Characterization of inositolpolyphosphate binding to myocardial membranes.
Although it is well-accepted that the phosphatidylinositol signalling transduction pathway, producing inositol-1,4,5-P3 (InsP3) and inositol-1,3,4,5-P4 (InsP4) as second messengers, functions in heart muscle, virtually nothing is known about the roles of the higher inositol polyphosphates such as inositolhexakisphosphate (InsP6). This study demonstrates that InsP6 has the ability to bind intracellularly, with different binding characteristics, to different myocardial membranes. Binding to purified sarcoplasmic reticulum (SR) membranes, purified sarcolemmal (SL) membranes as well as to viable mitochondria were characterized. Binding to all these membranes display high as well as low affinity binding sites, with differing affinities. Kd values of binding to SR were 32 and 383 nM, to SL 61 and 1312 nM, while those of mitochondrial binding were 230 and 2200 nM respectively. InsP4 binding was also investigated and displayed the following characteristics: to SR, one low affinity binding site (Kd = 203 nM) and to SL, a high as well as a low affinity binding site with Kd values of 41 and 2075 nM respectively. Presence of InsP3, the second messenger for SR calcium release, at concentrations of 1 nM, elevated the binding of InsP4 to SR and SL by a mean of 30% and 20% respectively. Fractionation of SR and SL membranes on sucrose density gradients, after solubilization with CHAPS, indicated that InsP6 bound to two separate protein peaks in both these membranes, while InsP4 bound to only one. In SR membranes, InsP4 bound preferentially to a protein separating at high sucrose density while it bound to a protein separating at low sucrose density in SL membranes.
Department of Medical Physiology and Biochemistry, Faculty of Medicine, University of Stellenbosch, Tygerberg, Republic of South Africa.
B
B
Huisamen
E
E
Ellis
M
M
van Dyk
A
A
Lochner
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002458:Cell Fractionation; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism*; D008297:Male; D051379:Mice; D008929:Mitochondria, Heart / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D010833:Phytic Acid / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012508:Sarcolemma / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
D007295:Inositol Phosphates; C081655:inositol-1,3,4,5-tetrakisphosphate; D010833:Phytic Acid; D015544:Inositol 1,4,5-Trisphosphate