JBC, Vol. 252, Issue 2, 596-601, Jan, 1977
Activation of guanylate cyclase in cerebral cortex of rat by hydroxylamine
T. Deguchi
Hydroxylamine actived guanylate cyclase in particulate fraction of cerebral
cortex of rat. Activation was most remarkable in crude mitochondrial
fraction. When the crude mitochondrial fraction was subjected to osmotic
shock and fractionated, guanylate cyclase activity recovered in the
subfractions as assayed with hydroxylamine was only one-third of the
starting material. Recombination of the soluble and the particulate
fractions, however, restored guanylate cyclase activity to the same level
as that of the starting material. When varying quantities of the
particulate and soluble fractions were combined, enzyme activity was
proportional to the quantity of the soluble fraction. Heating of the
soluble or particulate fraction at 55 degrees for 5 min inactivated
guanylate cyclase. The heated particulate fraction markedly activated
guanylate cyclase activity in the native soluble fraction, while the heated
soluble fraction did not stimulate enzyme activity in the particulate. The
particulate fraction preincubated with hydroxylamine at 37 degrees for 5
min followed by washing activated guanylate cyclase activity in the soluble
fraction in the absence of hydroxylamine. Further fractionation of the
crude mitochondrial fraction revealed that the factor(s) needed for the
activation by hydroxylamine is associated with the mitochondria. The
mitochondrial fraction of cerebral cortex activated guanylate cyclase in
supernatant of brain, liver, or kidney in the presence of hydroxylamine.
The mitochondrial fraction prepared from liver or kidney, in turn,
activated soluble guanylate cyclase in brain. Activation of guanylate
cyclase by hydroxylamine was compared with that of sodium azide. Azide
activated guanylate cyclase in the synaptosomal soluble fraction, while
hydroxylamine inhibited it. The particulate fraction preincubated with
azide followed by washing did not stimulate guanylate cyclase activity in
the absence of azide. The activation of guanylate cyclase by hydroxylamine
is not due to a change in the concentration of the substrate GTP, Addition
of hydroxylamine did not alter the apparent Km value of guanylate cyclase
for GTP. Guanylate cyclase became less dependent on manganese in the
presence of hydroxylamine. Thus the activation of guanylate cyclase by
hydroxylamine is due to the change in the Vmax of the reaction.
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