Abstract
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.
Highlights
Inositol trisphosphate (IP3) wapsreviously shown to formation of the fertilization envelope
Evidence is presented that IP3,the NAD metabolite,and a similarone producedfrom NADP allact through independent mechanisms inreleasing Ca2+, suggesting the existence of multiple Ca2+ stores in sea urchin eggs
Caz+Release fromSea Urchin Egg Microsomes excitation wavelength used, fura 2 fluorescence decreases in response to increasing Ca2+.Fig. l a shows IP3-induced Ca2+ release from the egg homogenate as indicated by the observed decrease in fura 2 fluorescence
Summary
Calcium Release from Crude Homogenates-In a previous study, we used L. pictus egg homogenates and microsomes as a cell-free system to study Ca2+uptake andrelease and found that IP, induced Ca2+release from a nonmitochondrial store (10). 4a shows that such preincubated NAD (N+S in the Fig. 4a) by anion exchange HPLC using a procedure known to provide induced Ca2+release without a lag and at a lower concentra- good separation of nucleotides (13). Releasing activity when added to purified microsomes, supernatant addition was still required for maximal Ca2+release. In c, preincubated NAD was added to aliquots of 5%purified microsomes previously mixed with 0.25,0.5,1, or 2%supernatant,with increasing supernatant concentration producing increasing Ca2+release. Since anion exchange and reverse phase HPLC separate according to different principles, these results, provide strong evidence that E-NAD is purified to homogeneity and contains a moiety that absorbs at 254 nm. The alkali product produced Ca2+release without a lag, desensitized homogenates only to NAD, and required the equivalent of 50 p~ original NAD for maximal Ca2+release Ca2’ Release from Sea Urchin Egg Microsomes trum of the HPLC-purified A-NAD was found t o be the TABLEI same as E-NAD andNAD
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