Separation of multiple isomers of inositol phosphates formed in GH3 cells

Dean, Nicholas M.; Moyer, James D.

doi:10.1042/bj2420361

Cited by 135 publications

(61 citation statements)

References 29 publications

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“…To characterize the products of the enzymic reactions involved in inositol polyphosphate degradation in platelets, we used a chromatographic system that allows separation of different isomers [24]. The major metabolite of Ins(1,4,5)P3 in platelet cytosol and membranes was found to be the product of Ins(1,4,5)P3 5-phosphatase, Ins(1,4)P2.…”

Section: Discussionmentioning

confidence: 99%

“…Platelets from fresh human blood or outdated platelets from the American Red Cross were washed twice with a phosphate buffer, pH 6.5, containing 113 mM-NaCl, 4.3 mM-K2HPO4, 4.3 mM-Na2HPO4, 24.4 mM-NaH2PO4, 5.5 mMglucose and 1 mM-EGTA. Platelets were then resuspended in a hypo-osmotic buffer (11 mM-Tris, 14 mm-Mes, 3 mM-MgCl2, 20 mM-,/-mercaptoethanol, pH 6.5) and lysed by one cycle of freezing and thawing, followed by nitrogen cavitation at 1000 lb/in2 (6.9 MPa) for 1 h.…”

Section: Experimental Preparation Of Platelet Subcellular Fractionsmentioning

confidence: 99%

“…were incubated as above, in a final volume of 100 pl, and contained 50000 cpm of Inositol polyphosphates were separated by h.p.l.c. on a Whatman Partisil 10 SAX column with ammonium phosphate, pH 3.8, flow rate 1 ml/min, in accordance with a method previously described [24]. The gradients used were: 0-0.1 M over 35 min for inositol, Ins(l)P and Ins (4) [3H]Ins(1,3,4)P3 standard was prepared by incubating [3H]Ins(1,3,4,5)P4 with platelet cytosol, yielding a product that was eluted immediately before Ins(1,4,5)P3.…”

Section: Experimental Preparation Of Platelet Subcellular Fractionsmentioning

confidence: 99%

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Subcellular localization of the enzymes that dephosphorylate myo-inositol polyphosphates in human platelets

Vedia

Nolan

Lapetina

1988

Biochemical Journal

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The phosphatase-induced hydrolysis of [3H]inositol 1,4-bisphosphate [Ins(1,4)P2)] and [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was studied in platelet subcellular fractions. The activity that hydrolyses Ins(1,4)P2 is cytosolic, whereas the activity that hydrolyses Ins(1,4,5)P3 is present in both particulate and cytosolic fractions. The cytosolic Ins(1,4)P2 phosphatase hydrolyses the 1-phosphate of Ins(1,4)P2, whereas the cytosolic and membrane-bound Ins(1,4,5)P3 phosphatases hydrolyse the 5-phosphate of Ins(1,4,5)P3. In the presence of ATP, it is possible to observe a cytosolic Ins(1,4,5)P3 3-kinase that phosphorylates Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate. Apparent Km values for the particulate and the cytosolic Ins(1,4,5)P3 phosphatases are 100 tM and 40,/M respectively. A large proportion of the membraneassociated Ins(1,4,5)P3 phosphatase can be extracted with 1 M-NaCl, and the Mr of this enzyme, as determined by hydrodynamic studies, is 49000, whereas that of the cytosolic enzyme is 59000. The Km values for the cytosolic Ins(1,4)P2 phosphatase is 40 ,UM; this enzyme has an Mr of49 000. The highest specific activity of the Ins(1,4,5)P3 phosphatase is present in a highly purified plasma-membrane fraction.

