An assay for dopamine-β-hydroxylase activity in tissues and serum

Goldstein, Menek; Freedman, Lewis S.; Bonnay, M

doi:10.1007/bf02136929

Cited by 123 publications

(27 citation statements)

References 3 publications

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“…In the first of coupled enzymatic reactions, tyramine is converted by the enzyme to octopamine; in the second reaction the octopamine is further converted by added phenylethanolamine: N-methyl transferase (EC 2.2.1.-) to N-methyl octopamine. The first reaction was done for 20 min and the second for 30 min (8). Tumors to be used for hydroxylase assay were homogenized in 5 mM Tris buffer (pH 6.8) containing 0.1% Triton X-100 and, the homogenates were centrifuged at 10,000 X g for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Dopamine-β-Hydroxylase Activity in Mouse Neuroblastoma Tumors and in Cell Cultures

Anagnoste¹,

Freedman²,

Goldstein³

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

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Dopamine-ft-hydroxylase activity is present in mouse neuroblastoma C-1300 tumors. The activity is proportional to the weight of the tumor. Serum activity is markedly increased in mice that bear the tumors. Treatment of mice with 5-bromodeoxyuridine causes marked inhibition of tumor growth and decrease of dopamine-p-hydroxylase activity in the serum. The histochemical studies reveal that 1-5% of the cells in mouse C-1300 neuroblastoma tumors contain catecholamines and that catecholamine-containing processes terminate mainly around blood vessels of the tumor. Dopamine-j3-hydroxylase is present in clonal neuroblastoma cell lines. The cell line with the greater tendency to form axon-like processes has a higher activity of this enzyme. Dopamine-g-hydroxylase (hydroxylase) (EC 1.14.2.1) is found in normal animals only in adrenal glands and in adrenergic neurons (1, 2). It catalyzes the hydroxylation of dopamine to norepinephrine in the only physiologically significant pathway for the formation of this neurotransmitter. We now present data on the activity of this enzyme in mouse C-1300 neuroblastoma tumors and in cultured cell lines derived from this tumor. have not appreciably changed during the year in which the experiments were performed. After trypsinization and centrifugation, cells (about 0.1 ml) were washed 3-times with 10 ml of tyrosine and phenylalanine-free Eagle's medium containing 10% calf serum and twice with saline solution; cells were then sonicated and aliquots were taken for enzyme assay. Cell numbers were determined in duplicate with the hemocytometer; after dilution in 0.4% trypan blue, 95-98% of cells regularly excluded the dye. Hydroxylase in tumor, cell cultures, and plasma was assayed (7-9) by a sensitive procedure. In the first of coupled enzymatic reactions, tyramine is converted by the enzyme to octopamine; in the second reaction the octopamine is further converted by added phenylethanolamine: N-methyl transferase (EC 2.2.1.-) to N-methyl octopamine. The first reaction was done for 20 min and the second for 30 min (8). Tumors to be used for hydroxylase assay were homogenized in 5 mM Tris buffer (pH 6.8) containing 0.1% Triton X-100 and, the homogenates were centrifuged at 10,000 X g for 10 min. Aliquots of the supernatant containing 0.1-0.2 mg of tissue in 0.1-0.2 ml were assayed. For assay of the enzyme in serum, aliquots of 0.05-0.1 ml of serum (diluted 1: 10 with water) were used, and for assay in cultured cells, 0.1-to 0.2-mg aliquots of the homogenate (50-100,gg of protein) were used. In all incubations, tyramine (0.4 Amol) served as a substrate for the enzyme and S-adenosyl-i-[methyl-4C]methionine (43 mCi/mol) served as methyl donor. METHODS

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Section: Methodsmentioning

confidence: 99%

Dopamine-β-Hydroxylase Activity in Mouse Neuroblastoma Tumors and in Cell Cultures

Anagnoste¹,

Freedman²,

Goldstein³

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

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“…The homogenate was centrifuged at 27,000 g for 15 min, and dopamine fl-hydroxylase (DBH) activity was assayed in 0-2 ml. of the supernatant according to the method of Goldstein, Freedman & Bonnay (1971) with some modifications (Garcia, Kirpekar, Prat & Wakade, 1974). DBH activity was expressed as n-mole of octopamine formed from tyramine per hour and 5 mm nerve (n-mole/hr.…”

Section: Ligation Of the Nervesmentioning

confidence: 99%

Presence and axonal transport of cholinoceptor, but not adrenoceptor sites on a cat noradrenergic neurone

