Effect of protein: lipid ratio on binding of nicotinic acid adenine dinucleotide phosphate (NAADP) to solubilised sea urchin egg homogenates D. Churamani, G. D. Dickinson & S. Patel (J.P. Fry), Department of Physiology, University College London, Gower Street, London WC1E 6BT.	Print Abstract Search PubMed for: Churamani D Dickinson GD Patel S Fry JP

Nicotinic acid adenine dinucleotide phosphate (NAADP) is fast emerging as an intracellular Ca²⁺ mobilising messenger in a variety of systems (Lee, 2003). In sea urchin egg homogenates, NAADP can activate the release of intracellular Ca²⁺ independently of inositol 1,4,5 triphosphate or ryanodine receptor activation. Little however is known about the biochemical properties of the NAADP receptor. In the present study we have examined the effects of detergents and lipids on radioligand binding to soluble NAADP receptors.

Solubilisation of NAADP receptors with Triton X-100 and CHAPS (1 % w/v) from sea urchin (Lytechinus pictus) egg homogenates, synthesis of [³²P]NAADP and radioligand binding to solubilised NAADP receptors was performed as previously described (Berridge et al., 2002).

Analysis of radioligand binding to soluble NAADP receptors that had been serially diluted in the presence of a fixed detergent concentration (1 % w/v Triton X-100), revealed that binding of a saturating concentration of [³²P]NAADP was markedly non-linear with respect to protein concentration. Thus, at receptor dilutions of 1/10, 1/80 and 1/160 [³²P]NAADP binding was 12±2 (100%), 0.4±0.3 (3±3%) and 0.3±0.2 (3±2%) fmol/incubation (n=3). We considered the possibility that inhibition of binding by dilution was due to decrease in the ratio of protein:lipid. Indeed, in the presence of phosphatidylcholine (PC; 0.25mg/ml), binding of [³²P]NAADP at receptor dilutions of 1/80 and 1/160 was 13±1% and 6±2%, respectively of that at a 1/10 receptor dilution (n=3), and therefore close to the values expected assuming a linear relationship between protein concentration and maximal binding (12.5% and 6.25% at 1/80 and 1/160 dilution, respectively). Comparison of [³²P]NAADP binding to receptors diluted (1/40) in the absence (12±2%) and presence (24±2%) of PC with that to receptors in which PC was added 5 min after receptor dilution (26±2%) indicated that the inhibitory effects of dilution on [³²P]NAADP binding were reversible (n=3).

In converse experiments, the effects of increasing detergent concentration on [³²P]NAADP binding to a fixed receptor concentration were examined. Both Triton X-100 and CHAPS inhibited binding of NAADP. [³²P]NAADP binding was 11±1 and 3±1 fmol/incubation in the presence of 0.1% and 1.6% w/v Triton X-100, respectively (n=4) and 9±1 and 4±0.4 fmol/incubation in the presence of 0.4% and 3.2% w/v CHAPS, respectively (n=4). Addition of PC reduced inhibition of [³²P]NAADP binding by high detergent concentrations. Binding of [³²P]NAADP was 9±1 fmol/incubation in the presence of Triton X-100 + PC and 6±1 fmol/incubation in the presence of CHAPS + PC (n=4).

In summary, we show that decreasing protein:lipid ratio, inhibits [³²P]NAADP binding to sea urchin egg homogenates, effects which are prevented by addition of an exogenous phospholipid. These data suggest that lipids may be important in regulating binding of NAADP to its receptor.

Berridge G., et al(2002) J. Biol Chem 277; 43717-43723.
Lee H.C. (2003) Curr Biol. 13; R186-8.