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Query: EC:3.1.3.1 (
alkaline phosphatase
)
47,916
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
A nonisotopic enzymolysis assay method of
phosphatidylinositol kinase
(PIK) has been developed. Phosphatidylinositol 4-phosphate (PIP), phosphorylated from phosphatidylinositol (PI) by PIK was hydrolyzed with phosphainositide-specific phospholipase C. The product, inositol 1, 4-bisphosphate (IP2), was separated from inositol 1-phosphate (IP) with Dowex-1 column chromatography. Then, the IP2 was hydrolyzed by
alkaline phosphatase
to yield PI, and the PI was determined by colorimetry. The PIK activity was defined as PI nmol/mg protein per min. The recovery was 91%, CV = 6.5%
...
PMID:[A nonisotopic enzymolysis assay method of phosphatidylinositol kinase]. 166 83
Phosphatidylinositol 4-phosphate (PIP) kinase (E.C. 2.7.1.68) has been purified about 1200-fold from rat liver plasma membranes, taking advantage of affinity chromatography on quercetin-Sepharose as a novel step. The purified PIP kinase showed no contamination by the following enzyme activities: phosphatidylinositol (PI) kinase (
EC 2.7.1.67
), protein kinase C (EC 2.7.1.-), diacylglycerol kinase (EC 2.7.1.-), phospholipase C (EC 3.1.4.11), protein-tyrosine kinase (EC 2.7.1.112),
alkaline phosphatase
(
EC 3.1.3.1
), triphosphoinositide phosphomonoesterase (EC 3.1.3.36), adenylate kinase (EC 2.7.4.3) and cAMP-dependent protein kinase (EC 2.7.1.37). The liver membrane enzyme requires high Mg2+ concentrations with a KM value of 10 mM. Ca2+ or Mn2+ could replace Mg2+ to a certain, though small, extent. Apparent KM values with respect to PIP and ATP were 10 and 65 microM, respectively. GTP was slightly utilized by the kinase as phosphate donor while CTP was not. Quercetin inhibited the enzyme with Ki = 34 microM. Extending our previous observations (Urumow, T. and Wieland, O.H. (1986) FEBS Lett. 207, 253-257 and Urumow, T. and Wieland, O.H. (1988) Biochim. Biophys. Acta 972, 232-238) [gamma S]pppG still stimulated the PIP kinase in extracts of solubilized liver membranes. 20-40% (NH4)2SO4 precipitation of the membrane extracts yielded a fraction that contained the bulk of enzyme activity but did not respond to stimulation by [gamma S]pppG any longer. This was restored by recombination with a protein fraction collected at 40-70% (NH4)2SO4 saturation, presumably containing a GTP binding protein and/or some other factor separated from the PIP kinase. In the reconstituted system [gamma S]pppG stimulated PIP kinase in a concentration dependent manner with maximal activation at 5 microM. This effect was not mimicked by [gamma S]pppA and was blocked by [beta S]ppG. These results strongly support our view that in liver membranes PIP kinase is regulated by a G-protein.
...
PMID:Purification and partial characterization of phosphatidylinositol-4-phosphate kinase from rat liver plasma membranes. Further evidence for a stimulatory G-protein. 215 97
PIK-A49 is a 49-kDa soluble protein that was isolated as an activator of the plasma membrane phosphatidylinositol (PI) 4-kinase from carrot cells (Yang, W., Burkhart, W., Cavallius, J., Merrick, W. C., and Boss, W. F. (1993) J. Biol. Chem. 268, 392-398). PIK-A49 is a multifunctional protein that binds and bundles F-actin and has translational elongation factor-1 alpha activity. In this paper, we have investigated the mechanism of activation of
PI 4-kinase
by PIK-A49. PIK-A49 decreased the Km of
PI 4-kinase
for ATP from 0.40 to 0.19 mM. GTP and GDP, which affect the elongation factor-1 alpha function of the protein, inhibited the activation of
PI 4-kinase
by PIK-A49. Phosphorylation of purified PIK-A49 by a calcium-dependent protein kinase enhanced activation of
PI 4-kinase
. When dephosphorylated by
alkaline phosphatase
, PIK-A49 no longer activated
PI 4-kinase
; however, rephosphorylation of PIK-A49 by calcium-dependent protein kinase fully restored activation. Western blots using anti-PIK-A49 serum showed that PIK-A49 was associated with the plasma membrane and the F-actin fraction isolated from plasma membranes, indicating that PIK-A49 would be in a position to regulate plasma membrane
PI 4-kinase
. Based on these data, we propose a mechanism for feed-forward regulation of polyphosphoinositide biosynthesis in response to increases in cytosolic calcium.
...
PMID:Regulation of phosphatidylinositol 4-kinase by the protein activator PIK-A49. Activation requires phosphorylation of PIK-A49. 810 30
Tamoxifen caused a dramatic stimulation of
phosphatidylinositol kinase
and phosphatidylinositol-phosphate (PIP) kinase activity in GH4C1 membrane preparation with an ED50 of 20 microM. Vanadate ions alone did not appreciably elevate the amount of polyphosphoinositides; however, when added together with tamoxifen it synergistically enhanced the formation of PIP and phosphatidylinositol-bisphosphate (PIP2). Vanadate caused the inhibition of
phosphomonoesterase
activity in the membranes that converts PIP2 to PIP and PIP to phosphatidylinositol. The synergism between tamoxifen and vanadate thus results from tamoxifen-induced stimulation of the phosphoinositide kinase reaction and vanadate inhibition of the backward
phosphomonoesterase
reaction. Tamoxifen had no effect on
phosphomonoesterase
activity. With optimal concentrations of the drugs, PIP was increased from 8.3 to 75%, and PIP2 was augmented from 0.36 to 8.5% of the total membrane phosphoinositides. Tamoxifen and vanadate are thus useful tools for the investigation of phosphoinositides metabolism.
...
PMID:Tamoxifen and vanadate synergize in causing accumulation of polyphosphoinositides in GH4C1 membranes. 824 36
