Purification and properties of a protein that accumulates in the fat body of pre-diapausing larvae of the southwestern corn borer, Diatraea grandiosella

Abstract

A low molecular weight protein, the diapause-associated protein, which selectively accumulates in the fat body of last instar pre-diapausing larvae of the southwestern corn borer, Diatraea grandiosella, was purified and characterised. Gel filtration on Sephadex G-100 and chromatofocusing on Polybuffer Exchanger 96 were used to purify the protein and rocket immunoelectrophoresis showed that an overall yield of 10% was obtained. Electrophoresis under non-dissociating and dissociating conditions and visualization with silver stain revealed that the purified protein was present as a single polypeptide chain. Double immunodiffusion of the purified protein against antiserum generated in rabbits to the purified protein, and to proteins present in a whole fat body extract, yielded single fused bands indicating that the isolated protein was pure, and identical to the protein present in the fat body. Gel filtration and dissociating electrophoresis indicated that the protein has a mol. wt of approx. 35,000. Cation exchange chromatography revealed that the protein contains about 12% aromatic amino acids (24 residues tyrosine/mol, 12 residues phenylalanine/mol). The protein also has a pI of 6.1 at 24°C, a Stokes' radius of 25.3 × 10 −8 cm, a diffusion coefficient of 8.44 × 10 −7 cm 2/sec, a partial specific volume of 0.73 ml/g, a frictional ratio of 1.01 and an average hydrophobicity of 1032.4 cal/mol. These properties indicate that the protein has a compact globular structure. Since substantial amounts of the protein accumulate in the fat body before diapause begins, the protein serves as a storage molecule. The specific function of the protein, however, remains to be determined.