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R H Michell

Publications and source records attributed to R H Michell.

At least 37 records · Page 2Linked to original sources
PubMed1990

Inositol phosphates in receptor-mediated cell signaling: metabolic origins and interrelationships.

We have investigated the metabolic interrelationships of the major inositol phosphates in vasopressin-stimulated WRK 1 mammary tumor cells which were labeled to equilibrium with [14C]inositol and briefly, just prior to stimulation, with [3H]inositol. A comparison of the 3H/14C ratios of these compounds with those of the cellular inositol lipids suggests that most of the known inositol mono-, bis-, tris-, and tetrakis-phosphates are derived from precursors with turnover rates similar to those of these lipids. However, Ins(3,4,5,6)P4 (which is the major inositol tetrakisphosphate to accumulate in stimulated WRK 1 cells), Ins(1,3,4,5,6)P5, and InsP6 had 3H/14C ratios of 0 in this experiment, indicating that they must have a different metabolic origin.

Animals↗
PubMed1989

Mitogen-induced phosphorylation of human B-lymphocyte proteins. Relationship to protein kinase C activation.

We have investigated the rapid phosphorylation of proteins in B-lymphocytes incubated with the tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), anti-Ig and combinations of TPA and the Ca2+ ionophore ionomycin. Two-dimensional electrophoretic analysis was used to identify the proteins phosphorylated in cells preincubated with [32P]Pi. TPA induced a characteristic pattern of labelled proteins, four of which (pp85, pp76, pp66 and pp63) showed a dose-dependent incorporation of 32P on serine residues. The phosphorylation of pp63 and pp66, in particular, correlated with the mitogenic dose-response curve. Addition of the Ca2+ ionophore ionomycin to B-cells also stimulated a characteristic incorporation of 32P into proteins, which included pp63 and pp66. With combined doses of TPA and ionomycin, these two proteins show an enhanced phosphorylation, which correlated well with the synergistic enhancement of proliferation shown by this combination of agents. Protein kinase C (PKC) was partially purified from B-cells and separated into alpha and beta subtypes. The activation of both PKCs was assessed with increasing doses of TPA and concentrations of Ca2+ of 0.1 microM and 2 microM. For both forms of PKC, in particular the beta form, higher concentrations of Ca2+ shifted the dose-response curve for TPA to the left and increased the maximum activation. Anti-Ig, which stimulated B-cells by cross-linking surface immunoglobulin and causing hydrolysis of PtdIns(4,5)P2, also caused increased phosphorylation of several proteins, which again included pp63 and pp66. These data suggest that PKC, particularly the beta form, is involved in the early part of the proliferation cascade for human B-lymphocytes. It is most probably activated in a synergistic manner by the increased Ca2+ and diacylglycerol levels which result from the earlier hydrolysis of PtdIns(4,5)P2.

Animals↗
PubMed1989

Determination of the steady-state turnover rates of the metabolically active pools of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in human erythrocytes.

When intact human erythrocytes are incubated at metabolic steady state in a chloride-free medium containing [32P]Pi, there is rapid labelling of the gamma-phosphate of ATP, followed by a slower labelling of the monoester phosphate groups of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] [King, Stephens, Hawkins, Guy & Michell (1987) Biochem. J. 244, 209-217]. We have analysed the early kinetics of the labelling of these phosphate groups, in order to determine: (a) the steady-state rates of the interconversions of phosphatidylinositol, PtdIns4P and PtdIns(4,5)P2; and (b) the fractions of the total cellular complement of PtdIns4P and PtdIns(4,5)P2 that participate in this steady-state turnover. The experimental data most closely fit a pattern of PtdIns4P and PtdIns(4,5)P2 turnover in which one-quarter of the total cellular complement of each lipid is in the metabolic pool that participates in rapid metabolic turnover, with rate constants of 0.028 min-1 for the interconversion of PtdIns and PtdIns4P, and of 0.010 min-1 for the PtdIns4P/PtdIns(4,5)P2 cycle. These rate constants represent metabolic fluxes of approx. 2.1 nmol of lipid/h per ml of packed erythrocytes between PtdIns and PtdIns4P and of approx. 5.7 nmol/h per ml of cells between PtdIns4P and PtdIns(4,5)P2.

Adenosine Triphosphate↗
PubMed1989

The use of cells doubly labelled with [14C]inositol and [3H]inositol to search for a hormone-sensitive inositol lipid pool with atypically rapid metabolic turnover.

