Isolation and characterization of a novel inositol hexakisphosphate binding protein from mammalian cell cytosol.
Inositol hexakisphosphate (IP6) is present in most mammalian cells, although its intracellular function is as yet undefined. We find that the total protein fraction from bovine brain cytosol contains a significant level of specific binding for IP6 precipitable with 40% saturated ammonium sulfate. A protein complex has been isolated from this fraction that specifically binds IP6 and is purified about 500-fold over the cytosol. The IP6 binding protein (IP6BP) chromatographs as a single peak of binding activity on a gel exclusion column, with a Stokes radius equivalent to 266 +/- 14 kDa. The IP6BP is a heterooligomeric complex composed of a number of subunits with molecular weights varying from 23,000 to 60,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Scatchard analyses of IP6 binding of both the crude ammonium sulfate fraction and the purified complex show the presence of a similar high-affinity binding site (Kd approximately 6.0 nM). Bmax for the purified fraction is 1.8 nmol of IP6/mg of protein or 0.48 mol of IP6 bound/mol of complex. Other inositol polyphosphates, such as inositol 1,3,4,5,6-pentakisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol 1,4,5-trisphosphate, are poor competitors for IP6 binding to the purified complex. The purification scheme, when applied to a rat liver cytosol fraction, yields a similar IP6BP. This complex has an apparent size of 512,000 using gel exclusion chromatography and contains an additional protein band with M(r) = 97,000 by SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)
Xie
J
J
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235.
Fleischer
B
B
D000818:Animals; D001667:Binding, Competitive; D001923:Brain Chemistry; D002417:Cattle; D003600:Cytosol / Q000737:chemistry*; D008099:Liver / Q000737:chemistry; D008297:Male; D008970:Molecular Weight; D010833:Phytic Acid / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats
D011506:Proteins; D010833:Phytic Acid