. Within hours the level of ornithine decarboxylase (ODC) increased, peaked at about 24 h (11-fold increase) and returned to subnormal levels within 48 h. The diet evoked a similar rapid increase in the cellular level of mRNA for the bifunctional enzyme of peroxisomal beta-oxidation (enoyl-CoA hydratase: beta-hydroxyacyl-CoA dehydrogenase (HD)) (12-fold), followed by increases in the specific content of HD protein (3-fold) and the capacity for beta-oxidation in peroxisomes (5.3-fold). The cellular level of long-chain acyl-CoA increased 2.1-fold. By contrast, no significant changes were observed in the specific activities of ornithine decarboxylase, peroxisomal beta-oxidation activity and microsomal omega-hydroxylation as well as the level of long-chain acyl-CoA in livers of rats fed (1 week) diets containing 20% (w/w) soybean oil with added 3 or 6% (w/w) of either elaidic acid (18:1(11) (trans)), brassidic acid (22:1(13) (trans)) or erucic acid (22:1(13) (cis)). Expression of normal levels of mRNA for the bifunctional enzyme was also found. Morphometric analyses revealed no proliferation of peroxisomes in these fatty acid-supplemented diets, in contrast to that observed with the partially hydrogenated fish oil diet. These results are consistent with the proposal (Flatmark, T., Christiansen, E.N. and Kryvi, H. (1983) Biochim. Biohys. Acta 753, 460-466) that components in dietary oils, different from C22:1 cis and trans fatty acids, are responsible for the pleiotropic responses evoked in target cells. Thus, the pattern of response induced by partially hydrogenated fish oil mimics those induced by xenobiotic compounds collectively termed peroxisome proliferators.

Energy-dependent accumulation of iron by isolated rat liver mitochondria. I. General features

Abstract 1. 1. Rat liver mitochondria isolated in sucrose-N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid (HEPES) medium rapidly accumulate iron from the suspending medium when 59Fe(III)-sucrose is used as a soluble and stable model complex. 2. 2. The accumulation proceeds by two different mechanisms, i.e. by an energy-independent and an energy-dependent (uncoupler and cyanide-sensitive) mechanism which have different pH and temperature dependencies. Both processes require Mg2+ and are inhibited by Pi and chelating agents (e.g. EDTA), and reveal maximal rates at an iron concentration of 0.2–0.3 mM. At pH 7.2 the energy-dependent accumulation is found to be 10–15 nmoles iron per mg of protein and is completed in less than 45 s. On the other hand, the energy-independent accumulation proceeds at an approximate rate of 25 nmoles iron per mg of protein per min, with a maximal load of 180–240 nmoles iron per mg of protein. 3. 3. Together with our previous studies (Romslo, I. and Flatmark, T. (1972) Abstr. Commun. Meet. Fed. Eur. Biochem. Soc. 8, No. 639) these results strongly suggest that the energy-dependent mechanism of accumulation is responsible for a movement of iron across the mitochondrial inner membrane.

Cytochrome b561 of the bovine adrenal chromaffin granules. A high potential b-type cytochrome

Simultaneous potentiometric and spectrophotometric assays of cytochrome b561 oxidation-reduction in chromaffin granule membrane preparations (depleted of catecholamines and ATP) have been carried out under strictly anaerobic conditions. The cytochrome oxidation-reduction is described by a one-electron transition with an apparent midpoint potential at pH 7.0 (E′m7) of +140 mV. This value is in good agreement with the rate and extent of its aerobic reduction by ascorbate; the K′m for the reductant was found to be 0.34 mM. The available evidence therefore confirms our previous suggestion that cytochrome b561 is a unique mammalian b-type cytochrome of chromaffin tissues.

Generation of Reactive Oxygen Species by Tyrosine Hydroxylase: A Possible Contribution to the Degeneration of Dopaminergic Neurons?

Abstract: It has been suggested that idiopathic parkinsonism, characterized by a loss of dopaminergic neurons of the nigrostriatal pathway, is due to the intracellular generation of reactive oxygen species, generated by a nonenzymatic or enzymatic partial reduction of dioxygen. Based on in vitro studies of the iron‐containing monooxygenase tyrosine hydroxylase (TH), evidence is presented that this enzyme system may also contribute to such an oxidative stress. Thus, the purified and Fe2+‐reconstituted recombinant human enzyme shows a time‐ and temperature‐dependent partial uncoupling of the hydroxylation of l‐tyrosine with the natural cofactor (6R)‐tetrahydrobiopterin, resulting in the formation of H2O2. The degree of uncoupling of the hydroxylation reaction is significantly higher when certain substrate analogues, notably the 7‐substituted isomer (7‐tetrahydrobiopterin) of the natural cofactor, is used. In the presence of H2O2 and Fe2+, the addition of TH increases the production of the highly reactive •OH radical, probably via a Fenton type of reaction. It is not clear whether this in vitro reaction can mediate cellular injury in vivo. However, it is known that the distribution of TH in the central and peripheral nervous system often corresponds to that of the neuronal degeneration in idiopathic parkinsonism, a finding that is compatible with a pathogenetic effect of TH.