Some, though not all, previous studies have suggested that the inositol lipid which is hydrolysed during transmembrane signalling in response to receptor activation might be drawn from a metabolically discrete and relatively small hormone-sensitive lipid pool that turns over more rapidly than the bulk of membrane inositol lipid. In order to seek evidence for the existence of this putative hormone-sensitive lipid pool, we have double-labelled cells by growing them for 3 days in a medium containing [14C]inositol and then supplying them with [3H]inositol for the final 2 h before stimulation. We anticipated that stimulation of these doubly labelled cells might provoke the formation, from the postulated hormone-sensitive pool, of small quantities of relatively 3H-enriched inositol phosphates, and that these could be harvested from cells (provided that the cytosolic inositol monophosphatase and inositol 1,4-bisphosphate/inositol 1,3,4-trisphosphate 1-phosphatase activities are first inhibited by Li+). Experiments of this type, using both vasopressin-stimulated WRK1 rat mammary tumour cells and 3T3 mouse fibroblasts stimulated by prostaglandin F2 alpha, have largely failed to demonstrate the formation of relatively 3H-enriched inositol phosphates. There was a tendency for phosphatidyl-inositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate to have slightly higher 3H: 14C ratios than phosphatidylinositol, but the 3H: 14C ratios of the inositol phosphates formed in stimulated cells were not substantially greater than the 3H: 14C ratios of the inositol lipids. We therefore conclude, at least for the two cell lines that we studied, that hormone-stimulated inositol lipid hydrolysis can call, either directly or indirectly, upon the majority of the inositol lipid complement of the stimulated cell.

Animals↗
PubMed1988

Preferential localization of rat liver D-myo-inositol 1,4,5-trisphosphate/1,3,4,5-tetrakisphosphate 5-phosphatase in bile-canalicular plasma membrane and 'late' endosomal vesicles.

Previous studies have shown that most of the inositol 1,4,5-trisphosphate/inositol 1,3,4,5-tetrakisphosphate 5-phosphatase activity of rat hepatocytes is associated with the plasma membrane [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147]. We now show that the specific activity of this enzyme is highest in the bile-canalicular domain of the plasma membrane, at the opposite pole of the hepatocyte from the presumed site of receptor-mediated formation of inositol 1,4,5-trisphosphate. In intact hepatocytes and in sealed membrane vesicles originating from the bile-canalicular domain of the plasma membrane, the 5-phosphatase activity was mostly latent and therefore located at the cytoplasmic surface. A substantial amount of 5-phosphatase was also found in rat liver endosomal fractions, particularly a 'late' endosomal subfraction, indicating that this enzyme may be transported between the sinusoidal plasma membrane and other cellular membranes.

Animals↗
PubMed1988

1D-myo-inositol 1,4,5-trisphosphate dephosphorylation by rat enterocytes involves an intracellular 5-phosphatase and non-specific phosphatase activity at the cell surface.

We studied the dephosphorylation of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) by permeabilized rat intestinal epithelial cells incubated in a medium resembling intracellular ionic strength and pH. Saponin-permeabilized cells rapidly dephosphorylated Ins(1,4,5)P3 to a mixture of three InsP2 (inositol bisphosphate) isomers, namely Ins(1,4)P2, Ins(1,5)P2 and Ins(4,5)P2. These products were identified by h.p.l.c. analysis after dephosphorylation of both 3H- and 32P-labelled Ins(1,4,5)P3. Ins(1,4)P2 accumulated to about half of the concentration attained by Ins(1,5)P2 and Ins(4,5)P2. Ins(1,4,5)P3 dephosphorylation was inhibited, by up to 75%, by 10 mM-glucose 6-phosphate. In these conditions Ins(1,4)P2 became the predominant product, indicating that glucose 6-phosphate inhibited non-specific dephosphorylation of Ins(1,4,5)P3, at least at the 1- and 4-phosphate groups. Ins(1,4)P2 was further dephosphorylated, and the major InsP (inositol monophosphate) product was Ins4P. Most of the glucose 6-phosphate-inhibitable Ins(1,4,5)P3 phosphatase activity was exposed on the cell surface. The glucose 6-phosphate-insensitive Ins(1,4,5)P3 5-phosphatase activity was not detected until the cells were permeabilized with saponin. This intracellular 5-phosphatase activity was: (i) predominantly associated with the particulate portion of the cell; (ii) strongly inhibited by 10 mM-2,3-bisphosphoglycerate; (iii) insensitive to 50 mM-Li+. Therefore the Ins(1,4,5)P3 5-phosphatase activity in enterocytes appears similar to the 5-phosphatase that has been characterized in a number of cell types.

