A purification strategy for inositol 1,4,5-trisphosphate 3-kinase from rat liver based upon heparin interaction chromatography

Conigrave, Arthur D.; Patwardhan, A. A.; Broomhead, Louise; Roufogalis, Basil D.

doi:10.1016/0898-6568(92)90070-o

Cited by 11 publications

(8 citation statements)

References 14 publications

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“…Indeed, injection of an enzyme that acts specifically on InsP 3 catabolism in the injected cell but cannot diffuse through gap junctions makes it possible to observe the effect of InsP 3 metabolism on Ca 2+ oscillations, while the non‐injected cell provides a natural control for unperturbed Ca 2+ oscillations. Moreover, 3‐kinase B has been isolated from rat hepatocytes [30] and shown to be stimulated by Ca 2+ [31], while the activity of InsP 3 5‐phosphatase has been shown to be unaffected by changes in [Ca 2+ ] in this cell type [32]. Thus, we have injected type I InsP 3 5‐phosphatase in only one cell of hepatocyte doublets.…”

Section: Experimental Results: Effect Of Injecting Insp3 5‐phosphatasmentioning

confidence: 99%

Ca²⁺ oscillations in hepatocytes do not require the modulation of InsP₃ 3‐kinase activity by Ca²⁺

et al. 2002

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Receptor-mediated production of inositol 1,4,5-trisphosphate (InsP 3 ) initiates Ca 2+ release and is responsible for cytosolic Ca 2+ oscillations. InsP 3 oscillations have also been observed in some cells. One of the enzymes controlling InsP 3 catabolism, the InsP 3 3-kinase, is stimulated by Ca 2+ ; this regulation is presumably part of the reason for InsP 3 oscillations that have been observed in some cells. Here, we investigate the possible role of Ca 2+ -activated InsP 3 catabolism on the characteristics of the InsP 3 -induced Ca 2+ oscillations. Numerical simulations show that if it is assumed that the Ca 2+ -independent InsP 3 catabolism is predominant, Ca 2+ oscillations remain qualitatively unchanged although the relative amplitude of the oscillations in InsP 3 concentrations becomes minimal. We tested this prediction in hepatocytes by masking the Ca 2+ -dependent InsP 3 catabolism by 3-kinase through the injection of massive amounts of InsP 3 5-phosphatase, which is not stimulated by Ca 2+ . We ¢nd that in such injected hepatocytes, Ca 2+ oscillations generated by modest agonist levels are suppressed, presumably because of the decreased dose in InsP 3 , but that at higher doses of agonist, oscillations reappear, with characteristics similar to those of untreated cells at low agonist doses. Altogether, these results suggest that oscillations in InsP 3 concentration due to Ca 2+ -stimulated InsP 3 catabolism do not play a major role for the oscillations in Ca 2+ concentration. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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Section: Experimental Results: Effect Of Injecting Insp3 5‐phosphatasmentioning

confidence: 99%

Ca²⁺ oscillations in hepatocytes do not require the modulation of InsP₃ 3‐kinase activity by Ca²⁺

et al. 2002

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“…However, since the molecular mass of these proteins does not match the predicted molecular mass for the B isoform and the degree of calcium/ calmodulin stimulation is not completely predictive of the IP3K isoform present (see below), further experimentation is necessary to identify these proteins. IP3K proteins in the 60-kDa range have been isolated from rat liver (12) and porcine muscle (43), but these proteins have much lower specific activity and are far less sensitive to calcium/calmodulin than the recombinant IP3K B isoform purified in this study. The cDNA for the B isoform has been expressed in E. coli, but the protein is insoluble or inactive unless expressed as a fusion protein (44).…”

Section: Purification Of the Ip3k Isoformsmentioning

confidence: 99%

“…Both calcium/calmodulin and protein phosphorylation mechanisms are documented to control its activity (8). Preparations of IP3K purified from rat brain (9 -11) or rat liver (12) can be activated 2-3-fold by addition of calcium/calmodulin, while the IP3K purified from other sources can be activated in the range of 4 -17-fold by calcium/calmodulin (13)(14)(15)(16). The IP3K is also a substrate for the cyclic AMP-dependent protein kinase, the calcium/calmodulin-dependent protein kinase II, and protein kinase C in vitro.…”

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confidence: 99%

Expression, Purification, and Regulation of Two Isoforms of the Inositol 1,4,5-Trisphosphate 3-Kinase

