IDENTIFICATION OF INOSITOL 1,4,5-TRISPHOSPHATE [IP3] RECEPTORS IN WHEAT (Triticum aestivum)

Maki, Nazli

Ph.D., Department of Biology
Supervisor: Doç. Dr. Hüseyin A. Öktem
Co-Supervisor: Prof. Dr. Meral Yücel

June 1998, 118 pages

Inositol 1,4,5-trisphosphate (IP3) is a second messenger of the phosphoinositol system which can obilize Ca2+ from intracellular stores. Allthough, binding characteristics of animal IP3 receptors have been studied extensively, up-to-date there is only one literature study concerning the binding profiles of IP3 receptors in plants. In the present study, IP3 receptors have been identified in crude membrane preparations and detergent-solubilized microsomal fractions from leaf and root tissues of 15-days old wheat seedlings employing [3H]IP3 binding assays. Equilibrium binding of [3H]IP3 was reached after 2 minutes of incubation in leaf and 4 minutes in root for both crude membrane and detergent-solubilized fractions at 4°C. Solubilization was achieved by the treatment of crude membranes fractions with 1.5% (v/v) Trition X-100 for 30 minutes at 4°C. [3H]IP3 binding appeared to be partially inactivated by 1.5% Triton X-100, however, when the detergent concentration was reduced to 0.1% (v/v), [3H]IP3 binding to solubilized fraction was retained. Specific binding of [3H]IP3 was found to be pH-dependent, in both leaf and root tissues. At neutral or acidic pH values, very little [3H]IP3 binding was observed and the optimum pH was found to be 8.0. The binding sites were further identified by using low molecular weight (3000) heparin in [3H]IP3 binding assays to both crude and detergent-solubilized fractions of leaf tissue. The results showed considerable amount of [3H]IP3 specific binding by the addition of heparin. Furthermore, binding experiments were conducted in the presence of varying concentrations of Ca2+(10-6M - 2´10-3M), Mg2+ (10-5M-10-2M), and ATP (10-5M-10-3M), on the [3H]IP3 binding sites in detergent-solubilized microsomal fractions isolated from leaf and root tissues of wheat. The results were compared with the [3H]IP3 binding results to the receptors in crude membrane preparations from rat cerebellum, which provided a usefull model system for studying IP3 binding sites. Under varying concentrations of Ca2+, Mg2+, and ATP, the binding of [3H]IP3 to its receptor were inhibited in detergent-solubilized fractions in both leaf and root tissues as well as in cerebellar membranes. The presented results were the first direct evidence for the presence of IP3 receptors in wheat, and would support the idea that IP3 functions as a second messenger in plant cells.

Keywords: Wheat, Inositol 1,4,5-trisphosphate, IP3 receptors