2,3-Diphosphoglycerate↗
PubMed1988

Dephosphorylation of 1D-myo-inositol 1,4-bisphosphate in rat liver.

Dephosphorylation of 1D-myo-inositol 1,4-bisphosphate [Ins(1,4)P2] in rat liver is catalysed by a cytosolic phosphatase that removes the 1-phosphate group. The Km for Ins(1,4)P2 is approx. 17 microM. Li+ (100 mM) causes 50% inhibition of Ins(1,4)P2 phosphatase activity when activity is measured at the very low substrate concentration of 10 nM, but on raising the substrate concentration to 100 microM there is a greater than 10-fold increase in sensitivity to Li+, suggesting that Li+ acts mainly, but not entirely, as an uncompetitive inhibitor of Ins(1,4)P2 phosphatase. In addition, rat liver cytosol shows Li+-sensitive phosphatase activity against 1D-myo-inositol 1-,3- and 4-monophosphates. The Ins(1,4)P2 1-phosphatase and inositol monophosphatase activities all share an apparent Mr of 47 x 10(3), as determined by gel-filtration chromatography. However, the Ins(1,4)P2 1-phosphatase is more sensitive to inactivation by heat, and can be separated from inositol monophosphatase activity by anion-exchange chromatography. We conclude that rat liver cytosol contains an Ins(1,4)P2 1-phosphatase that is distinct from, but in many ways similar to, inositol monophosphatase.

Animals↗
PubMed1988

Inositol lipids: receptor-stimulated hydrolysis and cellular lipid pools.

Our current knowledge of the process by which receptors stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) has its origin in the discovery by Hokin & Hokin (J. biol. Chem. 263, 967 (1953] that some pancreatic secretagogues not only elicit exocrine secretion but also stimulate the metabolism of membrane phospholipids. Despite the recent elucidation of many aspects of this widespread signalling system, there is still little information on the control of the supply of its substrate, PtdIns(4,5)P2. In particular, some studies have suggested that inositol-lipid-mediated signalling involves much or all of the inositol lipid complement of the stimulated cells, whereas other observations have equally clearly implicated the receptor-activated hydrolysis of an inositol phospholipid pool that comprises only a small fraction of the total cellular complement of these lipids. These studies, which have largely employed radiochemical analyses using single isotopes, are briefly reviewed. In addition, we report the first information obtained by a new procedure for analysing the metabolic characteristics of the inositol lipids that are broken down during stimulation. This technique employs cells that are doubly labelled in the inositol moiety of their lipids (to isotopic equilibrium with 14C and only briefly with 3H) to search for functional metabolic heterogeneity among the inositol lipids of stimulated cells. Using this method, we have found that the inositol phosphates liberated in stimulated cells during brief stimulation of V1a-vasopressin receptors or prostaglandin F2 alpha receptors come from phospholipid that has a turnover rate typical of the bulk of the cellular inositol lipids.

Animals↗
PubMed1988

Inositol 1:2(cyclic),4,5-trisphosphate is not a major product of inositol phospholipid metabolism in vasopressin-stimulated WRK1 cells.

1. A method has been devised for quenching cell incubations with an aqueous phenol/chloroform/EDTA mixture of neutral pH, to allow the analysis of acid-labile cell components. 2. Using this method, we have searched for the appearance of Ins(1:2cyclic,4,5)P3 [inositol 1:2(cyclic),4,5-trisphosphate] in WRK1 mammary tumour cells that were labelled to high specific radioactivity with [3H]inositol and then stimulated with 0.4 microM-vasopressin. 3. Vasopressin caused a very rapid accumulation of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate), followed by a slower decline towards the original concentration. An acid-labile and inositol-labelled compound with the chromatographic properties of Ins(1:2cyclic,4,5)P3 was present in unstimulated cells at less than 5% of the elevated concentration of Ins(1,4,5)P3. Its concentration rose 2-3-fold during stimulation for 3 min, at which time its concentration was about 5% of the elevated concentration of Ins(1,4,5)P3. 4. We conclude that Ins(1,4,5)P3 is the major product of phosphoinositidase C-catalysed phosphatidylinositol 4,5-bisphosphate hydrolysis in vasopressin-stimulated WRK1 cells. Ins(1:2cyclic,4,5)P3 is unlikely to be an important intracellular messenger in these cells, at least during the first few minutes of stimulation.

