Proprietes cinetiques de la dopamine‐β‐hydroxylase membranaire

Portugal, María Teresa Miras; Jordá, A.; Ruiz, Angel Santos

doi:10.1016/0014-5793(76)80983-0

Cited by 7 publications

(3 citation statements)

References 17 publications

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“…Soluble dopamine fJ-hydroxylase has a pH optimum of 6.0, with ferrocyanide as reductant and in the presence of sodium fumarate (Rosenberg et al, 1980), and that of the membrane enzyme may be even lower (Miras-Portugal et al, 1976). The increase in steady-state rate may therefore also be explained by the decrease in internal pH of the 'ghosts' that results from ATP hydrolysis (Phillips & Allison, 1978).…”

Section: Resultsmentioning

confidence: 99%

“…7). Tyramine accumulation by the 'ghosts' is virtually unaffected by 0.01% Triton X-100, but is completely abolished by 0.03%, so we explain the inhibition of hydroxylation as being due to the decreased tyramine concentration at the active site of the enzyme [apparent Kmi, 0.29 mm (Miras-Portugal et al, 1976); concentration of tyramine in the medium, 0.048 mm].…”

Section: Variation Oftyramine Concentrationmentioning

confidence: 91%

“…The apparent Km for tyramine for membranebound dopamine fJ-hydroxylase is 0.29 mm (Miras-Portugal et al, 1976). We investigated the dependence of the ferrocyanide-stimulated hydroxylation rate on tyramine concentration in the medium.…”

Section: Variation Oftyramine Concentrationmentioning

confidence: 99%

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Reduction of membrane-bound dopamine β-hydroxylase from the cytoplasmic surface of the chromaffin-granule membrane

Grouselle¹,

Phillips²

1982

Biochemical Journal

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Resealed bovine chromaffin-granule 'ghosts' were used for assaying the membrane-bound form of dopamine beta-hydroxylase. Hydroxylation of the substrate tyramine is dependent on its accumulation within the 'ghosts', where the active site of the enzyme is located. Free tyramine in the medium is at a low concentration, low ionic strength and a relatively high pH (7.0), so that even in the presence of a reducing agent (co-reductant) the unaccumulated amine is hydroxylated at a negligible rate. 'Ghosts' contain an endogenous co-reductant, which is shown to be catecholamine remaining in the membrane itself after granule lysis. Catecholamine that is free in solution in the medium or in the interior of the 'ghosts' is not effective as co-reductant, nor is ascorbate, in contrast with the situation with soluble dopamine beta-hydroxylase. Ferrocyanide is an active co-reductant, however, giving a hydroxylation rate approximately equal to the tyramine accumulation rate: it does not enter the 'ghosts', nor does the enzyme appear to utilize ferrocyanide sealed inside the 'ghosts'. A mechanism must therefore exist for transferring electrons across the membrane from the cytoplasmic surface to the matrix surface. NADH is not an electron donor for the enzyme, nor is cytochrome b-561 involved.

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Section: Resultsmentioning

confidence: 99%

Section: Variation Oftyramine Concentrationmentioning

confidence: 91%

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Reduction of membrane-bound dopamine β-hydroxylase from the cytoplasmic surface of the chromaffin-granule membrane

Grouselle¹,

Phillips²

1982

Biochemical Journal

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Properties of soluble and membrane bound dopamine-?-monooxygenase from bovine adrenal medulla cross-linked with dimethyl suberimidate

1980

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Bovine dopamine-beta-monooxygenase from chromaffin granules in its soluble and membrane-bound forms was cross-linked with the bifunctional reagent dimethyl suberimidate, and its structural and kinetic properties were studied. 1. The cross-linking reaction does not affect the activity of soluble dopamine-beta-monooxygenase; it produces a ten percent inactivation in the membrane-bound enzyme, possibly because the linkage to other membrane proteins hinders its activity. 2. The soluble dopamine-beta-monooxygenase reaction mixture was analyzed by sodium dodecyl sulfate gel electrophoresis, showing appreciable amounts of dimer and tetramer, but only small amounts of trimer. In membrane-bound dopamine-beta-monooxygenase, subjected to the same treatment, appreciable amounts of dimer and higher aggregates were found. 3. The kinetic properties of soluble dopamine-beta-monooxygenase after the crosslinking reaction are the same as those of the native enzyme, with a ping-pong kinetic mechanism and the same real Michaelis constants for tyramine and ascorbate: KmT = 0.36 mM and KmA = 0.32 mM. Membrane-bound dopamine-beta-monooxygenase does not present a ping-pong mechanism before or after cross-linking; its real Michaelis constants are slightly modified by the cross-linking reaction: KmT = 0.4 mM and KMA = 0.4 mM.

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