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 an d purified from cultured sugarcane (Saccharum officinarum L.) cell lin e H50-7209. Autophosphorylation assays were used to detect pp18 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresi s (SDS-PACE). Only pp18 was detected by a brief in situ phosphorylatio n 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 P-32 label durin g in vitro phosphorylation experiments with highest levels present at shorter incubation times. Two major isoforms of the protein were ident ified in two-dimensional isoelectric focusing/SDS-PACE of crude extrac ts and microsomal fractions. The levels of pp18 were enhanced approxim ately 4-fold by heat shock at 36 degrees C and the elevated pp18 decay ed after heat shock was discontinued. pp18 was purified to apparent ho mogeneity, could be phosphorylated on serine residues, and also exhibi ted kinase-like activity toward histone H1. The amino acid sequence ob tained from a cyanogen bromide digest was greater than 80% identical t o nucleoside diphosphate (NDP) kinases from a variety of organisms. Bi ochemical analysis of the purified protein confirmed the identity as N DP kinase. Thus, NDP kinase appears to be modulated by heat shock in p lants.