Expression of rat tyrosine hydroxylase in insect tissue culture cells and purification and characterization of the cloned enzyme.

Fitzpatrick, Paul F.; Chlumsky, Lawrence J.; Daubner, S. Colette; O’Malley, Karen L.

doi:10.1016/s0021-9258(19)39937-5

Cited by 49 publications

(28 citation statements)

References 38 publications

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“…Solvent Isotope Effects. The steady-state kinetic parameters of tyrosine hydroxylase are pH dependent (Fitzpatrick et al, 1990). The Fmax-pH profile is bell-shaped, suggesting the possibility of acid or base catalysis.…”

Section: Resultsmentioning

confidence: 96%

“…The physiological substrate tetrahydrobiopterin (BH4) was used as cosubstrate because its lower £M value allowed lower levels of tetrahydropterin to be used compared to those required with 6methyltetrahydropterin, decreasing the background rates due to tetrahydrobiopterin autoxidation. Also, with BH4 as substrate under the conditions of these experiments (pH 6.5, 30 °C), oxygen is saturating when tyrosine is the substrate (Fitzpatrick et al, 1990). The assay that was used followed the rate of production of dihydrobiopterin, to ensure the total rate of turnover was measured, independently of whether hydroxylation of the amino acid substrate occurred.…”

Section: Resultsmentioning

confidence: 99%

“…Sheep dihydropterin reductase was from Sigma. Rat tyrosine hydroxylase expressed in insect tissue culture cells was purified as described previously (Fitzpatrick et al, 1990). Bovine adrenal tyrosine hydroxylase was purified to between 50 and 80% homogeneity by the method of Oka et al (1982).…”

Section: Methodsmentioning

confidence: 99%

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Studies of the rate-limiting step in the tyrosine hydroxylase reaction: alternate substrates, solvent isotope effects, and transition-state analogs

Fitzpatrick¹

1991

Biochemistry

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Tyrosine hydroxylase catalyzes the formation of dihydroxyphenylalanine from tyrosine, utilizing a tetrahydropterin and molecular oxygen as cosubstrates. Several approaches were taken to examining the identity of the rate-limiting step in catalysis. Steady-state kinetic parameters were determined with a series of ring-substituted phenylalanines. The Vmax value was unchanged with substrates ranging in reactivity from tyrosine to 4-fluorophenylalanine. Neither 4-pyridylalanine N-oxide, a model of tyrosine phenoxide, nor 4-hydroxy-3-pyridylalanine N-oxide or alpha-amino-3-hydroxy-4-pyridone-1- propionic acid, models of a hydroxycyclohexadienone intermediate, was an effective inhibitor. There was no solvent isotope effect on either the Vmax or the V/KTyr value. These results establish that no chemistry occurs at the amino acid in the rate-limiting step and no exchangeable proton is in flight in the rate-limiting step. The results are consistent with a model in which the slow step in catalysis is formation of the hydroxylating intermediate.

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

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Studies of the rate-limiting step in the tyrosine hydroxylase reaction: alternate substrates, solvent isotope effects, and transition-state analogs

Fitzpatrick¹

1991

Biochemistry

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“…Recombinant sources of TyrH appear to afford the best chance of improving knowledge of that enzyme, which lags behind what is known about PAH. Any one of the human isoforms may be heterologously expressed in eukaryotic cells, − but in order to start with phosphate-free enzyme, E. coli expression appears to be preferred. ,, This is also the case for the single allelic form of the heavily studied rat PC12 TyrH, − which is known to be partially phosphorylated by heterologous expression in baculovirus. , …”

Section: Molecular Structure Isoforms and Expressionmentioning

confidence: 99%

“…This source of TyrH is purified from a catecholamine-rich tissue, and in some situations appears to copurify with a catecholamine, yielding a blue-green color from the Fe 3+ −catechol complex. , This appears to be due to breakage of the catecholamine-containing secretory vesicles during tissue disruption. Others have observed colorless enzyme, indicating that there is no iron-bound catecholamine, which can be easily understood if the TyrH-bound iron remains Fe 2+ or if the enzyme's iron content is low. Since this feedback mode of regulation may be physiologically significant, it is discussed in section II.B.3.5.…”

Section: Molecular Structure Isoforms and Expressionmentioning

confidence: 99%

Pterin-Dependent Amino Acid Hydroxylases

1996

Chem. Rev.

315

385

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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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Expression of rat tyrosine hydroxylase in insect tissue culture cells and purification and characterization of the cloned enzyme.

Cited by 49 publications

References 38 publications

Studies of the rate-limiting step in the tyrosine hydroxylase reaction: alternate substrates, solvent isotope effects, and transition-state analogs

Studies of the rate-limiting step in the tyrosine hydroxylase reaction: alternate substrates, solvent isotope effects, and transition-state analogs

Pterin-Dependent Amino Acid Hydroxylases

Tyrosine Hydroxylase

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