Characterization of a low molecular mass autophosphorylating protein in cultured sugarcane cells and its identification as a nucleoside diphosphate kinase.
A low molecular mass (18 kD) phosphoprotein (pp18) was characterized and purified from cultured sugarcane (Saccharum officinarum L.) cell line H50-7209. Autophosphorylation assays were used to detect pp18 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Only pp18 was detected by a brief in situ phosphorylation method, whereas additional putative protein kinases were detected by an extended method. pp18 was present in both microsomal membrane and soluble fractions and exhibited anomalous turnover of 32P label during in vitro phosphorylation experiments with highest levels present at shorter incubation times. Two major isoforms of the protein were identified in two-dimensional isoelectric focusing/SDS-PAGE of crude extracts and microsomal fractions. The levels of pp18 were enhanced approximately 4-fold by heat shock at 36 degrees C and the elevated pp18 decayed after heat shock was discontinued. pp18 was purified to apparent homogeneity, could be phosphorylated on serine residues, and also exhibited kinase-like activity toward histone H1. The amino acid sequence obtained from a cyanogen bromide digest was greater than 80% identical to nucleoside diphosphate (NDP) kinases from a variety of organisms. Biochemical analysis of the purified protein confirmed the identity as NDP kinase. Thus, NDP kinase appears to be modulated by heat shock in plants.
Moisyadi
S
S
Department of Plant Molecular Physiology, University of Hawaii at Manoa, Honolulu 96822.
Dharmasiri
S
S
Harrington
H M
HM
Lukas
T J
TJ
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010944:Plants / Q000201:enzymology*; D051381:Rats; D017386:Sequence Homology, Amino Acid
D010750:Phosphoproteins; D009701:Nucleoside-Diphosphate Kinase