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  • 1985-1989  (2)
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  • 1
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Tryptophan hydroxylase is activated in a crude extract by addition of ATP and Mg2+. This activation is reversible and requires in addition both Ca2+ and calmodulin. Thus, phosphorylation by an endogenous calmodulin-dependent protein kinase has long been suspected. Now that we have prepared a specific polyclonal antibody to rat brain tryptophan hydroxylase, we have been able to prove that this hypothesis is correct. After incubation of purified tryptophan hydroxylase with Ca2+/calmodulin-dependent protein kinase together with [γ-32P]ATP, Mg2+, Ca2+, and calmodulin, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotting of the enzymes onto nitrocellulose sheets, we could label the band of tryptophan hydroxylase by the antiserum and the peroxidase technique and show by autoradiography that 32P was incorporated into this band. By measuring the radioactivity, we calculated that about 1 mol of phosphate was incorporated per 8 mol of subunits of the enzyme (2 mol of native enzyme). Because the concentration of ATP which we employed (50μM) gives about half-maximal activation in crude extract compared to saturating ATP conditions (about 1 mM), this result indicates that the incorporation of at least 1 mol of phosphate/mol of tetramer of native tryptophan hydroxylase is required for maximal activation.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Superfusion of slices from the dorsal half of the lumbar enlargement of rat spinal cord with Krebs-Henseleit medium supplemented with 30 μM bacitracin allowed the collection of substance P-like immunoreactive material (SPLI), which was released at a rate of ∼ 10 pg/4 min. Tissue depolarization by an excess of K+ (30–60 mM) or veratridine (50 μM) induced a marked increase in SPLI outflow, provided that Ca2+ was present in the superfusing fluid. K+-or veratridine-induced SPLI overflow could be modulated in opposite directions by μ and δ opioid receptor agonists. Thus, the two preferential μ agonists Tyr-d-Ala-Gly-MePhe-Gly-ol (DAGO; 10 μM) and Tyr-d-Ala-Gly-MePhe-Met(O)5-OH (FK-33824; 0.1 μM) enhanced SPLI overflow from depolarized tissues, whereas the selective δ agonists Tyr-d-Thr-Gly-Phe-Leu-Thr (deltakephalin; 3 μM) and [2-d-penicillamine, 5-d-penicillamine]enkephalin (50 μM) reduced it. The effect of DAGO was antagonized by a low concentration (1 μM) of naloxone but not by the selective δ antagonist ICI-154129 (50 μM). In contrast, the latter drug prevented the inhibitory influence of δ agonists on K+-induced SPLI release. Complementary experiments with morphine (10 μM) and [2-d-alanine, 5-d-leucine]enkephalinamide (3 μM), in combination with 1 μM naloxone or 50 μM ICI-154129 for the selective blockade of μ or δ receptors, respectively, confirmed that the stimulation of μ receptors increased, whereas the stimulation of δ receptors reduced, SPLI overflow. The results suggest that, at the spinal level, the antinociceptive action of δ but not μ agonists might involve a presynaptic inhibition of substance P-containing primary afferent fibers.
    Type of Medium: Electronic Resource
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