High‐affinity inositol 1,3,4,5‐tetrakisphosphate receptor from cerebellum: solubilization, partial purification and characterization

Abstract

Proteins which bind with high affinity Ins 1,3,4,5-P4 or Ins 1,4,5-Ps were solubilized from porcine cerebellar membranes. Both binding activities were separated by heparin-agarose chromatography. The Ins 1,3,4,5-P4 receptor was partially purified with an approximately IOOO-fold enrichment as compared to the membrane preparation. In the receptor-enriched preparation the Ins 1,3,4,5-PJ3 binding protein had an afhnity (&) for Ins 1,3,4,5-P,, of 4.6 nM. Ins 1,3,4,5,6-P> displaced [3H]Ins 1,3,4,5-P,binding with a comparable affinity. The Ins 1,3,4,5-qbinding protein displayed high selectivity for Ins 1,3,4,5-P, over other inositolphosphates (ICa for Ins 1,4,5,6-P, 150 nM, for Ins-P, 1 PM and for Ins 1,3,4-P3 5 PM). Most Importantly, Ins 1,4,5-P3 did not displace [3H]Ins 1,3,4,5-P4binding at concentrations up to 10 PM. Binding of Ins 1,3,4&LP4 was maximal in the pH range between 4.5 and 6, was stable with Ca*+ concentration varied from 1 nM to 1 mM, and was suppressed by heparin (ICH, about 2 nM). The high affinity receptor for Ins 1,3,4,5-P, reported here, which is distinct from the Ins 1,4,5-PJ receptor might allow to evaluate the possible functional role of Ins I ,3,4,5-P, in the cellular signal transduction.

Binding sites for α‐trinositol (inositol 1,2,6‐trisphosphate) in porcine tissues; comparison with Ins(1,4,5)P3 and Ins(1,3,4,5)P4‐binding sites

“…The purpose of the present study was not a systematical analysis of influence of pH on binding. This has already been described for Ins(1,3,4,5)P4 binding activity (Donie et al, 1990). We checked for the alledged optimum binding at a pH close to a physiological value and at salt concentrations mimicking the intracellular milieu (Cullen & Irvine, 1992).…”

“…The Ins(1,3,4,5)P4 receptor protein was solubilized as described (Donie et al, 1990;Reiser, 1993). Frozen membranes were thawed and centrifuged (10 min, 4°C, 27,000 g).…”

Characterization of a high‐affinity Ins‐P4 (inositol 1,3,4,5‐tetrakisphosphate) receptor from brain by an anti‐peptide antiserum

“…Membranes from beef cerebellum, sheep cerebellum and porcine brain cortex were prepared by using the same protocol. The Ins-P 4 receptor protein from pig cerebellum was solubilized and purified as described [7,8].…”

Inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate receptor proteins: isolation and characterization from rat brain.

“…We (26,27) and others (28)(29)(30)(31) Preparation of Membranes and Membrane Solubilization. Rat cerebellar membranes were prepared as described (26,27).…”

“…Further characterization will determine the relationship of these protein subunits. Donie et al (29) partially purified [3H]IP4 binding activity from porcine cerebellar membranes using heparin-agarose. Their binding site displays low nanomolar affinity for IP4 with a somewhat different inositol phosphate specificity but a similar pH optimum.…”