Alonso¹,

Ceña²,

Garcı́a³

et al. 1982

The Journal of Physiology

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SUMMARY1. Noradrenaline release and radioligand binding studies were carried out in the cat hypogastric nerve ligated in vivo 2 cmr distal to the inferior mesenteric ganglion for different time periods, and in different effector organs.2. Large quantities ofnoradrenaline and dopamine fl-hydroxylase (DBH) accumulated in the segments of nerve immediately proximal (P,) and distal (D1) to the ligation, with rates of about 100 and 25 mm/24 hr for the orthograde and retrograde transport, respectively.3. Nicotine evoked the release of noradrenaline from P, and atrial slices; the secretary response to nicotine was completely antagonized by mecamylamine.[3H]a-bungarotoxin biding to membranes from P, allowed the estimation of a KD of 2-97 nm and a Bmax of 1639 f-mole/mg protein. F. G. ALONSO AND OTHERS 9. In conclusion, these data (a) further show that the ligated hypogastric nerve is a good model of noradrenergic nerve terminal free of effector cell; (b) provide direct evidence for the neural location of nicotinic receptors whose activation trigger noradrenaline release from noradrenergic neurones; (c) demonstrate the neural location and axonal transport of muscarinic receptor sites, but leave certain doubts about its functional role in this noradrenergic neurone; and (d) do not support the hypothesis that a and ,-adrenoceptors which modulate noradrenaline release from peripheral noradrenergic nerve terminals are neurally (or prejunctionally) located.

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“…The homogenate was centrifuged at 27,000 g for 10 min, and DBH was assayed in 0.2 ml of the supernatant according to the method of Goldstein, Freedman & Bonnay (1971) with some modifications (Garcia et al, 1974). In order to allow for any variation in the concentration of endogenous inhibitors of DBH in the nerve homogenate, a sample of purified bovine adrenal DBH (Aunis & Miras-Portugal, 1976) The hypogastric nerves with ganglia attached were incubated at 37°C for a 6 h period in oxygenated Krebs solution or in the presence of A23187, 33 gM.…”

Section: Assay Of Dopamine /B-hydroxylasementioning

confidence: 99%

The EFFECTS OF THE CALCIUM IONOPHORE, A23187, ON THE AXOPLASMIC TRANSPORT OF DOPAMINE Β‐HYDROXYLASE

Esquerro¹,

Garcı́a²,

Sánchez‐García³

1980

British J Pharmacology

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1The effects of the ionophore, A23187, on the intra-axonal transport of dopamine f3-hydroxylase (DBH) were investigated in the cat hypogastric nerve-inferior mesenteric ganglion preparation by monitoring, in vitro, the enzyme accumulation above a ligature, 2 to 2.5 cm distal to the ganglion.2 DBH accumulation in the proximal segment immediately above the ligature (P,) increased linearly up to 6 h, during incubation in normal Krebs solution at 37°C. The ionophore, A23187, interfered with the enzyme accumulation, but did not modify the previously accumulated DBH activity present in P1. 3 The blocking effects of A23187 on DBH transport were greatly impaired in the absence of extracellular calcium ions; an excess of calcium in the bathing solution (7.5 mM) itself blocked the enzyme transport by 50%.4 A23187 did not significantly modify the levels of adenosine triphosphate (ATP) in the segments P1 and P2 of the nerve proximal to the ligature. 5 Nerves incubated in an A23187-containing medium showed many mitochondria of normal shape and fine structure; however, typical microtubules or filaments were not seen in these preparations. 6 The results suggest that the ionophore A23187, by considerably raising the axoplasmic ionized calcium levels, interferes with the assembling of microtubules. In this manner, the ionophore would inhibit the transport of adrenergic vesicles and therefore of DBH along the axon. The results also provide additional evidence in favour of the view that for the transport system to work adequately, it is necessary to maintain the intra-axoplasmic ionized calcium concentration between certain critical levels.

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An assay for dopamine-β-hydroxylase activity in tissues and serum

Cited by 123 publications

References 3 publications

Dopamine-β-Hydroxylase Activity in Mouse Neuroblastoma Tumors and in Cell Cultures

Dopamine-β-Hydroxylase Activity in Mouse Neuroblastoma Tumors and in Cell Cultures

Presence and axonal transport of cholinoceptor, but not adrenoceptor sites on a cat noradrenergic neurone

The EFFECTS OF THE CALCIUM IONOPHORE, A23187, ON THE AXOPLASMIC TRANSPORT OF DOPAMINE Β‐HYDROXYLASE

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