Partial purification and characterization of phosphatidylinositol kinases from human platelets

Hiroyuki Kanoh, Yoshiko Banno, Masato Hirata, Yoshinori Nozawa

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Abstract

Most of human platelet phosphatidylinositol (PI) kinase activity (approx. 80%) was associated with the membrane fraction and its majority was released by the extraction with Triton X-100 after KCI treatment. Two major activity peaks (mPIK-I and mPIK-III) were obtained by Mono Q column chromatography. They were distinct from each other with regard to Mr (76000 and 80000 as determined by gel-filtration chromatography), apparent Km values for ATP, effect of arachidonic acid and phosphatidylserine and detergent requirement. Triton X-100 inhibited the activity of mPIK-I but rather weakly enhanced the mPIK-III activity, and sodium cholate remarkably inhibited both mPIK-I and mPIK-III activities. Their products were identified to be phosphatidylinositol 4-phosphate. On the other hand, about 20% of PI kinase activity was recovered from the cytosolic fraction and two activity peaks (cPIK-I and cPIK-II) were resolved on Mono Q column chromatography. There were no significant differences in biochemical properties between cPIK-I and cPIK-II. Both of them had Mr approx. 550000 as determined by gel-filtration chromatography and were activated by sodium cholate to a greater extent than by Triton X-100. The results suggest that the major PI kinases (mPIK-I and mPIK-III) are PI 4-kinase and mPIK-I is distinct from PI 4-kinases in other sources especially with regard to the effect of Triton X-100.

Original languageEnglish
Pages (from-to)120-126
Number of pages7
JournalBiochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
Volume1046
Issue number2
DOIs
Publication statusPublished - Sep 18 1990

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Endocrinology

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