Purification and characterization of a phosphatidylinositol 4-kinase activator in carrot cells.
A phosphatidylinositol 4-kinase activator (PIK-A49) has been purified from carrot cells grown in suspension culture. The activator was purified from a soluble fraction using DEAE-Sepharose CL-6B and S-Sepharose chromatography columns. PIK-A49 has a relative molecular mass of 49 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The A50 for the activation of the Triton X-100-solubilized phosphatidylinositol 4-kinase fraction was 0.1 microM. Maximal activation was 3-4-fold. The analysis of the sequences of seven peptide fragments containing a total of 142 amino acid residues indicated that PIK-A49 was 69% identical to an actin-binding protein (ABP-50) from Dictyostelium and > 90% identical to elongation factor-1 alpha (EF-1 alpha) from carrot, tomato, and Arabidopsis. PIK-A49 bound actin and facilitated actin polymerization. Poly(U)-directed polyphenylalanine synthesis assays indicated that PIK-A49 had EF-1 alpha activity. The EF-1 alpha activity was enhanced by rabbit EF-1 beta gamma. Activation of phosphatidylinositol 4-kinase by a protein that binds actin and that has EF-1 alpha activity provides additional complexity to the signal transduction mechanisms involving inositol phospholipid metabolism.
Yang
W
W
Department of Botany, North Carolina State University, Raleigh 27695-7612.
Burkhart
W
W
Cavallius
J
J
Merrick
W C
WC
Boss
W F
WF
D019870:1-Phosphatidylinositol 4-Kinase; D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D008840:Microfilament Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010944:Plants / Q000378:metabolism*; D011072:Poly U / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D008840:Microfilament Proteins; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D010455:Peptides; D010940:Plant Proteins; D011072:Poly U; C012001:polyphenylalanine; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase