The quantitative spectrum of inositol phosphate metabolites in avian erythrocytes, analysed by proton n.m.r. and h.p.l.c. with direct isomer detection

Abstract

The spectrum of inositol phosphate isomers present in avian erythrocytes was investigated in qualitative and quantitative terms. Inositol phosphates were isolated in micromolar quantities from turkey blood by anion-exchange chromatography on Q-Sepharose and subjected to proton n.m.r. and h.p.l.c. analysis. We employed a h.p.l.c. technique with a novel, recently described complexometric post-column detection system, called 'metal-dye detection' [Mayr (1988) Biochem. J. 254, 585-591], which enabled us to identify non-radioactively labelled inositol phosphate isomers and to determine their masses. The results indicate that avian erythrocytes contain the same inositol phosphate isomers as mammalian cells. Denoted by the 'lowest-locant rule' [NC-IUB Recommendations (1988) Biochem. J. 258, 1-2] irrespective of true enantiomerism, these are Ins(1,4)P2, Ins(1,6)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, Ins(1,3,4,5,6)P5, and InsP6. Furthermore, we identified two inositol trisphosphate isomers hitherto not described for mammalian cells, namely Ins(1,5,6)P3 and Ins(2,4,5)P3. The possible position of these two isomers in inositol phosphate metabolism and implications resulting from absolute abundances of inositol phosphates are discussed.

Translocation between membranes and cytosol of p42IP4, a specific inositol 1,3,4,5‐tetrakisphosphate/phosphatidylinositol 3,4,5‐trisphosphate‐receptor protein from brain, is induced by inositol 1,3,4,5‐tetrakisphosphate and regulated by a membrane‐associated 5‐phosphatase

“…Therefore we examined the possibility that addition of Mg 2+ enabled enzymatic degradation of Ins(1,3,4,5) P 4 , even at a temperature of 4 °C, and thus prevented the translocation of p42 IP4 . Analysis of the inositol polyphosphates in these samples by HPLC‐MDD [30,31] revealed that Ins(1,3,4,5) P 4 disappeared and concomitantly stoichiometric amounts of Ins(1,3,4) P 3 were formed (Fig. 4).…”

“…Inositol phosphates were extracted from the membrane suspension by a slight modification of the procedure described by Radenberg et al . [30]. All steps were performed at 4 °C.…”

Inositol phosphates from barley low-phytate grain mutants analysed by metal-dye detection HPLC and NMR

“…After two rounds of charcoal treatment [26], the grain extract was diluted with 1.3 litres of water and loaded on to a 70 ml Q-Sepharose FF column (1.6 cmi35 cm), operated on an FPLC system (Pharmacia). The column was eluted by a linear gradient of ammonium acetate (0-1.5 M) at a flow rate of 13 ml:min −" [28]. Fraction size was 21 ml and 1 ml of each fraction was lyophilized and resuspended in 5 µl of water prior to inositol phosphate analysis by TLC [23,29].…”

Metabolic Relations of Inositol 3,4,5,6-Tetrakisphosphate Revealed by Cell Permeabilization. Identification of Inositol 3,4,5,6-Tetrakisphosphate 1-Kinase and Inositol 3,4,5,6-Tetrakisphosphate Phosphatase Activities in Mesophyll Cells

“…Other investigators (Radenberg et al, 1989) have speculated that the d-and/or l-Ins (1,5,6) P 3 , which they identified in avian erythrocytes, may be a product(s) of dephosphorylation of Ins(3,4,5,6) P 4 and Ins(1,3,4,5) P 4 . Our approach affords an experimental test of this.…”

“…We conclude that the [ 3 H]glucitol derived from peak III is indicative of the presence of d-and/or l-Ins(2,4,5) P 3 , which elutes with d/l-Ins (1,4,6) P 3 on Partisphere SAX HPLC (14). d-and/or l-Ins(2,4,5) P 3 has been detected in avian erythrocytes (Radenberg et al, 1989), in which it is one of the major isomers in terms of chemical mass, and possibly in rat mammary tumor cells (Wong et al, 1992 (Fig. 3A) 3 H]inositol-labeled Arabidopsis were mixed with standards and resolved by HPLC.…”