Woodring

Garrison

1997

Journal of Biological Chemistry

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The level of inositol 1,4,5-trisphosphate in the cytoplasm is tightly regulated by two enzymes, the inositol 1,4,5,5-phosphatase and the inositol 1,4,5-trisphosphate 3-kinase. Two isoforms of the inositol 1,4,5-trisphosphate 3-kinase have been identified, the A form and the B form. The regulatory properties of the two isoforms were compared following overexpression and purification of the proteins from a v-src transformed mammalian cell line. The highly purified, recombinant inositol 1,4,5-trisphosphate 3-kinases were differentially regulated by calcium/calmodulin and via phosphorylation by protein kinase C or the cyclic AMP-dependent protein kinase. Both enzymes had similar affinities for inositol 1,4,5-trisphosphate (K m 2-5 M). Calcium/calmodulin stimulated the activity of isoform A about 2.5-fold, whereas the activity of isoform B was increased 20-fold. The cyclic AMP-dependent protein kinase phosphorylated the inositol 1,4,5-trisphosphate 3-kinase A to the extent of 0.9 mol/mol and isoform B to 1 mol/mol. Protein kinase C phosphorylated isoform A to the extent of 2 mol/mol and isoform B to 2.7 mol/mol. Phosphorylation of isoform A by the cyclic AMP-dependent protein kinase caused a 2.5-fold increase in its activity when assayed in the absence of calcium/calmodulin, whereas phosphorylation by protein kinase C decreased activity by 72%. The activity of isoform B in the absence of calcium/calmodulin was not affected by phosphorylation using either kinase. When assayed in the presence of calcium/calmodulin, phosphorylation of isoform A by the cyclic AMP-dependent protein kinase increased activity 1.5-fold, whereas phosphorylation of isoform B decreased activity by 45%. Phosphorylation of either isoform A or B by protein kinase C resulted in a 70% reduction of calcium/calmodulin-stimulated activity. Differential expression and regulation of the two inositol 1,4,5-trisphosphate 3-kinase isoforms provides multiple mechanisms for regulating the cytosolic level of inositol 1,4,5-trisphosphate in cells.The second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) 1 mediates the biological response of a large number of hormones and neurotransmitters in target cells by regulating calcium release from intracellular stores (1, 2). In keeping with its biological role, the levels of Ins(1,4,5)P 3 are tightly regulated by two mechanisms; dephosphorylation via an Ins(1,4,5)P 3 5-phosphatase to Ins(1,4)P 2 or by phosphorylation with the inositol 1,4,5-trisphosphate 3-kinase (IP3K) to Ins(1,3,4,5)P 4 . The former enzyme initiates the pathway which recycles the inositol moiety to the plasma membrane as phosphatidylinositols. The latter enzyme produces Ins(1,3,4,5)P 4 (1, 3, 4), which has been suggested to have roles in controlling calcium homeostasis, transferring calcium between intracellular stores, and/or regulating calcium entry across the plasma membrane (5). In addition, Ins(1,3,4,5)P 4 may also play a role in regulating cross-talk between the calcium and other signaling pathways as an Ins(1,3,4,5)P 4 binding protein...

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“…Data obtained with cell lysates have often confirmed that Caz+ is a stimulator of Ins(l,4,5)P3 3-kinase (see, e. g. Biden and Wollheim, 1986;Zilberman et al, 1987). Stimulation can be very potent, as in bovine brain (Takazawa et al, 1989;Li et al, 1989) or limited to 2-3-fold effects, such as those observed in rat hepatocytes (Biden et al, 1988), liver (Conigrave et al, 1992) or pig aortic smooth muscle (Yamagushi et al, 1988).…”

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Interaction of calmodulin with a putative calmodulin‐binding domain of inositol 1,4,5‐triphosphate 3‐kinase.

Erneux

Moreau

Vandermeers

et al. 1993

European Journal of Biochemistry

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Recombinant rat brain inositol 1,4,5‐triphosphate [Ins(1,4,5)P3] 3‐kinase was expressed in Escherichia coli as a β‐galactosidase fusion product. It could be adsorbed onto calmodulin‐Sepharose and eluted in Ca2+‐free medium as a 48‐kDa protein. Purification could be achieved in a single step. Molecular evidence for a calmodulin‐binding domain on Ins(1,4,5)P3 3‐kinase can be shown by the following approaches. (a) Inhibition of Ca2+/calmodulin stimulation by a synthetic peptide based on a candidate calmodulin‐binding domain. The inhibition was mimicked by a well‐characterized peptide derived from the sequence of smooth muscle myosin light‐chain kinase calmodulin‐binding site. (b) The construction of two mutants by site‐directed mutagenesis of Trp165 to Gly or Arg. Both mutants displayed kinase activity but were no longer Ca2+/calmodulin sensitive, supporting, therefore, the role of Trp165 in calmodulin binding.

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A purification strategy for inositol 1,4,5-trisphosphate 3-kinase from rat liver based upon heparin interaction chromatography

Cited by 11 publications

References 14 publications

Ca²⁺ oscillations in hepatocytes do not require the modulation of InsP₃ 3‐kinase activity by Ca²⁺

Ca²⁺ oscillations in hepatocytes do not require the modulation of InsP₃ 3‐kinase activity by Ca²⁺

Expression, Purification, and Regulation of Two Isoforms of the Inositol 1,4,5-Trisphosphate 3-Kinase

Interaction of calmodulin with a putative calmodulin‐binding domain of inositol 1,4,5‐triphosphate 3‐kinase.

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A purification strategy for inositol 1,4,5-trisphosphate 3-kinase from rat liver based upon heparin interaction chromatography

Cited by 11 publications

References 14 publications

Ca2+ oscillations in hepatocytes do not require the modulation of InsP3 3‐kinase activity by Ca2+

Ca2+ oscillations in hepatocytes do not require the modulation of InsP3 3‐kinase activity by Ca2+

Expression, Purification, and Regulation of Two Isoforms of the Inositol 1,4,5-Trisphosphate 3-Kinase

Interaction of calmodulin with a putative calmodulin‐binding domain of inositol 1,4,5‐triphosphate 3‐kinase.

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Ca²⁺ oscillations in hepatocytes do not require the modulation of InsP₃ 3‐kinase activity by Ca²⁺

Ca²⁺ oscillations in hepatocytes do not require the modulation of InsP₃ 3‐kinase activity by Ca²⁺