The subcellular distribution of triphosphoinositide phosphomonoesterase in guinea-pig brain

Upon subcellular fractionation of (murine) Friend erythroleukaemic cells (FELCs), purified plasma membranes were identified by their high enrichment in specific marker enzymes and typical plasma membrane lipids. When FELCs were incubated for short periods with 32Pi before cell fractionation, the lipid‐bound radioactivity was almost exclusively present in phosphatidylinositol‐4‐phosphate (DPI) and phosphatidylinositol‐4,5‐bisphosphate (TPI), and its distribution closely matched that of the plasma membrane markers. In addition, purified plasma membranes actively incorporated 32P from [γ‐32P]ATP into polyphosphoinositides, and the specific activities of the involved kinases were again mostly enriched in the plasma membrane fraction.

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“…Phosphorylation was started by addition of 10 ~1 38. 8 The relative specific activity in the homogenate = 1.0.…”

Section: Methodsmentioning

confidence: 99%

(poly)Phosphoinositide phosphorylation is a marker for plasma membrane in Friend erythroleukaemic cells

et al. 1982

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“…These kinases are known to exist in a variety of tissues (7,8,15,17,(20)(21)(22)30). There are also corresponding phosphomonoesterases (23,33,36), and the inositol lipids mentioned above are thought to be in equilibrium with each other through "futile cycles" constituted by these kinases and esterases. The close relation between this signaling system and cell growth regulation was suggested by the fact that several growth factors could induce PI turnover (10,14,34).…”

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confidence: 99%

Phosphatidylinositol kinase activity in virus-transformed and nontransformed cells.

Sugano¹,

Hanafusa²

1985

Mol. Cell. Biol.

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We assayed phosphatidylinositol (PI) kinase (EC 2.7.1.67) activity in detergent extracts of nontransformed or virus-transformed cells. Nontransformed chicken embryo fibroblasts (CEF) contain PI kinase activity with an apparent specific activity of 20 pmol/min per mg of protein. This activity sedimented as a single peak with a molecular weight of approximately 60,000 in a glycerol gradient, although immunoprecipitation with anti-p60src sera showed that the PI kinase activity is distinct from p60c-src. Extracts from CEF transformed by Rous sarcoma virus, Fujinami sarcoma virus, or avian sarcoma virus UR2 showed no elevation of PI kinase activity over nontransformed CEF. Removal of the oncogene products from extracts by immunoprecipitation did not change the level of PI kinase activity in extracts, suggesting that putative virus-coded PI kinases do not make a significant contribution to overall levels of PI kinase activity in transformed cells. Additionally, P140Wagf-Is was separated from cellular PI kinase by phosphocellulose chromatography. This partially purified fraction contained low PI kinase activity distinct from Pl40W-fPs; indicating that p140Wzg.-fiS has no detectable PI kinase activity.There has been a great deal of interest in an intracellular signaling system that utilizes inositol lipids and might be involved in regulating cell growth (1,26,29,32). Various stimuli induce the rapid hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) by phospholipase C to diacylglycerol and inositol trisphosphate which act as second messengers. Diacylglycerol activates protein kinase C, and inositol trisphosphate mobilizes intracellular calcium. This hydrolysis is followed by the phosphorylation of phosphatidylinositol (PI) and phosphatidylinositol-4-monophosphate (PIP). PIP2 is formed from PI through stepwise phosphorylation by PI kinase (EC 2.7.1.67) and PIP kinase (EC 2.7.1.68). These kinases are known to exist in a variety of tissues (7,8,15,17,(20)(21)(22)30). There are also corresponding phosphomonoesterases (23,33,36), and the inositol lipids mentioned above are thought to be in equilibrium with each other through "futile cycles" constituted by these kinases and esterases. The close relation between this signaling system and cell growth regulation was suggested by the fact that several growth factors could induce PI turnover (10,14,34). In addition, tumor-promoting phorbol esters can stimulate protein kinase C directly (5).The stimulation of this system in the transforming process of a tumor virus was first suggested by Diringer and Friis (9) PI. They suggested that various oncogene products possessing tyrosine kinase activity might also have PI kinase activity and that PI kinase activity is responsible for transformation.In this report, we asked two questions relevant to two important points of the above hypotheses. (i) If elevated PI turnover is a direct effect of the PI kinase activity of oncogene products, then what is the difference in the overall PI kinase activity between transformed and nontransfor...

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“…Since no Cetavlon activation of diphosphoinositide phosphatase was observed with dialysed homogenate and Mg2+, it is possible that a third factor present in the non-dialysed preparation can also activate this enzyme. The pH5-supernatant factor of Sheltawy et al (1972) appears to be different, since it resists dialysis .…”

Section: Effect Of Cetavlon In the Presence Of Mg2+mentioning

confidence: 96%

“…Salway et al (1967) and Harwood & Hawthorne (1969) studying brain found that triphosphoinositide phosphatase was localized in the particle-free cytoplasmic fraction. Under different assay conditions, using 'pH 5 supernatant' as activator, Sheltawy et al (1972) reported a distribution of the brain phosphatase similar to that of the plasma-membrane marker 5'-nucleotidase, but different from that of alkaline phosphatase. However, Lee & Huggins (1968a,b), using a very simple assay procedure for the enzyme, found enrichment in the microsomal fraction of kidney, the distribution being similar to that of glucose 6-phosphatase.…”

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confidence: 93%

“…Similarly, Santiago-Calvo et al (1964) found a high turnover of the phosphoinositides in various tissues. Phosphatidylinositol bisphosphate phosphatase (EC 3.1.3.36) has been investigated in brain (Dawson & Salway et al, 1967;Sheltawy et al, 1972) and kidney (Lee & Huggins, 1968a,b), but work on the subcellular distribution of this enzyme has led to conflicting observations. Salway et al (1967) and Harwood & Hawthorne (1969) studying brain found that triphosphoinositide phosphatase was localized in the particle-free cytoplasmic fraction.…”

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confidence: 99%

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Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system

Cooper

Hawthorne

1975

Biochemical Journal

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The properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergents sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the substrate and in the presence of Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and Li+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and densitygradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that ofthe liver Golgi marker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble. Sloane-Stanley (1953) showed that brain phosphoinositide fractions were rapidly attacked by brain homogenates with the release of free and combined phosphate as well as free and combined inositol. Rodnight (1956) later performed experiments to suggest that two enzymes, a phosphomonoesterase and a phosphodiesterase, were present. Work with pure triphosphoinositide (phosphatidyl-myoinositol 4,5-bisphosphate)$ indicated that the lipid was broken down first to diphosphoinositide (phosphatidyl-myo-inositol 4-phosphate) and then to phosphatidylinositol

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The subcellular distribution of triphosphoinositide phosphomonoesterase in guinea-pig brain

Cited by 35 publications

References 23 publications

(poly)Phosphoinositide phosphorylation is a marker for plasma membrane in Friend erythroleukaemic cells

(poly)Phosphoinositide phosphorylation is a marker for plasma membrane in Friend erythroleukaemic cells

Phosphatidylinositol kinase activity in virus-transformed and nontransformed cells.

Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system

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