Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1988 Apr 1;251(1):129–134. doi: 10.1042/bj2510129

Purification and characterization of inositol 1,4,5-trisphosphate 3-kinase from pig aortic smooth muscle.

K Yamaguchi 1, M Hirata 1, H Kuriyama 1
PMCID: PMC1148973  PMID: 2839156

Abstract

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase, which phosphorylates InsP3 to form inositol 1,3,4,5-tetrakisphosphate, was purified to apparent homogeneity by (NH4)2SO4 fractionation and sequential chromatographic steps on DEAE-sepharose, calmodulin-Affi-Gel and DEAE-5PW h.p.l.c. The purified enzyme had a specific activity of 24.4 nmol of inositol tetrakisphosphate formed/min per mg of protein, which represented a purification of approx. 195-fold with a 0.29% recovery, compared with the cytosol fraction of the muscle. SDS/polyacrylamide-gel electrophoresis showed a single protein-staining band of Mr 93,000. Moreover, the major protein peak, of Mr 84,000, was detected by TSK gel G3000SW gel-permeation chromatography of the purified sample. As this value was approximately consistent with the Mr determined by SDS/polyacrylamide-gel-electrophoretic analysis, the InsP3 3-kinase might be a monomeric enzyme. The purified enzyme had a Km for InsP3 of 0.4 microM, with an optimum pH range of 5.8-7.7. The enzyme was maximally activated by calmodulin, with a stoichiometry of 1:1.

