Synthesis and application of photoaffinity analogues of inositol 1,4,5-trisphosphate selectively substituted at the 1-phosphate group.
We have synthesized two photolabile arylazido-analogues of Ins(1,4,5)P3 selectively substituted at the 1-phosphate group for determination of Ins(1,4,5)P3-binding proteins. These two photoaffinity derivatives, namely N-(4-azidobenzoyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AbaIP3) and N-(4-azidosalicyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AsaIP3), bind to high affinity Ins(1,4,5)P3-specific binding sites at a 9-fold lower affinity (Kd = 66 and 70 nM) than Ins(1,4,5)P3 (Kd = 7.15 nM) in a fraction from rat pancreatic acinar cells enriched in endoplasmic reticulum (ER). Other inositol phosphates tested showed comparable (DL-myo-inositol 1,4,5-trisphosphothioate, Kd = 81 nM) or much lower affinities for the binding sites [Ins(1,3,4,5)P4, Kd = 4 microM; Ins(1,4)P2, Kd = 80 microM]. Binding of AbaIP3 was also tested on a microsomal preparation of rat cerebellum [Kd = 300 nM as compared with Ins(1,4,5)P3, Kd = 45 nM]. Ca2+ release activity of the inositol derivatives was tested with AbaIP3. It induced a rapid and concentration-dependent Ca2+ release from the ER fraction [EC50 (dose producing half-maximal effect) = 3.1 microM] being only 10-fold less potent than Ins(1,4,5)P3 (EC50 = 0.3 microM). From the two radioactive labelled analogues ([3H]AbaIP3 and 125I-AsIP3) synthesized, the radioiodinated derivative was used for photoaffinity labelling. It specifically labelled three proteins with apparent molecular masses of 49, 37 and 31 kDa in the ER-enriched fraction. By subfractionation of this ER-enriched fraction on a Percoll gradient the 37 kDa Ins(1,4,5)P3 binding protein was obtained in a membrane fraction which showed the highest effect in Ins(1,4,5)P3-inducible Ca2+ release (fraction P1). The other two Ins(1,4,5)P3-binding proteins, of 49 and 31 kDa, were obtained in fraction P2, in which Ins(1,4,5)P3-induced Ca2+ release was half of that obtained in fraction P1. We conclude from these data that the 37 kDa and/or the 49 and 31 kDa proteins are involved in Ins(1,4,5)P3-induced Ca2+ release from the ER of rat pancreatic acinar cells.
Schäfer
R
R
Max-Planck Institut for Biophysics, Frankfurt, Germany.
Nehls-Sahabandu
M
M
Grabowsky
B
B
Dehlinger-Kremer
M
M
Schulz
I
I
Mayr
G W
GW
D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007202:Indicators and Reagents; D015544:Inositol 1,4,5-Trisphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D014316:Tritium
D000345:Affinity Labels; D015220:Calcium Channels; D007202:Indicators and Reagents; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D014316:Tritium; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium