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[Study of the tissue and subcellular distribution of isatin-binding proteins with optical biosensor]
Iu D Ivanov1, N G Panova, O V Gnedenko
1Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, 10 Pogodinskaya Street, Moscow, 119992 Russia.
Abstract:
An original method for the integral evaluation of tissue and subcellular distribution of isatin binding proteins has been developed. This method is based on continuous monitoring of changes of optical characteristics that accompany complex formation between a ligand (immobilized on dextran bed of IAsys biosensor cell) and its soluble receptor. Solubilisation of tissue preparations and subcellular fractions with detergent (1% Triton X-100) is the important preconditions for the applicability of this method. The immobilisation of 5-aminoisatin was achieved by peptide bond formation between amino group of this isatin analogue and carboxyl group of the dextran bed of the biosensor cell. Addition of Triton X-100 treated preparations of membrane and soluble fractions of rat brain, liver, heart, and kidneys to the biosensor cell resulted in appearance of the characteristic response, indicating complex formation with the immobilised isatin analogue. The magnitude and a shape of kinetic curve vary in these samples. Isatin binding proteins predominated in membrane fractions of brain, liver and heart preparations whereas in the kidneys the highest isatin-binding response was detected in the soluble fraction. The distribution of isatin binding sites in the particulate fraction reduced in the following order: brainstem > brain hemispheres = cerebellum > heart > kidneys > liver. In the soluble fraction there was different rank of isatin binding activity: kidneys > heart > brainstem = brain hemispheres > liver > cerebellum. Liver outer mitochondrial membranes are characterised by the higher isatin-binding than mitochondria. Treatment of mitochondria with clorgyline and deprenyl, specifically inhibiting MAO A and B, respectively, significantly reduced the magnitude of the biosensor response and changed the shape of the kinetic curve. These data are consistent with the notion that within mitochondria MAOs are the major targets of isatin.