Activation of tryptophan hydroxylase from central serotonergic neurons by calcium and depolarization

Abstract

The activity of tryptophan hydroxylase in a low speed supernatant preparation from rat thalamus, midbrain and medulla pons. assayed in the presence of subsaturating concentrations of 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine (6MPH 4), was enhanced 100 per cent by addition of 10 mM CaCl 2 to the reaction medium. A similar increase in activity was also observed if CaCl 2 (10 or 100 mM final concn) was added directly to the enzyme and the unbound CaCl 2 then removed by gel filtration on Sephadex G-25. This increase in activity could not be reversed by the addition of ethylene glycol bis-(β-aminoethyl ether)- N, N′-tetraacetic acid (EGTA). It was associated with a modest increase in V max (16 per cent) and an approximate doubling of the affinity of the enzyme for substrate and cofactor (apparent K m for tryptophan decreased from 79 to 46 μM; apparent K m for 6MPH 4 decreased from 148 to 101 μM). An increase in activity was also observed when tryptophan hydroxylase was prepared from slices of the brain preparation which had been (1) depolarized with a medium containing 66 mM KCl; (2) incubated overnight in an Na +-free medium or (3) treated with ouabain (0.05 or 0.1 mM), procedures believed to increase free intraneuronal levels of Ca 2+. The increase in activity was, however, not observed when the slices were incubated in a Ca 2+-free, K-rich medium to which 100 μM EGTA had been added. A kinetic analysis of the enzyme prepared from slices depolarized in K + revealed an increase in V max and a decrease in the apparent K max for substrate (74–45 μM) and artificial cofactor (177–118 μM). The activation observed after depolarization could not be reversed with EGTA.