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

Haavik, Jan; Andersson, Karin; Petersson, Leif; Flatmark, Torgeir

doi:10.1016/0167-4838(88)90019-2

Cited by 87 publications

(98 citation statements)

References 63 publications

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“…In the final step of the purification, the enzyme was eluted as a single symmetrical peak on gel filtration (Fig. 1 C), corresponding to the tetrameric form of the enzyme [8]. In this experiment, the yield of the purified enzyme was about 10% and 55 mg pure THI was obtained.…”

Section: Purification Of' Recombinant Human Tyrosine Hydroxylase Isozmentioning

confidence: 99%

“…THI, TH2 and TH4 were purified to homogeneity using a modification of our procedure previously described for the bovine adrenal enzyme (see Methods) [8]. The three last steps in the purification of THI are shown in Fig.…”

Section: Purification Of' Recombinant Human Tyrosine Hydroxylase Isozmentioning

confidence: 99%

“…The tyrosine hydroxylase isozymes were purified from the bacterial pellets by a modification of the procedure previously described for the bovine adrenal enzyme [8]. In brief, the bacteria (from 84 1 of culture) were first disrupted by passage through a French press (type FA-073 from SLM Instruments, Urbana IL) at 69 MPa and diluted in 20 mM Tris/HCl, containing 5% (mass/vol.…”

Section: Tyrosine Hydroxylase Expression and Purijicationmentioning

confidence: 99%

“…Metal content was determined using a PerkinElmer model 402 atomic absorption spectrophotometer equipped with a graphite furnace (type HGA-76B from Perkin-Elmer). The concentration of the purified isozymes was measured using an absorption coefficient of A&&nm = 10.4 cm-' [8].…”

Section: Other Proceduresmentioning

confidence: 99%

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Recombinant human tyrosine hydroxylase isozymes

Haavik

Bourdellès

Martı́nez

et al. 1991

European Journal of Biochemistry

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109

131

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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 Escherichiu coli. The purified isozymes revealed high catalytic activity [when reconstituted with Fe(II)] and stability at neutral pH. The isozymes as isolated contained 0.04-0.1 atom iron and 0.02-0.06 atom zinc/enzyme subunit. All three isozymes were rapidly activated (1 3 -40-fold) by incubation with Fe(I1) salts (concentration of iron at half-maximal activation = 6-14 pM), and were inhibited by other divalent metal ions, e.g. Zn(II), Co(I1) and Ni(I1). They all bind stoichiometric amounts of Fe(I1) and Zn(I1) with high affinity (Kd = 0.2-3 pM at pH 5.4-6.5). Similar time courses were observed for binding of Fe(I1) and enzyme activation. In the absence of any free Fe(I1) or Zn(II), the metal ions were released from the reconstituted isozymes. The dissociation was favoured by acidic pH, as well as by the presence of metal chelators and dithiothreitol. The potency of metal chelators to remove iron from the hydroxylase correlated with their ability to inhibit the enzyme activity. These studies show that tyrosine hydroxylase binds iron reversibly and that its catalytic activity is strictly dependent on the presence of this metal.Tyrosine 3-monooxygenase (tyrosine hydroxylase) is a tetrahydropteridine-dependent enzyme which catalyses the rate-limiting step in the biosynthesis of catecholamines [ 11. Early studies showed that the enzyme activity is stimulated by ferrous iron and inhibited by certain iron chelators [2 -51.Subsequently, it was shown that the enzyme isolated from various tissues contained significant amounts of iron [6 -81, which seems to be involved in oxygen activation, an essential step in the catalytic reaction [6-81. However, partly due to the difficulties involved in obtaining pure tyrosine hydroxylase, no systematic study has been performed in order to determine the metal-binding properties of this enzyme.The recent expression of cloned tyrosine hydroxylase in high yield in eukaryotic cells using the baculovirus system [9, 101 and in prokaryotic cells [ 10 a] has, however, changed this situation. Human tyrosine hydroxylase exists as four different isozymes (TH1-TH4), generated through alternative splicing of pre-mRNA [ l l -131. Since these isozymes have different amino acid sequences in their regulatory N-terminal region and different distribution in the nervous system [14], it has been postulated that the occurrence of multiple molecular forms of the enzyme has important functional consequences. Previous studies, in which the isozymes have been expressed in low quantities in eukaryotic systems, have also demonstrated different kinetic properties of the various isozymes [15, 161. Correspondence to J. Haavik, Department of Biochemistry, UniAbbreviations. TH 1 -TH4, tyrosine hydroxylase isozymes 1 -4. Enzyme. Tyrosine 3-monooxygenase or tyrosine hydroxylase versity of Bergen, N-5009 Berge...

