A competition binding assay for determination of the inositol (1,4,5)-trisphosphate content of human leucocytes.
We developed a competition binding assay for estimation of the intracellular inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) and optimalized it for the measurement of the Ins(1,4,5)P3 content of human blood leucocytes. The present method is considerably cheaper and requires five times fewer cells than the commercial Ins(1,4,5)P3 kit. The mean Ins(1,4,5)P3 content of human blood monocytes, granulocytes, and lymphocytes amounted to 3.3 +/- 1.2 microM, 3.1 +/- 1.4 microM, and 4.6 +/- 1.5 microM, respectively. After stimulation with formyl-methionyl-leucyl-phenylalanine (f-MLP) the Ins(1,4,5)P3 content of human granulocytes and monocytes increased 2-3 times within 10 sec and then gradually decreased, returning to basal values at 60 sec. Lymphocytes did not respond to f-MLP with an increase in their Ins(1,4,5)P3 content.
Nibbering
P H
PH
Dept. of Infectious Diseases, University Hospital, Leiden, The Netherlands.
Zomerdijk
T P
TP
van Haastert
P J
PJ
van Furth
R
R
D001667:Binding, Competitive; D006098:Granulocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007962:Leukocytes / Q000032:analysis* / Q000187:drug effects; D008861:Microsomes / Q000032:analysis; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology
D009240:N-Formylmethionine Leucyl-Phenylalanine; D015544:Inositol 1,4,5-Trisphosphate