Time course of changes in nociception after 5,6-dihydroxytryptamine lesions of descending 5-HT pathways

Intrathecal injection of 5,6-dihydroxytryptamine (5,6-DHT) in rats produced selective lesions of the descending 5-HT pathways. Spinal 5-HT levels gradually fell to less than 10% of controls within 10 days of 5,6-DHT administration with no recovery evident within 4 weeks. The uptake of 14C-5-HT into crude spinal synaptosomes was similarly reduced. The uptake of 3H-NA into spinal synaptosomes was unaffected, as was the uptake of 14C-5-HT and 3H-NA into cortical synaptosomes. Following 5,6-DHT, tail-flick latencies were reduced by 20-30% during the first post-injection week, but returned to control levels during the second week. Intrathecal or systemic administration of the 5-HT receptor antagonist metergoline significantly reduced latencies of normal rats and of 5,6-DHT treated rats tested after the second week when the response was normalized. Metergoline did not, however, further reduce the latencies of lesioned rats during the first post-injection week. It is concluded that functional adaptation involving 5-HT neurotransmission compensated for the selective lesion of descending 5-HT pathways induced by 5,6-DHT.

Conformational Properties and Stability of Tyrosine Hydroxylase Studied by Infrared Spectroscopy EFFECT OF IRON/CATECHOLAMINE BINDING AND PHOSPHORYLATION

The conformation and stability of recombinant tetrameric human tyrosine hydroxylase isoenzyme 1 (hTH1) was studied by infrared spectroscopy and by limited tryptic proteolysis. Its secondary structure was estimated to be 42% α-helix, 35% β-extended structures (including β-sheet), 14% β-turns, and 10% nonstructured conformations. Addition of Fe(II) or Fe(II) plus dopamine to the apoenzyme did not significantly modify its secondary structure. However, an increased thermal stability and resistance to proteolysis, as well as a decreased cooperativity in the thermal denaturation transition, was observed for the ligand-bound forms. Thus, as compared with the apoenzyme, the ligand-bound subunits of hTH1 showed a more compact tertiary structure but weaker intersubunit contacts within the protein tetramer. Phosphorylation of the apoenzyme by cyclic AMP-dependent protein kinase did not change its overall conformation but allowed on iron binding a conformational change characterized by an increase (about 10%) in α-helix and protein stability. Our results suggest that the conformational events involved in TH inhibition by catecholamines are mainly related to modifications of tertiary and quaternary structural features. However, the combined effect of iron binding and phosphorylation, which activates the enzyme, also involves modifications of the protein secondary structure.

Soluble tyrosine hydroxylase (tyrosine 3-monooxygenase) from bovine adrenal medulla: large-scale purification and physicochemical properties

A new procedure that permits large-scale purification of tyrosine 3-monooxygenase (tyrosine hydroxylase) (L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) from the cytosolic fraction of bovine adrenal medulla is described. The homogenous enzyme revealed a subunit Mr of 60,000 and a specific activity of 425 nmol.min-1.mg-1. The N-terminal amino-acid sequence (27 residues) revealed 89% homology with the human pheochromocytoma enzyme as deduced from its cDNA sequence. The pure enzyme contained 0.66 +/- 0.09 mol iron, 0.13 mol zinc and 0.62 +/- 0.04 mol phosphate per mol subunit of Mr = 60,000. A broad light absorption band with its maximum around 700 nm (epsilon 700 nm = 1.3 (mM monomer)-1.cm-1) explains its blue-green color. EPR spectra at 3.6 K revealed high-spin Fe(III) (S = 5/2) in an environment of nearly axial symmetry (g values at 7.2-6.7, 4.7-5.3 and 1.9-2.0). A close correlation was observed between the absorbance at 700 nm and the intensity of the axial type of EPR spectrum. The absorption peak at 700 nm is compatible with a ligand-to-iron charge-transfer transition as a result of catecholate coordination to the iron. Physicochemical studies suggest that the enzyme does not undergo such major substrate- or cofactor-induced conformational changes as have been reported for the related enzyme, phenylalanine hydroxylase.