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Section: Purification Of' Recombinant Human Tyrosine Hydroxylase Isozmentioning

confidence: 99%

Section: Purification Of' Recombinant Human Tyrosine Hydroxylase Isozmentioning

confidence: 99%

Section: Tyrosine Hydroxylase Expression and Purijicationmentioning

confidence: 99%

Section: Other Proceduresmentioning

confidence: 99%

See 2 more Smart Citations

Recombinant human tyrosine hydroxylase isozymes

Haavik

Bourdellès

Martı́nez

et al. 1991

European Journal of Biochemistry

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109

131

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“…Native TH purified from vertebrate tissues contain bound catecholamines, and catecholamine binding prevents the release of the bound iron (47)(48)(49). Because E. coli contains no catecholamines, iron may be less strongly bound to TH enzymes expressed in bacteria than to native TH enzymes extracted from tissues.…”

Section: Fig 6 Inhibition Of Dth Activity By Dopaminementioning

confidence: 99%

Differential Regulation of Drosophila Tyrosine Hydroxylase Isoforms by Dopamine Binding and cAMP-dependent Phosphorylation

Vié

Cigna

Toci

et al. 1999

Journal of Biological Chemistry

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Tyrosine hydroxylase (TH) catalyzes the first step in dopamine biosynthesis in Drosophila as in vertebrates. We have previously reported that tissue-specific alternative splicing of the TH primary transcript generates two distinct TH isoforms in Drosophila, DTH I and DTH II (Birman, S., Morgan, B., Anzivino, M., and Hirsh, J. (1994) J. Biol. Chem. 269, 26559 -26567). Expression of DTH I is restricted to the central nervous system, whereas DTH II is expressed in non-nervous tissues like the epidermis. The two enzymes present a single structural difference; DTH II specifically contains a very acidic segment of 71 amino acids inserted in the regulatory domain. We show here that the enzymatic and regulatory properties of vertebrate TH are generally conserved in insect TH and that the isoform DTH II presents unique characteristics. The two DTH isoforms were expressed as apoenzymes in Escherichia coli and purified by fast protein liquid chromatography. The recombinant DTH isoforms are enzymatically active in the presence of ferrous iron and a tetrahydropteridine co-substrate. However, the two enzymes differ in many of their properties. DTH II has a lower K m value for the cosubstrate (6R)-tetrahydrobiopterin and requires a lower level of ferrous ion than DTH I to be activated. The two isoforms also have a different pH profile. As for mammalian TH, enzymatic activity of the Drosophila enzymes is decreased by dopamine binding, and this effect is dependent on ferrous iron levels. However, DTH II appears comparatively less sensitive than DTH I to dopamine inhibition. The central nervous system isoform DTH I is activated through phosphorylation by cAMPdependent protein kinase (PKA) in the absence of dopamine. In contrast, activation of DTH II by PKA is only manifest in the presence of dopamine. Site-directed mutagenesis of Ser 32 , a serine residue occurring in a PKA site conserved in all known TH proteins, abolishes phosphorylation of both isoforms and activation by PKA. We propose that tissue-specific alternative splicing of TH has a functional role for differential regulation of dopamine biosynthesis in the nervous and non-nervous tissues of insects.Tyrosine hydroxylase (TH 1 ) (tyrosine 3-monooxygenase, EC 1.14.16.2) is an eukaryotic enzyme catalyzing the first and rate-limiting step in dopamine and other catecholamine biosynthesis, i.e. the hydroxylation of the monophenol amino acid L-tyrosine to produce the ortho-diphenol L-dihydroxyphenylalanine (2, 3). The enzyme is active in the presence of ferrous iron, O 2 , and a tetrahydrobiopterin co-substrate. A single gene encodes TH, which is required for embryonic development and survival in mammals (4, 5). In vertebrates, TH activity is exquisitely regulated at each step of its expression: control of gene transcription, RNA alternative processing, mRNA stability, and direct modulation of the enzyme by catecholamine feedback inhibition and protein kinase activation (6 -8).In contrast, much less is known on the regulatory properties of tyrosine hydroxylase in insects. Muta...

