Suborganellar Localization of Proteinase Catalyzing the Limited Hydrolysis of Pumpkin Globulin

Abstract

Protein bodies were prepared from the cotyledons of pumpkin (Cucurbita sp.) seeds by employng a nonaqueous isolation method. Light microscophic examination and the marker enzyme assays showed that the isolated protein bodies were intact and contamination with other cell organelles or cytoplasmic components was negligible. A proteolytic enzyme catalyzing the limited hydrolysis of carboxymethylated .gamma.'' chain of globulin was present in the protein bodies. The specific activity in the protein body (18 U/mg protein) was higher than that in the whole cell extract (13 U/mg protein), indicating that the limited proteolytic enzyme was localized in the protein body. After lysis of the protein bodies using hypotonic buffer solution, the suborganellar components (matrix, membranes and crystalloids) were separated by sucrose density gradient centrifugation. The crystalloid was composed of only globulin, a major seed protein. The major proteins of matrix and membrane fractions have MW of .apprx. 10,000. Approximately 90% of the limited proteolytic activity was present in the matrix region.