Stereospecific recognition sites for (/sup 3/H)inositol(1,4,5)-triphosphate in particulate preparations of rat cerebellum
A very high density of stereospecific binding sites for inositol-(1,4,5)P3 have been identified in rat cerebellar membranes using (/sup 3/H)inositol-(1,4,5)P3 and a rapid centrifugation step to separate free and bound ligand. Binding was shown to be rapid and reversible and of relatively high affinity (KD 23 nM). Incubations were carried out at 4 degrees and under these conditions HPLC analysis demonstrated that there was no significant metabolism of (/sup 3/H)-(1,4,5)P3 in the presence or absence of ATP over 15 min. The specificity of the site has been carefully evaluated using both natural and novel synthetic inositol phosphates. The stereospecificity is very marked with the D-, DL- and L-isomers of Ins(1,4,5)P3 showing a 1:4:2000 ratio of affinity for the binding site. D-Ins(2,4,5)P3 was the only other phosphate to show relatively high affinity (KD 1500 nM). HPLC-pure Ins(1,3,4)P3 and Ins(1,3,4,5)P4 were substantially weaker and Ins(1,4)P2, Ins-2-P1, Ins-1-P1, Ins(1,2)-cyclic P1 and inositol were totally inactive at concentrations less than 50 microM. These data are discussed in relation to a putative receptor on the endoplasmic reticulum by which Ins(1,4,5)P3 can initiate the release of bound Ca/sup 2 +/.
- Research Organization:
- Univ. of Leicester, England
- OSTI ID:
- 6125862
- Journal Information:
- Biochem. Biophys. Res. Commun.; (United States), Vol. 146:3
- Country of Publication:
- United States
- Language:
- English
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PHOSPHOLIPIDS
BIOCHEMICAL REACTION KINETICS
RECEPTORS
AFFINITY
ATP
CELL MEMBRANES
CEREBELLUM
LIGANDS
LIQUID COLUMN CHROMATOGRAPHY
RATS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ANIMALS
BODY
BRAIN
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM
CHROMATOGRAPHY
ESTERS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIPIDS
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
NERVOUS SYSTEM
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
ORGANS
PROTEINS
REACTION KINETICS
RODENTS
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques