Identification and Microsequence Analysis of High Molecular Weight Proteins in Endosperm of the Rice Seed

Abstract

Summary Several high molecular weight (HMW) proteins (40 to 96 kD) extracted from endosperm of rice seed were separated by 20-PAGE, and visualized by CBB and silver staining. Molecular weight and pi standard markers were used to identify molecular weights and isoelectric focusing points of each protein, respectively. The proteins were electroblotted onto a PVDF membrane, and the partial amino acid sequences of HMW proteins were determined by automatic Edman degradation. The N-terminal sequences of six of these proteins (having molecular weights of about 74, 71, 60, 57, 51, and 43 kD) were determined, while the N-terminal of six other proteins (having molecular weights of 96, 69, 67, 54, 45, and 42 kD) could not be sequenced due to blockage of the N-terminus. Some internal amino acid sequences were also determined. Structural homology searches resulted in identification of 6 HMW proteins (enzymes), namely, starch debranching enzyme precursor, malate dehydrogenase, phosphoglycerate mutase, enolase, xylulose, and UDP-glucose pyrophosphorylase. In addition, three of the proteins appeared to be related to proteins in the heat shock family, i.e. chloroplast stroma protein (70 kD), HSP protein (70 kD), and chloroplast chaperonin (60 kD). Three proteins (60, 43 and 42 kD) were found to have no homology with those of known plant and animal proteins. All of these proteins were found to have a single polypeptide chain without intermolecular disulfide bonding.