Full text

PDF
129

Images in this article

132Fig. 4.
on p.132

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adelstein R. S., Klee C. B. Purification and characterization of smooth muscle myosin light chain kinase. J Biol Chem. 1981 Jul 25;256(14):7501–7509. [PubMed] [Google Scholar]
  2. Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
  3. Biden T. J., Wollheim C. B. Ca2+ regulates the inositol tris/tetrakisphosphate pathway in intact and broken preparations of insulin-secreting RINm5F cells. J Biol Chem. 1986 Sep 15;261(26):11931–11934. [PubMed] [Google Scholar]
  4. Cohen P. The subunit structure of rabbit-skeletal-muscle phosphorylase kinase, and the molecular basis of its activation reactions. Eur J Biochem. 1973 Apr 2;34(1):1–14. doi: 10.1111/j.1432-1033.1973.tb02721.x. [DOI] [PubMed] [Google Scholar]
  5. Connolly T. M., Bross T. E., Majerus P. W. Isolation of a phosphomonoesterase from human platelets that specifically hydrolyzes the 5-phosphate of inositol 1,4,5-trisphosphate. J Biol Chem. 1985 Jul 5;260(13):7868–7874. [PubMed] [Google Scholar]
  6. Downes C. P., Mussat M. C., Michell R. H. The inositol trisphosphate phosphomonoesterase of the human erythrocyte membrane. Biochem J. 1982 Apr 1;203(1):169–177. doi: 10.1042/bj2030169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. GRADO C., BALLOU C. E. Myo-inositol phosphates obtained by alkaline hydrolysis of beef brain phosphoinositide. J Biol Chem. 1961 Jan;236:54–60. [PubMed] [Google Scholar]
  8. Hansen C. A., Mah S., Williamson J. R. Formation and metabolism of inositol 1,3,4,5-tetrakisphosphate in liver. J Biol Chem. 1986 Jun 25;261(18):8100–8103. [PubMed] [Google Scholar]
  9. Harafuji H., Ogawa Y. Re-examination of the apparent binding constant of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid with calcium around neutral pH. J Biochem. 1980 May;87(5):1305–1312. doi: 10.1093/oxfordjournals.jbchem.a132868. [DOI] [PubMed] [Google Scholar]
  10. Hashimoto T., Hirata M., Itoh T., Kanmura Y., Kuriyama H. Inositol 1,4,5-trisphosphate activates pharmacomechanical coupling in smooth muscle of the rabbit mesenteric artery. J Physiol. 1986 Jan;370:605–618. doi: 10.1113/jphysiol.1986.sp015953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hawkins P. T., Stephens L., Downes C. P. Rapid formation of inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate in rat parotid glands may both result indirectly from receptor-stimulated release of inositol 1,4,5-trisphosphate from phosphatidylinositol 4,5-bisphosphate. Biochem J. 1986 Sep 1;238(2):507–516. doi: 10.1042/bj2380507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Higashida H., Brown D. A. Membrane current responses to intracellular injections of inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate in NG108-15 hybrid cells. FEBS Lett. 1986 Nov 24;208(2):283–286. doi: 10.1016/0014-5793(86)81033-x. [DOI] [PubMed] [Google Scholar]
  13. Hirata M., Sasaguri T., Hamachi T., Hashimoto T., Kukita M., Koga T. Irreversible inhibition of Ca2+ release in saponin-treated macrophages by the photoaffinity derivative of inositol-1, 4, 5-trisphosphate. Nature. 1985 Oct 24;317(6039):723–725. doi: 10.1038/317723a0. [DOI] [PubMed] [Google Scholar]
  14. Hirata M., Suematsu E., Koga T. Effect of calmodulin and calmodulin antagonists on the Ca2+ uptake by the intracellular Ca2+-accumulating system of guinea pig peritoneal macrophages treated with saponin. Mol Pharmacol. 1983 Jan;23(1):78–85. [PubMed] [Google Scholar]
  15. Irvine R. F., Letcher A. J., Heslop J. P., Berridge M. J. The inositol tris/tetrakisphosphate pathway--demonstration of Ins(1,4,5)P3 3-kinase activity in animal tissues. Nature. 1986 Apr 17;320(6063):631–634. doi: 10.1038/320631a0. [DOI] [PubMed] [Google Scholar]
  16. Irvine R. F., Letcher A. J., Lander D. J., Downes C. P. Inositol trisphosphates in carbachol-stimulated rat parotid glands. Biochem J. 1984 Oct 1;223(1):237–243. doi: 10.1042/bj2230237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Irvine R. F., Moor R. M. Micro-injection of inositol 1,3,4,5-tetrakisphosphate activates sea urchin eggs by a mechanism dependent on external Ca2+. Biochem J. 1986 Dec 15;240(3):917–920. doi: 10.1042/bj2400917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kennedy M. B., Greengard P. Two calcium/calmodulin-dependent protein kinases, which are highly concentrated in brain, phosphorylate protein I at distinct sites. Proc Natl Acad Sci U S A. 1981 Feb;78(2):1293–1297. doi: 10.1073/pnas.78.2.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kimura Y., Hirata M., Yamaguchi K., Koga T. Activation by calmodulin of inositol-1,4,5-trisphosphate 3-kinase in guinea pig peritoneal macrophages. Arch Biochem Biophys. 1987 Sep;257(2):363–369. doi: 10.1016/0003-9861(87)90578-9. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Rossier M. F., Dentand I. A., Lew P. D., Capponi A. M., Vallotton M. B. Interconversion of inositol (1,4,5)-trisphosphate to inositol (1,3,4,5)-tetrakisphosphate and (1,3,4)-trisphosphate in permeabilized adrenal glomerulosa cells is calcium-sensitive and ATP-dependent. Biochem Biophys Res Commun. 1986 Aug 29;139(1):259–265. doi: 10.1016/s0006-291x(86)80107-3. [DOI] [PubMed] [Google Scholar]
  22. Somlyo A. V., Bond M., Somlyo A. P., Scarpa A. Inositol trisphosphate-induced calcium release and contraction in vascular smooth muscle. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5231–5235. doi: 10.1073/pnas.82.15.5231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Streb H., Irvine R. F., Berridge M. J., Schulz I. Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature. 1983 Nov 3;306(5938):67–69. doi: 10.1038/306067a0. [DOI] [PubMed] [Google Scholar]
  24. Suematsu E., Hirata M., Hashimoto T., Kuriyama H. Inositol 1,4,5-trisphosphate releases Ca2+ from intracellular store sites in skinned single cells of porcine coronary artery. Biochem Biophys Res Commun. 1984 Apr 30;120(2):481–485. doi: 10.1016/0006-291x(84)91279-8. [DOI] [PubMed] [Google Scholar]
  25. Suematsu E., Hirata M., Sasaguri T., Hashimoto T., Kuriyama H. Roles of Ca2+ on the inositol 1,4,5-trisphosphate-induced release of Ca2+ from saponin-permeabilized single cells of the porcine coronary artery. Comp Biochem Physiol A Comp Physiol. 1985;82(3):645–649. doi: 10.1016/0300-9629(85)90446-3. [DOI] [PubMed] [Google Scholar]
  26. Yamaguchi K., Hirata M., Kuriyama H. Calmodulin activates inositol 1,4,5-trisphosphate 3-kinase activity in pig aortic smooth muscle. Biochem J. 1987 Jun 15;244(3):787–791. doi: 10.1042/bj2440787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yamamoto H., van Breemen C. Inositol-1,4,5-trisphosphate releases calcium from skinned cultured smooth muscle cells. Biochem Biophys Res Commun. 1985 Jul 16;130(1):270–274. doi: 10.1016/0006-291x(85)90412-7. [DOI] [PubMed] [Google Scholar]
  28. Yazawa M., Sakuma M., Yagi K. Calmodulins from muscles of marine invertebrates, scallop and sea anemone. J Biochem. 1980 May;87(5):1313–1320. doi: 10.1093/oxfordjournals.jbchem.a132869. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES