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J. Biol. Chem., Vol. 262, Issue 20, 9561-9568, Jul, 1987
DL Clapper, TF Walseth, PJ Dargie and HC Lee
Inositol trisphosphate (IP3) was previously shown to release Ca2+ from a
nonmitochondrial store in sea urchin eggs. In this study, egg homogenates
and purified microsomes were monitored with either fura 2 or Ca2+-sensitive
minielectrodes to determine whether other stimuli would induce Ca2+
release. Pyridine nucleotides (whose concentrations are known to change at
fertilization) were found to release nearly as much Ca2+ as did IP3.
Average releases/ml of homogenate were: 0.6 microM IP3, 10.9 nmol of Ca2+;
50 microM NADP, 7.3 nmol of Ca2+; and 100 microM NAD, 6.5 nmol of Ca2+ (n =
6). Specificity was demonstrated by screening a series of other
phosphorylated metabolites, and none was found to reproducibly release
Ca2+. Calcium release induced by IP3 or NADP was immediate, whereas a lag
of 1-4 min occurred with NAD. This lag before NAD-induced Ca2+ release led
to the discovery that a soluble egg factor (Mr greater than 100,000)
converts NAD into a highly active metabolite that releases Ca2+ without a
lag. The NAD metabolite (E-NAD) was purified to homogeneity by high
pressure liquid chromatography and produced half-maximal Ca2+ release at
about 40 nM. Injection of E-NAD into intact eggs produced both an increase
in intracellular Ca2+ (as assayed with indo-1) and a cortical reaction.
Following Ca2+ release by each of the active agents (IP3, NAD, and NADP),
the homogenates resequestered the released Ca2+ but were desensitized to
further addition of the same agent. A series of desensitization experiments
showed that homogenates desensitized to any two of these agents still
responded to the third, indicating the presence of three independent Ca2+
release mechanisms. This is further supported by experiments using Percoll
density gradient centrifugation in which NADP-sensitive microsomes were
partially separated from those sensitive to IP3 and NAD.
Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate
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