SUBCELLULAR-LOCALIZATION OF SPECIFIC INOSITOL 1,3,4,5-TETRAKIS([H-3]PHOSPHATE) BINDING-SITES IN RAT-LIVER MEMBRANE-FRACTIONS - A COMPARATIVE-EVALUATION OF PH SENSITIVITY AND BINDING CHARACTERISTICS

Inositol 1,3,4,5-tetrakis([H-3]phosphate) ([H-3]IP4) binding sites wer e investigated in plasma membranes, nuclei and microsomes derived from the rat liver. The pH optimum for maximum [3H]P-4 binding was not the same for plasma membranes, pH 7.5, nuclei, pH 6.5, and microsomes, pH 8.0. Evidence is presented demonstrating that inositol 1,3,4,5-tetrak is(phosphate) (IP4) was the most effective inositol phosphate in displ acing the binding of the [H-3]IP4 in all the membrane fractions studie d. Furthermore, the rank order of inhibition in various membrane fract ions was identical; i.e., IP5, Ins(3,4,5,6), and IP3. This suggests th at similar types of putative IP4 receptor proteins are dealt with in t he plasma membranes, nuclei, and microsomes. Scatchard analysis of sat uration isotherms revealed a single binding site in the plasma membran es and in the microsomes, whereas two binding sites marked by distinct K-D and B-max values were found in the nuclei. The density of putativ e IP4 binding sites in the plasma membranes corresponded to that of th e high-affinity ones in the nuclei. Microsomes contained fewer binding sites as compared with plasma membranes or nuclei. On the basis of th e pH sensitivity of [H-3]IP4 binding and the K-D and B-max values in v arious membrane compartments, it is proposed that inositol 1,3,4,5-tet rakis(phosphate) receptor proteins are similar but not identical in me mbrane fractions in rat liver. Plasma membrane [H-3]IP4 binding was di splaced with IP4 and IP6, revealing IC50 values of 8 +/- 2 and 150 +/- 20 nM, respectively, indicating that rat liver plasma membrane IP4 re ceptor is not clathrin assembly protein AP-2. This study provides a pH -dependent regulatory mechanism for the mode of functioning of IP4 in different membrane fractions, which may be relevant for pH and calcium interactions during cellular growth promotion.