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Biochem J
2010 Aug 01;4293:485-95. doi: 10.1042/BJ20091956.
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Calcium- and polyphosphate-containing acidic granules of sea urchin eggs are similar to acidocalcisomes, but are not the targets for NAADP.
Ramos IB, Miranda K, Pace DA, Verbist KC, Lin FY, Zhang Y, Oldfield E, Machado EA, De Souza W, Docampo R.
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Acidocalcisomes are acidic calcium-storage compartments described from bacteria to humans and characterized by their high content in poly P (polyphosphate), a linear polymer of many tens to hundreds of Pi residues linked by high-energy phosphoanhydride bonds. In the present paper we report that millimolar levels of short-chain poly P (in terms of Pi residues) and inorganic PPi are present in sea urchin extracts as detected using 31P-NMR, enzymatic determinations and agarose gel electrophoresis. Poly P was localized to granules randomly distributed in the sea urchin eggs, as shown by labelling with the poly-P-binding domain of Escherichia coli exopolyphosphatase. These granules were enriched using iodixanol centrifugation and shown to be acidic and to contain poly P, as determined by Acridine Orange and DAPI (4'',6''-diamidino-2-phenylindole) staining respectively. These granules also contained large amounts of calcium, sodium, magnesium, potassium and zinc, as detected by X-ray microanalysis, and bafilomycin A1-sensitive ATPase, pyrophosphatase and exopolyphosphatase activities, as well as Ca2+/H+ and Na+/H+ exchange activities, being therefore similar to acidocalcisomes described in other organisms. Calcium release from these granules induced by nigericin was associated with poly P hydrolysis. Although NAADP (nicotinic acid-adenine dinucleotide phosphate) released calcium from the granule fraction, this activity was not significantly enriched as compared with the NAADP-stimulated calcium release from homogenates and was not accompanied by poly P hydrolysis. GPN (glycyl-L-phenylalanine-naphthylamide) released calcium when added to sea urchin homogenates, but was unable to release calcium from acidocalcisome-enriched fractions, suggesting that these acidic stores are not the targets for NAADP.
Figure 1. Transmission electron microscopy of A. punctulata eggsThe preparation was fixed and processed for standard electron microscopy. Cortical granules (cg, thin arrow), yolk platelets (YP) and ‘clear granules’ containing residual electron-dense material (thick arrow), smaller clear vesicles (white arrowheads) and mitochondria (black arrowheads) are indicated. Scale bars in (A) are 3 μm, (B) 1 μm and (C–E) 0.5 μm.
Figure 2. Electron microscopy and X-ray microanalysis of whole intact eggs and homogenates(A and B) Electron energy-filtered transmission electron microscopy of an unfixed and unstained egg (A) or an egg homogenate (B) from A. punctulata. Dense granules are identified by black arrows. (C) Typical X-ray microanalysis spectrum of dense granules found in egg homogenates [black arrow in (B)]. (D) Typical X-ray microanalysis spectrum of a less-electron-dense vesicle (probably a yolk platelet) [white arrow in (B)] found in egg homogenates. Scale bars in (A) and (B) are 3 μm.
Figure 3. Detection of PPi and poly P in sea urchin eggs(A) PPi, SC poly P and LC poly P were assayed as described in the Materials and methods section, and levels are expressed as mM poly P (in terms of Pi residues). Data are from four experiments, and show means±S.E.M. Different letters indicate significant differences (one-way ANOVA, P<0.05). (B) Agarose gel electrophoresis of poly P stained with Toluidine Blue. Lane 1, poly P extract; lane 2, poly P extract after rPPX1 treatment. P75, P45 and P24 are poly P standards of 75+, 45 and 25 phosphate units. (C) 242.8-MHz (1H decoupled) 31P-NMR spectra of a perchloric acid extract of sea urchins eggs. See assignments in Table 1. (D) Differential interferential microscopy of the sea urchin egg shown in (E). (E) Localization of poly P using the recombinant PPBD of E. coli PPX linked with an Xpress epitope tag. Images were deconvolved for 15 cycles using Softwarx deconvolution software. Other conditions are described in the Materials and methods. Scale bars in (D) and (E) are 15 μm.
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