Identification of a 200 kDa polypeptide as type 3 phosphatidylinositol 4-kinase from bovine brain by partial protein and cDNA sequencing.

Two phosphatidylinositol 4-kinase isozymes, type 3 and type 2, have been separated on hydroxylapatite after solubilizing bovine brain microsomes with Triton X-114. Employing a newly developed renaturation procedure following SDS-PAGE, we demonstrate that a 200 kDa polypeptide carries the enzyme activity of this type 3 isoform. Chromatography on hydroxylapatite, Heparin-Sepharose, Superdex 200 and finally SDS-PAGE results in an approximately 30,000-fold purification. Tryptic peptides generated from the 200 kDa polypeptide after SDS-PAGE have been sequenced and the obtained data have been used for constructing and synthesizing degenerated oligonucleotides. Polymerase chain reaction as well as screening of cDNA libraries allowed several clones to be isolated from which a 4.7 kb contiguous sequence can be built up. The open reading frame covers 4.4 kb with a 0.3 kb untranslated 3' end which yields a deduced amino acid sequence of 1,467 amino acids. The C-terminal part of ca. 300 amino acids represents the catalytic domain. Sequence alignment of this domain with the mammalian counterpart, the human type 2 phosphatidylinositol 4-kinase, the yeast kinases STT4 and PIK1, as well as with the catalytic domains of bovine, human, mouse and yeast phosphatidylinositol 3-kinases reveals a high degree of identity: 26 of these approximately 300 amino acids are invariable in all of these eight catalytic domains. Five motifs indicate nuclear localization and DNA binding properties of the enzyme. Two leucine zipper motifs (amino acids 358-386, 862-882) are detectable. Furthermore, a helix loop helix motif (amino acids 716-729) as well as two nuclear localization signals (amino acids 838-854, 345-349) indicate the presence of the type 3 isoform in the nucleus.

Monoclonal Antibodies to Plant Growth Regulators: III. Zeatinriboside and Dihydrozeatinriboside.

Four high affinity monoclonal antibodies, which recognize two plant growth regulators from the cytokinin group, namely trans-zeatin riboside and dihydrozeatin riboside and their derivatives are reported. Six hybridomas were produced from three independent fusions of Balb/c spleen cells with P3-NS1-Ag 4-1 (abbreviated NS1) or X63-Ag 8.653 (X63) myeloma cells. The mice had been hyperimmunized with zeatin riboside-bovine serum albumin conjugate or dihydrozeatin riboside-bovine serum albumin conjugate for 3 months. The hybridomas secrete antibodies of the IgG 1 or IgG 2b subclass and allow the detection of femtomole amounts of the free cytokinins, their ribosides, and ribotides in plant extracts. The use of these monoclonals in radio- and enzyme-linked immunosorbent assay is also discussed.