JBC, Vol. 251, Issue 20, 6205-6209, Oct, 1976
Activation and inhibition of fat cell adenylate cyclase by fluoride
V. C. Manganiello and M. Vaughan
Incubation (30 degrees) of fat cell particulate fractions with fluoride
before assay in the effective absence of fluoride results in activation of
adenylate cyclase. Whereas the effect of fluoride (1.3 to 7 mM) when added
to the assay was maximal in less than 2 min, 10 or 15 min of incubation
before assay was usually required to produce maximal activation with any
given concentration of fluoride. Under both conditions 3 to 5 mM fluoride
produced maximal activation. After incubation with fluoride for 5 to 20 min
cyclase activity was constant for at least 15 min of assay without
fluoride; maximal activity was greater than that produced by fluoride added
to the assay system and the concentration required to produce significant
activation was lower. Fluoride activation in the assay or during prior
incubation could be prevented by pyrophosphate. When added during the early
minutes of assays with fluoride, 1.5 mM pyrophosphate, which had little
effect on the activity of enzyme previously incubated with fluoride,
rapidly reduced activity to essentially basal levels; when added after 10
min its effect was equally rapid but much smaller. It appears that
activation is initially reversible by removal of fluoride as it is by
addition of pyrophosphate, but becomes largely irreversible by these means
with continued exposure to fluoride. Fluoride in the assay system inhibited
cyclase activated by isoproterenol or choleragen or by incubation with
fluoride prior to assay; inhibition, dependent on fluoride concentration,
was maximal at 5.3 mM. Since maximal activity produced by incubation with
fluoride before assay exceeded that of nonincubated preparations assayed
with fluoride, and was reduced to the latter level when assayed in the
presence of fluoride, we infer that inhibition is reversible at a time when
fluoride activation is relatively irreversible. Pyrophosphate (1.5 mM),
which prevented fluoride activation, did not reduce fluoride inhibition of
isoproterenol-, fluoride-, or choleragen-activated cyclase. When 3 mM MnCl2
was present in the assay, inhibition by fluoride was not observed. In
descriptive terms, MnCl2 appeared to cause rapid reversal of fluoride
inhibition. Thus, fluoride inhibits, in an apparently similar manner, fat
cell adenylate cyclase whether it is activated by isoproterenol, fluoride,
or choleragen. Although fluoride activation and inhibition can apparently
be dissociated or modified differentially, until the mechanism(s) of action
of fluoride is elucidated it cannot be concluded that these are totally
independent processes.
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