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Section: Discussionmentioning

confidence: 99%

Section: Experimental Preparation Of Platelet Subcellular Fractionsmentioning

confidence: 99%

Section: Experimental Preparation Of Platelet Subcellular Fractionsmentioning

confidence: 99%

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Subcellular localization of the enzymes that dephosphorylate myo-inositol polyphosphates in human platelets

Vedia

Nolan

Lapetina

1988

Biochemical Journal

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“…Pooled samples were spiked with 5 /iL of a solution containing 1 mmol/L each of cAMP, GMP, ADP, GDP, ATP, and GTP, and 100-^AL aliquots were injected onto the column. The HPLC analysis of inositol phosphates was performed according to a previously described method 20 21 VSMCs were loaded with a l-/wnol/L fura 2-AM solution for 15 minutes at 37°C. The cultures were washed with PSS to remove unincorporated fura 2-AM and incubated for an additional 5 minutes at 37°C to allow diffusion of nonhydrolyzed dye from the cell.…”

mentioning

confidence: 99%

Concerted effects of lipoproteins and angiotensin II on signal transduction processes in vascular smooth muscle cells.

Bochkov

Ткачук

Hahn

et al. 1993

Arterioscler Thromb

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Low-density (LDL) and high-density (HDLj) lipoproteins dose-dependently activate phosphoinositide turnover and elevate cytosolic free Ca 2+ concentrations ([Ca 1+ ]|) in cultured vascular smooth muscle cells (VSMCs) from either human (microarterioles and aorta) or rat (aorta) sources. High-performance liquid chromatography analysis of cell extracts revealed comparable spectra of inositol phosphate isomers generated in response to either LDL, HDL,, or angiotensin II (Ang II 10 Epidemiologic studies also show that hypertension is a major risk factor for development of atherosclerosis. 4 Different hypotheses have been proposed to explain the association between the two diseases, with the common etiologic denominators being either disturbances in plasma lipoprotein concentrations and cellular cholesterol homeostasis, 5 intracellular calcium overload, 6 or altered responses to growth factors and vasoregulatory hormones.1 -3 These theories are not mutually exclusive, however, and linkage between the latter two is predictable because growth factors and vasoregulatory hormones are known to mobilize intracellular calcium. The relation between lipoprotein/cholesterol disturbances and the calcium-based or agonist growth factor-based theories is less conspicuous, but it

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“…The column was calibrated using authentic labelled standards obtained commercially or prepared according to published methods [19,20].…”

Section: Separation Of Water-soluble Inositol Phosphatesmentioning

confidence: 99%

Phosphatidylinositol turnover is not a general regulator of neuroblastoma cell differentiation: comparison between two differentiating agents, retinoic acid and γ‐interferon

Lanciotti¹,

Cornaglia-Ferraris²,

Ponzoni³

1989

FEBS Letters

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The turnover of phosphatidylinositol (PI) is believed to constitute a crucial step in the signaling pathways for stimulation of cells by a variety of bioactive substances, including differentiating agents; however decisive evidence for the idea has not been obtained. In the present paper, we investigated the involvement of PI turnover in cell differentiation using a human neuroblastoma cell line, LAN-1, which can be induced to differentiate along the neuronal pathway by both retinoic acid (RA) and ~,-interferon (?-IFN). Analysis of labelled phosphatidylinositol metabolites from prelabelled cells indicated a rapid decrease of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol within 1 min of induction of LAN-1 cell differentiation by RA, while no changes were observed in ~,-IFN-treated cells. These findings indicate the occurrence of decreased inositol phospholipid turnover in RA-treated LAN-I cells and suggest that phosphoinositide-derived metaholites may not constitute general regulators of cellular differentiation.

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Separation of multiple isomers of inositol phosphates formed in GH3 cells

Cited by 135 publications

References 29 publications

Subcellular localization of the enzymes that dephosphorylate myo-inositol polyphosphates in human platelets

Subcellular localization of the enzymes that dephosphorylate myo-inositol polyphosphates in human platelets

Concerted effects of lipoproteins and angiotensin II on signal transduction processes in vascular smooth muscle cells.

Phosphatidylinositol turnover is not a general regulator of neuroblastoma cell differentiation: comparison between two differentiating agents, retinoic acid and γ‐interferon

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