A high-affinity inositol 1,3,4,5-tetrakisphosphate receptor protein from brain is specifically labelled by a newly synthesized photoaffinity analogue, N-(4-azidosalicyl)aminoethanol(1)-1-phospho-D-myo-inositol 3,4,5-trisphosphate.
A photolabile arylazido analogue of Ins(1,3,4,5)P4 selectively substituted at the 1-phosphate group was synthesized by coupling 2-aminoethanol(1)-1-phospho-D-myo-inositol 4,5-bisphosphate with N-hydroxysuccinimidyl-4-azidosalicylic acid [Schäfer, Nehls-Sahabandu, Grabowsky, Dehlinger-Kremer, Schulz & Mayr (1990) Biochem. J. 272, 817-825] and subsequently phosphorylating the product by bovine brain Ins(1,4,5)P3 3-kinase. The product, N-(4-azidosalicyl)-aminoethanol(1)-1-phospho-D-myo-inositol 3,4,5-trisphosphate [AsaIns(1,3,4,5)P4] was radioiodinated and purified by anion-exchange chromatography. AsaIns(1,3,4,5)P4 bound to a high-affinity Ins(1,3,4,5)P4 receptor from pig cerebellum with an affinity only 3-fold lower than that of Ins(1,3,4,5)P4. Photoirradiation of 125I-AsaIns(1,3,4,5)P4 in the presence of the receptor preparation revealed that the radioactive label was specifically associated with a protein band of apparent molecular mass 42 kDa, which Donié & Reiser [(1991) Biochem. J. 275, 453-457] had previously tentatively assigned to the Ins(1,3,4,5)P4 receptor protein. The radioactive label was displaced from the receptor when the binding reaction with 125I-AsaIns(1,3,4,5)P4 was carried out in the presence of 5 microM-Ins(1,3,4,5)P4.
Reiser
G
G
Physiologisch-chemisches Institut der Universität Tübingen, Federal Republic of Germany.
Schäfer
R
R
Donié
F
F
Hülser
E
E
Nehls-Sahabandu
M
M
Mayr
G W
GW
D000345:Affinity Labels; D000818:Animals; D002531:Cerebellum / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007294:Inositol / Q000031:analogs & derivatives / Q000378:metabolism*; D007295:Inositol Phosphates / Q000096:biosynthesis* / Q000378:metabolism*; D010777:Photochemistry; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear; D013379:Substrate Specificity; D013552:Swine
D000345:Affinity Labels; D007295:Inositol Phosphates; C071966:N-(4-azidosalicyl)aminoethanol(1)-1-phospho-D-myo-inositol 3,4,5-triphosphate; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; C061107:inositol-1,3,4,5-tetrakisphosphate receptor; D007294:Inositol