Arginine Vasopressin↗
PubMed1988

Partial purification and some properties of rat brain inositol 1,4,5-trisphosphate 3-kinase.

An enzyme which catalyses the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was purified approx. 180-fold from rat brain cytosol by (NH4)2SO4 precipitation, chromatography through hydroxyapatite, anion-exchange fast protein liquid chromatography and gel-filtration chromatography. Gel filtration on Sepharose 4B CL gives an Mr of 200 x 10(3) for the native enzyme. The inositol tetrakisphosphate (InsP4) produced by the enzyme has the chromatographic, chemical and metabolic properties of Ins(1,3,4,5)P4. Ins(1,4,5)P3 3-kinase displays simple Michaelis-Menten kinetics for both its substrates, having Km values of 460 microM and 0.44 microM for ATP and Ins(1,4,5)P3 respectively. When many of the inositol phosphates known to occur in cells were tested, only Ins(1,4,5)P3 was a substrate for the enzyme; the 2,4,5-trisphosphate was not phosphorylated. Inositol 4,5-bisphosphate and glycerophosphoinositol 4,5-bisphosphate were phosphorylated much more slowly than Ins(1,4,5)P3. CTP, GTP and adenosine 5'-[gamma-thio]triphosphate were unable to substitute for ATP. When assayed under conditions of first-order kinetics, Ins(1,4,5)P3 kinase activity decreased by about 40% as the [Ca2+] was increased over the physiologically relevant range. This effect was insensitive to the presence of calmodulin and appeared to be the result of an increase in the Km of the enzyme for Ins(1,4,5)P3. Preincubation with ATP and the purified catalytic subunit of cyclic AMP-dependent protein kinase did not affect the rate of phosphorylation of Ins(1,4,5)P3 when the enzyme was assayed at saturating concentrations of Ins(1,4,5)P3 or at concentrations close to its Km for this substrate.

Adenosine Triphosphate↗
PubMed1987

The pathway of myo-inositol 1,3,4-trisphosphate dephosphorylation in liver.

We studied the dephosphorylation pathway for Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) by liver homogenates and soluble and particulate subfractions incubated in media resembling physiological ionic strength and pH. Ins(1,3,4)P3 was dephosphorylated to two InsP2 (inositol bisphosphate) isomers, one of which is Ins(3,4)P2 [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147]. The second InsP2 is the 1,3 isomer. Ins(3,4)P2 is dephosphorylated to inositol 3-phosphate by an enzyme activity located in both soluble and particulate fractions. The phosphatase(s) that attacks Ins(1,3)P2 was largely soluble, but we have not determined which phosphate(s) is removed. When the initial substrate concentration was 1 nM, the rate of dephosphorylation of Ins(1,4)P2 greater than Ins(1,3)P2 greater than Ins(3,4)P2. None of these bisphosphates was phosphorylated when incubated with liver homogenates and 5 mM-ATP, but their rates of dephosphorylation were then decreased.

Animals↗
PubMed1987

Turkey erythrocytes possess a membrane-associated inositol 1,4,5-trisphosphate 3-kinase that is activated by Ca2+ in the presence of calmodulin.

Turkey erythrocytes contain soluble and particulate kinase activities which catalyse the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. The particle-bound activity accounts for approximately one-quarter of the total cellular Ins(1,4,5)P3 kinase, when assayed at a [Ca2+] of 10 nM. The particle-bound Ins(1,4,5)P3 kinase is not washed from the membrane by 0.6 M-KCl, yet may be solubilized by a variety of detergents. This suggests that it is an intrinsic membrane protein. The product of the membrane-bound Ins(1,4,5)P3 kinase is inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], identifying the enzyme as an Ins(1,4,5)P3 3-kinase. In the presence of calmodulin, the membrane-associated Ins(1,4,5)P3 3-kinase is activated as [Ca2+] is increased over the range 0.2-1.0 microM. Under these conditions, the rates of dephosphorylation of Ins(1,3,4,5)P4 and Ins(1,4,5)P3 by phosphatases in the membrane fraction are unchanged.

Animals↗