phosphorus-radioisotopes and inositol-1-3-4-5-tetrakisphosphate

A series of 32P-labeled D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] analogues was enzymically prepared from the corresponding D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] analogues using recombinant rat brain Ins(1,4,5)P3 3-kinase and [gamma-32P]ATP. Ins(1,4,5)P3 analogues with bulky groups at the 2-OH position, substitutions of phosphates by thiophosphates and D-6-deoxy-myo-Ins(1,4,5)P3 were tested. Using [3H]Ins(1,4,5)P3 and ATP gamma S, a [3H]Ins(1,3,4,5)P4 analogue with a thiophosphate at the D-3 position was prepared. The D-4 and/or D-5 phosphate group seemed to be important for 3-kinase activity, while the OH group at position 6 was not crucial. The addition of bulky groups at the 2-OH position did not prevent phosphorylation. The labeled Ins(1,3,4,5)P4 analogues were purified and their degradation by type-I Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase was compared with the degradation of Ins(1,3,4,5)P4. Substitution of the phosphate group at positions 1 or 3 by a thiophosphate, or the addition of bulky groups at the 2-OH position did not prevent degradation. D-6-Deoxy-myo-inositol 1,3,4,5-tetrakisphosphate could not be degraded by the 5-phosphatase, indicating the importance of the 6-OH group for 5-phosphatase action. D-6-Deoxy-myo-inositol 1,3,4,5-tetrakisphosphate could be an important tool in elucidating the cellular functions of Ins(1,3,4,5)P4.

Topics: Adenosine Triphosphate; Animals; Brain; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Inositol Polyphosphate 5-Phosphatases; Molecular Structure; Phosphoric Monoester Hydrolases; Phosphorus Radioisotopes; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Rats; Recombinant Proteins; Structure-Activity Relationship; Substrate Specificity

In the present study we describe the characterization and localization of Ins(1,3,4,5)P4-binding sites in human platelet membranes. Specific binding sites for Ins(1,3,4,5)P4 have been identified on mixed, plasma and intracellular membranes from neuraminidase-treated platelets using highly purified carrier-free [32P]Ins(1,3,4,5)P4. The displacement of Ins(1,3,4,5)P4 from these sites by Ins(1,4,5)P3 and InsP6 occurs at greater than two orders of magnitude higher concentrations and with Ins(1,3,4,5,6)P5 at about 40-fold higher concentrations than with Ins(1,3,4,5)P4. The membranes were further separated by free-flow electrophoresis into plasma and intracellular membranes. The Ins(1,3,4,5)P4-binding sites separated with plasma membranes, and showed similar affinities and specificities as mixed membranes, whereas Ins(1,4,5)P3-binding sites were predominantly in the intracellular membranes. These results suggest a predominantly plasma membrane location for putative Ins(1,3,4,5)P4 receptors in human platelets.

Topics: Animals; Binding Sites; Binding, Competitive; Blood Platelets; Cattle; Cell Membrane; Humans; Inositol Phosphates; Phosphorus Radioisotopes; Receptors, Cytoplasmic and Nuclear

Avian erythrocytes were incubated with myo-[3H]inositol for 6-7 h and with [32P]Pi for the final 50-90 min of this period. An acid extract was prepared from the prelabelled erythrocytes, and the specific radioactivities of the gamma-phosphate of ATP and of both the myo-inositol moieties (3H, d.p.m./nmol) and the individual phosphate groups (32P, d.p.m./nmol) of [3H]Ins[32P](1,3,4,6)P4,[3H]Ins[32P](1,3,4,5)P4, [3H]Ins[32P](3,4,5,6)P4 and [3H]Ins[32P](1,3,4,5,6)P5 were determined. The results provide direct confirmation that one of the cellular InsP4 isomers is Ins(1,3,4,5)P4 which is synthesized by sequential phosphorylation of the 1,4,5 and 3 substitution sites of the myo-Ins moiety, precisely as previously deduced [Batty, Nahorski & Irvine (1985) Biochem. J. 232, 211-215; Irvine, Letcher, Heslop & Berridge (1986) Nature (London) 320, 631-634]. This is compatible with the proposed synthetic route from PtdIns via PtdIns4P, PtdIns(4,5)P2 and Ins(1,4,5)P3. The data also suggest that, in avian erythrocytes, the principle precursor of Ins(1,3,4,5,6)P5 is Ins(3,4,5,6)P4. Furthermore, if the gamma- (and/or beta-) phosphate of ATP is the precursor of the phosphate moieties of Ins(3,4,5,6)P4, then this isomer must be derived from the phosphorylation of Ins(3,4,6)P3. If the gamma- (and/or beta-) phosphate of ATP similarly acts as the ultimate precursor to all of the phosphates of Ins(1,3,4,6)P4, then, in intact avian erythrocytes, the main precursor of Ins(1,3,4,6)P4 is Ins(1,4,6)P3. This contrasts with the expectation, based on results with cell-free systems, that Ins(1,3,4,6)P4 is synthesized by the direct phosphorylation of Ins(1,3,4)P3.

Topics: Adenosine Triphosphate; Animals; Chickens; Chromatography, High Pressure Liquid; Erythrocyte Membrane; Erythrocytes; Humans; Inositol; Inositol Phosphates; Kinetics; Phosphates; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Structure-Activity Relationship

Previous studies with antigen-stimulated rat basophilic leukemia (RBL-2H3) cells indicated the formation of multiple isomers of each of the various categories of inositol phosphates. The identities of the different isomers have been elucidated by selective labeling of [3H]inositol 1,3,4,5-tetrakisphosphate with [32P]phosphate in the 3'-or 4',5'-positions and by following the metabolism of different radiolabeled inositol phosphates in extracts of RBL-2H3 cells. We report here that inositol 1,3,4,5-tetrakisphosphate, when incubated with the membrane fraction of extracts of RBL-2H3 cells, was converted to inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate. Further dephosphorylation of the inositol polyphosphates proceeded rapidly in whole extracts of cells, although the process was significantly retarded when ATP (2 mM) levels were maintained by an ATP-regenerating system. The degradation of inositol 1,4,5-trisphosphate proceeded with the sequential formation of inositol 1,4-bisphosphate, the inositol 4-monophosphate (with smaller amounts of the 1-monophosphate), and finally inositol. Inositol 1,3,4-trisphosphate, on the other hand, was converted to inositol 1,3-bisphosphate and inositol 3,4-bisphosphate and subsequently to inositol 4-monophosphate and inositol 1-monophosphate (stereoisomeric forms were undetermined). The possible implications of the apparent interconversion between inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in regulating histamine secretion in the RBL-2H3 cells are discussed.

Topics: Animals; Cell Line; Chromatography, High Pressure Liquid; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Leukemia, Experimental; Phosphorus Radioisotopes; Rats; Sugar Phosphates; Tritium