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Tyrosine Hydroxylase

Kaufman¹

1995

Advances in Enzymology - And Related Areas of Molecular Biology

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a b o r a t o r y of N e u r o c h e m i s t r y , N a t i o n a l I n s t i t u t e of M e n t a l H e a l t h , B e t h e s d a , Maryland the first step in the biosynthesis of norepinephrine and epinephrine involved the conversion of tyrosine to 3,4dihydroxyphenylalanine (dopa), followed by decarboxylation of the dopa to dopamine, which was then hydroxylated in the p position of the side chain and methylated on the amino group to form norepinephrine and epinephrine, respectively (Fig. 1). Although the evidence in favor of the pathway proposed by Blaschko continued to mount (5-8), and it is now widely accepted as the only quantitatively important one, the enzyme responsible for the ring hydroxylation SEYMOUR KAUFMAN droxylation of phenylalanine (17, 18), this product was also shown to be found during the TH-catalyzed conversion of tyrosine to dopa. (For a further discussion of this aspect of the reaction, see Section XI and Fig. 7.) Intracellular Localization and Regional DistributionAs already mentioned, TH is present in brain, adrenal medulla, and sympathetically innervated tissues. Its intracellular location in these tissues, however, has been the subject of controversy. Udenfriend and co-workers (12) reported that in guinea pig brain all of the activity, and in beef adrenal medulla, most of the activity, was particle bound. They further concluded that the hydroxylase found in the soluble fraction of adrenal medulla was originally present in particles from which it had been released by the homogenization process, a conclusion that was strengthened by the finding that about 90% of the activity was sedimentable after centrifugation for 1 h at 144,000 g. These findings appeared to support the proposal that all of the enzymes involved in catecholamine biosynthesis are localized within a catecholamine-containing granule (19).There is a growing body of evidence that contradicts the conclusion that TH is present intracellularly within a granule (20, 21). For the enzyme from both adrenal medulla and nerve tissue, it has been found that a major fraction of the activity is present in the highspeed supernatant fraction and that the distribution of hydroxylase activity is a function of the composition of the homogenization medium; homogenization in isotonic KC1 leads to more enzyme in the supernatant fraction than does homogenization in isotonic sucrose (21). At least part of the uncertainty about the intracellular localization of TH can be traced to the tendency of the enzyme from bovine adrenal medulla, and probably from other tissues as well, to aggregate and to adsorb to subcellular organelles (21).Although there is now a near consensus in favor of the view that the high molecular weight form of the enzyme in adrenal medulla is an artifact that is formed during cell disruption and that it is not in equilibrium with the native, nonaggregated form (22), that conclusion does not apply to brain where the enzyme appears to exist in two distinct forms, a soluble and a membrane-bound form. In fact, in the earliest studies...

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

Cited by 87 publications

References 63 publications

Recombinant human tyrosine hydroxylase isozymes

Recombinant human tyrosine hydroxylase isozymes

Differential Regulation of Drosophila Tyrosine Hydroxylase Isoforms by Dopamine Binding and cAMP-dependent Phosphorylation

Tyrosine Hydroxylase

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